| Objective:Through the whole transcriptome sequencing of cervical cancer tissue specimens with three different lymph node metastasis states(lymph node metastasis(N+):3 cases;lymphatic vessel invasion(LVSI):3 cases;lymph node non-metastasis(NO):3 cases).The differentially expressed genes(DEGs)between N+,LVSI and NO tissue samples were screened from the sequencing results,and finally angiostatin-2(Vasohibin-2,VASH2)was obtained,and the Gene Expression Omnibus(GEO)was analyzed using bioinformatics methods.The high-throughput sequencing microarray data in the database is analyzed,and the upstream miRNA that may regulate VASH2-miR-106a-5p,is obtained through the ceRNA mechanism and online tool prediction.Furthermore,the expression levels of miR-106a-5p and VASH2 were detected in cervical cancer cell lines,N+and NO cervical cancer tissue specimens,and the effect of VASH2 on cell migration and invasion ability was studied in cervical cancer cell lines.Finally,explore the potential molecular mechanism of VASH2 gene to promote cell migration and invasion in cervical cancer cell lines.Methods:1.1.The cervical cancer tissues of 3 cases of N+,3 cases of LVSI,and 3 cases of NO were detected by whole transcriptome sequencing to detect differential gene expression,and then verified in GEO database and TCGA database using bioinformatics methods to obtain Differentially expressed genes(DEGs)between N+and NO in cervical cancer tissues,and screen out the gene VASH2 involved in angiogenesis from DEGs,and finally obtain the miRNA that may regulate VASH2-miR-106a-5p through online tool prediction;2.Collect postoperative tissue specimens of cervical cancer patients who underwent surgery at the Third Affiliated Hospital of Kunming Medical University from June 2019 to November 2019;3.Using RT-qPCR and Western-blotting to detect various human cervical cancer cell lines(Hela,C-33A,Ca Ski,SiHa,MS751)and normal cervical epithelial cell lines(HcerEpic),as well as 6 cases of N+and 6 cases of N0.Detect the mRNA and protein expression levels of VASH2,and the expression levels of miR-106a-5p in cervical cancer tissues;4.Use immunohistochemical staining(IHC)to detect the protein expression level of VASH2 in the above 6 cases of N+and 6 cases of NO cervical cancer tissues;5.Combined with the expression of VASH2 in cervical cancer cell lines,use lentiviral stable expression vectors to construct exogenously overexpressing cervical cancer cell lines and control cells(and interfering with endogenous VASH2 expression)cell;6.Use the CCK-8 test to detect the proliferation ability of the cells in the above-mentioned cervical cancer cell lines that overexpress VASH2 exogenously and interfere with the expression of endogenous VASH2 and their control cells,use the scratch test to detect the migration ability of cells,and use Transwell Experiment to detect the invasion ability of cells,and use the lymphatic vessel formation experiment to detect the ability of cells to form new lymphatic vessels;7.Use Western-blotting to detect the expression of key molecules in the process of epithelial cell interstitial(EMT)in the above-mentioned cervical cancer cell lines that overexpress VASH2 exogenously and interfere with endogenous VASH2 expression and their control cells,and use RT-qPCR detects the expression of TGF-β,and explores the potential molecular mechanism of VASH2 promoting the proliferation,migration,invasion and lymphatic vessel formation of cervical cancer cells.Results:1.The analysis of the whole transcriptome sequencing results of 9 samples obtained 8 types of differential expression trends and 48 differential trend genes.VASH2 mRNA expression in N0,LVSI,and N+tissues gradually increased and the differences were statistically significant.After analyzing the GSE26511 microarray data set in the GEO database,it was found that compared with cervical cancer NO tissue,the expression of VASH2 in cervical cancer N+tissue was significantly up-regulated and the difference was statistically significant,log2FC=2.01;After analyzing the data of cancer NO and N+tissues,it was found that the expression of VASH2 in cervical cancer N+tissues was significantly up-regulated and the difference was statistically significant.Finally,use the ceRNA mechanism and the online software TargetScan to predict the upstream miRNA that may target and regulate VASH2.Among them,miR-106a-5p is highly conservative and participates in the