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Comprehensive analysis of lncRNA-mRNAs co-expression network identifies potential lncRNA biomarkers in cutaneous squamous cell carcinoma

Comprehensive analysis of lncRNA-mRNAs co-expression network identifies potential lncRNA... Background: Cutaneous squamous cell carcinoma (cSCC) is the second most common type of skin cancer, the prog- nosis for patients with metastatic cSCC remains relatively poor. Thus, there is an urgent need to identify new diagnos- tic, prognostic, and therapeutic targets and pathways in cSCC. Results: It detected a total of 37,507 lncRNA probes and 32,825 mRNA probes and found 3593 differentially expressed lncRNAs and 3236 differentially expressed mRNAs. It has been found that mRNAs ACY3, NR1D1, MZB1 has co-expression relationship with six lncRNAs, GXYLT1P3, LINC00348, LOC101928131, A-33-p3340852, A-21-p0003442 and LOC644838. Conclusions: The aim of this study is to identify cSCC-specific lncRNAs and indicated that six unstudied lncRNAs may serve an important role in endoplasmic reticulum stress apoptosis, autophagy and the progression of cSCC by modulating ACY3, NR1D1 and MZB1. Keywords: Cutaneous squamous cell carcinoma, Long non-coding RNA, Gene regulatory networks, Microarray analysis, Genome Introduction cumulative ultraviolet radiation (UVR) exposure [1]. Cutaneous squamous cell carcinoma (cSCC) is the sec- There were still over 2.1% of cSCC patients that devel - ond most common type of skin cancer, accounting for oped into lymphatic metastases [2]. Although clinical 20% of nonmelanoma skin cancers It is reported that features and dermoscopic findings could strongly suggest approximately 1.8 million incidences of cSCC in a global the diagnosis of cSCC, it is still necessary to get a patho- context in 2017. The etiology of cSCC is multifactorial, logical examination to confirm the diagnosis [3]. Moreo - including environmental, immunological, and genetic ver, there have no specific biomarkers for the diagnosis of factors. Of all these risk factors, the most important is cSCC. The therapeutic options for cSCC includes surgi - cal excision, radiation therapy, chemotherapy and immu- notherapy [4]. However, the prognosis for patients with *Correspondence: kunchen181@aliyun.com metastasis cSCC remains relatively poor. Thus, there is Yu Hu and Rong Li contributed equally to this work. 1 an urgent need to identify new diagnostic, prognostic, Department of Physiotherapy, Institute of Dermatology, Chinese Academy of Medical Sciences, Peking Union Medical College, and therapeutic targets and pathways in cSCC. 210042 Nanjing, China Full list of author information is available at the end of the article © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hu et al. BMC Genomics (2022) 23:274 Page 2 of 12 Long non-coding RNAs (lncRNAs) are a novel class participants had signed written informed consent prior of RNA molecules containing more than 200 nucleo- to recruitment. tides which have little or no protein-coding capabil- ity [5]. LncRNAs are involved in gene transcription and RNA extraction and quality control post-transcriptional translation by mediating target gene Total RNA was extracted from the cSCC samples and activation. Previous studies suggest that lncRNAs play normal skin tissues using a RNeasy Mini Kit (Qiagen, critical roles in numerous biological processes, including Hilden, Germany) according to the manufacturer’s pro- epigenetic regulation, cell apoptosis cell cycle and cell dif- tocol. RNA purity and concentration were quantified ferentiation regulation [6]. Recently, much attention has through Nano Drop ND-1000 (Thermo Fisher Scientific, focused on the role of lncRNAs in cancer since it could Waltham, MA) and RNA integrity was assessed by dena- affect various aspects of carcinogenesis, including cancer turing agarose gel electrophoresis for quality control. proliferation, invasion, metastasis and prognosis [2]. A recent review has reported that several dysregulation of RNA labeling and hybridization lncRNAs play an vital part in cSCC [3]. The expression The lncRNA Human Gene Expression Microarray V4.0 of HOTAIR, PICSAR, LINC00319, THOR, MALAT1 and (CapitalBio Corp, Beijing, China) was applied for this LINC10148 was upregulated in cSCCs and the expres- detection. The detection starts with the total RNA and sion of GAS5, TINCR and LINC00520 was downregu- performs amplification and fluorescent labeling in  vitro. lated in cSCCs [7, 8]. However, these certain lncRNAs In brief, the First Strand cDNA was synthesized from were mostly identified in in vitro or vivo studies and few total RNA and T7 Oligo(dT)Primer (containing a T7 lncRNAs studies clarified their roles in cSCC. The role of RNA polymerase promoter sequence) and T7-specific lncRNAs and their overall contributions to the pathogen- primers by using First Strand Enzyme Mix. Then the esis of cSCC are still unknown. Thus, it is of great impor - Second Strand DNA was synthesized by converting the tance to explore the potential biological effects of specific RNA strand in the DNA-RNA hybrid into Second Strand lncRNAs on cSCC, which could be used as biomarker for cDNA using Second Strand Enzyme Mix. cRNA was syn- early diagnosis and prognosis prediction. thesized from Second Strand cDNA by using T7 Enzyme In order to explore the expression profiles of dysregu - Mix. High yields of Cy3- and Cy5-labeled cDNAs were lated lncRNAs in cSCC and to reveal the potential roles produced using the CapitalBio cRNA Amplification and of lncRNAs in the pathogenesis of cSCC, we performed Labeling Kit (CapitalBio, Beijing, China). The labeled microarray analysis to identify dysregulated lncRNAs products were used for microarray hybridization. and mRNAs. Also, we intended to construct a lncRNA- mRNA correlation network for cSCC, which helps to Microarray assay establish bridges between lncRNAs and mRNAs to reveal The global profiling of human lncRNA and mRNA the potential functional involvement of lncRNAs in cSCC expression were performed with the CapitalBio Tech- pathobiology. Furthermore, the expressions of these dif- nology Human lncRNA Array V4 (CapitalBio, Beijing, ferentially expressed lncRNAs are verified by quantita - China). The acquired tiff format images were obtained tive real-time PCR (qRT-PCR). The aim of this study is to using Agilent Feature Extraction (V10.7). Quantile nor- identify cSCC-specific lncRNAs and to clarify the molec - malization, quality control and subsequent data pro- ular mechanism of cSCC, which could provide potential cessing were performed using the Agilent GeneSpring biomarkers and therapeutic targets for cSCC. software (V13.0). The differentially expressed lncRNAs and mRNAs were identified via Volcano plot filtering with the threshold set of fold change ≥2.0 and P-value < 0.05. Further hierarchical clustering analysis was per- Materials and methods formed to present lncRNA and mRNA expression Samples patterns. Primary carcinoma tissues and adjacent tissues were obtained from six patients with cutaneous squamous cell Gene ontology and pathway enrichment analysis carcinoma who underwent surgical treatment at Institute Gene ontology (GO) analysis (http:// www. geneo ntolo gy. of Dermatology, Chinese Academy of Medical Sciences. org) is frequently used in functional enrichment studies All samples from patients were pathologically diagnosed of large-scale genes and categorizes the roles of mRNAs as cSCC and stored in RNA later at -80℃ until RNA into