The Experimental Research On Application Of TPX2Gene In Cervical Cancer Diagnosis And Treatment

Objective: This topic proposed three parts for experimental research. The first part fromthe level of cell and tissue level clear TPX2expressed in cervical different physiological orpathological condition, the difference of the understanding from the normal cervicalepithelium to CIN and cervical invasive cancer TPX2protein expression levels in theorganization, and combined with case analysis, to explore its clinical stage with cervicalcancer, the relationship between pathologic differentiation degree and lymph node metastases.The second part discusses the small interfering RNA targeting silence TPX2gene in cervicalsquamous cell carcinomas SiHa cell biology. The third part based on RNAi construct stabletransfection HeLa cell line and explore RNAi TPX2inhibition of cell biology behavior ofprotein expression and the influence of the activation of p53, p21and the possibility of Rbcell signal transduction pathways.Methods: HPV positive type of anthropogenic SiHa cervical squamous carcinoma celllines, cervical adenocarcinoma HeLa cell line and with the approval of the medical ethicscommittee review and confirmed by pathological diagnosis of97cases of different cervicaltissue samples as experiment materials. The first part of the experiment using Western Blot-Western Blot analysis, rt-pcr and immunohistochemical study of cervical TPX2expressionunder different physiological or pathological status. The second part of the experiment usingRNAi to specific inhibition of TPX2gene of SiHa cells, by flow cytometry, determined byMTT and invasive test after TPX2inhibition effects on cell biology behavior. Third part buildrestructuring bear load TPX2-shRNA eukaryotic plasmid vector, and transfection HeLa,build stable transfection HeLa cell line and the Q-PCR, Western Blot immunoblot analysis,determined by MTT, flow cytometry and TUNEL method to observe TPX2-shRNA impacton HeLa biology as well as the E6, TPX2, p53, p21and Rb may interaction mechanismbetween the type of high-risk HPV infection after the E6, TPX2activating p53, p21, Rb cell signal, and the possibility of mediating cell signal transduction.Results: The first part of the experimental results for TPX2protein and TPX2mRNA inCIN and cervical squamous carcinoma, cervical adenocarcinoma tissue, cervical squamouscancer cells SiHa and cervical glands cancer HeLa have different degree of expression, butwithin normal cervical epithelial tissue, TPX2hardly express; Development as the illness,from CIN â…  to CIN â…¢TPX2protein expression of different cervical tissue graduallystrengthened, cervical invasive cancer clinical phase II TPX2expression level higher than thatof phase I, lymph node metastasis positive TPX2expression level is higher than the negative,pathology, differentiation of low TPX2expression level is higher than the high. The secondpart of the experimental results of TPX2-siRNA transfection cell, with the extension oftransfection time, cell apoptosis rate increased; In slow after transfection cell proliferation,cell proliferation is restrained, the loss of cell invasion, cell cycle regulation, mainly for G1phase cells decline, with an increase in S phase and G2cells. The third part of the experimentsuccessfully build stable transfection restructuring TPX2-shRNA plasmids of HeLa cell lineand played a specific inhibition in the expression of intracellular TPX2, Western Blot and Q-PCR experiments demonstrate TPX2-shRNA in cut TPX2at the same time also can cut theE6, transcription and translation level of p53, p21and Rb also have cut effect, activation ofp53, p21, TPX2-shRNA has been proved Rb cell signal ability, specific RNAi silence afterTPX2mediated signal transduction mechanisms may be TPX2recovery after being targetedsilence wild-type p53function of activity, promote cell apoptosis. RNAi TPX2silence afterthe regulation of cell cycle there are mainly two kinds of way, on the one hand, p53, throughregulating its downstream target genes p21gene transcription and translation, and stimulatethe p21WAF1/CIP1expression regulation of cell cycle, p21WAF1/CIP1can cause Rbprotein phosphorylation regulation of cell cycle;, on the other hand, from the perspective ofthe cell signal transduction pathway of Rb mediated, Rb gene by regulating the cell cycle G1/S phase limit point, Rb protein phosphorylation status determines the cell proliferation ability,it cannot be combined with transcription factor E2F phosphorylation Rb, Rb protein intophosphorylated release has active free E2F, regulation of cell proliferation. E6gene productthrough its E6protein has tumor suppressor function of phosphorylation Rb protein into protein phosphorylation Rb. Wild-type p53with the phosphorylation of Rb, by relying on theapoptosis induced by p53Rb tumor suppressor function through protection, promptingphosphorylation Rb protein into the phosphorylation Rb. In addition, the E7protein ofhigh-risk type HPV could make E2F free combination of Rb, transcription activation S period.Effect of RNAi, the better, the degree of cell proliferation activity is restrained, the moreobvious, the more cell apoptosis, pause in S phase and G2/M phase cells proportion is larger.Conclusion: In cervical squamous cell carcinoma, CIN and cervical adenocarcinomatissues expression had TPX2, TPX2almost no expression in normal cervical tissue; In thecervical epithelial cells from normal to the process of the development of cancer, TPX2expression level increased gradually; The clinical stage, pathological classification andmetastasis of cervical cancer with TPX2expression levels are closely related, TPX2expression level is higher, the later clinical stage, pathological differentiation, the worse, themore prone to lymph node metastasis. RNAi specificity after silence SiHa and HeLa TPX2can not only regulate the cell cycle, cell cycle in S phase and G2/M phase, and can inhibitcell proliferation activity and invasive ability, TPX2-shRNA can make cells apoptosis.Successfully built a stable transfection TPX2-shRNA recombinant plasmid of HeLa cell lineand confirmed a high-risk type HPV infection after TPX2at the transcription and translationlevel are closely associated with the E6, TPX2-shRNA can activate the p53, p21and Rb cellsignaling pathways, route of mediating cell signal transduction, regulation of cell cycle,inducing cell apoptosis; E6and TPX2through the inhibition of p53, p21and Rb anti-cancerfunction, make the function of the cell cycle checkpoint defects, the normal state of cancerouscells. RNAi interference can effectively inhibit TPX2expression, protection of the normalfunctions of p53, p21and Rb state, based on the RNAi technology TPX2gene expressionmay have inhibitory effect on the occurrence and progress of cervical cancer, TPX2gene isexpected to become a new kind of cervical cancer early diagnosis and gene therapy ofcandidate targets, providing a new method for the clinical diagnosis and treatment of cervicalcancer.