| Cervical cancer is a common female genital malignant cancer which has high incidence and mortality. At present,the major therapeutic methods include surgery, chemotherapy and radiotherapy.But the patients in advanced stage lose the opportunity of surgery,and some patients can’t tolerate the injury to normal tissues induced by chemotherapy and radiotherapy.Gene therapy,which is famous for high effiency,specialty and stability,give us a new sight for treatment.Forkhead box protein(FOX) is a kind of transcription factors and is divided into nineteen subfamilies named FOXA-S,among which FOXO was investigated intensively.There are four members in human FOXO subfamily included FOXO1, FOXO3,FOXO4 and FOXO6.FOXO1 protein,which exists in almost all the tissues, acts as a kind of tumor suppressors. In recent years,researches showed that micro RNA regulated cell proliferation,differentiation,apoptosis in malignant tumors by targeting FOXO1,which means that mi RNA may act as oncogene or antioncogene in tumor development.Micro RNA(mi RNA),as a post-transcription regulator,is a kind of short 21-23 nt non-coding single strand small RNAs. Micro RNA profile in tumor tissues is significantly different from that in normal tissues,which was confirmed by a large number of researches. Micro RNA-135b(mi R-135b) was first discovered in mouse,and then was confirmed to generally exist in vertebrate.mi R-135 b is coded by MIRN135 B,which is located in 1q32.1.The precursor turns to mature mi RNA after cleaved by Dicer.mi R-135 b regulates cell proliferation,invasion and metastasis in many malignant tumors.Howerer, the biological function of mi R-135 b in cervical cancer has not been investigated so far.This research investigated the effect and related mechanism of mi R-135 b on the biological function of cervical cancer cells by observing expression of mi R-135 b in cervical cancer cells,the effect of mi R-135 b on cell proliferation,apoptosis,migration and invasion.The research makes up for the blanks of biological function of mi R-135 b in cervical cancer,which maybe a new therapeutic target of cervical cancer and bring hope to the patients in advanced stage or relapse and metastasis.Objective: This research was carried out to investigate the effection and related mechnisims of mi R-135 b on cervical cancer cells in order to provide a new research strategy for cancer therapy.Methods : 1. Real-time PCR was used to detect the mi R-135 b expression in human normal cervical epithelium cell line(End1/E6E7) and six cervical cancer cell lines such as C33 A, HCC94, He La, HT-3, Si Ha and Ca SKi. 2.To detect the effect of mi R-135 b on cell proliferation,cervical cancer cells were transfected with mi R-135 b inhibitor(anti-mi R-135b) or negative control(anti-mi RNC).Cell viability was detected by MTT assay;Cell proliferation was determined by Brd U-ELISA;Cell cycle was observed through flow cytometry;The m RNA and protein expression of p21,p27 and cyclin D1 was detected by real time PCR and western blot respectively. 3. To determine the role of mi R-135 b in regulating cell apoptosis,cervical cancer cells were transfected with anti-mi R-135 b or anti-mi R-NC.Cell apoptosis was observed through Hoechst 33258 fluorescence staining and flow cytometry stained by Annexin V-PI;The release of cytochrome C was detected by western blot;Expression of cleaved caspase-3,8,9 and Bax/Bcl-2 was detected by western blot;JC-1 staining was used to detect mitochondrial membrane potential(MMP). 4. To investigate the effect of mi R-135 b on cell migration and invasion,cervical cancer cells were transfected with anti-mi R-135 b or anti-mi R-NC. Transwell assay was used to determine the ability of invasion;Wound healing assay was played to observe the ability of migration;The expression of MMP-2 and MMP-9 was detected by western blot. 5.To discuss the molecular mechanisms of mi R-135b’s effect on cell proliferation,apoptosis and invasion in cervical cancer cells further, FOXO1 was predicted to be the target gene of mi R-135 b by online database Target Scan 6.2, and the m RNA and protein expression of FOXO1 was detected by real-time PCR and western blot respectively in cervical cancer cells after transfected with anti-mi R-135 b or anti-mi R-NC, luciferase reporter assay was performed to determine the target site of mi R-135b;Then