three domains, including biological process, cel- extraction. Besides, six normal skin tissues were obtained lular component and molecular function. GO analysis from six healthy individuals. The study protocol was was performed to identify the potential functions of dif- approved by the ethics committee of the Institute of Der- ferentially expressed genes. Pathway analysis was applied matology, Chinese Academy of Medical Sciences. All Hu  et al. BMC Genomics (2022) 23:274 Page 3 of 12 to reveal potential biological pathways associated with Quantitative RT‑PCR differentially expressed genes according to the KEGG Complementary DNA was synthesized using the (Kyoto Encyclopedia of Genes and Genomes) database Reverse Transcription Kit (Takara, China). qRT-PCR (http:// www. genome. ad. jp/ kegg/). was performed using the SYBR Green One Step qPCR Kit (Biotool) on the ABI 7500 Real-Time PCR machine (Applied Biosystems). Primers used for qPT-PCR are listed in Table  1. For quantitative results, 2 − ΔΔCt LncRNA‑mRNA correlation analysis method was used to calculate the relative expression level To reveal the association between the lncRNAs with of each lncRNA. direct regulated expression of target mRNAs, we con- ducted the co-expression analysis. The co-expressed lncRNAs and mRNAs were selected with the standard Statistical analysis of Pearson correlation > 0.99 or < −0.99 and P value < All statistical analyses were performed using the SPSS 0.05. The top 1000 gene pairs were used to construct version 17.0 software (SPSS, Inc., Chicago, USA). Data the coding-non-coding gene co-expression network were analyzed by two-tailed Student’s t test. P<0.05 was using bioinformatics Cytoscape software. considered as a statistically significant difference. Results Target prediction Identification of differentially expressed lncRNAs Target prediction can be divided into cis-prediction and mRNAs in cSCC and trans-prediction. Cis-prediction could predict the The microarray analysis revealed that the global pro - co-expressed lncRNAs and mRNAs through the posi- filing of differentially expressed lncRNAs and mRNAs tional comparison of lncRNAs and mRNAs. Trans- between cSCC and healthy control. From the lncRNA prediction could predict the possible relationships expression profiles, 3593 differentially expressed lncR - through sequence alignment. Based on the results of NAs and 3236 differentially expressed mRNAs were per - the correlation analysis of lncRNA and mRNA (Cor- formed with the threshold of fold-change ≥2.0 and P≤ relation>0.99 or Correlation<-0.99, and P value<0.05), 0.05, with the results are shown in Table S 1. Hierarchical cis-prediction looks for lncRNA-mRNA pairs whose Clustering was applied to group lncRNAs and mRNAs genome position is within 10 kb. Trans-prediction was based on their expression levels (Fig.  1A and B). Among conducted using blat tool to select lncRNA-mRNA all the differentially expressed LncRNAs and mRNAs, pairs with similar sequences by comparing lncRNA 1335 lncRNAs and 1411 mRNAs were upregulated and and mRNA (3’UTR) sequences. 2258 lncRNAs and 1825 mRNAs were downregulated Table 1 Primers used for reverse transcription-quantitative PCR lncRNA Forward primer (5′→3′) Reverse primer (5′→3′) PVT1TGA GAA CTG TCC TTA CGT GACC AGA GCA CCA AGA CTG GCT CT CTD-2521M24.9CTC GTT CTT AGG CAG CAT CTG TGT CTGG AGA CTG AAA GGT GGT GTT GAT TG AL353997.3GTG AAA CCT CAG ATG CCC ATT TGT AAC CCT CTT TGG TGC CTG GTG CTATG MIR4720GGA ACC TGG CAC CAC ACT AAACC GGG AAT GAA CAC GAA TAC GGA ATA AGC BX004987.5CAT CCT GGC TAA CTC AGT GAA ACC CCAC ACC ATT ATC CTG CCT CAG TCT C CTD-2619J13.13CAA TGT CTG CCT GCC TAT CCA CTG GAA GAG AAA GAG GAG ATT GGC TGA GG LINC00478TGC CTT AAC TGA TGA CAT TCC ACC ACAGC CAG GAT GAG CCA GGA GAAG GAPDHTGC CTT AAC TGA TGA CAT TCC ACC ACAGC CAG GAT GAG CCA GGA GAAG lncRNA long non-coding RNA (See figure on next page.) Fig. 1 Differentially expressed lncRNAs and mRNAs in CSCC. The expression of lncRNAs and mRNAs were compared between CSCC and normal cutaneous tissue specimens. Hierarchical Clustering was applied to group lncRNAs and mRNAs based on their expression levels (A and B). The horizontal axis represents fold of change in expression (on a log2 scale) and the vertical axis represents P-value (on a negative log10 scale). The scatter plot and volcano plot of lncRNAs (C and E) and mRNAs (D, F) illustrate the difference in the expression levels of each transcript. The red and green dots represent transcripts significantly upregulated and downregulated in cSCC, respectively. lncRNA, long non-coding RNA; cSCC, cutaneous squamous cell carcinoma Hu et al. BMC Genomics (2022) 23:274 Page 4 of 12 Fig. 1 (See legend on previous page.) Hu  et al. BMC Genomics (2022) 23:274 Page 5 of 12 in cSCC cancer tissues compared to normal skin tissues. the three most significant molecular functions of the dys - The scatter plot and volcano plot of these probe-matched regulated mRNAs (Fig. 2A). gene expressions of LncRNA and mRNA shows the up- Pathway analysis was carried out based on the KEGG, regulated and down-regulated lncRNAs (Fig.  1C and E) BioCyc and Panther database. The top 20 enrichment and mRNAs (Fig. 1D, F) profiling across groups. The red score values of the enriched pathways by KEGG pathway dots and green dots stand for up-regulated and down- (Fig. 2B) analysis and Panther pathway analysis (Fig. 2 C). regulated expressions, respectively. The dysregulated mRNAs were most associated with “Cytokine-cytokine receptor interaction”, “Jak-STAT sign- Gene ontology and pathway analyses aling pathway” and “Retinol metabolism” in KEGG path- GO analysis was conducted to classify differentially way. The dysregulated mRNAs were most associated with expressed mRNA into three categories, including biologi- “Plasminogen activating cascade”, “Blood coagulation”, cal processes, cellular components and molecular func- “Cadherin signaling pathway”, “Nicotine degradation” tion. Among the involved biological processes, cellular and “Jak-STAT signaling pathway” in Panther pathway. process, single-organism process, biological regulation, regulation of biological processes and metabolic pro- LncRNA‑mRNA co‑expression network cesses, were the five most significant processes associated We first constructed genes co-expression networks with the dysregulated mRNAs. Among the cellular com- between the cSCC group and normal skin group with ponents, cell, cell parts and organelle were the three most the all differential lncRNAs and mRNAs in this study. significant component processes associated with the dys - Among these, the lncRNAs and mRNAs were selected regulated mRNAs. Among molecular functions, binding, with the threshold set of Pearson’s correlation coeffi- catalytic activity and molecular function regulator were cients >0.99 or <-0.99 and P-value <0.05. Subsequently, Fig. 2 Gene ontology and KEGG pathway analyses. A Top 20 GO terms with significantly differential expression from GO analysis are categorized into biological process (blue), cellular component (green) and molecular function (red). B Top20 KEGG pathways with significantly differential expression are shown. C Top20 PANTHER pathways with significantly differential expression are shown. Vertical axis represents LgP values indicate significance of the enrichment. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; lncRNA, long non-coding RNA; cSCC, cutaneous squamous cell carcinoma Hu et al. BMC Genomics (2022) 23:274 Page 6 of 12 The target mRNA of LncRNA prediction and functional the top 1000 enriched gene pairs of lncRNAs and analysis mRNAs, correlation coefficient was constructed to To investigate whether the differentially expressed build the co-expression network by using cytoscape lncRNAs regulate genes and determine the signaling program, as shown in Fig. 3. In the co-expression net- pathways associated with cSCC, target prediction was work, A_33_P3340852 was obtained the highest num- performed to predict the possible targets of the dysregu- ber of interactions. lated lncRNAs. Based on the results of the co-expression network of lncRNAs and mRNAs (Correlation>0.99 or Fig. 3 Construction of the lncRNA-mRNA co-expression network. Green nodes represented for LncRNA, yellow rodes represent for the correlated mRNA, each circle’s size indicates relative number of related genes. The blue lines indicate negative correlations and the red lines are positive correlations Hu  et al. BMC Genomics (2022) 23:274 Page 7 of 12 Fig. 4 The target mRNA of LncRNA prediction and functional analysis. A Top 20 GO terms of the targeted mRNA with significantly differential expression from GO analysis are categorized into biological process (blue), cellular component (green) and molecular function (red). B Top20 KEGG pathways of the targeted mRNA with significantly differential expression are shown. Vertical axis represents LgP values indicate significance of the enrichment.GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; lncRNA, long non-coding RNA; CSCC, cutaneous squamous cell carcinoma Correlation<-0.99, and P < 0.05), cis-prediction looks of lncRNAs with top 1000 correlation coefficient were for lncRNA-mRNA pairs whose genome position is shown in Fig. 4. within 10  kb, and trans-prediction compares lncRNA Then we performed GO enrichment and KEGG path - and mRNA (3’UTR) sequences, and selects lncRNA- way analyses on the target mRNAs (Fig. 4). Through GO mRNA pairs with similar sequences. The target genes analysis, we found that the target expressed mRNAs were enriched for cellular process, single-organism process, Hu et al. BMC Genomics (2022) 23:274 Page 8 of 12 Fig. 5 Validation of the expression lncRNAs by qRT-PCR. The expression of top 7 differentially lncRNAs are compared between six paired cSCC tissues ,matched adjacent and normal tissue samples. There are significant difference between cSCC tissues and normol tissue. *P < 0.05 ,**P < 0.01 and ****P < 0.0001. RT-qPCR, reverse transcription-quantitative PCR; N, distant normal cutaneous tissue ; P, para-tumor tissue; T, tumor tissue biological regulation linked with biological processes, RT‑qPCR validation of differentially expressed lncRNAs and cell, cell part, organelle involved in cellular compo- The top 7 DE lncRNAs (Table  1) were selected for RT- nents, as well as binding, catalytic activity, nucleic acid qPCR analysis to verify the microarray results in six binding transcription factor activity in molecular func- paired cSCC tissues, matched adjacent and normal tissue tions (Fig.  4A). For the KEGG pathway analysis, the top samples. The RT-qPCR results demonstrated that three 20 enrichment score values of the enriched pathways lncRNAs (PVT1,CTD-2521M24.9 and AL353997.3) included measles, tight junction, circadian rhythm, were upregulated in cSCC and four lncRNAs hedgehog signaling pathway and histidine metabolism (MIR4720,BX004987.5,CTD-2619J13.13andLINC00478) (Fig. 4B). were downregulated in cSCC. As shown in Fig. 5, the rel- ative values of the expression levels detected by RT-qPCR were found to be consistent with the microarray data. Hu  et al. BMC Genomics (2022) 23:274 Page 9 of 12 This result suggests that the transcript identification and downregulated lncRNAs and three upregulated lncR- abundance estimates were highly reliable. NAs, were confirmed expressions by real-time PCR. Results of the qRT-PCR were consistent with those Discussion trends of high-throughput sequencing, proving its reli- Recent studies have revealed the significant role of ability. Of these verified lncRNAs in the present report, lncRNA in various types of cancer for tumorigenesis some have been identified as oncogenic lncRNA in car - and development, such as non-small-cell lung cancer, cinogenesis. For instance, the p29508 probe detected liver cancer, bladder cancer and breast cancer [9–11]. lncRNA PVT1, a top overexpressed lncRNA in micro- Moreover, numerous lncRNAs play an important role array analysis [27]. Accumulating evidence suggests that in the onset and progression of diseases by affecting the PVT1 locus as an epigenetic enhancer in colorectal cell proliferation ,invasion and other functions [12]. As cancer (CRC) and it has a regulatory effect on regulat - the second most common cancer worldwide ,cSCC has ing the expression of MYC. Furthermore, PVT1 lncRNA been reported with an annual accidence with over one expression mediated through aberrant methylation may million individuals [13–15]. UV irradiation, particu- also impact TGFβ/SMAD and Wnt/β-Catenin pathways larly UVB, is one of the classical cellular stressors caus- by the key CRC gene. Thus, high expression of the PVT1 ing DNA damage in skins, which receives chronic sun lncRNA may serve a pivotal role in the pathogenesis of exposure, and ultimately leading to skin development of CRC and has demonstrated roles in several aspects, as a cSCC [16]. Therefore, many present studies investigated prognostic biomarker and a potential therapeutic target. the potential oncogenic role of molecules and pathways Moreover, upregulated PVT1 can activate Wnt/β-catenin in cSCC and tried to evaluate the promising diagnostic signaling pathway by regulating expression of both Pygo2 biomarkers value for cSCC [17–20]. It is currently aimed and ATG14 and thus promote autophagy related com- to elucidate DNA binding/differentiation 4 (ID4) func - plex in pancreatic cancer [28]. Similarly,it has already tion in cSCC development [21]. The results indicate that been reported in anothor study that lncRNA PVT1 UVB irradiation leads to abnormal downregulated ID4 inhibite the proliferation of gastric cancer by combining via DNA methylation and ID4 acts as a tumor suppres- with enhancer of zeste homolog 2 (EZH2) to repress p15 sor gene in tumorigenesis. In addition, reports indicates and p16 [29]. Thus, we hypothesize that lncRNA PVT1 that the phosphoinositide3-kinases (PI3Ks)/Akt pathway upregulation may serve as an oncogenic gene in cSCC by plays an essential role in advanced cSCC, which inhibit combing EZH2 to activate Wnt/β-catenin pathways and viability and growth of cSCC and can also prove to be promote tumorigenic process of proliferation, autophagy valid target in cSCC [22]. Some dysregulated transcrip- and apoptosis aspects. Besides, LINC00478 has been tion factors (MYC, RELA, ETS1, SP1, TP63, TP53, AP1, identified to be highly link to accurate classification of TCF3, SOX2, OCT-3/4) and downregulated epidermal subtypes of breast cancer by facilitating expression of rel- differentiation genes (LCE1D, FLG, KRT77, KRT10, evant miRNA profiles [30]. ALOEX3 etc.) are all altered at the transcript level in LncRNA is a class of nucleotides that transcription cSCC [23–26]. However, the underlying molecular mech- length are more than 200 nt and which have little or no anism of LncRNAs in cSCC are rarely mentioned and protein-coding capability [5, 31, 32]. These molecules still remains unclear. play crucial roles in several aspects of biological func- To investigate whether lncRNA is involved in the tions