| Cervical cancer is one of the most common malignant tumors of the female reproductive system.In 2018,the number of new cases of cervical cancer in women worldwide was about 570,000,with 311,000 deaths,accounting for 6.6% and 7.5% of female cancer incidence and death,respectively.Cervical cancer is a disease in which multiple factors,multiple genes and multiple links work together to form a complex molecular regulatory mechanism.Although the research on the pathogenesis of cervical cancer has made some progress,there is still a lack of clinically effective predictors,specific indicators for detecting tumor metastasis and prognosis,and individualized treatment methods.Therefore,further research is needed on the biological and clinical characteristics of cervical cancer.Long non-codingRNA(lncRNA)is an important group of non-codingRNA.LncRNA can regulate cell growth,proliferation,cell cycle and apoptosis and other biological behaviors,and is closely related to the occurrence,development and prevention of diseases.NEAT1 is a lncRNA located in the nucleus.Studies have shown that NEAT1 participates in regulating gene expression by regulating and stabilizing mRNA in the nucleus,and NEAT1 plays an important role in the occurrence and progression of tumors.However,the research on the related mechanism of NEAT1 in cervical cancer has not been fully explored.In addition,the dysregulation of lncRNA expression can have a significant impact on tumor cell proliferation and apoptosis,and can participate in cancer metastasis.It can also affect the chemotherapy resistance and radiotherapy resistance of tumors by participating in different related mechanisms.In cervical cancer,the signaling pathways that NEAT1 can participate in and the related molecular mechanisms are still lacking.Exploring the impact of NEAT1 on the key proteins of a variety of important signaling pathways can further explore the internal mechanism of NEAT1.This will also provide a new target for accurate clinical diagnosis and treatment of cervical cancer.Objective In this study,the expression level of lncRNA NEAT1 in the tumor tissues and adjacent normal tissues of cervical cancer patients was detected,and the difference in the expression of NEAT1 in cervical cancer tissues and its correlation with the prognosis of patients were analyzed through the TCGA database.Through further detection of NEAT1 expression in a variety of cervical cancer cell lines,and through siRNA interference and inhibition of NEAT1 expression in HeLa cells and C33 A cells.Through a series of cytological experiments,the effects of NEAT1 on the proliferation,migration and invasion of cervical cancer cells are studied,and the related molecular mechanisms of its effects are explored to provide new ideas and targets for the clinical diagnosis and treatment of cervical cancer.Materials and Methods1.The difference in the expression of lncRNA NEAT1 in cervical cancer tumor tissues and normal tissues adjacent to the cancer was analyzed by the Cancer Genome Atlas(TCGA).GEPIA2 online tool was used to conduct ROC analysis to explore the correlation between NEAT1 expression and the prognosis of cervical cancer patients.2.The expression level of lncRNA NEAT1 in 20 cases of cervical cancer patients’ tumor tissues and normal tissues adjacent to the cancer were detected by qPCR,and the differences in expression were analyzed.3.The expression levels of lncRNA NEAT1 in four different cervical cancer cell lines(HeLa,C33 A,SiHa and Ca Ski)and normal human cervical epithelial cell lines(H8)were detected by qPCR.4.Interference and silence the expression of NEAT1 in HeLa and C33 A cells by transfection of siRNA-NEAT1.5.The MTT experiment was used to explore the effect of lncRNA NEAT1 expression on the proliferation of cervical cancer cells.6.The clone formation experiment was used to explore the influence of the expression level of lncRNA NEAT1 on the colony-forming ability of cervical cancer cells.7.Transwell experiment and wounding healing assay are used to explore the influence of lncRNA NEAT1 expression level on the invasion and migration ability of cervical cancer cells.8.The effect of lncRNA NEAT1 expression level on the tumor formation of cervical cancer cells was explored through the tumor formation experiment in NOD/SCID mice.9.Western Blot experiment was used to detect the effect of lncRNA NEAT1 expression level on cell proliferation and metastasis-related protein expression levels in cervical cancer cells,including E-cadherin,N-cadherin,Vimentin,β-catemin,T-Akt and p-Akt.Results1.TCGA database analysis showed that the expression of lncRNA NEAT1 in cervical cancer tissue was significantly higher than that in normal cervical tissue,and the difference was