| Objective:To explore the regulation mechanism of differential lncRNAs on EMT in ovarian cancer.In this study,5 cases of ovarian cancer and 5 cases of normal ovarian tissue were selected and differentially expressed lncRNAs were screened by high-throughput sequencing.Bioinformatics was used to analyze the lncRNAs related to EMT and predict the downstream mechanism.Expression was verified in ovarian cancer tissues,and its malignant tumor function was verified at the cellular and animal levels.Based on biogenic analysis,miRNA downstream of the target molecule lncRNA was predicted,and it was inferred that lncRNA may play an important role in promoting EMT in the malignant progression of ovarian cancer through miRNA adsorption and regulation of downstream target genes.Methods:1.High-throughput sequencing was used to detect differentially expressed lncRNAs in 5 cases of ovarian cancer and 5 cases of normal ovarian cancer,which were correlated with the overall survival of patients with TCGA ovarian cancer(hazard ratio>1 or<1)by means of bioinformatics analysis.It was found that EMT-related pathway was activated in ovarian cancer,which was closely related to the occurrence and development of ovarian cancer.QPCR was used to analyze the expression of the five EMT-related lncRNAs in ovarian and normal ovarian tissues,as well as in ovarian cancer cell lines.2.LncRNA AC005224.4 overexpressed plasmid and interference sequence were instantaneously transfected into ovarian cancer cells SKOV3 and CAOV-3,and the proliferation ability,migration ability and invasion ability of ovarian cancer cells were detected by cck-8 assay,tranwell migration test and tranwell invasion test.Western Blot was used to detect the expression of EMT-related proteins to determine the effect of changes in the expression level of lncRNA AC005224.4 on the malignant biological function of ovarian cancer cell lines.3.The SKOV3 cells were infected with lentivirus lv-si-nc(control lentivirus)and lv-si-ac005224.4(interference lentivirus)respectively.The cells were inoculated in the armpit of the mice,and the tumors were produced gradually over time.The volume and weight of the tumors were detected.The effects of lncRNA AC005224.4 on EMT related proteins(E-cadherin,N-cadherin,snail and vimentin)in tumor were detected.4.LncRNA AC005224.4 was found to be associated with important EMT transcription factors such as SNAI2 by simultaneous screening of co-expressed genes from our high-throughput screening and TCGA.Combined with starBase v3.0,HMDD v3.0 and TargetScan Release 7.2 databases,lncRNA AC005224.4 targeted miRNAs,ovarian cancer disease related miRNAs and SNAI2 gene targeted miRNAs were respectively obtained.The intersection of the three was taken to obtain 7 miRNAs:Hsa-mir-98,hsa-let-7g,hsa-let-7a,hsa-let-7b,hsa-let-7c,hsa-let-7f,hsa-mir-140-3p.QPCR detected the expression of the above 7 miRNAs and SNAI2 in ovarian cancer tissues,and q-PCR detected the expression of mir-140-3p and SNAI2 in ovarian cancer cell lines.The targeted binding of IncRNA AC005224.4 to mir-140-3p and mir-140-3p to SNAI2 was detected by double luciferase assay.The effects of lncRNA AC005224.4 expression on mir-140-3p and SNAI2 were detected and analyzed.The influence of mir-140-3p expression on SNAI2 was detected.Western Blot was used to detect the effect of mir-140-3p and SNAI2 expression on EMT protein expression.Results:1.In this study,lncRNA was sequenced and analyzed in 5 cases of ovarian cancer and 5 cases of normal ovarian tissues by high-throughput sequencing technology.We screened the lncRNAs differential expression(log2fc>1,P<0.05),which was related to the overall survival of TCGA patients with ovarian cancer(hazard ratio>1 or<1).Five lncRNAs related to EMT were found in ovarian cancer:AC005224.4,AC009093.2,UG0898H09,AC015912.3 and AL139081.1.LncRNA AC005224.4 was highly expressed in ovarian tissue and ovarian cancer cell lines by qPCR.2.Cck-8 results showed that overexpression of lncRNA AC005224.4 in SKOV3 and caov-3 cell lines promoted the proliferation of ovarian cancer cells,while interference with lncRNA AC005224.4 inhibited the proliferation of ovarian cancer cells.Transwell migration results showed that overexpression of lncRNA AC005224.4 in SKOV3 and CAOV-3 cell lines promoted ovarian cancer cell migration,while interference with lncRNA AC005224.4 inhibited ovarian cancer cell migration.Transwell invasion test results showed that overexpression of lncRNA AC005224.4 in SKOV3 and CAOV-3 cell lines promoted the invasion of ovarian cancer cells,while interference with lncRNA AC005224.4 inhibited the invasion of ovarian cancer cells.Overexpression of lncRNA AC005224.4 in SKOV3 and CAOV-3 cell lines inhibited the expression of E-cadherin in ovarian cancer cells,and promoted the expression of N-cadherin,Snail and Vimentin.Interference with lncRNA AC005224.4 can promote E-cadherin expression in ovarian cancer cells and inhibit the expression of N-cadherin,Snail and Vimentin.3.The xenograft tumor model of nude mice was constructed.In the lv-si-AC005224.4 group(interfering lentivirus),tumor volume and tumor weight were significantly lower than that of the control group.WB results in the tumor showed that E-cadherin expression was significantly up-regulated in the lv-si-AC005224.4 group,while N-cadherin,Snail and Vimentin expression were significantly down-regulated in the Iv-si-NC group.4.The expression of mir-140-3p was the lowest in ovarian cancer and ovarian cancer cells.Compared with normal ovarian tissue,SNAI2 is highly expressed in ovarian tissue.Overexpression of lncRNA AC005224.4 inhibited mir-140-3p expression in SKOV3 cells,and interfered with lncRNA AC005224.4 to promote mir-140-3p expression in SKOV3 cells.Overexpression of mir-140-3p can inhibit SNAI2 expression in nest cancer cells,and down-regulation of mir-140-3p can promote SNAI2 expression in ovarian cancer cells.Double luciferase assay confirmed the interaction site between lncRNA AC005224.4 and mir-140-3p,and the targeting site between mir-140-3p and the 3’-utr region of SNAI2.Overexpression of mir-140-3p can promote the expression of E-cadherin in ovarian cancer cells and inhibit the expression of N-cadherin,Snail and Vimentin.Overexpression of SNAI2 inhibited the expression of E-cadherin in ovarian cancer cells and promoted the expression of N-cadherin,Snail and Vimentin.Conclusions:1.Differential expression of lncRNAs related to EMT was found by combining high-throughput sequencing and bioinformatics analysis in ovarian cancer tissues and normal ovarian tissue,in which lncRNA AC005224.4 showed high expression in ovarian cancer tissues and cells.2.LncRNA AC005224.4 promotes the proliferation,invasion and migration of ovarian cancer cells,Promote the EMT process of ovarian cancer cells.3.Inhibition of the expression of lncRNA ac005224.4 can inhibit the proliferation of tumor and the expression of EMT related protein in transplanted ovarian cancer tissue at the animal level.4.LncRNA AC005224.4/mir-140-3p/SNAI2 regulation axis promotes ovarian cancer invasion and metastasis through EMT. |
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