The Mechanism Of Let-7a Mediated TGF-β / Smad Signaling Pathway In The Regulation Of Cervical Cancer

PARTI THE EXPRESSIION LEVEL OF LET-7A IN CERVICAL CANCERObjective The aim of this study was to explore the expression level of let-7a for preparation of the molecular mechanism study in cervical cancerMethods Collection of the cervical cancer patient and normal healthy people blood samples with heparin anticoagulant tube from chongqing medical university first affiliated hospital,each of the group was 10 cases,Cervical cancer ranged from 25 to 65 years old was subject to the gynecologic examinationand pathological diagnosis.The total blood RNA was extracted by Cellulose column chromatography, Using the Real-time RT-PCR technique detected the expression of let-7a on the two group specimens.Results Compared with the normal healthy blood specimens,the expression of let-7a in cervical cancer blood specimens was significantly lower.Conclusion The expression of let-7a in cervical cancer was dropped.PART Ⅱ THE EXPRESSION OF TGF-B1 AND SMAD4 PROTEIN IN CERVICAL CANCERObjective the object of this study was to determine the expression of TGF-β1 and Smad4 protein in cervical cancer.Methods Collection of cervical squamous carcinoma, adenocarcinoma and cervical intraepithelial neoplasia biopsy from chongqing medical university first affiliated hospital pathology department,The expression of TGF-β1 and Smad4 protein were determined by Immunohistochemistry and immunofluorescence detection.Results Immunohistochemistry and immunofluorescence showed that the expression of TGF-β1 and Smad4 protein in cervical squamous carcinoma and adenocarcinoma biopsy were significantly increased, compared with the cervical intraepithelial neoplasia biopsy.PART Ⅲ EFFECT OF LET-7A ON CERVICAL CANCER PROLIFERATION THROUGH THE TGF-B/SMAD SIGNALING PATHWAYSObjective the object of this research was to access the influence of let-7a on the growth of cervical cancer cell and explore its molecular mechanismMethods Culturing the siha, hela and hacat cell which was preserved in Chongqing medical university basic medical faculty, using the the Real-time RT-PCR technique detected the expression of let-7a.The let-7a mimics and let-7a mimics NC were transfected into hela,siha and caski cells with LipofectamineTM 2000, then verified the expression of let-7a by Real time RT-PCR. The living cells of let-7a mimics, let-7a mimics NC and untreated group cells were counted by MTT assay and cell growth curves were drew to analyze the cell proliferation.After transient transfection, the expression of P53,TGF-β1 and smad4 protein of let-7a mimics, let-7a mimics nc and untreated group cells were determined by Western blot. TGF-β1 siRNA and siRNA NC were transfected into hela cells with LipofectamineTM 2000, then detected the expression of TGF-β1 protein by western blot, the expression of let-7a in all groups were determined by Real time RT-PCR.Results Compared the expression of let-7a in hacat cell,let-7a was reduced in cervical cancer cell lines,The result was the same with the blood, indicated that let- 7a is potential to be an important factors involved in cervical cancer.Real time RT-PCR showed that let-7a was significantly raised in let-7a mimics cells compared with untreated cells, and there was no obvious change between the let-7a mimics NC cells and untreated cells, suggesting that let-7a was successful transfected into the cells by LipofectamineTM 2000.MTT result displayed that the proliferation of let-7a mimics cells were inhibited significantly compared with that in untreated cells.Western blot showed that a increase expression in P53 protein and a coordinated decrease in TGF-β1 and Smad4 protein in let-7a mimics hela cells,However, in siha and caski let-7a mimics cells western blot found all the expression of the three proteins were decreased. Furthermore,western blot indicated that TGF-β1 siRNA can significantly suppress the expression of TGF-β1 protein, and the let-7a was increase in TGF-β1 siRNA hela cells group.Conclusion Our results strongly showed that the growth inhibition effect of let-7a could be explanined in part through reducing the expression of TGF-β1,Smad4 protein and regulating the expression of P53 protein levels.