statistically significant(P<0.01).ROC analysis showed that the overall survival of patients in the NEAT1 high expression group was significantly lower than that of the NEAT1 low expression group,and the difference was statistically significant(P<0.01).2.The qPCR results of tissue samples from 20 patients with cervical cancer also showed that the expression of NEAT1 in the tumor tissues of cervical cancer patients was significantly higher than that in normal tissues adjacent to the cancer.The relative expression level of NEAT1 in tumor tissues was 10.012±3.757,and the relative expression level of NEAT1 in normal tissues adjacent to cancer was3.830±1.927,the difference was statistically significant(P<0.01).3.The qPCR results showed that the expression levels of NEAT1 in HeLa,C33 A,SiHa and Ca Ski cells were 2.920±0.216,2.987±0.212,2.780±0.154 and2.633±0.075,respectively,and the expression level of NEAT1 in H8 cells was1.000±0.080.The expression of NEAT1 in cervical cancer cell lines was significantly higher than that in normal human cervical epithelial cell lines,and the difference was statistically significant(P<0.01).4.Hela and C33 A cells that silence NEAT1 expression were successfully established by siRNA transfection.The expression level of NEAT1 in cells transfected with si-NEAT1 was significantly lower than that in cells transfected with si-NC,and the difference was statistically significant(P<0.01).5.The results of the MTT experiment showed that the relative cell proliferation number of the si-NEAT1 group in HeLa cells was 1.426±0.292 when the experiment was conducted for 120 hours,which was significantly lower than the relative cell proliferation number of the si-NC group cells of 2.803±0.204.The relative cell proliferation number of C33 A cells in the si-NEAT1 group was0.513±0.110,which was significantly lower than the relative cell proliferation number of cells in the si-NC group of 2.048±0.048.The difference was statistically significant(P<0.01).6.The clone formation experiment results showed that among HeLa cells,the number of clones in the si-NEAT1 group was 196.80±51.57,which was significantly lower than the number of clones in the si-NC group,which was353.40±23.59.In C33 A cells,the number of clones in the si-NEAT1 group was195.6±19.59,which was significantly lower than that of the si-NC group at290.00±28.44.The difference was statistically significant(P<0.01).7.The results of transwell experiments showed that the cell counts of HeLa and C33 A cells passing through the chamber in the si-NEAT1 group were177.60±18.45 and 239.8±22.88,respectively,and the cell counts of HeLa and C33 A cells passing through the chamber in the si-NC group were 620.80±35.71 and 372.20±38.46,respectively.The difference was statistically significant(P<0.01).The wounding healing assay results showed that the healing rates of HeLa and C33 A cells in the si-NEAT1 group were 0.483±0.021 and 0.382±0.014,respectively,and the healing rates of HeLa and C33 A cells in the si-NC group were 0.798±0.036 and 0.800±0.030,respectively.The difference was statistically significant(P<0.05).8.The results of in vivo tumor formation experiments showed that at the end of the experiment,the tumor mass in the shRNA-NEAT1 group was 0.106±0.027 g,which was significantly lower than the tumor mass in the shRNA-NC group,which was 0.223±0.031 g.The difference was statistically significant(P<0.01).9.Western Blot experiment results showed that the expression of E-cadherin in HeLa cells and C33 A cells in the siRNA-NEAT1 group was significantly higher than its expression in HeLa cells in the siRNA-NC group.The expressions of N-cadherin,Vimentin,p-Akt,β-catenin in HeLa cells and C33 A cells in the siRNA-NEAT1 group were significantly reduced.The difference was statistically significant(P<0.01).Conclusion LncRNA NEAT1 is significantly up-regulated in the tumor tissues of cervical cancer patients and cervical cancer cell lines,and the prognostic survival rate of cervical cancer patients with high NEAT1 expression is significantly lower than that of cervical cancer patients with low NEAT1 expression.NEAT1 has the function of oncogene.After silencing NEAT1 expression,the proliferation,invasion and migration ability of HeLa cells and C33 A cells were significantly reduced,and the tumorigenesis ability in vivo was significantly reduced.NEAT1 is closely related to the activation of the epithelial-mesenchymal transition related signaling pathway,Wnt/β-catenin signaling pathway and PI3K/Akt signaling pathway in cervical cancer cells.Therefore,this study can show that NEAT1 has a good application prospect in the diagnosis,treatment,and prognosis of cervical cancer. |
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