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Expression of eIF6 and its relationship with cell proliferation in colorectal adenocarcinoma

Expression of eIF6 and its relationship with cell proliferation in colorectal adenocarcinoma IntroductionColorectal adenocarcinoma (CRAC) is a common tumor that originates from glandular epithelium and exhibits high proliferation characteristics [1]. With the changes in people’s lifestyles, its incidence has been on the rise, posing a serious threat to human health [2]. Surgery is the main treatment for CRAC, and conservative therapy is often used for late-stage patients. CRAC is a complex biological process involving multiple gene regulations and abnormal functions of multiple regulatory pathways [3]. The stability of protein synthesis is crucial in tumor formation, and changes in translation-related factors have an important effect on protein synthesis because translation is involved in regulating protein synthesis [4]. The eukaryotic translation initiation factor (eIF) family is an important factor in regulating protein synthesis [5]. Changes in protein synthesis mechanisms and translation control can selectively translate some mRNAs, regulating abnormal cell proliferation, invasion, and migration and affecting tumor signal transduction [6]. eIFs maintain the formation of cell mRNA-ribosome complexes [7]. Studies on the expression of eIFs in tumor formation found that multiple eIFs may be potential therapeutic targets for tumors [8]. Eukaryotic translation initiation factor 6 (eIF6) is an important member of the eIF family; it regulates the interaction of other translation initiation complexes with eIFs and has “initiation” characteristics in tumor formation [9]. Some scholars found the increased expression of eIF6 in some epithelial-derived malignant tumors and identified related pathways it regulates, such as the mTOR signaling pathway [10]. In this study, we observed the expression of eIF6 in CRAC, explored its relationship with cell proliferation, and analyzed its clinical significance.Materials and methodsClinical dataAll included cases were diagnosed with CRAC from January 2017 to November 2022 in Tianjin Hospital(China). Inclusion criteria were as follows: 1) patients who underwent curative surgery and had a histopathological diagnosis of the tumor tissues and 2) complete clinical and follow-up data. Exclusion criteria were as follows: 1) patients who received preoperative radiotherapy or chemotherapy, 2) patients with Lynch syndrome, and 3) patients with gastrointestinal dysplasia. Sixty-four cases were selected. Tumor tissues and normal colorectal mucosal tissues (>3 cm away from the tumor margin) were retained and, immediately after the surgery, were taken, fixed in neutral-buffered formalin, and embedded in paraffin as specimens.Ethical approvalThe experiments were approved by the Medical Ethics Committee of Tianjin Hospital(China) (no. 2022-150) and conducted in compliance with the Declaration of Helsinki.Cell cultureColon cancer cell lines SW480, HCT15, and SW620 and normal colon cell line NCM460 were obtained from the Shanghai GenePharma Co., Ltd. (Shanghai, China). RPMI 1640 medium containing fetal bovine serum and the secondary antibodies of IHC were acquired from Shanghai Zhaoyuan Biotechnology Co., Ltd. (Shanghai, China). eIF6 pAb (PB9582), proliferating cell nuclear antigen (PCNA) (A00125), and β-actin (BM0627) were purchased from Boster Biological Technology Co., Ltd. (Wuhan China). ECL reagent was bought from Thermo Fisher Scientific Inc. (MA, USA).Immunohistochemical experimenteIF6 and PCNA expression was detected in paraffin-embedded tissues by immunohistochemistry using EnVision method. Given that eIF6 and PCNA were concentrated liquids, they were diluted to the ratio (1:200 for both eIF6 and PCNA), which was proven to be the optimal value for pre-experiment, prior to application in the formal assay. In brief, 4 μm sections were cut and baked for 25 min before being dipped in xylene I and II for 15 min each for dewaxing. The sections were then placed in graded alcohol for 2 min and rinsed in clean water. After the antigen was retrieved in a pressure cooker, the sections were washed three times in PBS, blocked with 3 % H2O2 for 10 min, and cleaned again three times in PBS. The diluted eIF6 or PCNA was dipped onto the sections, which were then placed in wet boxes and incubated overnight at 4 °C in a fridge. The next day, the sections were taken out and rinsed three times with PBS. The all-purpose secondary antibody was added prior to 30 min incubation at 37 °C in an incubator. After being washed with PBS three times, the sections were added with DAB and the color development was observed under a microscope. The color development was terminated by rinsing with water after it had appeared. Hematoxylin counterstaining was performed, followed by washing, differentiation, bluing in water, dehydration, transparency, and mounting.The positive site for eIF6 was the cytoplasm and/or the cell membrane, and the