as chromatin remodeling, gene transcription level carcinogenic process of the development of cSCC, we regulation and protein modification during the occur - performed lncRNA microarray to compare the com- rence and development of many diseases [33, 34]. An prehensive lncRNA expression profiles in three paired increasing number of studies described that dysregu- cSCC tissues and normal skin samples. In this study, lated lncRNA expression is associated with various through Hierarchical Clustering, we identified differen - cancers and may contribute to tumorigenesis, metasta- tial expression of lncRNAs and mRNAs in cSCC tissues sis and prognosis of many tumors [35]. Recent studies and predicted the potential biological function, cellu- demonstrated that lncRNAs can be utilized as a disease lar progress and enriched pathways by GO, KEGG and biomarkers and biomarkers for survival prediction in cer- Path-net analysis. It detected a total of 37,507 lncRNA vical cancer, colorectal cancer and skin cancers [36–38]. probes and 32,825 mRNA probes and found 3593 dif- Available evidence has reported that comprehensive ferentially expressed lncRNAs and 3236 differentially analysis of interaction between lncRNAs and mRNAs expressed mRNAs. The selection criteria, fold change may provide a reference for further explore its biologi- >2 and P < 0.05, ensured the significance of the dif - cal functions and the potential underlying mechanisms ferential expression data. Among these differentially in cancers [39]. In order to better study whether lncRNA expression lncRNAs, the top 7 lncRNAs, including four is involved in cSCC tumorigenesis, we constructed Hu et al. BMC Genomics (2022) 23:274 Page 10 of 12 co-expression network of top 1000 pairs of lncRNAs co-expression network ,which mainly were focused and mRNAs to annotate clear biological functions and on endoplasmic reticulum stress of apoptosis ,cellular regulatory mechanism of lncRNAs [40]. After construc- signal transduction and autophagy in tumorgenesis, as tion of the co-expression network, it has been found aforementioned. Since each results were individually that mRNAs ACY3, NR1D1, MZB1 has co-expression analyzed and relationships of co-expressed lncRNA- relationship with six lncRNAs, GXYLT1P3, LINC00348, mRNA and pathway analysis are independent, this LOC101928131, A-33-p3340852, A-21-p0003442 and outcome greatly supports the reliability of the cur- LOC644838 which largely have not been studied before. rent study. Therefore, it was hypothesized that six Otherwise, NR1D1is reported to showed significant lncRNAs, GXYLT1P3, LINC00348, LOC101928131, correlation with regulation of autophagy activities by A-33-p3340852, A-21-p0003442 and LOC644838, in microphages, metabolic process of stress response, signal the co-expression network may serve a considerable transduction and inflammatory pathways [41, 42]. The role in the progression of cSCC by regulating apoptosis results of study suggested that MZB1 expression plays an induced by endoplasmic reticulum stress, cellular sig- critical role in advanced stage of breast cancer and may nal transduction and autophagy. be a poor prognostic marker,which interact with endo- In conclusion, the present study revealed a com- plasmic reticulum stress related pathways [43]. ACY3 has prehensive analysis of lncRNA-mRNA co-expression rarely been reported but also may contribute to patho- profiles of patients with cutaneous squamous cell car - genesis of Huntington disease (HD) by altering binding cinoma. GO, KEGG and PANTHER analyses provided of transcriptional factors [44]. Within the co-expression the function of mRNAs and suggested the possible bio- network, that NR1D1 was noted that are connected with logical effects of lncRNAs. The results indicated that six the maximum number (six) of lncRNAs, indicating that lncRNAs, which have not been studied in cSCC,may it may play a significant role in the development and serve an important role in endoplasmic reticulum stress progression of cSCC. The dysregulation of these three apoptosis, autophagy and the progression of cSCC by transcripts mainly contribute to modulating apoptosis modulating ACY3, NR1D1 and MZB1. However, there induced by endoplasmic reticulum stress, physiologi- are still some defects which need to be improved to cal processes of cellular signal transduction and meta- carry out more comprehensive research, including bolic regulation, inflammatory signaling pathways and small study size containing only six tissues and the lack autophagy. of experimental verification in liquid biopsies (plasma/ In the present study ,the enrichment analy- serum)of cSCC.These data argue for further intensive sis of GO,KEGG and Panther pathway identified research to provide a more comprehensive under- that“Cytokine-cytokine receptor interaction”, “Jak-STAT standing of the molecular mechanisms underlying the signaling pathway”,“Plasminogen activating cascade ”and metabolism of cSCC. “Cadherin signaling pathway” were the top four signifi - cantly changed pathways. It has been reported that Jak- Supplementary Information STAT signaling pathway can contribute to resistance of The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s12864- 022- 08481-0. cervical squamous cell carcinoma by mediating key acti- vator protein as STAT1/STAT2 [19]. Jak-STAT signal- Additional file 1. ing pathway inhibition is involved in anti-tumor activity Additional file 2. in several pre-clinical studies and continued activation of STAT1 and STAT2,which are essential components of JAK/STAT pathway, are associated with suppressing Acknowledgements Not applicable. tumor apoptosis [45, 46]. Meanwhile, the present study found that exogenous IL-6 can induce activation of JAK/ Authors’ contributions STAT signaling and to increase autophagy [47]. Further- YH,RL,DH and KC conceived and designed the study. YH,RL,DH and HC collected the samples and acquired the data. LC,XZ and LL interpreted or ana- more, Cadherin signaling pathway can act as an intracel- lyzed data. YH and RL prepared the manuscript, which was revised for impor- lular signal transducer and was involved in invasion and tant intellectual content by DH and KC. All authors reviewed and approved metastasis by Wnt/β-catenin pathways inducing EMT the final manuscript. DH and KC confirm the authenticity of all the raw data. expression profiles in tumor progression [48]. Funding In fact, four of significant enriched pathways, This study was supported by grants from the National Natural Science Foun- including the“Cytokine-cytokine receptor interac- dation of China (82073445 and 81903247), the Natural Science Foundation of Jiangsu Province (BK20190145 and BK20210049), the Nanjing Incubation tion”, “Jak-STAT signaling pathway”,“Plasminogen acti- Program for National Clinical Research Center (2019060001) and CAMS Inno- vating cascade” and “Cadherin signaling pathway”, vation Fund for Medical Sciences(CIFMS-2021-I2M-1-001). were coincided with the pathogenesis mechanism of Hu  et al. BMC Genomics (2022) 23:274 Page 11 of 12 Availability of data and materials Potential Prospects for Treatment Evaluation and Prognostic Prediction. All data generated or analyzed during this study are included in this published Front Oncol 2020;10:590352. article. 12. Hu W, Li H, Wang S. 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Comprehensive analysis of lncRNA-mRNAs co-expression network identifies potential lncRNA biomarkers in cutaneous squamous cell carcinoma

