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Long noncoding RNA ZEB2-AS1 facilitates laryngeal squamous cell carcinoma progression by miR-6840-3p/PLXNB1 axis

Long noncoding RNA ZEB2-AS1 facilitates laryngeal squamous cell carcinoma progression by... OncoTargets and Therapy Dovepress open access to scientific and medical research Open Access Full Text Article ORIGINAL RE SE ARCH Long noncoding RNA ZEB2-AS1 facilitates laryngeal squamous cell carcinoma progression by miR-6840-3p/PLXNB1 axis This article was published in the following Dove Press journal: OncoTargets and Therapy Purpose: To investigate the role of zinc finger E-box-binding homeobox 2 antisense RNA 1 Qiushi Xu (ZEB2-AS1) in regulating laryngeal squamous cell carcinoma (LSCC) progression. Hongyu Liu Patients and methods: In this retrospective study, we included all patients who underwent a Bing Yu 2 surgical operation at The First Hospital of Qiqihaer City for LSCC. Then, we compared the Wenjing Chen expression of ZEB2-AS1 in LSCC tissues and paired healthy tissues. Besides, we also performed Lili Zhai a series of functional assays, CCK8 assays, colony formation assays, and transwell assays to XueYing Li examine the functions of LSCC cells after knockdown of ZEB2-AS1. Through bioinformatics Yanchun Fang analysis, we predicted that ZEB2-AS1 binds to miR-6840-3p and targets PLXNB1. Ear Nose and Throat Department, Results: We indicated that the expression of ZEB2-AS1 was higher in LSCC tissues Affiliated Qiqihar Hospital, Southern compared to the paired adjacent tissues, and ZEB2-AS1 was also highly expressed in Medical University, The First Hospital of LSCC cell lines. Furthermore, we discovered that ZEB2-AS1 promoted cell proliferation, Qiqihaer City, Guangzhou, Heilongjiang 161000, People’s Republic of China; migration and invasion and was associated with poor prognosis. To find the mechanism, we Pathology Department, Affiliated performed bioinformatics analysis. We identified that ZEB2-AS1 binds to miR-6840-3p and Qiqihar Hospital, Southern Medical University, The First Hospital of Qiqihaer targets PLXNB1. Additionally, miR-6840-3p overexpression or knockdown of PLXNB1 City, Guangzhou, Heilongjiang 161000, decreased the abilities of cell migration and invasion. People’s Republic of China Conclusion: These findings demonstrated that overexpression of ZEB2-AS1 promotes LSCC progression. Overexpression of miR-6840-3p or downregulation of PLXNB1 can abrogate ZEB2-AS1-mediated LSCC malignant development. Keywords: ZEB2-AS1, LSCC, miR-6840-3p, PLXNB1, migration, invasion Introduction Laryngeal squamous cell carcinoma (LSCC) is the most common malignancy with 1,2 a poor diagnosis. It has a higher incidence among the head and neck malignan- cies. In the past years, there are many research focused on the molecular mechan- isms about the development and progression of LSCC. However, it is still unclear that how LSCC progression. And, it is crucial to identify new therapies for LSCC patients. lncRNA was found to have little or no coding potential. However, lncRNA was proven to play an important role in many biological processes including develop- 5–8 ment, immunology, differentiation and cancers. Moreover, lncRNA was identi- Correspondence: Yanchun Fang Pathology Department, Affiliated Qiqihar fied to have the potential as a biomarker or therapy target. So, it is necessary to Hospital, Southern Medical University, explore the role of lncRNA in promoting cancer development. The First Hospital of Qiqihaer City, Guangzhou, Heilongjiang 161000, People’s Zinc finger E-box-binding homeobox 2 antisense RNA 1 (ZEB2-AS1) was a Republic of China Email yanchun_fang@163.com noncoding RNA newly found in hepatocellular carcinoma. In addition, it has submit your manuscript | www.dovepress.com OncoTargets and Therapy 2019:12 7337–7345 7337 DovePress © 2019 Xu et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work http://doi.org/10.2147/OTT.S212749 you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Xu et al Dovepress been proved to involve in promoting many cancer progres- Cell counting kit-8 assays 11 12 sion including lung cancer, gastric cancer, bladder To measure the cell proliferation abilities, cells were trans- 13 14 cancer and pancreatic cancer. However, the functional fected with indicated plasmids and then performed CCK8 role and underlying mechanism in LSCC remain unde- assay (7 sea biotech, Shanghai, China). fined. In our study, we found that ZEB2-AS1 was highly expressed in LSCC tissues and cell lines. And, abnormal Colony formation assay expression of ZEB2-AS1 in LSCC cell line promotes the TU212 cells transfected with indicated plasmids were put abilities of cell proliferation, migration and invasion. into 6-well plate. Every well was put into 1000 cells. After lncRNA was found to facilitate cancer cell progression 13–15 days culturing, colonies were stained and counted. 6,15 through miR-RNA as a ceRNA. Through bioinfor- matics analysis, we found that ZEB2-AS1 binds to Luciferase reporter assay miR-6840-3p directly. Moreover, we discovered that over- pMIR-PLXNB1-3ʹUTR (WT or Mutant) plasmids or expression of ZEB2-AS1 inhibits miR-6840-3p while inhi- pMIR-ZEB2-AS1 (WT or Mutant) plasmids and bition of miR-6840-3p promotes ZEB2-AS1 expression. miR-6840-3p mimics were co-transfected in TU212 cells Moreover, we also discovered that miR-6840-3p targets to couple with pRL-TK vectors (Promega, USA). After 24 PLXNB1. hrs, the dual Glo™ Luciferase Assay System (Promega) Plexin-B1 is a transmembrane receptor for semaphoring was used to measure luciferase activity according to the 4D. Previous studies have demonstrated that PLXNB1 is manufacturer’s protocols. 