positive site for PCNA was the nucleus. Observation was first performed under low magnification (×100). The fields where tumor cells using the technique by Wu et al. [11], namely, a 2D assessment method. Percentage of stain: ≤5 % was scored 0 point, 6–25 % 1 point, 26–50 % 2 points, 51–75 % 3 points, and >75 % 4 points. Color intensity: no color was scored 0 point, light yellow 1 point, yellow 2 points, and brown 3 points. The final score resulted from the product of the above two ranged from 0 to 12, with 0–3 points being negative and 4–12 positive.Western bloteIF6 (1:2000) and PCNA (1:2500) expression was detected in cell line assays by Western blot experiments, and β-actin was applied as an internal reference. After the extraction of total protein, the concentration was determined by BCA assay. In brief, 10 μL of loading butter (5×) was evenly mixed with 40 μL of each protein. The mixture was boiled in a water bath for 8 min and then kept on standby at −4 °C in a fridge. The separating and stacking gels were prepared, and 20 μL of protein solution was mixed with the loading buffer (5×). With the gel removed, the glass plates were secured by clips, the surface with notches facing inside. An adequate amount of specimen was added into the well. With the gel removed, the glass plates were secured by clips, the surface with notches facing inside. The electrophoresis buffer was then added. A voltage of 80 V was set for the stacking gel, and 120 V for the separating gel. Electrotransfer and immunohybridization were performed once the loading buffer overflowed. After the film was taken out, the ultrahigh-sensitivity substrate was added for 2 min incubation. The film was then placed in an X-ray film cassette for 3 min exposure and 20 s development. Grayscale analysis was conducted by using the Image J software, and the ratio of protein to β-actin was used for semiquantitative analysis.Bioinformatics analysisThe GEPIA database (http://gepia.cancer-pku.cn) was used to predict the expression trends of eIF6 and PCNA in colon cancer by downloading the colon adenocarcinoma dataset and the data of eIF6 and PCNA in the control group, extracting the eIF6 and PCNA expression data, and analyzing their differences.Statistical analysisSAS 6.12 was applied for statistical analysis of the data. The quantitative data were expressed as mean ± standard deviation (x̄ ± s), and the normality and homogeneity of variance were checked beforehand. Multiple group comparisons were performed using ANOVA with Student-Newman–Keuls test for post hoc pairwise comparisons. Categorical data were examined by chi-square test. Linear correlation analysis was performed, and differences with p<0.05 were considered statistically significant.ResultsBasic data analysisThe group consisted of 64 cases with 38 males and 26 females aged 44–89 years (median of 70 years). Among these patients, cancer was found in the colon (ileocecal junction, ascending colon, transverse colon, descending colon, and sigmoid colon) in 37 cases and in the rectum in 27 cases. The maxim tumor diameter (MTD) was 1.5–10 cm and 5.0 ± 0.9 cm in average. In addition, 29 cases were highly differentiated, 24 moderately differentiated, and 11 poorly differentiated (including mucinous adenocarcinoma). Lymph node metastases (LNMets) were detected in only 32 cases (at 1–15 nodes), and intravascular cancer thrombosis was identified in only 12 cases (Figure 1).Figure 1:Images of CRAC and normal mucosa (HE stained). (A) Normal colonic mucosa is glandular epithelial cells with a cup-shaped cell morphology and a nucleus located at the base (×100). (B) Partial enlargement of Figure A (×200). (C) Highly differentiated adenocarcinoma: tumor cells consist of heterogeneous glandular ducts with elongated ducts, disorganized cell arrangement and infiltrative growth (×100). (D) Partial enlargement of Figure C (×200). (E) Moderately differentiated adenocarcinoma: The tumor consists of cells composed of heterogeneous glandular ducts that form a sieve-like structure with a back-to-back, coalescent wall, markedly disorganized cellular arrangement, and darkly stained nuclei (×100). (F) Partial enlargement of Figure E (×200). (G) Mucinous adenocarcinoma (poorly differentiated adenocarcinoma): The tumor consists of mucus-producing heterogeneous glands with disturbed cell polarity and infiltrative growth (×100). (H) Partial enlargement of Figure G (×200). (I) intravascular cancer thrombosis: Cancer cells visible in the blood vessels (×100). (J) Partial enlargement of Figure I (×200). (K) Metastatic adenocarcinoma of the lymph nodes: Adenocarcinoma cells are seen in the lymph sinuses (×100). (L) Partial enlargement of Figure K (×200).Results of bioinformatics analysisOur prediction based on GEPIA (http://gepia.cancer-pku.cn) showed a trend of high expression of eIF6 (in 274 tumor cases and 349 cases with normal mucosa) and PCNA (in 275 tumor cases and 349 cases with normal mucosa) in colon cancer (Figure 2).Figure 2:Expression predictions of eIF6 and PCNA (http://gepia.cancer-pku.cn). (A) Expression prediction of eIF6 by GEPIA. (B) Expression prediction of PCNA by GEPIA.Comparison of eIF6 and PCNA expression between colorectal adenocarcinoma and normal mucosaeIF6 and PCNA expression was statistically significant in the two groups, namely, their expression was higher in CRAC than in normal mucosa as shown in Table 1 and Figures 3 and 4.Table 1:Comparison of eIF6 and PCNA expression between CRAC and normal mucosa.GroupCaseseIF6X2p-ValuePCNAX2p-ValuePositiveNegativePositiveNegativeCRAC6442(65.63 %)22(34.37 %)43.2000<0.000154(84.38 %)10(15.63 %)19.9481<0.0001Normal mucosa646(9.38 %)58(90.62 %)30(46.88 %)34(53.13 %)Figure 3:Expression of eIF6 in CRAC and normal mucosa (IHC EnVision method). (A) Negative expression of eIF6 in normal mucosa: no color of eIF6 (×100). (B) Partial enlargement of Figure A (×200). (C) Positive expression of eIF6 in CRAC: eIF6 is brownish in the cytoplasm and cell membrane of CRAC (×100). (D) Partial enlargement of Figure C (×200). (E) Distribution of immunohistochemical score on eIF6 in CRAC and normal mucosa.Figure 4:Expression of PCNA in CRAC and normal mucosa (IHC EnVision method). (A) Expression of PCNA in normal mucosa: PCNA is scattered in the nucleus of colonic mucosal epithelial cells with yellow color expression (×100). (B) Partial enlargement of Figure A (×200). (C) Expression of PCNA in CRAC: PCNA shows brownish color in the nucleus of colon adenocarcinoma epithelial cells (×100). (D) Partial enlargement of Figure C (×200). (E) Distribution of immunohistochemical score on PCNA in CRAC and normal mucosa.Comparison of eIF6 and PCNA expression among different clinicopathologic characteristics of colorectal adenocarcinomaeIF6 and PCNA expression showed significant difference among different values of MTD and depth of infiltration (DOI) in colon adenocarcinoma (p<0.05). However, their expression was not statistically different among different genders, ages, degrees of differentiation, LNMets, intravascular cancer thrombosis, TNM stages, and numbers of lymph node metastases (p>0.05) (Table 2).Table 2:Comparison of eIF6 and PCNA expression among different clinicopathologic characteristics of colorectal adenocarcinoma.GroupCaseseIF6X2p-ValuePCNAX2p-ValuePositiveNegativePositiveNegativeGender Male3827(71.05 %)11(28.95 %)1.22150.269133(86.84 %)5(13.16 %)0.43180.5111 Female2615(57.69 %)11(42.31 %)21(80.77 %)5(19.23 %)Age <6094(44.44 %)5(55.56 %)2.08270.14908(88.89 %)1(11.11 %)0.16190.6875 ≥605538(69.01 %)17(30.91 %)46(83.64 %)9(16.36 %)MTD <5 cm3418(52.94 %)16(47.06 %)5.17820.0229a25(73.53 %)9(26.47 %)6.47160.0110a ≥5 cm3024(80.00 %)6(20.00 %)29(96.67 %)1(3.33 %)DOI Uninvolving serosa199(47.37 %)10(52.63 %)3.99250.0457a13(68.42 %)6(31.58 %)5.21700.0224a Through serosa4533(73.33 %)12(26.67 %)41(91.11 %)4(8.89 %)Intravascular cancer thrombosis0.34810.5552 No5235(67.31 %)17(32.69 %)44(84.62 %)8(15.38 %)0.01220.9122 Yes127(58.33 %)5(41.67 %)10(83.33 %)2(16.67 %)Degree of differentiation High-moderate5236(69.23 %)16(30.77 %)1.59840.206145(86.54 %)7(13.46 %)0.98460.3211 Low126(50.00 %)6(50.00 %)9(75.00 %)3(25.00 %)LNMets No3219(59.38 %)13(40.63 %)1.10820.292526(81.25 %)6(18.75 %)0.47410.4911 Yes3223(71.88 %)9(28.13 %)28(87.50 %)4(12.50 %)TNM stage I–II3118(58.06 %)13(41.94 %)1.52340.217125(80.65 %)6(19.35 %)0.63440.4257 III–IV3324(72.73 %)9(27.27 %)29(87.88 %)4(12.12 %)Number of lymph node metastases 1–31814(77.78 %)4(22.22 %)0.70910.399716(88.89 %)2(11.11 %)0.12270.7262 ≥4149(64.29 %)5(35.71 %)12(85.71 %)2(14.29 %)aP<0.05.Correlation between eIF6 and PCNA in CRACThe score of eIF6 ranged from 0 to 12 in CRAC, and that of PCNA ranged from 2 to 12. Positive correlation was found between eIF6 and PCNA by linear correlation analysis (X2=5.05, r=0.77, regression equation is Y=0.2873 + 5.662, and p=0.0283) (Figure 5).Figure 5:Correlation between eIF6 and PCNA in CRAC.Comparison of eIF6 and PCNA expression among SW480, HCT15, SW620, and NCM460eIF6 and PCNA expression was significantly higher in the cell lines of SW480, HCT15, and SW620 than in NCM460 (Table 3 and Figure 6).Table 3:Comparison of eIF6 and PCNA expression among SW480, HCT15, SW620, and NCM460.GroupneIF6Fp-ValuePCNAFp-ValueSW48031.36 ± 0.11a1.98 ± 0.17aHCT1531.35 ± 0.09a26.840.00021.87 ± 0.12a31.63<0.0001SW62031.25 ± 0.09a1.83 ± 0.21aNCM46030.79 ± 0.080.89 ± 0.09avs NCM460, p<0.05 (SNK-based pairwise comparison).Figure 6:Comparison of eIF6 and PCNA expression among SW480, HCT15, SW620, and NCM460. (A) Comparison of eIF6 in different cell lines. (B) Comparison of PCNA in different cell lines. *vs. NCM460, p<0.05.DiscussionThe formation of CRAC cells is associated with atypical proliferation, and the abnormal regulation of proliferation-related genes provides a strong basis for oncogenesis [12, 13]. The eIF family includes eIF1 to eIF6, some of which have multiple subtypes with different functions. eIFs also regulate translation mainly at initiation, which is a limiting stage in protein synthesis. Incorrect regulation tends to induce exceptions of translation and atypical