Effects Of MiR-24-3p On The Proliferation,Migration And Invasion Of Cevrial Cancer Cells And Their Mechanisms

Objective: To detect the expression of miR-24-3p in cervical cancer cells and explore its effect on the proliferation,migration and invasion of cervical cancer cells as well as its molecular mechanism.Methods:1)Online software and experiments were used to analyze and detect the expression of miR-24-3p in different tumors,normal tissues and three cell lines of cervical cancer: The tumor-miRNA-pathway online software was used to analyze the differential expression of miR-24-3p in various tumors and normal tissues,and miRNA-24-3p was detected in three cell lines of cervical cancer(SiHa,CaSki and C33A)by qRT-PCR.2)The effects of miR-24-3p on cell proliferation,migration and invasion of SiHa and CaSki cells: miR-24-3p was overexpressed or interfered in SiHa and CaSki cells using miR-24-3p mimics or inhibitors,CCK8 assay was used to detect cell proliferation,and Transwell assay was used to measure cell migration and invasion.3)The prediction,screening and verification of miR-24-3p’s targets:The target genes of miR-24-3p were screened in TargetScan,miRtarbase,miRDB and starBase to obtain target gene collection and functional annotation in Venn and Metascape,respectively.Targetscan7.2 was used to find the binding sites between miR-24-3p and target gene,the relationship between miR-24-3p and target gene was verified byluciferase reporter assays,Western blotting and qRT-PCR.4)To investigate whether AMOTL2 is involved in the promotive effects of miR-24-3p on cervical cancer cell proliferation and migration:The small interfering RNA fragments of AMOTL2 was co-transfected into the CaSki cells with inhibitors of miR-24-3p,and the cell proliferation and migration were detected by CCK8 and Transwell assays,respectively.Without or with small interfering RNA fragments of AMOTL2,miR-24-3p inhibitors were transfected into the CaSki cells,and the cell proliferation and migration were detected by CCK8 and Transwell assays,respectively.5)To analyze and verify the signaling pathway of miR-24-3p in cervical cancer cells: The target gene set above was intersected with cervical cancer pathogenic genes in Genecards to obtain a new aggregation for KEGG signal pathway enrichment analysis,being combined with the literature,we chose the Hippo signaling pathway and verified it.miR-24-3p was overexpressed or interfered in SiHa and CaSki cells using miR-24-3p mimics or inhibitors,then Western blot was used to measure the expression and phosphorylation level of YAP which is a key factor of Hippo signaling pathway,additionally,qRT-PCR was used to detect the mRNA levels of YAP downstream targets such as ANKRD1,CYR61 and CTGF.6)The effect of miR-24-3p on tumor growth,AMOTL2 protein,phosphorylated YAP and phosphorylated LATS1/2 in vivo: CaSki cells,which were transfected with NC or miR-24-3p antagomir,were inoculated subcutaneously in nude mice to observe the tumor volume every seven days.After 35 days,nude mice were sacrificed and the tumors were removed,then the tumor tissues were fixed and embedded in paraffin and sectioned.The tissue sections were stained for the detection of AMOTL2,p-YAP,and p-LATS1/2 by immunohistochemistry.7)The prediction and screening of upstream LncRNAs regulating miR-24-3p: starBase and DIANA databases were used to predict the LncRNAs binding miR-24-3p;LncRNAs were identified with low expression in cervical cancer and were performed an overall survival curve analysis according to GEPIA.Results:1)Tumor-miRNA-pathway from online software analysis showed that miR-24-3p was highly expressed in bladder cancer,cervical cancer,melanoma and esophageal cancer compared with corresponding normal tissues.In our study,its expression was higher in SiHa and CaSki cells than that in C33 A cells.SiHa and CaSki cells were selected for subsequent research.2)miR-24-3p promoted the cell proliferation,migration and invasion in cervical cancer cells.The CCK8 and Transwell assays showed that the cell proliferation,migration and invasion in