17,18 involved in many cellular processes. And also, there are some reports that proved that PLXNB1 plays a vital role in Western blot 19,20 the progression of glioma. However, the biological role Western blot assay was used to determine the expression of PLXNB1 in LSCC is unknown. Through a series of of the indicated protein. The assay was performed accord- experiments, we revealed that PLXNB1 overexpression ing to the paper reported. associates with LSCC proliferation and invasion. Statistical analysis To analyze the results, we used GraphPad Prism 6 soft- Materials and methods ware. The data were shown as means ± SD. Student’s t-test Samples or one-way ANOVA was used to determine significant LSCC tissues and paired healthy tissues were isolated differences. Survival rate was analyzed by Kaplan–Meier from LSCC patients after surgery at The First Hospital analysis and log-rank test. of Qiqihaer City. The tissues were frozen in liquid nitro- gen. This work was conducted in accordance with the Results Declaration of Helsinki and approved by the Ethics ZEB2-AS1 expression is elevated in LSCC Committee of The First Hospital of Qiqihaer City. All written informed consents were received from patients. tissues and cell lines As previous study reports, ZEB2-AS1 promotes many cancer progression. However, the role of ZEB2-AS1 in Cell lines LSCC is unknown. Firstly, we performed qRT-PCR and LSCC cell lines (TU212, Hep-2) and normal bronchial found that the expression of ZEB2-AS1 is higher in LSCC epithelial cell line (16HBE) were obtained from the samples compared to near healthy tissues (Figure 1A). Chinese Academy of Science of Shanghai (Shanghai, Among them, 20 (20/27, 74%) pair samples showed a China). significantly higher expression of ZEB2-AS1 in LSCC tissues compared to adjacent normal tissues (Figure 1B). Real-time PCR Next, we examined the expression of ZEB2-AS1 in RNA was isolated from LSCC tissues and cell lines by advanced samples. We found that higher expression asso- Trizol reagent (Invitrogen, Carlsbad, CA, USA) according ciated with advanced cancer samples which indicated that to the manufacturer’s instructions. qRT-PCR was per- ZEB2-AS1 expression associated with more malignant formed according to the manufacturer’s instructions. tumor (Figure 1C). Consistently, we also examined the submit your manuscript | www.dovepress.com OncoTargets and Therapy 2019:12 DovePress Dovepress Xu et al Figure 1 ZEB2-AS1 expressed at a higher level in LSCC tissues and cell lines compared to normal tissues and cell lines. (A) qRT-PCR was performed to examine the expression of ZEB2-AS1 in LSCC tissues and near healthy tissues. Forty-five pairs of LSCC tissues and normal tissues were collected for analysis. (B) Among the 27 pairs of LSCC tissues and normal tissues, 20 cases (20/27, 74%) showed increased expression of ZEB2-AS1 in LSCC tissues. (C) qRT-PCR assay was performed to examine ZEB2- AS1 expression in LSCC tissues with early stage or advanced stage. And, 20 early-stage LSCC tissues and 25 advanced LSCC tissues were used for qRT-PCR. (D) ZEB2-AS1 expression in LSCC cell lines and normal cell line were detected using qRT-PCR. LSCC cell lines: TU212, Hep-2. Normal bronchial epithelial cell line: 16HBE. (E) Overall survival and log-rank test were analyzed by Kaplan–Meier analysis in 45 LSCC patients with ZEB2-AS1 high expression level or ZEB2-AS1 low expression level (P=0.015). *P<0.05. All experiments were repeated three times. expression of ZEB2-AS1 in LSCC cell lines and noticed a higher ZEB2-AS1 expression which associates with that the expression of ZEB2-AS1 was higher in LSCC cell patients’ prognosis. lines (TU211 and Hep-2) than normal bronchial epithelial cell line (16HBE) (Figure 1D). Then, we wanted to inves- Knockdown of ZEB2-AS1 significantly tigate the prognostic significance of ZEB2-AS1. We ana- decreases LSCC cell migration, invasion lyzed the overall survival rate by performing the Kaplan– and proliferation Meier curve. We divided the 45 patients into higher ZEB2- To define the role of ZEB2-AS1 in LSCC cells, we per- AS1 expression and lower ZEB2-AS1 expression group formed a series of functional experiments. We constructed based on the median expression level of ZEB2-AS1. shRNAs to decrease the expression of ZEB2-AS1. As Consistently, we found that lower ZEB2-AS1 expression examined by qRT-PCR, we found that the expression of group possessed better overall survival (Figure 1E). ZEB2-AS1 was significantly decreased by the shZEB2-AS1 Collectively, these data indicate that LSCC samples show submit your manuscript | www.dovepress.com OncoTargets and Therapy 2019:12 7339 DovePress Xu et al Dovepress plasmids (Figure 2A). Then, we found that LSCC cell (Figure 2E–F). These data prove that ZEB2-AS1 partici- proliferation ability was obviously decreased in pates in the regulation of LSCC progression. ZEB2-AS1 knockdown group examined by CCK8 assay Overexpression of ZEB2-AS1 promotes (Figure 2B). And, we also performed transwell assays. LSCC cells proliferation, migration and Consistent with the previous study, we found that ZEB2-AS1 knockdown decreased LSCC cell migration invasion and invasion abilities (Figure 2C–D). Colony formation As shown above, we found that knockdown of ZEB2-AS1 assay was always used to examine the proliferation ability decreases LSCC progression. So, we wanted to know the of cancer cells. So, we performed colony formation assay effect of overexpression of ZEB2-AS1. Firstly, we con- and indicated that LSCC cell proliferation ability signifi- structed ZEB2-AS1 overexpressed plasmid to elevate the cantly decreased after knockdown of ZEB2-AS1 expression of ZEB2-AS1 (Figure 3A). Next, we found that Figure 2 Knockdown of ZEB2-AS1 decreased LSCC cell abilities of progression. (A) Knockdown effect of ZEB2-AS1 in TU212 cells was examined using qRT-PCR. (B) Growth curves of TU212 cells transfected with negative control plasmids or ZEB2-AS1 shRNA plasmids. (C and D) The abilities of migration and invasion of TU212 cells after knockdown of ZEB2-AS1 were detected. (E and F) The number of colonies after overexpression of ZEB2-AS1 were counted compared to negative control. *P<0.05. All experiments were repeated three times. submit your manuscript | www.dovepress.com OncoTargets and Therapy 2019:12 DovePress Dovepress Xu et al Figure 3 Overexpression of ZEB2-AS1 promotes LSCC cell progression. (A) Expression of ZEB2-AS1 in TU212 cell line overexpressed with ZEB2-AS1 was determined by qRT-PCR. (B) TU212 cells were transfected with negative control plasmid or ZEB2-AS1 overexpression plasmid. Then, the growth rates of cells were determined using CCK-8 assay. (C and D) Transwell assay was used to detect the migration and invasive abilities of TU212 cells after transfected with control or ZEB2-AS1 overexpression plasmid. (E and F) Colony formation assay revealed that LSCC cells overexpressed of ZEB2-AS1 promoted the number of colonies. *P<0.05. All experiments were repeated three times. overexpression of ZEB2-AS1 significantly promoted cell group (Figure 3E–F). Taken together, these data indicate proliferation examined by CCK8 assays (Figure 3B). that ZEB2-AS1 significantly promotes the proliferation, migration and invasion abilities of LSCC cells. Moreover, we further examined the migration and invasion abilities of LSCC cells. We found that the migration and ZEB2-AS1 inhibits miR-6840-3p invasion abilities were increased in ZEB2-AS1 overex- expression to promote LSCC progression pressed LSCC cells (Figure 3C–D). We performed colony formation