cell proliferation [14]. As the latest member of the family, eIF6 exists in yeast and mammalian cells and affects the maturity of ribosomal subunit 60S. In yeast, eIF6 is mainly located in the nucleosome; in mammalian cells, however, most eIF6 genes exist in the cytoplasm and a small number (30 % or so) are found in the nucleus, such as HeLa, A431, and NIH/3T3 fibroblast [15]. eIF6 plays a remarkably role in the generation of ribosomal subunit 60S as a part of the preribosomal particle in the nucleolus [16]. It also acts as a translation factor in the nucleosome. Hence, the subcellular location is crucial to the functional regulation of eIF6. The eIF6 complex has three stable structures: module A, module B, and module C [17, 18]. Therefore, eIF6 is possibly a housekeeping gene involved in MFC polymerization, trimer recruitment, and AUG codon recognition. Scholars have recently discovered the abnormal expression of eIF6 in tumors [19]. Cai et al. [20] tested the expression of eIF6 in lung cancer and found that high eIF6 expression is an important factor facilitating oncogenesis. Some researchers also found that the expression level of eIF6 is significantly higher in WHO grade IV gliomas than in grade II and III gliomas; they believe that the high expression of eIF6 is positively correlated with malignancy in gliomas, and patients with high eIF6 expression have low overall survival rates [21].By using bioinformatic analysis, our study has predicted the increasing trend of eIF6 and PCNA in CRAC. IHC assay reveals that eIF6 and PCNA expression is significantly higher in CRAC than in normal mucosa, which is further proved by the cell line culture experiment. These findings indicate that high eIF6 and PCNA expression facilitates the onset of CRAC. Their expression is significantly higher in the MTD≥5 cm group than in the MTD<5 cm group in CRAC, suggesting that eIF6 and PCNA are associated with MTD. In other words, eIF6 and PCNA are involved in tumor growth. Furthermore, their expression is significantly higher in the Through Serosa group than in the Uninvolving Serosa group. This finding suggests that eIF6 and PCNA are associated with tumor invasion, that is, their high expression is a promoter of tumor cell invasion. No differences in eIF6 and PCNA expression were found in terms of degree of differentiation, LNMets, intravascular cancer thrombosis, TNM stage, and number of lymph node metastases. This finding shows that eIF6 and PCNA are not involved in tumor differentiation or lymphatic spread. The results also reveal a positive correlation between eIF6 and PCNA, implying a positive collaboration between them. eIF6 regulates the proliferation of CRAC cells by mediating PCNA. Nevertheless, the specific regulatory impact of eIF6 on tumor cells must be further confirmed by in vitro experiments using CCK8. In addition, eIF6 regulates ribosome assembly and biogenesis, thus controlling the binding of ribosome subunits 40S and 60S and engaging in the assembly of 80S [22]. In turn, it regulates protein synthesis and participates in atypical tumor proliferation. Nuclear–cytoplasmic trafficking is triggered by eIF6 phosphorylation, which is in accordance with the fact that the bioactivity of proteins can be regulated by phosphorylation. The in vitro phosphorylation of eIF6 at Ser-175 and Ser-174 by the nuclear isomer of casein kinase gene CK1α or CK1δ fosters the formation of 60S preribosomal particles in the cytoplasm [23, 24]. Nevertheless, CaMK and calcineurin mediate dephosphorylation, which helps eIF6 migrate back into the nucleolus and resume the biosynthesis of ribosomal subunit 60S. Therefore, phosphorylated eIF6 and unphosphorylated eIF6 are possible factors in the dynamic balance during oncogenesis. This function is also the typical role of eIF6 in regulating oncogenesis. eIF6 involvement in tumor progression is possibly affected by regulating the expression of related factors, including multiple growth factors and downstream regulatory factors, such as TGFs, MMPs, and angiogenesis factors [25]. Multiple factors can effectively activate the related transcriptional regulatory elements, thus promoting the proliferation of tumor cells. Given that CRAC proliferation is a constant process, different factors may regulate this phenomenon in different ways at different stages of tumor progression, though some scholars have proven that eIF6 regulates cell proliferation via pathways such as WNT, mediates the imbalance of tumor-related factors, and thus regulates tumor progression [26].ConclusionsHigh eIF6 expression was observed in CRAC and was involved in the formation and progression of the tumor. A positive correlation was found between eIF6 and PCNA. Further research on the various regulatory pathways of CRAC controlled by eIF6 is an important focus for future studies. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png ONCOLOGIE de Gruyter

Expression of eIF6 and its relationship with cell proliferation in colorectal adenocarcinoma

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Publisher
