| Purpose of studyThe cervical cancer is one of major reasons for the motality and modality in the female,and was diagnosed as locally advanced disease in about 80% of patients with cervical cancer in developing countries.The cisplatin-based concurrent chemoradiotherapy has been the standard treatment for cervical cancer from 1999 and the 5-year survival rate of patients with locally advanced cervical cancer has been increased 9-18% compared with that of patients with radiotherapy only.The 5-year overall survival has remained 50-70%for patients with local advanced cervical cancer.So that,the factors and mechanisms involved in proliferation of cancer cell are urgent to be understood.Previous literatures have shown that Wnt signaling is an important regulatory pathway for the proliferation and differentiation of stem cells and tumor cells.Aberrant activation of Wnt signaling promotes tumor cell proliferation.Recently,microRNA is also found to be involved in tumor cell proliferation.Among them,miR-182 can promote cell proliferation in various tumors.Our group preliminarily predicted that miR-182 may interact with APC in cervical cancer with bioinformatics analysis and preliminary experiments.miR-182 negatively regulates APC,a key molecule of Wnt signaling pathway.Therefore,we use human cervical cancer cell lines HeLa and SiHa cells asmodels in vitro to observe the effect of miR-182 on the proliferation of cervical cancer cells and their effect on Wnt signaling via targeting APC.Our research will provide further understanding of pathogenesis of cervical cancer and new target for treatment.Methods(1)Cell culture for cevical cancer HeLa and SiHa cells;(2)Bioinformatics methods for the analysis and prediction of possible target genes of miR-182;(3)Luciferase reporter gene assay for the validation experiment that miR-182 inhibits APC;(4)Transient transfection for upregulation or downregulation of miR-182 in cervical cancer cells;(5)CCK-8 test for the observation of tumor cell survival or its proliferation activity;(6)Clony formation assay for observing the survival and clony formation of tumor cell;(7)qPCR method for quantitative detection at mRNA level;(8)Western blot for quantitative detection at protein level;(9)Statictical Student’s t test for difference of the experimental and control group.Results Part 1.The influence of miR-182 expression level on cervical cancer cell proliferation miR-182 mimic and miR-182 inhibitor molecular were transiently transfected to HeLa and SiHa cells.In miR-182 mimic group,the level of miR-182 expression incresed by 87-fold in HeLa and 456-fold in SiHa cells respectively,compared with that of control groups.In miR-182 inhibitor group,the miR-182 level fell by 40% in HeLa and 96% in SiHa cells,compared with that of control groups.The CCK-8 assay showed that the proliferation activity of HeLa and SiHa cells in miR-182 mimic group increased by 25.4% and 48.8% respectively,compared with control group;and the viability of HeLa and SiHa cell decreased by about 57.5% and 14.9%respectively in miR-182 inhibitor group.The number of HeLa and SiHa clones in miR-182 mimic group were significantlyhigher than those of control group(HeLa,434±29 vs 304±33,P<0.01;SiHa,365±9 vs237±6,P<0.01).The numbers of clony formation of HeLa and SiHa in miR-182 inhibitor group were significantly lower than those in control group(HeLa,168±24 vs 233±29,P<0.05;SiHa,172±25 vs 231±26,P<0.05).Part 2.Verifying that miR-182 targets and inhibits APC through bioinfomic analysis and luciferase reporter assayThe potential target genes and functions of miR-182 were predicted through the Target Scan software.The results showed that miR-182 could complementarily bind 3’-UTR of APC and verified the negative correlation between miR-182 and APC molecules in 60cells(P=0.013,r-0.323).With qPCR methods,the APC gene expression leve in miR-182 mimic group was significantly reduced by 85% compared with the control group.The luciferase reporter assay was used to detect the effect of miR-182 on APC translation.The miR-182 mimic decreased the expression of APC by 76.6% after 24-hour and 30.2% after48-hour compared with the control group.Part 3.Effects of miR-182 expression level on downstream molecules of Wnt /β-catenin signalingThe effects of miR-182 on the expression of CCND1 and MYC were further investigated by qPCR and Western blot.In miR-182 mimic group,CCND1 gene expression increased by 32-fold in HeLa cells and 2-fold in SiHa cells and the MYC gene expression increased by 10-fold and 2-fold in HeLa and SiHa cells respectively.In miR-182 inhibitor group,CCND1 gene level decreased by 97% in Hela cells and by 83% in SiHa cells and MYC gene level also fell by96% in Hela cells and 50% in SiHa cells.The Westem blot result showed that the expression of CCNDl and MYC in Hela and Siha cells were changed according to the miR-182 expression level.Conclusions The present study showed that the upregulation of miR-182 could significantly enhance the proliferation and clony formation of cervical cancer cells.The mechanism involved should be that miR-182 targets and inhibits the expression of APC,and then resuts in the aberrant activation of Wnt signaling pathway. |
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