assay using control and ZEB2-AS1 overex- through regulating PLXNB1 expression pressed LSCC cells and found that the number of colonies In the previous data, we found that ZEB2-AS1 can pro- increased significantly in ZEB2-AS1 highly expressed mote the proliferation, migration and invasion of LSCC submit your manuscript | www.dovepress.com OncoTargets and Therapy 2019:12 7341 DovePress Xu et al Dovepress and colleagues found that lncRNA LOC554202 can pro- cells. So, we want to explore the mechanism that ZEB2- mote LSCC carcinoma progression by miR-31. AS1 regulates LSCC progression. To find the mechanism, They we performed bioinformatics analysis. We found that demonstrated that LOC554202 is highly expressed in ZEB2-AS1 can form a complementary base pair with LSCC tissues while miR-31 is lowly expressed in LSCC miR-6840-3p (Figure 4A). In addition, we found that tissues compared to adjacent tissues. And also, it is overexpression of ZEB2-AS1 significantly inhibited reported that lncRNA UCA1 can activate Wnt/β-catenin miR-6840-3p expression (Figure 4B). In contrast, we signaling pathway to elevate the proliferation, invasion also indicated that miR-6840-3p overexpression decreased and migration abilities of LSCC cells. ZEB2-AS1 expression while miR-6840-3p inhibition pro- ZEB2-AS1 is found to regulate many cancer progres- moted ZEB2-AS1 expression (Figure 4C). Next, we per- sion. For instance, ZEB2-AS1 was found to promote formed luciferase assay to prove that ZEB2-AS1 binds to breast cancer progression mainly regulating the prolifera- miR-6840-3p. We showed that ectopic expression of tion and epithelial–mesenchymal transition of breast can- miR-6840-3p significantly decreased luciferase intensity cer. And, Wu et al demonstrated that ZEB2-AS1 binds to in ZEB2-AS1 WT construct while ZEB2-AS1 mutant did miR-143-5p and promotes gastric cancer cells proliferation not change (Figure 4D). and migration via HIF-1a axis. However, the role of In addition, we wanted to find the potential target gene ZEB2-AS1 in LSCC remains unclear. So we explore the regulated by ZEB2-AS1 and miR-6840-3p. We performed biological role of ZEB2-AS1 in LSCC progression. In our bioinformatics analysis again using TargetScan7 program. study, we found that ZEB2-AS1 expresses at a higher level We showed that miR-6840-3p bound to PLXNB1 through in LSCC tissues compared to near-normal tissues and complementary base pair mechanism (Figure 4E). positively correlated with LSCC progression. Moreover, Through qRT-PCR experiment, we found that overexpres- we found that ZEB2-AS1 regulates the expression of miR- sion of miR-6840-3p decreased PLXNB1 expression. We 6840-3p and PLXNB1 in LSCC cell line. However, we did also proved that by Western blot assay (Figure 4F). Then, not explore the role of ZEB2-AS1/miR-6840-3p/PLXNB1 we wanted to explore the relationship between ZEB2-AS1, axis in other cancer cell lines. So we concluded that this miR-6840-3p and PLXNB1 expression. We indicated that mechanism is suited for LSCC. But, to know if this overexpression of ZEB2-AS1 promoted PLXNB1 expres- mechanism suit for other cancer needs more evidence. sion and knockdown of ZEB2-AS1 decreased PLXNB1 miRNAs are proven to participant in many cancer expression. Moreover, the increased expression of ZEB2- regulating. And there are many miRNAs that are involved 24–26 AS1 can be rescued by ectopic expression of miR-6840-3p in regulating LSCC progression. However, the role of (Figure 4G). And, we also performed luciferase assay and miR-6840-3p has not been reported in any cancer. In our found that overexpression of miR-6840-3p or knockdown study, we showed that ZEB2-AS1 can bind to miR-6840- of ZEB2-AS1 expression decreased luciferase intensity of 3p directly and inhibit the expression of miR-6840-3p. PLXNB1 WT construct while inhibition of miR-6840-3p And, we also found that miR-6840-3p mimic can also coupled with knockdown ZEB2-AS1 expression rescued inhibit ZEB2-AS1 expression. Moreover, miR-6840-3p PLXNB1 WT luciferase intensity. And also, the luciferase inhibits the expression of PLXNB1. So, we concluded intensity of PLXNB1 mutant did not change (Figure 4H). that ZEB2-AS1 is upstream while miR-6840-3p is As a result, we also performed transwell assay to examine downstream. the migration and invasion abilities. We showed that PLXNB1 is also found to be expressed in many cancer knockdown of ZEB2-AS1 decreased LSCC cell migration cells. Cao et al found that PLXNB1 can promote the cell and invasion while inhibition of miR-6840-3p or overex- proliferation, migration and invasion of cutaneous squa- pression of PLXNB1 rescues the abilities of migration and mous cell carcinoma. In addition, PLXNB1 is proven to invasion (Figure 4I–J). Collectively, we show that ZEB2- regulate Rho/αvβ3/PI3K/Akt signaling pathway, which is AS1 regulates LSCC progression through binding to miR- involved in regulating glioma invasiveness and 6840-3p from PLXNB1 mRNA. angiogenesis. In our study, we found that PLXNB1 is a target gene for ZEB2-AS1 and miR-6840-3p. ZEB2-AS1 Discussion overexpression or inhibition of miR-6840-3p significantly In the past decades, lncRNA is proven to play a crucial promotes PLXNB1 expression. And also, overexpression role in regulating LSCC progression. For example, Yang of PLXNB1 restores the decreased migration and invasion submit your manuscript | www.dovepress.com OncoTargets and Therapy 2019:12 DovePress Dovepress Xu et al Figure 4 ZEB2-AS1 binds to miR-6840-3p and regulates the expression of PLXNB1. (A) Predicted target site between miR-6840-3p and ZEB2-AS1 was shown. (B) Expression level of miR-6840-3p in TU212 cells transfected with ZEB2-AS1 overexpression plasmid was determined using qRT-PCR assay. (C) qRT-PCR assay was used to determine ZEB2-AS1 expression after miR-6840-3p overexpression or miR-6840-3p inhibition in TU212 cell line. (D) TU212 cells were co-transfected with the WT or Mut plasmid and the indicated miRNAs. Then, luciferase reporter assay was used to determine luciferase intensity. (E) Predicted target site between miR-6840-3p and PLXNB1. (F) Expression of PLXNB1 after overexpression of miR-6840-3p in TU212 cell line was examined by qRT-PCR and Western blot assays. qRT-PCR assay was normalized to 18S, Western blot assay was normalized to β-actin. (G) Luciferase activity assay was used to determine the luciferase intensity in TU212 cells transfected with different vectors. (H) Relative expression of PLXNB1 after ZEB2-AS1 overexpression or ZEB2-AS1 knockdown or ZEB2-AS1 overexpression with inhibition of miR-204-3p in TU212 cell lines. (I and J) Rescue assay was used to examine the abilities of migration and invasion after transfected with control or different plasmids in TU212 cells. *P<0.05. All experiments were repeated three times. submit your manuscript | www.dovepress.com OncoTargets and Therapy 2019:12 7343 DovePress Xu et al Dovepress 12. Wu FX, Gao HY, Liu KG, et al. The IncRNA ZEB2-AS1 is upregu- abilities of LSCC cells caused by decreased expression of lated in gastric cancer and affects cell proliferation and invasion via ZEB2-AS1. These data indicated that PLXNB1 plays a miR-143-5p/HIF-1 alpha axis. Onco Targets Ther. 2019;12:657–667. crucial role in regulating LSCC progression through doi:10.2147/OTT.S175521 13. Wu XQ, Yan TZ, Wang ZW, Wu X, Cao GH, Zhang C. LncRNA ZEB2-AS1 and miR-6840-3p axis. ZEB2-AS1 promotes bladder cancer cell proliferation and inhibits In conclusion, we showed that ZEB2-AS1 participants apoptosis by regulating miR-27b. Biomed Pharmacother. 