de Gruyter
Copyright
© 2023 the author(s), published by De Gruyter, Berlin/Boston
eISSN
1765-2839
DOI
10.1515/oncologie-2023-0007
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Abstract

IntroductionColorectal adenocarcinoma (CRAC) is a common tumor that originates from glandular epithelium and exhibits high proliferation characteristics [1]. With the changes in people’s lifestyles, its incidence has been on the rise, posing a serious threat to human health [2]. Surgery is the main treatment for CRAC, and conservative therapy is often used for late-stage patients. CRAC is a complex biological process involving multiple gene regulations and abnormal functions of multiple regulatory pathways [3]. The stability of protein synthesis is crucial in tumor formation, and changes in translation-related factors have an important effect on protein synthesis because translation is involved in regulating protein synthesis [4]. The eukaryotic translation initiation factor (eIF) family is an important factor in regulating protein synthesis [5]. Changes in protein synthesis mechanisms and translation control can selectively translate some mRNAs, regulating abnormal cell proliferation, invasion, and migration and affecting tumor signal transduction [6]. eIFs maintain the formation of cell mRNA-ribosome complexes [7]. Studies on the expression of eIFs in tumor formation found that multiple eIFs may be potential therapeutic targets for tumors [8]. Eukaryotic translation initiation factor 6 (eIF6) is an important member of the eIF family; it regulates the interaction of other translation initiation complexes with eIFs and has “initiation” characteristics in tumor formation [9]. Some scholars found the increased expression of eIF6 in some epithelial-derived malignant tumors and identified related pathways it regulates, such as the mTOR signaling pathway [10]. In this study, we observed the expression of eIF6 in CRAC, explored its relationship with cell proliferation, and analyzed its clinical significance.Materials and methodsClinical dataAll included cases were diagnosed with CRAC from January 2017 to November 2022 in Tianjin Hospital(China). Inclusion criteria were as follows: 1) patients who underwent curative surgery and had a histopathological diagnosis of the tumor tissues and 2) complete clinical and follow-up data. Exclusion criteria were as follows: 1) patients who received preoperative radiotherapy or chemotherapy, 2) patients with Lynch syndrome, and 3) patients with gastrointestinal dysplasia. Sixty-four cases were selected. Tumor tissues and normal colorectal mucosal tissues (>3 cm away from the tumor margin) were retained and, immediately after the surgery, were taken, fixed in neutral-buffered formalin, and embedded in paraffin as specimens.Ethical approvalThe experiments were approved by the Medical Ethics Committee of Tianjin Hospital(China) (no. 2022-150) and conducted in compliance with the Declaration of Helsinki.Cell cultureColon cancer cell lines SW480, HCT15, and SW620 and normal colon cell line NCM460 were obtained from the Shanghai GenePharma Co., Ltd. (Shanghai, China). RPMI 1640 medium containing fetal bovine serum and the secondary antibodies of IHC were acquired from Shanghai Zhaoyuan Biotechnology Co., Ltd. (Shanghai, China). eIF6 pAb (PB9582), proliferating cell nuclear antigen (PCNA) (A00125), and β-actin (BM0627) were purchased from Boster Biological Technology Co., Ltd. (Wuhan China). ECL reagent was bought from Thermo Fisher Scientific Inc. (MA, USA).Immunohistochemical experimenteIF6 and PCNA expression was detected in paraffin-embedded tissues by immunohistochemistry using EnVision method. Given that eIF6 and PCNA were concentrated liquids, they were diluted to the ratio (1:200 for both eIF6 and PCNA), which was proven to be the optimal value for pre-experiment, prior to application in the formal assay. In brief, 4 μm sections were cut and baked for 25 min before being dipped in xylene I and II for 15 min each for dewaxing. The sections were then placed in graded alcohol for 2 min and rinsed in clean water. After the antigen was retrieved in a pressure cooker, the sections were washed three times in PBS, blocked with 3 % H2O2 for 10 min, and cleaned again three times in PBS. The diluted eIF6 or PCNA was dipped onto the sections, which were then placed in wet boxes and incubated overnight at 4 °C in a fridge. The next day, the sections were taken out and rinsed three times with PBS. The all-purpose secondary antibody was added prior to 30 min incubation at 37 °C in an incubator. After being washed with PBS three times, the sections were added with DAB and the color development was observed under a microscope. The color development was terminated by rinsing with water after it had appeared. Hematoxylin counterstaining was performed, followed by washing, differentiation, bluing in water, dehydration, transparency, and mounting.The positive site for eIF6 was the cytoplasm and/or the cell membrane, and the positive site for PCNA was