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Springer Journals
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Copyright © The Author(s) 2022
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10.1186/s12864-022-08481-0
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Abstract

Background: Cutaneous squamous cell carcinoma (cSCC) is the second most common type of skin cancer, the prog- nosis for patients with metastatic cSCC remains relatively poor. Thus, there is an urgent need to identify new diagnos- tic, prognostic, and therapeutic targets and pathways in cSCC. Results: It detected a total of 37,507 lncRNA probes and 32,825 mRNA probes and found 3593 differentially expressed lncRNAs and 3236 differentially expressed mRNAs. It has been found that mRNAs ACY3, NR1D1, MZB1 has co-expression relationship with six lncRNAs, GXYLT1P3, LINC00348, LOC101928131, A-33-p3340852, A-21-p0003442 and LOC644838. Conclusions: The aim of this study is to identify cSCC-specific lncRNAs and indicated that six unstudied lncRNAs may serve an important role in endoplasmic reticulum stress apoptosis, autophagy and the progression of cSCC by modulating ACY3, NR1D1 and MZB1. Keywords: Cutaneous squamous cell carcinoma, Long non-coding RNA, Gene regulatory networks, Microarray analysis, Genome Introduction cumulative ultraviolet radiation (UVR) exposure [1]. Cutaneous squamous cell carcinoma (cSCC) is the sec- There were still over 2.1% of cSCC patients that devel - ond most common type of skin cancer, accounting for oped into lymphatic metastases [2]. Although clinical 20% of nonmelanoma skin cancers It is reported that features and dermoscopic findings could strongly suggest approximately 1.8 million incidences of cSCC in a global the diagnosis of cSCC, it is still necessary to get a patho- context in 2017. The etiology of cSCC is multifactorial, logical examination to confirm the diagnosis [3]. Moreo - including environmental, immunological, and genetic ver, there have no specific biomarkers for the diagnosis of factors. Of all these risk factors, the most important is cSCC. The therapeutic options for cSCC includes surgi - cal excision, radiation therapy, chemotherapy and immu- notherapy [4]. However, the prognosis for patients with *Correspondence: kunchen181@aliyun.com metastasis cSCC remains relatively poor. Thus, there is Yu Hu and Rong Li contributed equally to this work. 1 an urgent need to identify new diagnostic, prognostic, Department of Physiotherapy, Institute of Dermatology, Chinese Academy of Medical Sciences, Peking Union Medical College, and therapeutic targets and pathways in cSCC. 210042 Nanjing, China Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hu et al. BMC Genomics (2022) 23:274 Page 2 of 12 Long non-coding RNAs (lncRNAs) are a novel class participants had signed written informed consent prior of RNA molecules containing more than 200 nucleo- to recruitment. tides which have little or no protein-coding capabil- ity [5]. LncRNAs are involved in gene transcription and RNA extraction and quality control post-transcriptional translation by mediating target gene Total RNA was extracted from the cSCC samples and activation. Previous studies suggest that lncRNAs play normal skin tissues using a RNeasy Mini Kit (Qiagen, critical roles in numerous biological processes, including Hilden, Germany) according to the manufacturer’s pro- epigenetic regulation, cell apoptosis cell cycle and cell dif- tocol. RNA purity and concentration were quantified ferentiation regulation [6]. Recently, much attention has through Nano Drop ND-1000 (Thermo Fisher Scientific, focused on the role of lncRNAs in cancer since it could Waltham, MA) and RNA integrity was assessed by dena- affect various aspects of carcinogenesis, including cancer turing agarose gel electrophoresis for quality control. proliferation, invasion, metastasis and prognosis [2]. A recent review has reported that several dysregulation of RNA labeling and hybridization lncRNAs play an vital part in cSCC [3]. The expression The lncRNA Human Gene Expression Microarray V4.0 of HOTAIR, PICSAR, LINC00319, THOR, MALAT1 and (CapitalBio Corp, Beijing, China) was applied for this LINC10148 was upregulated in cSCCs and the expres- detection. The detection starts with the total RNA and sion of GAS5, TINCR and LINC00520 was downregu- performs amplification and fluorescent labeling in  vitro. lated in cSCCs [7, 8]. However, these certain lncRNAs In brief, the First Strand cDNA was synthesized from were mostly identified in in vitro or vivo studies and few total RNA and T7 Oligo(dT)Primer (containing a T7 lncRNAs studies clarified their roles in cSCC. The role of RNA polymerase promoter sequence) and T7-specific lncRNAs and their overall contributions to the pathogen- primers by using First Strand Enzyme Mix. Then the esis of cSCC are still unknown. Thus, it is of great impor - Second Strand DNA was synthesized by converting the tance to explore the potential biological effects of specific RNA strand in the DNA-RNA hybrid into Second Strand lncRNAs on cSCC, which could be used as biomarker for cDNA using Second Strand Enzyme Mix. cRNA was syn- early diagnosis and prognosis prediction. thesized from Second Strand cDNA by using T7 Enzyme In order to explore the expression profiles of dysregu - Mix. High yields of Cy3- and Cy5-labeled cDNAs were lated lncRNAs in cSCC and to reveal the potential roles produced using the CapitalBio cRNA Amplification and of lncRNAs in the pathogenesis of cSCC, we performed Labeling Kit (CapitalBio, Beijing, China). The labeled microarray analysis to identify dysregulated lncRNAs products were used for microarray hybridization. and mRNAs. Also, we intended to construct a lncRNA- mRNA correlation network for cSCC, which helps to Microarray assay establish bridges between lncRNAs and mRNAs to reveal The global profiling of human lncRNA and mRNA the potential functional involvement of lncRNAs in cSCC expression were performed with the CapitalBio Tech- pathobiology. Furthermore, the expressions of these dif- nology Human lncRNA Array V4 (CapitalBio, Beijing, ferentially expressed lncRNAs are verified by quantita - China). The acquired tiff format images were obtained tive real-time PCR (qRT-PCR). The aim of this study is to using Agilent Feature Extraction (V10.7). Quantile nor- identify cSCC-specific lncRNAs and to clarify the molec - malization, quality control and subsequent data pro- ular mechanism of cSCC, which could provide potential cessing were performed using the Agilent GeneSpring biomarkers and therapeutic targets for cSCC. software (V13.0). The differentially expressed lncRNAs and mRNAs were identified via Volcano plot filtering with the threshold set of fold change ≥2.0 and P-value < 0.05. Further hierarchical clustering analysis was per- Materials and methods formed to present lncRNA and mRNA expression Samples patterns. Primary carcinoma tissues and adjacent tissues were obtained from six patients with cutaneous squamous cell Gene ontology and pathway enrichment analysis carcinoma who underwent surgical treatment at Institute Gene ontology (GO) analysis (http:// www. geneo ntolo gy. of Dermatology, Chinese Academy of Medical Sciences. org) is frequently used in functional enrichment studies All samples from patients were pathologically diagnosed of large-scale genes and categorizes the roles of mRNAs as cSCC and stored in RNA later at -80℃ until RNA into three domains, including biological process, cel- extraction. Besides, six