2017;96:299–304. doi:10.1016/j.biopha.2017.08.060 in promoting LSCC cells proliferation, migration and inva- 14. Gao H, Gong NN, Ma ZB, et al. LncRNA ZEB2-AS1 promotes sion via miR-6840-3p/PLXNB1 axis. pancreatic cancer cell growth and invasion through regulating the miR-204/HMGB1 axis. Int J Biol Macromol. 2018;116:545–551. doi:10.1016/j.ijbiomac.2018.05.044 Disclosure 15. Zhang MB, Li Y, Wang HB, Yu WH, Lin S, Guo JQ. LncRNA The authors report no conflicts of interest in this work. SNHG5 affects cell proliferation, metastasis and migration of color- ectal cancer through regulating miR-132-3p/CREB5. Cancer Biol Ther. 2019;20:524–536. doi:10.1080/15384047.2018.1537579 References 16. Malik MFA, Ye L, Jiang WG. Reduced expression of semaphorin 4D and plexin-B in breast cancer is associated with poorer prognosis and 1. Akbaba S, Held T, Lang K, et al. Salvage radiotherapy for recurrent the potential linkage with oestrogen receptor. Oncol Rep. hypopharyngeal and laryngeal squamous cell carcinoma (SCC) after 2015;34:1049–1057. doi:10.3892/or.2015.4015 first-line treatment with surgery alone: a 10-year single-centre experi- 17. Roy AD, Yin T, Choudhary S, Rodionov V, Pilbeam CC, Wu YI. ence. Radiat Oncol. 2019;14. doi:10.1186/s13014-019-1238-8 Optogenetic activation of Plexin-B1 reveals contact repulsion 2. Boxberg M, Kuhn PH, Reiser M, et al. Tumor budding and cell nest between osteoclasts and osteoblasts. Nat Commun. 2017;8:15831. size are highly prognostic in laryngeal and hypopharyngeal squamous 18. Williamson M, de Winter P, Masters JR. Plexin-B1 signalling pro- cell carcinoma further evidence for a unified histopathologic grading motes androgen receptor translocation to the nucleus. Oncogene. system for squamous cell carcinomas of the upper aerodigestive tract. 2016;35:1066–1072. doi:10.1038/onc.2015.160 Am J Surg Pathol. 2019;43:303–313. doi:10.1097/PAS.000000000 19. Chang YW, Li L, Zhang LP, et al. Plexin-B1 indirectly affects glioma invasiveness and angiogenesis by regulating the RhoA/alpha v beta 3 3. Malm IJ, Rooper LM, Bishop JA, et al. Molecular and immunologic signaling pathway and SRPK1. Tumor Biol. 2016;37:11225–11236. analysis of laryngeal squamous cell carcinoma in smokers and non- doi:10.1007/s13277-016-4849-9 smokers. Am J Otolaryngol. 2019;40:213–217. doi:10.1016/j. 20. Zhang Y, Li Q, Zhuang R, et al. Plexin-B1: a potential diagnostic biomar- amjoto.2018.11.009 ker for glioma and a future target for glioma immunotherapy. J 4. Cao SJ, Huang YY, Zhang Q, et al. Molecular mechanisms of Neuroimmunol. 2012;252:113–117. doi:10.1016/j.jneuroim.2012.08.005 apoptosis and autophagy elicited by combined treatment with orido- 21. Liu SZ, Duan WC. Long noncoding RNA LINC00339 promotes nin and cetuximab in laryngeal squamous cell carcinoma. Apoptosis. laryngeal squamous cell carcinoma cell proliferation and invasion 2019;24:33–45. doi:10.1007/s10495-018-1497-0 via sponging miR-145. J Cell Biochem. 2019;120:8272–8279. 5. Kong XL, Duan Y, Sang YT, et al. LncRNA-CDC6 promotes breast doi:10.1002/jcb.v120.5 cancer progression and function as ceRNA to target CDC6 by spong- 22. Yang SJ, Wang J, Ge WS, Jiang YF. Long non-coding RNA ing microRNA-215. J Cell Physiol. 2019;234:9105–9117. doi:10.10 LOC554202 promotes laryngeal squamous cell carcinoma progres- 02/jcp.27587 sion through regulating miR-31. J Cell Biochem. 2018;119:6953– 6. Liang HL, Zhang CY, Guan HY, Liu J, Cui YB. LncRNA DANCR 6960. doi:10.1002/jcb.26902 promotes cervical cancer progression by upregulating ROCK1 via 23. Sun SG, Gong C, Yuan K. LncRNA UCA1 promotes cell prolifera- sponging miR-335-5p. J Cell Physiol. 2019;234:7266–7278. doi:10. tion, invasion and migration of laryngeal squamous cell carcinoma 1002/jcp.27484 cells by activating Wnt/-catenin signaling pathway. Exp Ther Med. 7. Zhou T, Qin GW, Yang LH, Xiang DK, Li SN. LncRNA XIST 2019;17:1182–1189. doi:10.3892/etm.2018.7097 regulates myocardial infarction by targeting miR-130a-3p. J Cell 24. Wang Y, Zhang D, Yu CH, et al. MicroRNA-708-5p contributes to Physiol. 2019;234:8659–8667. doi:10.1002/jcp.26327 the malignant behavior of laryngeal squamous cell carcinoma by 8. Shi X, Cui ZG, Liu XD, et al. LncRNA FIRRE is activated by MYC directly targeting metastasis suppressor-1. Int J Clin Exp Med. and promotes the development of diffuse large B-cell lymphoma via 2019;12:597–603. Wnt/beta-catenin signaling pathway. Biochem Biophys Res Commun. 25. Zhang F, Cao H. MicroRNA-143-3p suppresses cell growth and 2019;510:594–600. doi:10.1016/j.bbrc.2019.01.105 invasion in laryngeal squamous cell carcinoma via targeting the k- 9. Shen ZS, Li Q, Deng HX, et al. Long non-coding RNA profiling in Ras/Raf/MEK/ERK signaling pathway. Int J Oncol. 2019;54:689– laryngeal squamous cell carcinoma and its clinical significance: 701. doi:10.3892/ijo.2018.4655 potential biomarkers for LSCC. PLoS One. 2014;9. doi:10.1371/jour- 26. Han L, Tang MM, Xu XJ, et al. MiR-143-3p suppresses cell prolif- nal.pone.0108237 eration, migration, and invasion by targeting melanoma-associated 10. Lan T, Chang L, Wu L, Yuan YF. Downregulation of ZEB2-AS1 antigen A9 in laryngeal squamous cell carcinoma. J Cell Biochem. decreased tumor growth and metastasis in hepatocellular carcinoma. 2019;120:1245–1257. doi:10.1002/jcb.v120.2 Mol Med Rep. 2016;14:4606–4612. doi:10.3892/mmr.2016.5836 27. Cao J, Zhang C, Chen T, et al. Plexin-B1 and semaphorin 4D 11. Guo Y, Hu Y, Hu MM, He JB, Li BL. Long non-coding RNA ZEB2- cooperate to promote cutaneous squamous cell carcinoma cell pro- AS1 promotes proliferation and inhibits apoptosis in human lung liferation, migration and invasion. J Dermatol Sci. 2015;79:127–136. cancer cells. 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Long noncoding RNA ZEB2-AS1 facilitates laryngeal squamous cell carcinoma progression by miR-6840-3p/PLXNB1 axis