the nucleus. Observation was first performed under low magnification (×100). The fields where tumor cells using the technique by Wu et al. [11], namely, a 2D assessment method. Percentage of stain: ≤5 % was scored 0 point, 6–25 % 1 point, 26–50 % 2 points, 51–75 % 3 points, and >75 % 4 points. Color intensity: no color was scored 0 point, light yellow 1 point, yellow 2 points, and brown 3 points. The final score resulted from the product of the above two ranged from 0 to 12, with 0–3 points being negative and 4–12 positive.Western bloteIF6 (1:2000) and PCNA (1:2500) expression was detected in cell line assays by Western blot experiments, and β-actin was applied as an internal reference. After the extraction of total protein, the concentration was determined by BCA assay. In brief, 10 μL of loading butter (5×) was evenly mixed with 40 μL of each protein. The mixture was boiled in a water bath for 8 min and then kept on standby at −4 °C in a fridge. The separating and stacking gels were prepared, and 20 μL of protein solution was mixed with the loading buffer (5×). With the gel removed, the glass plates were secured by clips, the surface with notches facing inside. An adequate amount of specimen was added into the well. With the gel removed, the glass plates were secured by clips, the surface with notches facing inside. The electrophoresis buffer was then added. A voltage of 80 V was set for the stacking gel, and 120 V for the separating gel. Electrotransfer and immunohybridization were performed once the loading buffer overflowed. After the film was taken out, the ultrahigh-sensitivity substrate was added for 2 min incubation. The film was then placed in an X-ray film cassette for 3 min exposure and 20 s development. Grayscale analysis was conducted by using the Image J software, and the ratio of protein to β-actin was used for semiquantitative analysis.Bioinformatics analysisThe GEPIA database (http://gepia.cancer-pku.cn) was used to predict the expression trends of eIF6 and PCNA in colon cancer by downloading the colon adenocarcinoma dataset and the data of eIF6 and PCNA in the control group, extracting the eIF6 and PCNA expression data, and analyzing their differences.Statistical analysisSAS 6.12 was applied for statistical analysis of the data. The quantitative data were expressed as mean ± standard deviation (x̄ ± s), and the normality and homogeneity of variance were checked beforehand. Multiple group comparisons were performed using ANOVA with Student-Newman–Keuls test for post hoc pairwise comparisons. Categorical data were examined by chi-square test. Linear correlation analysis was performed, and differences with p<0.05 were considered statistically significant.ResultsBasic data analysisThe group consisted of 64 cases with 38 males and 26 females aged 44–89 years (median of 70 years). Among these patients, cancer was found in the colon (ileocecal junction, ascending colon, transverse colon, descending colon, and sigmoid colon) in 37 cases and in the rectum in 27 cases. The maxim tumor diameter (MTD) was 1.5–10 cm and 5.0 ± 0.9 cm in average. In addition, 29 cases were highly differentiated, 24 moderately differentiated, and 11 poorly differentiated (including mucinous adenocarcinoma). Lymph node metastases (LNMets) were detected in only 32 cases (at 1–15 nodes), and intravascular cancer thrombosis was identified in only 12 cases (Figure 1).Figure 1:Images of CRAC and normal mucosa (HE stained). (A) Normal colonic mucosa is glandular epithelial cells with a cup-shaped cell morphology and a nucleus located at the base (×100). (B) Partial enlargement of Figure A (×200). (C) Highly differentiated adenocarcinoma: tumor cells consist of heterogeneous glandular ducts with elongated ducts, disorganized cell arrangement and infiltrative growth (×100). (D) Partial enlargement of Figure C (×200). (E) Moderately differentiated adenocarcinoma: The tumor consists of cells composed of heterogeneous glandular ducts that form a sieve-like structure with a back-to-back, coalescent wall, markedly disorganized cellular arrangement, and darkly stained nuclei (×100). (F) Partial enlargement of Figure E (×200). (G) Mucinous adenocarcinoma (poorly differentiated adenocarcinoma): The tumor consists of mucus-producing heterogeneous glands with disturbed cell polarity and infiltrative growth (×100). (H) Partial enlargement of Figure G (×200). (I) intravascular cancer thrombosis: Cancer cells visible in the blood vessels (×100). (J) Partial enlargement of Figure I (×200). (K) Metastatic adenocarcinoma of the lymph nodes: Adenocarcinoma cells are seen in the lymph sinuses (×100). (L) Partial enlargement of Figure K (×200).Results of bioinformatics analysisOur prediction based on GEPIA (http://gepia.cancer-pku.cn) showed a trend of high expression of eIF6 (in 274 tumor cases and 349 cases with normal mucosa) and PCNA (in 275 tumor cases and 349 cases with normal mucosa) in colon cancer (Figure 2).Figure 2:Expression predictions of eIF6 and PCNA (http://gepia.cancer-pku.cn). (A) Expression