normal skin tissues were obtained lular component and molecular function. GO analysis from six healthy individuals. The study protocol was was performed to identify the potential functions of dif- approved by the ethics committee of the Institute of Der- ferentially expressed genes. Pathway analysis was applied matology, Chinese Academy of Medical Sciences. All Hu  et al. BMC Genomics (2022) 23:274 Page 3 of 12 to reveal potential biological pathways associated with Quantitative RT‑PCR differentially expressed genes according to the KEGG Complementary DNA was synthesized using the (Kyoto Encyclopedia of Genes and Genomes) database Reverse Transcription Kit (Takara, China). qRT-PCR (http:// www. genome. ad. jp/ kegg/). was performed using the SYBR Green One Step qPCR Kit (Biotool) on the ABI 7500 Real-Time PCR machine (Applied Biosystems). Primers used for qPT-PCR are listed in Table  1. For quantitative results, 2 − ΔΔCt LncRNA‑mRNA correlation analysis method was used to calculate the relative expression level To reveal the association between the lncRNAs with of each lncRNA. direct regulated expression of target mRNAs, we con- ducted the co-expression analysis. The co-expressed lncRNAs and mRNAs were selected with the standard Statistical analysis of Pearson correlation > 0.99 or < −0.99 and P value < All statistical analyses were performed using the SPSS 0.05. The top 1000 gene pairs were used to construct version 17.0 software (SPSS, Inc., Chicago, USA). Data the coding-non-coding gene co-expression network were analyzed by two-tailed Student’s t test. P<0.05 was using bioinformatics Cytoscape software. considered as a statistically significant difference. Results Target prediction Identification of differentially expressed lncRNAs Target prediction can be divided into cis-prediction and mRNAs in cSCC and trans-prediction. Cis-prediction could predict the The microarray analysis revealed that the global pro - co-expressed lncRNAs and mRNAs through the posi- filing of differentially expressed lncRNAs and mRNAs tional comparison of lncRNAs and mRNAs. Trans- between cSCC and healthy control. From the lncRNA prediction could predict the possible relationships expression profiles, 3593 differentially expressed lncR - through sequence alignment. Based on the results of NAs and 3236 differentially expressed mRNAs were per - the correlation analysis of lncRNA and mRNA (Cor- formed with the threshold of fold-change ≥2.0 and P≤ relation>0.99 or Correlation<-0.99, and P value<0.05), 0.05, with the results are shown in Table S 1. Hierarchical cis-prediction looks for lncRNA-mRNA pairs whose Clustering was applied to group lncRNAs and mRNAs genome position is within 10 kb. Trans-prediction was based on their expression levels (Fig.  1A and B). Among conducted using blat tool to select lncRNA-mRNA all the differentially expressed LncRNAs and mRNAs, pairs with similar sequences by comparing lncRNA 1335 lncRNAs and 1411 mRNAs were upregulated and and mRNA (3’UTR) sequences. 2258 lncRNAs and 1825 mRNAs were downregulated Table 1 Primers used for reverse transcription-quantitative PCR lncRNA Forward primer (5′→3′) Reverse primer (5′→3′) PVT1TGA GAA CTG TCC TTA CGT GACC AGA GCA CCA AGA CTG GCT CT CTD-2521M24.9CTC GTT CTT AGG CAG CAT CTG TGT CTGG AGA CTG AAA GGT GGT GTT GAT TG AL353997.3GTG AAA CCT CAG ATG CCC ATT TGT AAC CCT CTT TGG TGC CTG GTG CTATG MIR4720GGA ACC TGG CAC CAC ACT AAACC GGG AAT GAA CAC GAA TAC GGA ATA AGC BX004987.5CAT CCT GGC TAA CTC AGT GAA ACC CCAC ACC ATT ATC CTG CCT CAG TCT C CTD-2619J13.13CAA TGT CTG CCT GCC TAT CCA CTG GAA GAG AAA GAG GAG ATT GGC TGA GG LINC00478TGC CTT AAC TGA TGA CAT TCC ACC ACAGC CAG GAT GAG CCA GGA GAAG GAPDHTGC CTT AAC TGA TGA CAT TCC ACC ACAGC CAG GAT GAG CCA GGA GAAG lncRNA long non-coding RNA (See figure on next page.) Fig. 1 Differentially expressed lncRNAs and mRNAs in CSCC. The expression of lncRNAs and mRNAs were compared between CSCC and normal cutaneous tissue specimens. Hierarchical Clustering was applied to group lncRNAs and mRNAs based on their expression levels (A and B). The horizontal axis represents fold of change in expression (on a log2 scale) and the vertical axis represents P-value (on a negative log10 scale). The scatter plot and volcano plot of lncRNAs (C and E) and mRNAs (D, F) illustrate the difference in the expression levels of each transcript. The red and green dots represent transcripts significantly upregulated and downregulated in cSCC, respectively. lncRNA, long non-coding RNA; cSCC, cutaneous squamous cell carcinoma Hu et al. BMC Genomics (2022) 23:274 Page 4 of 12 Fig. 1 (See legend on previous page.) Hu  et al. BMC Genomics (2022) 23:274 Page 5 of 12 in cSCC cancer tissues compared to normal skin tissues. the three most significant molecular functions of the dys - The scatter plot and volcano plot of these probe-matched regulated mRNAs (Fig. 2A). gene expressions of LncRNA and mRNA shows the up- Pathway analysis was carried out based on the KEGG, regulated and down-regulated lncRNAs (Fig.  1C and E) BioCyc and Panther database. The top 20 enrichment and mRNAs (Fig. 1D, F) profiling across groups. The red score values of the enriched pathways by KEGG pathway dots and green dots stand for up-regulated and down- (Fig. 2B) analysis and Panther pathway analysis (Fig. 2 C). regulated expressions, respectively. The dysregulated mRNAs were most associated with “Cytokine-cytokine receptor interaction”, “Jak-STAT sign- Gene ontology and pathway analyses aling pathway” and “Retinol metabolism” in KEGG path- GO analysis was conducted to classify differentially way. The dysregulated mRNAs were most associated with expressed mRNA into three categories, including biologi- “Plasminogen activating cascade”, “Blood coagulation”, cal processes, cellular components and molecular func- “Cadherin signaling pathway”, “Nicotine degradation” tion. Among the involved biological processes, cellular and “Jak-STAT signaling pathway” in Panther pathway. process, single-organism process, biological regulation, regulation of biological processes and metabolic pro- LncRNA‑mRNA co‑expression network cesses, were the five most significant processes associated We first constructed genes co-expression networks with the dysregulated mRNAs. Among the cellular com- between the cSCC group and normal skin group with ponents, cell, cell parts and organelle were the three most the all differential lncRNAs and mRNAs in this study. significant component processes associated with the dys - Among these, the lncRNAs and mRNAs were selected regulated mRNAs. Among molecular functions, binding, with the threshold set of Pearson’s correlation coeffi- catalytic activity and molecular function regulator were cients >0.99 or <-0.99 and P-value <0.05. Subsequently, Fig. 2 Gene ontology and KEGG pathway analyses. A Top 20 GO terms with significantly differential expression from GO analysis are categorized into biological process (blue), cellular component (green) and molecular function (red). B Top20 KEGG pathways with significantly differential expression are shown. C Top20 PANTHER pathways with significantly differential expression are shown. Vertical axis represents LgP values indicate significance of the enrichment. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; lncRNA, long non-coding RNA; cSCC, cutaneous squamous cell carcinoma Hu et al. BMC Genomics (2022) 23:274 Page 6 of 12 The target mRNA of LncRNA prediction and functional the top 1000 enriched gene pairs of lncRNAs and analysis mRNAs, correlation coefficient was constructed to To investigate whether the differentially expressed build the co-expression network by using cytoscape lncRNAs regulate genes and determine the signaling program, as shown in Fig. 3. In the co-expression net- pathways associated with cSCC, target prediction was work, A_33_P3340852 was obtained the highest num- performed to predict the possible targets of the dysregu- ber of interactions. lated lncRNAs. Based on the results of the co-expression network of lncRNAs and mRNAs (Correlation>0.99 or Fig. 3 Construction of the lncRNA-mRNA co-expression network. Green nodes represented for LncRNA, yellow rodes represent for the correlated mRNA, each circle’s size indicates relative number of related genes. The blue lines indicate negative correlations and the red lines are positive correlations Hu  et al. BMC Genomics (2022) 23:274 Page 7 of 12 Fig. 4 The target mRNA of LncRNA prediction and functional analysis. A Top 20 GO terms of the targeted mRNA with significantly differential expression from GO analysis are categorized into biological process (blue), cellular component (green) and molecular function (red). B Top20 KEGG pathways of the targeted mRNA with significantly differential expression are shown. Vertical axis represents LgP values indicate significance of the enrichment.GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; lncRNA, long non-coding RNA; CSCC, cutaneous squamous cell carcinoma Correlation<-0.99, and P < 0.05), cis-prediction looks of lncRNAs with top 1000 correlation coefficient were for lncRNA-mRNA pairs whose genome position is shown in Fig. 4. within 10  kb, and trans-prediction compares lncRNA Then we performed GO enrichment and KEGG path - and mRNA (3’UTR) sequences, and selects lncRNA- way analyses on the target mRNAs (Fig. 4). Through GO mRNA pairs with similar sequences. The target genes analysis, we found that the target expressed mRNAs were enriched for cellular process, single-organism process, Hu et al. BMC Genomics (2022) 23:274 Page 8 of 12 Fig. 5 Validation of the expression lncRNAs by qRT-PCR. The expression of top 7 differentially lncRNAs are compared between six paired cSCC tissues ,matched adjacent and normal tissue samples. There are significant difference between cSCC tissues and normol tissue. *P < 0.05 ,**P < 0.01 and ****P < 0.0001. RT-qPCR, reverse transcription-quantitative PCR; N, distant normal cutaneous tissue ; P, para-tumor tissue; T, tumor tissue biological regulation linked with biological processes, RT‑qPCR validation of differentially expressed lncRNAs and cell, cell part, organelle involved in cellular compo- The top 7 DE lncRNAs (Table  1) were selected for RT- nents, as well as binding, catalytic activity, nucleic acid qPCR analysis to verify the microarray results in six binding transcription factor activity in molecular func- paired cSCC tissues, matched adjacent and normal tissue tions (Fig.  4A). For the KEGG pathway analysis, the top samples. The RT-qPCR results demonstrated that three 20 enrichment score values of the enriched pathways lncRNAs (PVT1,CTD-2521M24.9 and AL353997.3) included measles, tight junction, circadian rhythm, were upregulated in cSCC and four lncRNAs hedgehog signaling pathway and histidine metabolism (MIR4720,BX004987.5,CTD-2619J13.13andLINC00478) (Fig. 4B). were downregulated in cSCC. As shown in Fig. 5, the rel- ative values of the expression levels detected by RT-qPCR were found to be consistent with the microarray data. Hu  et al. BMC Genomics (2022) 23:274 Page 9 of 12 This result suggests that the transcript identification and downregulated lncRNAs and three upregulated lncR- abundance estimates were highly reliable. NAs, were confirmed expressions by real-time PCR. Results of the qRT-PCR were consistent with those Discussion trends of high-throughput sequencing, proving its reli- Recent studies have revealed the significant role of ability. Of these verified lncRNAs in the present report, lncRNA in various types of cancer for tumorigenesis some have been identified as oncogenic lncRNA in car - and development, such as non-small-cell lung cancer, cinogenesis. For instance, the p29508 probe detected liver cancer, bladder cancer and breast cancer [9–11]. lncRNA PVT1, a top overexpressed lncRNA in micro- Moreover, numerous lncRNAs play an important role array analysis [27]. Accumulating evidence suggests that in the onset and progression of diseases by affecting the PVT1 locus as an epigenetic enhancer in colorectal cell proliferation ,invasion and other functions [12]. As cancer (CRC) and it has a regulatory effect on regulat - the second most common cancer worldwide ,cSCC has ing the expression of MYC. Furthermore, PVT1 lncRNA been reported with an annual accidence with over one expression mediated through aberrant methylation may million individuals [13–15]. UV irradiation, particu- also impact TGFβ/SMAD and Wnt/β-Catenin pathways larly UVB, is one of the classical cellular stressors caus- by the key CRC gene. Thus, high expression of the PVT1 ing DNA damage in skins, which receives chronic sun lncRNA may serve a pivotal role in the pathogenesis of exposure, and ultimately leading to skin development of CRC and has demonstrated roles in several aspects, as a cSCC [16]. Therefore, many present studies investigated prognostic biomarker and a potential therapeutic target. the potential oncogenic role of molecules and pathways Moreover, upregulated PVT1 can activate Wnt/β-catenin in cSCC and tried to evaluate the promising diagnostic signaling pathway by regulating expression of both Pygo2 biomarkers value for cSCC [17–20]. It is currently aimed and ATG14 and thus promote autophagy related com- to elucidate DNA binding/differentiation 4 (ID4) func - plex in pancreatic cancer [28]. Similarly,it has already tion in cSCC development [21]. The results indicate that been reported in anothor study that lncRNA PVT1 UVB irradiation leads to abnormal downregulated ID4 inhibite the proliferation of gastric cancer by combining via DNA methylation and ID4 acts as a tumor suppres- with enhancer of zeste homolog 2 (EZH2) to repress p15 sor gene in tumorigenesis. In addition, reports indicates and p16 [29]. Thus, we hypothesize that lncRNA PVT1 that the phosphoinositide3-kinases (PI3Ks)/Akt pathway upregulation may serve as an oncogenic gene in cSCC by plays an essential role in advanced cSCC, which inhibit combing EZH2 to activate Wnt/β-catenin pathways and viability and growth of cSCC and can also prove to be promote tumorigenic process of proliferation, autophagy valid target in cSCC [22]. Some dysregulated transcrip- and apoptosis aspects. Besides, LINC00478 has been tion factors (MYC, RELA, ETS1, SP1, TP63, TP53, AP1, identified to be highly link to accurate classification of TCF3, SOX2, OCT-3/4) and downregulated epidermal subtypes of breast cancer by facilitating expression of rel- differentiation genes (LCE1D, FLG, KRT77, KRT10, evant miRNA profiles [30]. ALOEX3 etc.) are all altered at the transcript level in LncRNA is a class of nucleotides that transcription cSCC [23–26]. However, the underlying molecular mech- length are more than 200 nt and which have little or no anism of LncRNAs in cSCC are rarely mentioned and protein-coding capability [5, 31, 32]. These molecules still remains unclear. play crucial roles in several aspects of biological func- To investigate whether lncRNA is involved in the tions as chromatin remodeling, gene transcription level carcinogenic process