OncoTargets and therapy , Volume 12 – Sep 6, 2019

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Abstract

OncoTargets and Therapy Dovepress open access to scientific and medical research Open Access Full Text Article ORIGINAL RE SE ARCH Long noncoding RNA ZEB2-AS1 facilitates laryngeal squamous cell carcinoma progression by miR-6840-3p/PLXNB1 axis This article was published in the following Dove Press journal: OncoTargets and Therapy Purpose: To investigate the role of zinc finger E-box-binding homeobox 2 antisense RNA 1 Qiushi Xu (ZEB2-AS1) in regulating laryngeal squamous cell carcinoma (LSCC) progression. Hongyu Liu Patients and methods: In this retrospective study, we included all patients who underwent a Bing Yu 2 surgical operation at The First Hospital of Qiqihaer City for LSCC. Then, we compared the Wenjing Chen expression of ZEB2-AS1 in LSCC tissues and paired healthy tissues. Besides, we also performed Lili Zhai a series of functional assays, CCK8 assays, colony formation assays, and transwell assays to XueYing Li examine the functions of LSCC cells after knockdown of ZEB2-AS1. Through bioinformatics Yanchun Fang analysis, we predicted that ZEB2-AS1 binds to miR-6840-3p and targets PLXNB1. Ear Nose and Throat Department, Results: We indicated that the expression of ZEB2-AS1 was higher in LSCC tissues Affiliated Qiqihar Hospital, Southern compared to the paired adjacent tissues, and ZEB2-AS1 was also highly expressed in Medical University, The First Hospital of LSCC cell lines. Furthermore, we discovered that ZEB2-AS1 promoted cell proliferation, Qiqihaer City, Guangzhou, Heilongjiang 161000, People’s Republic of China; migration and invasion and was associated with poor prognosis. To find the mechanism, we Pathology Department, Affiliated performed bioinformatics analysis. We identified that ZEB2-AS1 binds to miR-6840-3p and Qiqihar Hospital, Southern Medical University, The First Hospital of Qiqihaer targets PLXNB1. Additionally, miR-6840-3p overexpression or knockdown of PLXNB1 City, Guangzhou, Heilongjiang 161000, decreased the abilities of cell migration and invasion. People’s Republic of China Conclusion: These findings demonstrated that overexpression of ZEB2-AS1 promotes LSCC progression. Overexpression of miR-6840-3p or downregulation of PLXNB1 can abrogate ZEB2-AS1-mediated LSCC malignant development. Keywords: ZEB2-AS1, LSCC, miR-6840-3p, PLXNB1, migration, invasion Introduction Laryngeal squamous cell carcinoma (LSCC) is the most common malignancy with 1,2 a poor diagnosis. It has a higher incidence among the head and neck malignan- cies. In the past years, there are many research focused on the molecular mechan- isms about the development and progression of LSCC. However, it is still unclear that how LSCC progression. And, it is crucial to identify new therapies for LSCC patients. lncRNA was found to have little or no coding potential. However, lncRNA was proven to play an important role in many biological processes including develop- 5–8 ment, immunology, differentiation and cancers. Moreover, lncRNA was identi- Correspondence: Yanchun Fang Pathology Department, Affiliated Qiqihar fied to have the potential as a biomarker or therapy target. So, it is necessary to Hospital, Southern Medical University, explore the role of lncRNA in promoting cancer development. The First Hospital of Qiqihaer City, Guangzhou, Heilongjiang 161000, People’s Zinc finger E-box-binding homeobox 2 antisense RNA 1 (ZEB2-AS1) was a Republic of China Email yanchun_fang@163.com noncoding RNA newly found in hepatocellular carcinoma. In addition, it has submit your manuscript | www.dovepress.com OncoTargets and Therapy 2019:12 7337–7345 7337 DovePress © 2019 Xu et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work http://doi.org/10.2147/OTT.S212749 you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Xu et al Dovepress been proved to involve in promoting many cancer progres- Cell counting kit-8 assays 11 12 sion including lung cancer, gastric cancer, bladder To measure the cell proliferation abilities, cells were trans- 13 14 cancer and pancreatic cancer. However, the functional fected with indicated plasmids and then performed CCK8 role and underlying mechanism in LSCC remain unde- assay (7 sea biotech, Shanghai, China). fined. In our study, we found that ZEB2-AS1 was highly expressed in LSCC tissues and cell lines. And, abnormal Colony formation assay expression of ZEB2-AS1 in LSCC cell line promotes the TU212 cells transfected with indicated plasmids were put abilities of cell proliferation, migration and invasion. into 6-well plate. Every well was put into 1000 cells. After lncRNA was found to facilitate cancer cell progression 13–15 days culturing, colonies were stained and counted. 6,15 through miR-RNA as a ceRNA. Through bioinfor- matics analysis, we found that ZEB2-AS1 binds to Luciferase reporter assay miR-6840-3p directly. Moreover, we discovered that over- pMIR-PLXNB1-3ʹUTR (WT or Mutant) plasmids or expression of ZEB2-AS1 inhibits miR-6840-3p while inhi- pMIR-ZEB2-AS1 (WT or Mutant) plasmids and bition of miR-6840-3p promotes ZEB2-AS1 expression. miR-6840-3p mimics were co-transfected in TU212 cells Moreover, we also discovered that miR-6840-3p targets to couple with pRL-TK vectors (Promega, USA). After 24 PLXNB1. hrs, the dual Glo™ Luciferase Assay System (Promega) Plexin-B1 is a transmembrane receptor for semaphoring was used to measure luciferase activity according to the 4D. Previous studies have demonstrated that PLXNB1 is manufacturer’s protocols. 