cells were cotransfected with mi R-135 b inhibitor and si RNA against FOXO1 or si RNA-control,cell proliferation was determined by Brdu-ELISA, the m RNA and protein expression of p21,p27 and cyclin D1 was detected by real time PCR and western blot respectively;expression of cleaved caspase-3,8,9 and Bax/Bcl-2 was detected by western blot; western blot was also performed to detect the release of cytochrome C and expression of MMP-2 and MMP-9.Results: 1.The expression of mi R-135 b in cervical cancer cell lines mi R-135 b was evidently up-regulated in all six cervical cancer cell lines,especially in Si Ha and HT-3 cells, compared to that in End1/E6E7. These results indicated that mi R-135 b was increased in cervical cancer. 2.The effect of mi R-135 b on cell proliferation of cervical cancer cells(1) Inhibition of mi R-135 b significantly reduced the viabilities of HT-3 and Si Ha cells;(2)Proliferation of cells transfected with anti-mi R-135 b was decreased remarkably compared with cells transfected with anti-mi R-NC;(3) Down-regulation of mi R-135 b significantly increased the percentage of cells in the G0/G1 phase and decreased the percentage of cells in the S phase in both HT-3 and Si Ha cells compared with cells transfected with anti-mi R-NC.(4) The m RNA levels and protein expressions of p21 and p27 were increased in HT-3 and Si Ha cells transfected with mi R-135 b inhibitor.However, the m RNA level and protein expression of cyclin D1 were evidently decreased by down-regulation of mi R-135 b. 3. The effect of mi R-135 b on cell apoptosis of cervical cancer cells(1) The apoptotic cells in cells transfected with anti-mi R-135 b were increased compared with the negative control;(2)Cytochrome C transferred from mitochondria to cytoplasm by downregulation of mi R-135b;(3) Results showed that down-regulation of mi R-135 b led to up-regulation of Bax and down-regulation of Bcl-2, activated caspase-3,8 and 9 as well.(4)Mitochondrial membrane potential(MMP) decreased in cells transfected with anti-mi R-135 b compared with the negative control. 4. The effect of mi R-135 b on cell migration and invasion of cervical cancer cells(1) mi R-135 b inhibitor lowered down the ability of cell migration remarkably in a dose-dependent manner compared with the cells transfected with negative control;(2) The ability of invation in cells transfected with mi R-135 b inhibitor was decreased compared with the cells transfected with negative control;(3) Down-regulation of mi R-135 b could decrease the expression of MMP-2 and MMP-9 protein. 5.The molecular mechanisms of mi R-135b’s effect on cell proliferation,apoptosis and migration in cervical cancer cells(1) FOXO1 is a direct target of mi R-135 b in cervical cancer cells predicted by the online database Target Scan 6.2;(2) The m RNA and protein levels of FOXO1 were remarkably increased after down-regulation of mi R-135b;(3) Down-regulation of mi R-135 b significantly enhanced the luciferase activity of p Mir-FOXO13’-UTR WT. Mutation of the mi R-135b-binding site in the FOXO1 3’-UTR abolished the effect of mi R-135b;(4)Down-regulation of FOXO1 in cells transfected with the mi R-135 b inhibitor promoted the growth of cervical cancer cell, downregulated p21, p27 and upregulated cyclin D1 at m RNA and protein levels; Results from western blot showed that expression of cleaved caspase-3,8,9 and Bax decreased and Bcl-2 increased in cells cotransfected with mi R-135 b inhibitor and si RNA agains FOXO1; decreased FOXO1 expression inhitited transference of cytochrome C from mitochondria to cytoplasm in cells transfected with mi R-135 b inhibitor;Furthermore, the expression of MMP-2 and MMP-9 obviously increased in cells cotransfected with mi R-135 b inhibitor and si RNA against FOXO1.Conclusion: FOXO1 was the target gene of mi R-135 b and the target site located in 3’UTR.mi R-135 b regulates biological function of cervical cancer cells by targeting FOXO1.Down-regulation of mi R-135 b could inhibit cell growth,induce apoptosis, decrease migration and invasion of cervical cancer cells.So mi R-135 b acts as an oncogene in cervical cancer.These results collectively suggested that mi R-135bFOXO1 axis was considered as a promising prognostic and therapeutic target for future cervical cancer treatment. |
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