prediction of eIF6 by GEPIA. (B) Expression prediction of PCNA by GEPIA.Comparison of eIF6 and PCNA expression between colorectal adenocarcinoma and normal mucosaeIF6 and PCNA expression was statistically significant in the two groups, namely, their expression was higher in CRAC than in normal mucosa as shown in Table 1 and Figures 3 and 4.Table 1:Comparison of eIF6 and PCNA expression between CRAC and normal mucosa.GroupCaseseIF6X2p-ValuePCNAX2p-ValuePositiveNegativePositiveNegativeCRAC6442(65.63 %)22(34.37 %)43.2000<0.000154(84.38 %)10(15.63 %)19.9481<0.0001Normal mucosa646(9.38 %)58(90.62 %)30(46.88 %)34(53.13 %)Figure 3:Expression of eIF6 in CRAC and normal mucosa (IHC EnVision method). (A) Negative expression of eIF6 in normal mucosa: no color of eIF6 (×100). (B) Partial enlargement of Figure A (×200). (C) Positive expression of eIF6 in CRAC: eIF6 is brownish in the cytoplasm and cell membrane of CRAC (×100). (D) Partial enlargement of Figure C (×200). (E) Distribution of immunohistochemical score on eIF6 in CRAC and normal mucosa.Figure 4:Expression of PCNA in CRAC and normal mucosa (IHC EnVision method). (A) Expression of PCNA in normal mucosa: PCNA is scattered in the nucleus of colonic mucosal epithelial cells with yellow color expression (×100). (B) Partial enlargement of Figure A (×200). (C) Expression of PCNA in CRAC: PCNA shows brownish color in the nucleus of colon adenocarcinoma epithelial cells (×100). (D) Partial enlargement of Figure C (×200). (E) Distribution of immunohistochemical score on PCNA in CRAC and normal mucosa.Comparison of eIF6 and PCNA expression among different clinicopathologic characteristics of colorectal adenocarcinomaeIF6 and PCNA expression showed significant difference among different values of MTD and depth of infiltration (DOI) in colon adenocarcinoma (p<0.05). However, their expression was not statistically different among different genders, ages, degrees of differentiation, LNMets, intravascular cancer thrombosis, TNM stages, and numbers of lymph node metastases (p>0.05) (Table 2).Table 2:Comparison of eIF6 and PCNA expression among different clinicopathologic characteristics of colorectal adenocarcinoma.GroupCaseseIF6X2p-ValuePCNAX2p-ValuePositiveNegativePositiveNegativeGender Male3827(71.05 %)11(28.95 %)1.22150.269133(86.84 %)5(13.16 %)0.43180.5111 Female2615(57.69 %)11(42.31 %)21(80.77 %)5(19.23 %)Age <6094(44.44 %)5(55.56 %)2.08270.14908(88.89 %)1(11.11 %)0.16190.6875 ≥605538(69.01 %)17(30.91 %)46(83.64 %)9(16.36 %)MTD <5 cm3418(52.94 %)16(47.06 %)5.17820.0229a25(73.53 %)9(26.47 %)6.47160.0110a ≥5 cm3024(80.00 %)6(20.00 %)29(96.67 %)1(3.33 %)DOI Uninvolving serosa199(47.37 %)10(52.63 %)3.99250.0457a13(68.42 %)6(31.58 %)5.21700.0224a Through serosa4533(73.33 %)12(26.67 %)41(91.11 %)4(8.89 %)Intravascular cancer thrombosis0.34810.5552 No5235(67.31 %)17(32.69 %)44(84.62 %)8(15.38 %)0.01220.9122 Yes127(58.33 %)5(41.67 %)10(83.33 %)2(16.67 %)Degree of differentiation High-moderate5236(69.23 %)16(30.77 %)1.59840.206145(86.54 %)7(13.46 %)0.98460.3211 Low126(50.00 %)6(50.00 %)9(75.00 %)3(25.00 %)LNMets No3219(59.38 %)13(40.63 %)1.10820.292526(81.25 %)6(18.75 %)0.47410.4911 Yes3223(71.88 %)9(28.13 %)28(87.50 %)4(12.50 %)TNM stage I–II3118(58.06 %)13(41.94 %)1.52340.217125(80.65 %)6(19.35 %)0.63440.4257 III–IV3324(72.73 %)9(27.27 %)29(87.88 %)4(12.12 %)Number of lymph node metastases 1–31814(77.78 %)4(22.22 %)0.70910.399716(88.89 %)2(11.11 %)0.12270.7262 ≥4149(64.29 %)5(35.71 %)12(85.71 %)2(14.29 %)aP<0.05.Correlation between eIF6 and PCNA in CRACThe score of eIF6 ranged from 0 to 12 in CRAC, and that of PCNA ranged from 2 to 12. Positive correlation was found between eIF6 and PCNA by linear correlation analysis (X2=5.05, r=0.77, regression equation is Y=0.2873 + 5.662, and p=0.0283) (Figure 5).Figure 5:Correlation between eIF6 and PCNA in CRAC.Comparison of eIF6 and PCNA expression among SW480, HCT15, SW620, and NCM460eIF6 and PCNA expression was significantly higher in the cell lines of SW480, HCT15, and SW620 than in NCM460 (Table 3 and Figure 6).Table 3:Comparison of eIF6 and PCNA expression among SW480, HCT15, SW620, and NCM460.GroupneIF6Fp-ValuePCNAFp-ValueSW48031.36 ± 0.11a1.98 ± 0.17aHCT1531.35 ± 0.09a26.840.00021.87 ± 0.12a31.63<0.0001SW62031.25 ± 0.09a1.83 ± 0.21aNCM46030.79 ± 0.080.89 ± 0.09avs NCM460, p<0.05 (SNK-based pairwise comparison).Figure 6:Comparison of eIF6 and PCNA expression among SW480, HCT15, SW620, and NCM460. (A) Comparison of eIF6 in different cell lines. (B) Comparison of PCNA in different cell lines. *vs. NCM460, p<0.05.DiscussionThe formation of CRAC cells is associated with atypical proliferation, and the abnormal regulation of proliferation-related genes provides a strong basis for oncogenesis [12, 13]. The eIF family includes eIF1 to eIF6, some of which have multiple subtypes with different functions. eIFs also regulate translation mainly at initiation, which is a limiting stage in protein synthesis. Incorrect regulation tends to induce exceptions of translation and atypical cell proliferation [14]. As