17,18 involved in many cellular processes. And also, there are some reports that proved that PLXNB1 plays a vital role in Western blot 19,20 the progression of glioma. However, the biological role Western blot assay was used to determine the expression of PLXNB1 in LSCC is unknown. Through a series of of the indicated protein. The assay was performed accord- experiments, we revealed that PLXNB1 overexpression ing to the paper reported. associates with LSCC proliferation and invasion. Statistical analysis To analyze the results, we used GraphPad Prism 6 soft- Materials and methods ware. The data were shown as means ± SD. Student’s t-test Samples or one-way ANOVA was used to determine significant LSCC tissues and paired healthy tissues were isolated differences. Survival rate was analyzed by Kaplan–Meier from LSCC patients after surgery at The First Hospital analysis and log-rank test. of Qiqihaer City. The tissues were frozen in liquid nitro- gen. This work was conducted in accordance with the Results Declaration of Helsinki and approved by the Ethics ZEB2-AS1 expression is elevated in LSCC Committee of The First Hospital of Qiqihaer City. All written informed consents were received from patients. tissues and cell lines As previous study reports, ZEB2-AS1 promotes many cancer progression. However, the role of ZEB2-AS1 in Cell lines LSCC is unknown. Firstly, we performed qRT-PCR and LSCC cell lines (TU212, Hep-2) and normal bronchial found that the expression of ZEB2-AS1 is higher in LSCC epithelial cell line (16HBE) were obtained from the samples compared to near healthy tissues (Figure 1A). Chinese Academy of Science of Shanghai (Shanghai, Among them, 20 (20/27, 74%) pair samples showed a China). significantly higher expression of ZEB2-AS1 in LSCC tissues compared to adjacent normal tissues (Figure 1B). Real-time PCR Next, we examined the expression of ZEB2-AS1 in RNA was isolated from LSCC tissues and cell lines by advanced samples. We found that higher expression asso- Trizol reagent (Invitrogen, Carlsbad, CA, USA) according ciated with advanced cancer samples which indicated that to the manufacturer’s instructions. qRT-PCR was per- ZEB2-AS1 expression associated with more malignant formed according to the manufacturer’s instructions. tumor (Figure 1C). Consistently, we also examined the submit your manuscript | www.dovepress.com OncoTargets and Therapy 2019:12 DovePress Dovepress Xu et al Figure 1 ZEB2-AS1 expressed at a higher level in LSCC tissues and cell lines compared to normal tissues and cell lines. (A) qRT-PCR was performed to examine the expression of ZEB2-AS1 in LSCC tissues and near healthy tissues. Forty-five pairs of LSCC tissues and normal tissues were collected for analysis. (B) Among the 27 pairs of LSCC tissues and normal tissues, 20 cases (20/27, 74%) showed increased expression of ZEB2-AS1 in LSCC tissues. (C) qRT-PCR assay was performed to examine ZEB2- AS1 expression in LSCC tissues with early stage or advanced stage. And, 20 early-stage LSCC tissues and 25 advanced LSCC tissues were used for qRT-PCR. (D) ZEB2-AS1 expression in LSCC cell lines and normal cell line were detected using qRT-PCR. LSCC cell lines: TU212, Hep-2. Normal bronchial epithelial cell line: 16HBE. (E) Overall survival and log-rank test were analyzed by Kaplan–Meier analysis in 45 LSCC patients with ZEB2-AS1 high expression level or ZEB2-AS1 low expression level (P=0.015). *P<0.05. All experiments were repeated three times. expression of ZEB2-AS1 in LSCC cell lines and noticed a higher ZEB2-AS1 expression which associates with that the expression of ZEB2-AS1 was higher in LSCC cell patients’ prognosis. lines (TU211 and Hep-2) than normal bronchial epithelial cell line (16HBE) (Figure 1D). Then, we wanted to inves- Knockdown of ZEB2-AS1 significantly tigate the prognostic significance of ZEB2-AS1. We ana- decreases LSCC cell migration, invasion lyzed the overall survival rate by performing the Kaplan– and proliferation Meier curve. We divided the 45 patients into higher ZEB2- To define the role of ZEB2-AS1 in LSCC cells, we per- AS1 expression and lower ZEB2-AS1 expression group formed a series of functional experiments. We constructed based on the median expression level of ZEB2-AS1. shRNAs to decrease the expression of ZEB2-AS1. As Consistently, we found that lower ZEB2-AS1 expression examined by qRT-PCR, we found that the expression of group possessed better overall survival (Figure 1E). ZEB2-AS1 was significantly decreased by the shZEB2-AS1 Collectively, these data indicate that LSCC samples show submit your manuscript | www.dovepress.com OncoTargets and Therapy 2019:12 7339 DovePress Xu et al Dovepress plasmids (Figure 2A). Then, we found that LSCC cell (Figure 2E–F). These data prove that ZEB2-AS1 partici- proliferation ability was obviously decreased in pates in the regulation of LSCC progression. ZEB2-AS1 knockdown group examined by CCK8 assay Overexpression of ZEB2-AS1 promotes (Figure 2B). And, we also performed transwell assays. LSCC cells proliferation, migration and Consistent with the previous study, we found that ZEB2-AS1 knockdown decreased LSCC cell migration invasion and invasion abilities (Figure 2C–D). Colony formation As shown above, we found that knockdown of ZEB2-AS1 assay was always used to examine the proliferation ability decreases LSCC progression. So, we wanted to know the of cancer cells. So, we performed colony formation assay effect of overexpression of ZEB2-AS1. Firstly, we con- and indicated that LSCC cell proliferation ability signifi- structed ZEB2-AS1 overexpressed plasmid to elevate the cantly decreased after knockdown of ZEB2-AS1 expression of ZEB2-AS1 (Figure 3A). Next, we found that Figure 2 Knockdown of ZEB2-AS1 decreased LSCC cell abilities of progression. (A) Knockdown effect of ZEB2-AS1 in TU212 cells was examined using qRT-PCR. (B) Growth curves of TU212 cells transfected with negative control plasmids or ZEB2-AS1 shRNA plasmids. (C and D) The abilities of migration and invasion of TU212 cells after knockdown of ZEB2-AS1 were detected. (E and F) The number of colonies after overexpression of ZEB2-AS1 were counted compared to negative control. *P<0.05. All experiments were repeated three times. submit your manuscript | www.dovepress.com OncoTargets and Therapy 2019:12 DovePress Dovepress Xu et al Figure 3 Overexpression of ZEB2-AS1 promotes LSCC cell progression. (A) Expression of ZEB2-AS1 in TU212 cell line overexpressed with ZEB2-AS1 was determined by qRT-PCR. (B) TU212 cells were transfected with negative control plasmid or ZEB2-AS1 overexpression plasmid. Then, the growth rates of cells were determined using CCK-8 assay. (C and D) Transwell assay was used to detect the migration and invasive abilities of TU212 cells after transfected with control or ZEB2-AS1 overexpression plasmid. (E and F) Colony formation assay revealed that LSCC cells overexpressed of ZEB2-AS1 promoted the number of colonies. *P<0.05. All experiments were repeated three times. overexpression of ZEB2-AS1 significantly promoted cell group (Figure 3E–F). Taken together, these data indicate proliferation examined by CCK8 assays (Figure 3B). that ZEB2-AS1 significantly promotes the proliferation, migration and invasion abilities of LSCC cells. Moreover, we further examined the migration and invasion abilities of LSCC cells. We found that the migration and ZEB2-AS1 inhibits miR-6840-3p invasion abilities were increased in ZEB2-AS1 overex- expression to promote LSCC progression pressed LSCC cells (Figure 3C–D). We performed colony formation assay using