overexpressing miR-24-3p group were higher than the control group(P<0.001,0.001,0.01);while miR-24-3p were decreased,the cell proliferation,migration and invasion ability were lower than those of the control group(P<0.001,0.01,0.01).During the animal experiment,the growth rate of tumor in the miR-24-3p antagomir group was lower than that of the NC group(P<0.05),which was consistent with the conclusion of the in vitro experiment.3)AMOTL2 was the target gene of miR-24-3p.The intersection of 95 target genes of miR-24-3p was acquired for functional enrichment analysis by miRDB,miRtarbase and TargetScan,GO functional annotation results showed that the target genes are enriched in twenty biological processes including protein autophosphorylation,positive regulation of kinase activity,regulation of cellular stress response,regulation of cellular protein localization,reproductive development,growth regulation,negative regulation of cell proliferation,and so on.The optimal candidate target gene AMOTL2 was selected from the intersection of previous gene set and the predicted target gene in programNum equal or greater than to 7 of starBase.Targetscan7.2 prediction results showed that miR-24-3p had binding sites with AMOTL2 3’UTR region,and the sequences were the same in humans,chimp and rhesus.Dual-luciferase report assay showed that miR-24-3p bound AMOTL2 3 ’UTR directly.Western blot and qRT-PCR showed that miR-24-3p could negatively regulate the AMOTL2 protein level,nevertheless,there was no significant change in mRNA level.In vivo,the AMOTL2 protein level of miR-24-3p antagomir group was higher than that of the control group,which was consistent with in vitro test.4)miR-24-3p promoted the cell proliferation and migration in cervical cancer by targeting AMOTL2.After interfering with the expression of AMOTL2,CCK8 and Transwell assays showed that the proliferation,migration and invasion of CaSki cells increased,compared with the control group(P<0.001),suggesting that AMOTL2 was a negative regulator for cervical cancer cells;miR-24-3p inhibitor was transfected into CaSki cells,then the cell proliferation and migration ability was weakened,while siAMOTL2 was co-transfected with miR-24-3p inhibitor at the same time,the cell proliferation and migration ability was actually higher than those of the group transfected with miR-24-3p inhibitor alone(P<0.001),suggesting that miR-24-3p could promote the proliferation and migration of cervical cancer cells by negatively regulating AMOTL2.5)miR-24-3p suppressed the Hippo signaling pathway in cervical cancer cellsThere were 36 cervical cancer pathogenic genes in Genecards asmiR-24-3p target genes,which were enriched in various signaling pathways.It is found that AMOTL2 the target gene of miR-24-3p was vital regulatory factor of the Hippo signaling pathway.Western blot and qRT-PCR showed that miR-24-3p decreased levels of p-YAP but promoted the transcription of ANKRD1,CYR61 and CTGF which were target genes of YAP downstream.At the same time,our animal experiments showed that down-regulating of miR-24-3p up-regulated the levels of AMOTL2,p-YAP and p-LATS1/2.All suggested that miR-24-3p could inhibit the Hippo signaling pathway.6)ILF3-AS1 might be one of LncRNAs which can regulate miR-24-3p.Eight LncRNAs were selected according to starBase and DIANA databases,including MIRLET7 BHG,NRSN2-AS1,GNAS-AS1,ILF3-AS1,PAXIP1-AS1,LINC00662,NEAT1 and CEBPA-AS1.Then LINC00662,NEAT1 and ILF3-AS1 were verified to down-regulate in cervical cancer according to GEPIA.It suggested that the high expression of ILF3-AS1 may be a sign of a longer survival of cervical cancer patients.Conclusion:1)miR-24-3p can promote the proliferation,migration and invasion of cervical cancer cells.2)Angiomotin like 2(AMOTL2)is the target gene of miR-24-3p in cervical cancer cells;by targeting and downregulating AMOTL2,miR-24-3p promotes the proliferation and migration of cervical cancer cells.3)miR-24-3p can inhibit the YAP/Hippo signaling pathway in cervical cancer cells.4)LncRNA ILF-AS1 might be upstream regulator of miR-24-3p.