| BackgroundCervical cancer is the second leading cause of death in women with cancer,the world's new cases each year more than 500,000 cases,the new cases of about 270,000 deaths.One of the primary causes is human papillomavirus(HPV)infection,and HR-HPV(high risk-HPV)oncoprotein expression can cause abnormal regulation of non-coding genes such as microRNAs(miRNAs).MiRNAs are mainly aligned with the nucleotide sequence of the target mRNA and complementarily regulate the level of gene expression after transcription.Cancer-related EMT helps to increase the invasion and metastasis of tumor cells,affecting the treatment effect,advanced cervical cancer patients prone to metastasis.Therefore,epithelial-mesenchymal transition(EMT)provides a new way to study the development of cervical cancer.A variety of cytokines can induce tumor cells to produce EMT,where TGF? is the most commonly used cytokine.In this study,epithelial-mesenchymal transition of cervical cancer cells was successfully induced by TGF?,and several differentially expressed miRNAs were detected by chip detection of induced cells and parent cells.After repeated experiments,we selected microRNA-374c-5p as the research object and explored its mechanism of invasion and metastasis of cervical cancer.Objective:To screen the microRNAs associated with the epithelial-mesenchymal transition model of cervical cancer cells and to explore the possible mechanism of their target genes.Methods:MiRNAs were screened for differential expression by chip detection and qRT-PCR screening.The changes of invasion and migration ability of cells were detected by Transwell assay.The expression of E-cadherin and Vimentin were detected by immunoblotting and immunofluorescence assay.The expression of microRNA in the clinical samples of cervical cancer patients was detected by in situ hybridization.The expression of target genes was detected by immunohistochemistry.The targeting relationship of miRNAto target gene was verified by luciferase.Result:The microRNA-374c-5p was most significantly down-regulated after TGF?1 induction,and the expression of microRNA-374c-5p was significantly higher than that of the untreated cervical cancer cells.It was found that elevated expression of microRNA-374c-5p could significantly inhibit the invasion and metastasis of cervical cancer cells.The expression of E-cadherin was up-regulated,the expression of Vimentin was down-regulated and the EMT process was reversed.The results of qRT-PCR also found similar results in the mRNA level of microRNA-374c-5p.The expression of E-cadherin was down-regulated,the expression of Vimentin was up-regulated and the EMT process was observed in the parental cells.Transwell assay showed that the invasion and migration ability of the parental cells was significantly increased after decreasing the expression level of microRNA-374c-5p.In the study of the target gene of microRNA-374c-5p,it was found that microRNA-374c-5p could target FOXC1 by target gene prediction software and luciferase reporter assay system.The expression of microRNA-374c-5p was lower in the clinical samples of metastatic cervical cancer patients,while FOXC1 was overexpressed.In contrast,the expression of microRNA-374c-5p was higher in the clinical samples of cervical cancer without metastasis,while FOXC1 present lower expression.Further statistics showed a negative correlation between the expression of microRNA-374c-5p and FOXC1(r=-0.725,P<0.001).In the clinical prognosis of patients with cervical cancer in the study,we found that microRNA-374c-5p presented lower expression,FOXC1 presented higher expression of cervical cancer patients with poor prognosis(P<0.05).Conclusion:1.Changing the expression level of microRNA-374c-5p can effectively affect the invasion and migration ability of cells and the progress of EMT.2.MicroRNA-374c-5p further affects the expression of E-cadherin,Vimentin and snail by affecting the expression of FOXC1,which affects the invasion and metastasis of tumor cells.3.The clinical data showed that microRNA-374c-5p was negatively correlated with FOXC1 expression,which was closely related to the metastasis and clinical prognosis of cervical cancer patients.Innovation:1.Epithelial-mesenchymal transition was successfully induced by TGF?-1.The effect of microRNA-374c-5p and FOXC1 on epithelial-mesenchymal transition was successfully demonstrated by the related functional experiments.2.MicroRNA-374c-5p may serve as a new marker for determining whether metastases or not.It may be a marker for clinical diagnosis,treatment and prognosis of cervical cancer. |
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