regulation of the EMT pathway,and is most likely to regulate the expression of VASH2 in cervical cancer;2.A total of 76 postoperative cervical cancer tissue samples that met the criteria for admission and drainage were collected,including 17 N+tissue samples and 59 NO tissue samples;3.The expression level of miR-106a-5p in cervical cancer cell lines C-33A,Ca-Ski and SiHa is higher than that of normal cervical epithelial cell line HcerEpic and the difference is statistically significant;VASH2 in Ca Ski,SiHa and MS751 cells Compared with HcerEpic,the expression levels of mRNA and protein were significantly up-regulated and the difference was statistically significant;compared with NO tissues,the expression levels of miR-106a-5p in N+tissues had no significant difference;but compared to NO tissues,the expression levels of VASH2 in N+tissues mRNA and protein expression levels were significantly up-regulated and the difference was statistically significant;4.VASH2 was expressed in 6 cases of N+tissues with deep staining intensity and a high proportion of positive cells,while it was almost not expressed in 6 cases of NO tissues;5.According to the expression of VASH2 in cervical cancer cell lines,we successfully constructed the cervical cancer cell line HeLa-VASH2 that overexpresses VASH2 exogenously and the control cell HeLa-VASH2-vector;at the same time,we successfully constructed the endogenous interference VASH2 Expressed cervical cancer cell lines Ca Ski-VASH2RNAi#1,Ca Ski-VASH2RNAi#2,MS751-VASH2RNAi#1,MS751-VASH2RNAi#2 and control cells Ca Ski-VASH2RNAi-vector,MS751-VASH2RNAi-vector;6.CCK-8 experiment found that the cell viability of HeLa-VASH2 was significantly higher than that of HeLa-VASH2-vector,while the cell viability of Ca Ski-VASH2RNAi#1,Ca Ski-VASH2RNAi#2 and MS751-VASH2RNAi#1,MS751-VASH2RNAi#2 was higher than that of Ca Ski-VASH2RNAi-vector and MS751-VASH2RNAi-vector were significantly weakened;the scratch experiment found that the migration ability of HeLa-VASH2 cells was significantly higher than that of HeLa-VASH2-vector,while Ca Ski-VASH2RNAi#1,Ca Ski-VASH2RNAi#2 and MS751-VASH2RNAi-vector Compared with Ca Ski-VASH2RNAi-vector and MS75 1-VASH2RNAi-vector,the migration ability of VASH2RNAi#1 and MS751-VASH2RNAi#2 cells was significantly weakened;Transwell experiment found that HeLa-VASH2 cell invasion ability was significantly enhanced compared with HeLa-VASH2-vector,while Ca Ski-VASH2RNAi#1,Ca Ski-VASH2RNAi#2;and MS751-VASH2RNAi#1,MS751-VASH2RNAi#2 cell invasion ability was significantly weaker than Ca Ski-VASH2RNAi-vector and MS751-VASH2RNAi-vector;7.The protein expression level of E-cadherin in HeLa-VASH2 cells is significantly lower than that of HeLa-VASH2-vector,while in Ca Ski-VASH2RNAi#1,Ca Ski-VASH2RNAi#2 and MS751-VASH2RNAi#1,MS751-VASH2RNAi#The protein expression in 2 is significantly higher than CaSki-VASH2RNAi-vector and MS751-VASH2RNAi-vector;on the contrary,the protein expression levels of N-cadherin,Vimentin and VEGF-C in HeLa-VASH2 cells are significantly higher than HeLa-VASH2-vector,while the protein expression levels in Ca Ski-VASH2RNAi#1,CaSki-VASH2RNAi#2,MS751-VASH2RNAi#1,MS751-VASH2 RNAi#2 are significantly lower than CaSki-VASH2RNAi-vector and MS751-VASH2RNAi-vector;TGF-β mRNA expression level in HeLa-VASH2 cells was significantly higher than HeLa-VASH2-vector,and in Ca Ski-VASH2RNAi#1,Ca Ski-VASH2RNAi#2;MS751-VASH2RNAi#1,MS751-VASH2RNAi#2 The mRNA expression level of CaSki-VASH2RNAi-vector and MS751-VASH2RNAi-vector is significantly lower.Conclusion(s):1.The expression level of VASH2 in cervical cancer cell lines Ca Ski,SiHa,MS751 is significantly higher than that of normal cervical epithelial HcerEpic cells,and the expression in cervical cancer N+tissue is significantly higher than NO tissue,indicating that VASH2 may be involved in the regulation of cervical cancer lymph nodes Transfer;2.The expression level of miR-106a-5p in cervical cancer cell lines C-33A,Ca-Ski and SiHa was significantly higher than that of HcerEpic cells,but there was no difference in expression between NO tissue and N+tissue,indicating miR-106a-5p May not participate in cervical cancer lymph node metastasis and cannot regulate VASH2;3.VASH2 may promote the proliferation,migration,invasion and lymphatic vessel formation of cervical cancer cells in vitro through the EMT pathway mediated by the TGF-β pathway. |
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