the latest member of the family, eIF6 exists in yeast and mammalian cells and affects the maturity of ribosomal subunit 60S. In yeast, eIF6 is mainly located in the nucleosome; in mammalian cells, however, most eIF6 genes exist in the cytoplasm and a small number (30 % or so) are found in the nucleus, such as HeLa, A431, and NIH/3T3 fibroblast [15]. eIF6 plays a remarkably role in the generation of ribosomal subunit 60S as a part of the preribosomal particle in the nucleolus [16]. It also acts as a translation factor in the nucleosome. Hence, the subcellular location is crucial to the functional regulation of eIF6. The eIF6 complex has three stable structures: module A, module B, and module C [17, 18]. Therefore, eIF6 is possibly a housekeeping gene involved in MFC polymerization, trimer recruitment, and AUG codon recognition. Scholars have recently discovered the abnormal expression of eIF6 in tumors [19]. Cai et al. [20] tested the expression of eIF6 in lung cancer and found that high eIF6 expression is an important factor facilitating oncogenesis. Some researchers also found that the expression level of eIF6 is significantly higher in WHO grade IV gliomas than in grade II and III gliomas; they believe that the high expression of eIF6 is positively correlated with malignancy in gliomas, and patients with high eIF6 expression have low overall survival rates [21].By using bioinformatic analysis, our study has predicted the increasing trend of eIF6 and PCNA in CRAC. IHC assay reveals that eIF6 and PCNA expression is significantly higher in CRAC than in normal mucosa, which is further proved by the cell line culture experiment. These findings indicate that high eIF6 and PCNA expression facilitates the onset of CRAC. Their expression is significantly higher in the MTD≥5 cm group than in the MTD<5 cm group in CRAC, suggesting that eIF6 and PCNA are associated with MTD. In other words, eIF6 and PCNA are involved in tumor growth. Furthermore, their expression is significantly higher in the Through Serosa group than in the Uninvolving Serosa group. This finding suggests that eIF6 and PCNA are associated with tumor invasion, that is, their high expression is a promoter of tumor cell invasion. No differences in eIF6 and PCNA expression were found in terms of degree of differentiation, LNMets, intravascular cancer thrombosis, TNM stage, and number of lymph node metastases. This finding shows that eIF6 and PCNA are not involved in tumor differentiation or lymphatic spread. The results also reveal a positive correlation between eIF6 and PCNA, implying a positive collaboration between them. eIF6 regulates the proliferation of CRAC cells by mediating PCNA. Nevertheless, the specific regulatory impact of eIF6 on tumor cells must be further confirmed by in vitro experiments using CCK8. In addition, eIF6 regulates ribosome assembly and biogenesis, thus controlling the binding of ribosome subunits 40S and 60S and engaging in the assembly of 80S [22]. In turn, it regulates protein synthesis and participates in atypical tumor proliferation. Nuclear–cytoplasmic trafficking is triggered by eIF6 phosphorylation, which is in accordance with the fact that the bioactivity of proteins can be regulated by phosphorylation. The in vitro phosphorylation of eIF6 at Ser-175 and Ser-174 by the nuclear isomer of casein kinase gene CK1α or CK1δ fosters the formation of 60S preribosomal particles in the cytoplasm [23, 24]. Nevertheless, CaMK and calcineurin mediate dephosphorylation, which helps eIF6 migrate back into the nucleolus and resume the biosynthesis of ribosomal subunit 60S. Therefore, phosphorylated eIF6 and unphosphorylated eIF6 are possible factors in the dynamic balance during oncogenesis. This function is also the typical role of eIF6 in regulating oncogenesis. eIF6 involvement in tumor progression is possibly affected by regulating the expression of related factors, including multiple growth factors and downstream regulatory factors, such as TGFs, MMPs, and angiogenesis factors [25]. Multiple factors can effectively activate the related transcriptional regulatory elements, thus promoting the proliferation of tumor cells. Given that CRAC proliferation is a constant process, different factors may regulate this phenomenon in different ways at different stages of tumor progression, though some scholars have proven that eIF6 regulates cell proliferation via pathways such as WNT, mediates the imbalance of tumor-related factors, and thus regulates tumor progression [26].ConclusionsHigh eIF6 expression was observed in CRAC and was involved in the formation and progression of the tumor. A positive correlation was found between eIF6 and PCNA. Further research on the various regulatory pathways of CRAC controlled by eIF6 is an important focus for future studies.

Journal

ONCOLOGIEde Gruyter

Published: Jul 1, 2023

Keywords: clinical characteristic; colorectal adenocarcinoma; eukaryotic translation initiation factor 6; neoplasm; proliferation

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