The Effects Of MiR-5195-3p On The Proliferation,Migration And Invasion Of Cervical Cancer Cells And Its Potential Mechanisims

Objective This study intends to use normal epithelial cells HaCaT as control cells and human cervical cancer cell lines SiHa,Caski,HeLa,and C33A as research objects to explore the effects of miR-5195-3p on the proliferation,migration,and invasion of human cervical cancer cell lines and their implications mechanism.Methods1.Expression levels of miR-5195-3p in human cervical cancer cell lines SiHa,Caski,HeLa and C33A:HaCaT cells were used as the control group,and cervical cancer cells SiHa,Caski,HeLa,and C33A were used as the experimental group.The miR-5195-3p expression levels in both groups were measured by Real-time quantitative PCR.2.Effects of miR-5195-3p on cell proliferation,migration and invasion in human cervical cancer cell lines:Cationic liposome-mediated methods were used to deliver exogenous miR-5195-3p and knock down endogenous miR-5195-3p to construction of recombinant cervical cancer cells Caski/5195-mimic and SiHa/5195-inhibitor.The miR-5195-3p overexpression in Caski and miR-5195-3p knockdown in SiHa were verified by real-time quantitative PCR.MTT,Clone formation experiments,Scratch healing experiments,and Transwell chamber tests were utilized to assess the proliferation,migration,and invasion capacity of recombinant cervical cancer cells Caski/5195-mimic and SiHa/5195-inhibitor.3.Prediction and verification of miR-5195-3p target genes:Searching TargetScan Human7.2?http://www.targetscan.org/vert₇2/?,StarBasev3.0?http://starbase.sysu.edu.cn/agoClipRNA.php?andmiRTarBase?http://mirtarbase.mbc.nctu.edu.tw/php/index.php?to predict the target genes of miR-5195-3p,Draw Venn Diagram?http://bioinformatics.psb.ugent.be/webtools/Venn/?screen the target genes,and use double luciferase reporter gene experiments to confirm the combination of miR-5195-3p and the target genes.RT-qPCR and Western Blot further affirmed that miR-5195-3p targets and regulates the target genes in cervical cancer cells.4.Expression changes of recombinant cells Caski/5195-mimic and SiHa/5195-inhibitor epithelial-mesenchymal transition related molecules and matrix metalloproteinase?MMP?:RT-qPCR and Western blot were applied to detect Caski/5195-mimic and SiHa/5195-inhibitor Changes in mRNA and protein expression levels of MMP7,MMP9 and epithelial marker E-cadherin,interstitial marker Vimentin and EMT transcription factor snail.5.Effects of miR-5195-3p on cervical cancer proliferation and migration in vivo:The experiments were divided into two groups,Caski/stable-NC?negative control group?and Caski/5195-3p agmoir?over-expressing miR-5195-3p group?,with 5×10⁶ cells per group.The two groups of cells were seeded subcutaneously in female nude mice,and the tumor size was measured every four days after one week.Nude mice were sacrificed after 31 days,and the tumor tissue was stripped and embedded in paraffin,sectioned and HE stained.IHC was used to examine the expression of ICAT,Ki-67,MMP7,MMP9,E-cadherin and Vimentin in each group.Results1.RT-qPCR results showed that compared with normal epithelial cells HaCaT,miR-5195-3p had lower expression levels in cervical cancer cells Caski,SiHa,HeLa,C33A?P<0.05?,and the Caski cells with lower miR-5195-3p expression,the SiHa cells with higher miR-5195-3p expression were selected as follow-up research objects.2.RT-qPCR results demonstrated that miR-5195-3p mimics was significantly increased the expression of miR-5195-3p?P<0.01?;miR-5195-3p inhibitors was reduced the expression of miR-5195-3p?P<0.05?.Contrasted with negative control group,MTT experiments and clone formation experiments results indicated that the proliferation and clone formation ability of recombinant cells Caski/5195-mimic were distinctly reduced?P<0.01?;The results of Scratch healing and Transwell experiments showed that the migration and invasion ability of Caski/5195-mimic were obviously decreased compared with the negative control group?P<0.01?.Insteadly,the proliferation,migration and invasion capability of recombinant cells SiHa/5195-inhibitor were significantly enhanced contrasted with the negative control group?P<0.01,P<0.01,P<0.01?.3.TargetScan Human7.2?http://www.targetscan.org/vert₇2/?,StarBasev3.0?http://starbase.sysu.edu.cn/agoClipRNA.php?,miRTarBase?http://mirtarbase.mbc.nctu.edu.tw/php/index.php?,et al,these websites predict that the 3'UTR region of ICAT and miR-5195-3p have binding sites.The results of the double luciferase reportergene experiment confirmed that miR-5195-3p directly binds to the 3'UTR region of ICAT.RT-qPCR and Western blot results demonstrated that contrasted with the negative control group,the mRNA and protein levels of ICAT in Caski/5195-mimic cells were significantly down-regulated?P<0.05?,and in SiHa/5195-inhibitor cells were distinctly up-regulated?P<0.05?.4.Western blot results indicated that compared with the negative control group,the protein expression of the MMP7,MMP9,interstitial markers Vimentin and the EMT transcription factor snail were down-regulated,and the epithelial marker E-cadherin was up-regulated in the recombinant cells Caski/5195-mimic;the protein expression of MMP7,MMP9,the interstitial marker Vimentin and the EMT transcription factor snail were increased,and the expression of the epithelial marker E-cadherin was decreased in the recombinant cells SiHa/5195-inhibitor.5.After knocking down of endogenous ICAT expression,the proliferation?migration and invasion ability of recombinant cells SiHa/siICAT were significantly reduced?P<0.001,P<0.001,P<0.01?;After exogenously overexpressing ICAT,the proliferation,migration and invasion ability of recombinant cells Caski/AdICAT were obviously improved?P<0.001,P<0.01,P<0.01?.6.After knocking down the endogenous expression of ICAT in the recombinant cells SiHa/5195-inhibitor,the proliferation?migration and invasion ability of the SiHa/5195-inhibitor cells were decreased?P<0.001,P<0.001,P<0.001?;After exogenously overexpressing the miR-5195-3p in the recombinant cells Caski/AdICAT,the proliferation?migration and invasion ability of the Caski/AdICAT cells were declined?P<0.001,P<0.01,P<0.001?.7.Animal experiments illustrated that compared with the negative control group,the tumor volume and weight of the Caski/5195-3p agmoir group were reduced?P<0.001,P<0.05?;the results of the immunohistochemical indicated that E-cadherin was up-regulated,while the expressions of Vimentin,Ki-67,ICAT,MMP7 and MMP9 were all down-regulated in the Caski/5195-3p agmoir group.Conclusions1.Compared with the normal epithelial cells HaCaT,the miR-5195-3p has lower expression in cervical cancer cells SiHa and Caski.2.miR-5195-3p can inhibit the proliferation,migration and invasion ability,as well as the EMT process of cervical cancer cells SiHa and Caski.3.ICAT is one of the downstream target genes of miR-5195-3p in cervical cancer cells SiHa and Caski.4.miR-5195-3p inhibits the proliferation,migration and invasion of cervical cancer cells SiHa and Caski by down-regulating the ICAT,and its mechanism is related to the EMT pathway.