control and ZEB2-AS1 overex- through regulating PLXNB1 expression pressed LSCC cells and found that the number of colonies In the previous data, we found that ZEB2-AS1 can pro- increased significantly in ZEB2-AS1 highly expressed mote the proliferation, migration and invasion of LSCC submit your manuscript | www.dovepress.com OncoTargets and Therapy 2019:12 7341 DovePress Xu et al Dovepress and colleagues found that lncRNA LOC554202 can pro- cells. So, we want to explore the mechanism that ZEB2- mote LSCC carcinoma progression by miR-31. AS1 regulates LSCC progression. To find the mechanism, They we performed bioinformatics analysis. We found that demonstrated that LOC554202 is highly expressed in ZEB2-AS1 can form a complementary base pair with LSCC tissues while miR-31 is lowly expressed in LSCC miR-6840-3p (Figure 4A). In addition, we found that tissues compared to adjacent tissues. And also, it is overexpression of ZEB2-AS1 significantly inhibited reported that lncRNA UCA1 can activate Wnt/β-catenin miR-6840-3p expression (Figure 4B). In contrast, we signaling pathway to elevate the proliferation, invasion also indicated that miR-6840-3p overexpression decreased and migration abilities of LSCC cells. ZEB2-AS1 expression while miR-6840-3p inhibition pro- ZEB2-AS1 is found to regulate many cancer progres- moted ZEB2-AS1 expression (Figure 4C). Next, we per- sion. For instance, ZEB2-AS1 was found to promote formed luciferase assay to prove that ZEB2-AS1 binds to breast cancer progression mainly regulating the prolifera- miR-6840-3p. We showed that ectopic expression of tion and epithelial–mesenchymal transition of breast can- miR-6840-3p significantly decreased luciferase intensity cer. And, Wu et al demonstrated that ZEB2-AS1 binds to in ZEB2-AS1 WT construct while ZEB2-AS1 mutant did miR-143-5p and promotes gastric cancer cells proliferation not change (Figure 4D). and migration via HIF-1a axis. However, the role of In addition, we wanted to find the potential target gene ZEB2-AS1 in LSCC remains unclear. So we explore the regulated by ZEB2-AS1 and miR-6840-3p. We performed biological role of ZEB2-AS1 in LSCC progression. In our bioinformatics analysis again using TargetScan7 program. study, we found that ZEB2-AS1 expresses at a higher level We showed that miR-6840-3p bound to PLXNB1 through in LSCC tissues compared to near-normal tissues and complementary base pair mechanism (Figure 4E). positively correlated with LSCC progression. Moreover, Through qRT-PCR experiment, we found that overexpres- we found that ZEB2-AS1 regulates the expression of miR- sion of miR-6840-3p decreased PLXNB1 expression. We 6840-3p and PLXNB1 in LSCC cell line. However, we did also proved that by Western blot assay (Figure 4F). Then, not explore the role of ZEB2-AS1/miR-6840-3p/PLXNB1 we wanted to explore the relationship between ZEB2-AS1, axis in other cancer cell lines. So we concluded that this miR-6840-3p and PLXNB1 expression. We indicated that mechanism is suited for LSCC. But, to know if this overexpression of ZEB2-AS1 promoted PLXNB1 expres- mechanism suit for other cancer needs more evidence. sion and knockdown of ZEB2-AS1 decreased PLXNB1 miRNAs are proven to participant in many cancer expression. Moreover, the increased expression of ZEB2- regulating. And there are many miRNAs that are involved 24–26 AS1 can be rescued by ectopic expression of miR-6840-3p in regulating LSCC progression. However, the role of (Figure 4G). And, we also performed luciferase assay and miR-6840-3p has not been reported in any cancer. In our found that overexpression of miR-6840-3p or knockdown study, we showed that ZEB2-AS1 can bind to miR-6840- of ZEB2-AS1 expression decreased luciferase intensity of 3p directly and inhibit the expression of miR-6840-3p. PLXNB1 WT construct while inhibition of miR-6840-3p And, we also found that miR-6840-3p mimic can also coupled with knockdown ZEB2-AS1 expression rescued inhibit ZEB2-AS1 expression. Moreover, miR-6840-3p PLXNB1 WT luciferase intensity. And also, the luciferase inhibits the expression of PLXNB1. So, we concluded intensity of PLXNB1 mutant did not change (Figure 4H). that ZEB2-AS1 is upstream while miR-6840-3p is As a result, we also performed transwell assay to examine downstream. the migration and invasion abilities. We showed that PLXNB1 is also found to be expressed in many cancer knockdown of ZEB2-AS1 decreased LSCC cell migration cells. Cao et al found that PLXNB1 can promote the cell and invasion while inhibition of miR-6840-3p or overex- proliferation, migration and invasion of cutaneous squa- pression of PLXNB1 rescues the abilities of migration and mous cell carcinoma. In addition, PLXNB1 is proven to invasion (Figure 4I–J). Collectively, we show that ZEB2- regulate Rho/αvβ3/PI3K/Akt signaling pathway, which is AS1 regulates LSCC progression through binding to miR- involved in regulating glioma invasiveness and 6840-3p from PLXNB1 mRNA. angiogenesis. In our study, we found that PLXNB1 is a target gene for ZEB2-AS1 and miR-6840-3p. ZEB2-AS1 Discussion overexpression or inhibition of miR-6840-3p significantly In the past decades, lncRNA is proven to play a crucial promotes PLXNB1 expression. And also, overexpression role in regulating LSCC progression. For example, Yang of PLXNB1 restores the decreased migration and invasion submit your manuscript | www.dovepress.com OncoTargets and Therapy 2019:12 DovePress Dovepress Xu et al Figure 4 ZEB2-AS1 binds to miR-6840-3p and regulates the expression of PLXNB1. (A) Predicted target site between miR-6840-3p and ZEB2-AS1 was shown. (B) Expression level of miR-6840-3p in TU212 cells transfected with ZEB2-AS1 overexpression plasmid was determined using qRT-PCR assay. (C) qRT-PCR assay was used to determine ZEB2-AS1 expression after miR-6840-3p overexpression or miR-6840-3p inhibition in TU212 cell line. (D) TU212 cells were co-transfected with the WT or Mut plasmid and the indicated miRNAs. Then, luciferase reporter assay was used to determine luciferase intensity. (E) Predicted target site between miR-6840-3p and PLXNB1. (F) Expression of PLXNB1 after overexpression of miR-6840-3p in TU212 cell line was examined by qRT-PCR and Western blot assays. qRT-PCR assay was normalized to 18S, Western blot assay was normalized to β-actin. (G) Luciferase activity assay was used to determine the luciferase intensity in TU212 cells transfected with different vectors. (H) Relative expression of PLXNB1 after ZEB2-AS1 overexpression or ZEB2-AS1 knockdown or ZEB2-AS1 overexpression with inhibition of miR-204-3p in TU212 cell lines. (I and J) Rescue assay was used to examine the abilities of migration and invasion after transfected with control or different plasmids in TU212 cells. *P<0.05. All experiments were repeated three times. submit your manuscript | www.dovepress.com OncoTargets and Therapy 2019:12 7343 DovePress Xu et al Dovepress 12. Wu FX, Gao HY, Liu KG, et al. The IncRNA ZEB2-AS1 is upregu- abilities of LSCC cells caused by decreased expression of lated in gastric cancer and affects cell proliferation and invasion via ZEB2-AS1. These data indicated that PLXNB1 plays a miR-143-5p/HIF-1 alpha axis. Onco Targets Ther. 2019;12:657–667. crucial role in regulating LSCC progression through doi:10.2147/OTT.S175521 13. Wu XQ, Yan TZ, Wang ZW, Wu X, Cao GH, Zhang C. LncRNA ZEB2-AS1 and miR-6840-3p axis. ZEB2-AS1 promotes bladder cancer cell proliferation and inhibits In conclusion, we showed that ZEB2-AS1 participants apoptosis by regulating miR-27b. Biomed Pharmacother. 