of the development of cSCC, we regulation and protein modification during the occur - performed lncRNA microarray to compare the com- rence and development of many diseases [33, 34]. An prehensive lncRNA expression profiles in three paired increasing number of studies described that dysregu- cSCC tissues and normal skin samples. In this study, lated lncRNA expression is associated with various through Hierarchical Clustering, we identified differen - cancers and may contribute to tumorigenesis, metasta- tial expression of lncRNAs and mRNAs in cSCC tissues sis and prognosis of many tumors [35]. Recent studies and predicted the potential biological function, cellu- demonstrated that lncRNAs can be utilized as a disease lar progress and enriched pathways by GO, KEGG and biomarkers and biomarkers for survival prediction in cer- Path-net analysis. It detected a total of 37,507 lncRNA vical cancer, colorectal cancer and skin cancers [36–38]. probes and 32,825 mRNA probes and found 3593 dif- Available evidence has reported that comprehensive ferentially expressed lncRNAs and 3236 differentially analysis of interaction between lncRNAs and mRNAs expressed mRNAs. The selection criteria, fold change may provide a reference for further explore its biologi- >2 and P < 0.05, ensured the significance of the dif - cal functions and the potential underlying mechanisms ferential expression data. Among these differentially in cancers [39]. In order to better study whether lncRNA expression lncRNAs, the top 7 lncRNAs, including four is involved in cSCC tumorigenesis, we constructed Hu et al. BMC Genomics (2022) 23:274 Page 10 of 12 co-expression network of top 1000 pairs of lncRNAs co-expression network ,which mainly were focused and mRNAs to annotate clear biological functions and on endoplasmic reticulum stress of apoptosis ,cellular regulatory mechanism of lncRNAs [40]. After construc- signal transduction and autophagy in tumorgenesis, as tion of the co-expression network, it has been found aforementioned. Since each results were individually that mRNAs ACY3, NR1D1, MZB1 has co-expression analyzed and relationships of co-expressed lncRNA- relationship with six lncRNAs, GXYLT1P3, LINC00348, mRNA and pathway analysis are independent, this LOC101928131, A-33-p3340852, A-21-p0003442 and outcome greatly supports the reliability of the cur- LOC644838 which largely have not been studied before. rent study. Therefore, it was hypothesized that six Otherwise, NR1D1is reported to showed significant lncRNAs, GXYLT1P3, LINC00348, LOC101928131, correlation with regulation of autophagy activities by A-33-p3340852, A-21-p0003442 and LOC644838, in microphages, metabolic process of stress response, signal the co-expression network may serve a considerable transduction and inflammatory pathways [41, 42]. The role in the progression of cSCC by regulating apoptosis results of study suggested that MZB1 expression plays an induced by endoplasmic reticulum stress, cellular sig- critical role in advanced stage of breast cancer and may nal transduction and autophagy. be a poor prognostic marker,which interact with endo- In conclusion, the present study revealed a com- plasmic reticulum stress related pathways [43]. ACY3 has prehensive analysis of lncRNA-mRNA co-expression rarely been reported but also may contribute to patho- profiles of patients with cutaneous squamous cell car - genesis of Huntington disease (HD) by altering binding cinoma. GO, KEGG and PANTHER analyses provided of transcriptional factors [44]. Within the co-expression the function of mRNAs and suggested the possible bio- network, that NR1D1 was noted that are connected with logical effects of lncRNAs. The results indicated that six the maximum number (six) of lncRNAs, indicating that lncRNAs, which have not been studied in cSCC,may it may play a significant role in the development and serve an important role in endoplasmic reticulum stress progression of cSCC. The dysregulation of these three apoptosis, autophagy and the progression of cSCC by transcripts mainly contribute to modulating apoptosis modulating ACY3, NR1D1 and MZB1. However, there induced by endoplasmic reticulum stress, physiologi- are still some defects which need to be improved to cal processes of cellular signal transduction and meta- carry out more comprehensive research, including bolic regulation, inflammatory signaling pathways and small study size containing only six tissues and the lack autophagy. of experimental verification in liquid biopsies (plasma/ In the present study ,the enrichment analy- serum)of cSCC.These data argue for further intensive sis of GO,KEGG and Panther pathway identified research to provide a more comprehensive under- that“Cytokine-cytokine receptor interaction”, “Jak-STAT standing of the molecular mechanisms underlying the signaling pathway”,“Plasminogen activating cascade ”and metabolism of cSCC. “Cadherin signaling pathway” were the top four signifi - cantly changed pathways. It has been reported that Jak- Supplementary Information STAT signaling pathway can contribute to resistance of The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s12864- 022- 08481-0. cervical squamous cell carcinoma by mediating key acti- vator protein as STAT1/STAT2 [19]. Jak-STAT signal- Additional file 1. ing pathway inhibition is involved in anti-tumor activity Additional file 2. in several pre-clinical studies and continued activation of STAT1 and STAT2,which are essential components of JAK/STAT pathway, are associated with suppressing Acknowledgements Not applicable. tumor apoptosis [45, 46]. Meanwhile, the present study found that exogenous IL-6 can induce activation of JAK/ Authors’ contributions STAT signaling and to increase autophagy [47]. Further- YH,RL,DH and KC conceived and designed the study. YH,RL,DH and HC collected the samples and acquired the data. LC,XZ and LL interpreted or ana- more, Cadherin signaling pathway can act as an intracel- lyzed data. YH and RL prepared the manuscript, which was revised for impor- lular signal transducer and was involved in invasion and tant intellectual content by DH and KC. All authors reviewed and approved metastasis by Wnt/β-catenin pathways inducing EMT the final manuscript. DH and KC confirm the authenticity of all the raw data. expression profiles in tumor progression [48]. Funding In fact, four of significant enriched pathways, This study was supported by grants from the National Natural Science Foun- including the“Cytokine-cytokine receptor interac- dation of China (82073445 and 81903247), the Natural Science Foundation of Jiangsu Province (BK20190145 and BK20210049), the Nanjing Incubation tion”, “Jak-STAT signaling pathway”,“Plasminogen acti- Program for National Clinical Research Center (2019060001) and CAMS Inno- vating cascade” and “Cadherin signaling pathway”, vation Fund for Medical Sciences(CIFMS-2021-I2M-1-001). were coincided with the pathogenesis mechanism of Hu  et al. BMC Genomics (2022) 23:274 Page 11 of 12 Availability of data and materials Potential Prospects for Treatment Evaluation and Prognostic Prediction. All data generated or analyzed during this study are included in this published Front Oncol 2020;10:590352. article. 12. Hu W, Li H, Wang S. 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BMC GenomicsSpringer Journals

Published: Apr 7, 2022

Keywords: Cutaneous squamous cell carcinoma; Long non-coding RNA; Gene regulatory networks; Microarray analysis; Genome

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