2017;96:299–304. doi:10.1016/j.biopha.2017.08.060 in promoting LSCC cells proliferation, migration and inva- 14. Gao H, Gong NN, Ma ZB, et al. LncRNA ZEB2-AS1 promotes sion via miR-6840-3p/PLXNB1 axis. pancreatic cancer cell growth and invasion through regulating the miR-204/HMGB1 axis. Int J Biol Macromol. 2018;116:545–551. doi:10.1016/j.ijbiomac.2018.05.044 Disclosure 15. Zhang MB, Li Y, Wang HB, Yu WH, Lin S, Guo JQ. LncRNA The authors report no conflicts of interest in this work. SNHG5 affects cell proliferation, metastasis and migration of color- ectal cancer through regulating miR-132-3p/CREB5. Cancer Biol Ther. 2019;20:524–536. doi:10.1080/15384047.2018.1537579 References 16. Malik MFA, Ye L, Jiang WG. Reduced expression of semaphorin 4D and plexin-B in breast cancer is associated with poorer prognosis and 1. Akbaba S, Held T, Lang K, et al. Salvage radiotherapy for recurrent the potential linkage with oestrogen receptor. Oncol Rep. hypopharyngeal and laryngeal squamous cell carcinoma (SCC) after 2015;34:1049–1057. doi:10.3892/or.2015.4015 first-line treatment with surgery alone: a 10-year single-centre experi- 17. Roy AD, Yin T, Choudhary S, Rodionov V, Pilbeam CC, Wu YI. ence. Radiat Oncol. 2019;14. doi:10.1186/s13014-019-1238-8 Optogenetic activation of Plexin-B1 reveals contact repulsion 2. Boxberg M, Kuhn PH, Reiser M, et al. Tumor budding and cell nest between osteoclasts and osteoblasts. Nat Commun. 2017;8:15831. size are highly prognostic in laryngeal and hypopharyngeal squamous 18. Williamson M, de Winter P, Masters JR. Plexin-B1 signalling pro- cell carcinoma further evidence for a unified histopathologic grading motes androgen receptor translocation to the nucleus. Oncogene. system for squamous cell carcinomas of the upper aerodigestive tract. 2016;35:1066–1072. doi:10.1038/onc.2015.160 Am J Surg Pathol. 2019;43:303–313. doi:10.1097/PAS.000000000 19. Chang YW, Li L, Zhang LP, et al. Plexin-B1 indirectly affects glioma invasiveness and angiogenesis by regulating the RhoA/alpha v beta 3 3. Malm IJ, Rooper LM, Bishop JA, et al. Molecular and immunologic signaling pathway and SRPK1. Tumor Biol. 2016;37:11225–11236. analysis of laryngeal squamous cell carcinoma in smokers and non- doi:10.1007/s13277-016-4849-9 smokers. Am J Otolaryngol. 2019;40:213–217. doi:10.1016/j. 20. Zhang Y, Li Q, Zhuang R, et al. Plexin-B1: a potential diagnostic biomar- amjoto.2018.11.009 ker for glioma and a future target for glioma immunotherapy. J 4. Cao SJ, Huang YY, Zhang Q, et al. Molecular mechanisms of Neuroimmunol. 2012;252:113–117. doi:10.1016/j.jneuroim.2012.08.005 apoptosis and autophagy elicited by combined treatment with orido- 21. Liu SZ, Duan WC. Long noncoding RNA LINC00339 promotes nin and cetuximab in laryngeal squamous cell carcinoma. Apoptosis. laryngeal squamous cell carcinoma cell proliferation and invasion 2019;24:33–45. doi:10.1007/s10495-018-1497-0 via sponging miR-145. J Cell Biochem. 2019;120:8272–8279. 5. Kong XL, Duan Y, Sang YT, et al. LncRNA-CDC6 promotes breast doi:10.1002/jcb.v120.5 cancer progression and function as ceRNA to target CDC6 by spong- 22. Yang SJ, Wang J, Ge WS, Jiang YF. Long non-coding RNA ing microRNA-215. J Cell Physiol. 2019;234:9105–9117. doi:10.10 LOC554202 promotes laryngeal squamous cell carcinoma progres- 02/jcp.27587 sion through regulating miR-31. J Cell Biochem. 2018;119:6953– 6. Liang HL, Zhang CY, Guan HY, Liu J, Cui YB. LncRNA DANCR 6960. doi:10.1002/jcb.26902 promotes cervical cancer progression by upregulating ROCK1 via 23. Sun SG, Gong C, Yuan K. LncRNA UCA1 promotes cell prolifera- sponging miR-335-5p. J Cell Physiol. 2019;234:7266–7278. doi:10. tion, invasion and migration of laryngeal squamous cell carcinoma 1002/jcp.27484 cells by activating Wnt/-catenin signaling pathway. Exp Ther Med. 7. Zhou T, Qin GW, Yang LH, Xiang DK, Li SN. LncRNA XIST 2019;17:1182–1189. doi:10.3892/etm.2018.7097 regulates myocardial infarction by targeting miR-130a-3p. J Cell 24. Wang Y, Zhang D, Yu CH, et al. MicroRNA-708-5p contributes to Physiol. 2019;234:8659–8667. doi:10.1002/jcp.26327 the malignant behavior of laryngeal squamous cell carcinoma by 8. Shi X, Cui ZG, Liu XD, et al. LncRNA FIRRE is activated by MYC directly targeting metastasis suppressor-1. Int J Clin Exp Med. and promotes the development of diffuse large B-cell lymphoma via 2019;12:597–603. Wnt/beta-catenin signaling pathway. Biochem Biophys Res Commun. 25. Zhang F, Cao H. MicroRNA-143-3p suppresses cell growth and 2019;510:594–600. doi:10.1016/j.bbrc.2019.01.105 invasion in laryngeal squamous cell carcinoma via targeting the k- 9. Shen ZS, Li Q, Deng HX, et al. Long non-coding RNA profiling in Ras/Raf/MEK/ERK signaling pathway. Int J Oncol. 2019;54:689– laryngeal squamous cell carcinoma and its clinical significance: 701. doi:10.3892/ijo.2018.4655 potential biomarkers for LSCC. PLoS One. 2014;9. doi:10.1371/jour- 26. Han L, Tang MM, Xu XJ, et al. MiR-143-3p suppresses cell prolif- nal.pone.0108237 eration, migration, and invasion by targeting melanoma-associated 10. Lan T, Chang L, Wu L, Yuan YF. Downregulation of ZEB2-AS1 antigen A9 in laryngeal squamous cell carcinoma. J Cell Biochem. decreased tumor growth and metastasis in hepatocellular carcinoma. 2019;120:1245–1257. doi:10.1002/jcb.v120.2 Mol Med Rep. 2016;14:4606–4612. doi:10.3892/mmr.2016.5836 27. Cao J, Zhang C, Chen T, et al. Plexin-B1 and semaphorin 4D 11. Guo Y, Hu Y, Hu MM, He JB, Li BL. Long non-coding RNA ZEB2- cooperate to promote cutaneous squamous cell carcinoma cell pro- AS1 promotes proliferation and inhibits apoptosis in human lung liferation, migration and invasion. J Dermatol Sci. 2015;79:127–136. cancer cells. 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