The Research On The LRP16 Gene Expression And Regulation In Endometrial Carcinoma And Its Polyclonal Antibody Preparation

(Chapter 1) The Research on the Expression of LRP16 Gene in theEndometrial Carcinoma TissuesObjective: ①To establish the method of gaining the LRP16 mRNA expressed inthe endometrial carcinoma tissues; ②To find the different characteristics ofLRP16 gene expression in the endometrial carcinoma tissues and the normalendometrial tissues beside the carcinoma, then analysis the changes of LPR16gene expression in the course of the endometrial carcinoma invasion.Methods: The fresh tissue samples of the endometrial carcinoma were collectedduring the operations. And part of the clinical and pathological diagnosisinformation was also collected. Northern Blot was used to analysis the LRP16mRNA levels of the samples.Results: ① The method of gaining the LRP16 mRNA expressed in theendometrial carcinoma tissues was successfully established. ②With β-actin usedas the endogenous confer, LRP16 mRNA levels significantly increased in 2 of the6 endometrial carcinoma tissue samples compared with the normal endometrialtissues beside the carcinoma.Conclusion: ① As a constitutive gene, the LRP16 gene was widely expressed inthe endometrial tissues. ②The LRP16 gene expressed highly in one third of theendometrial cancer patients. The fact indicated that the LRP16 gene mightover-express in the endometrial carcinoma tissues.(Chapter 2) Antibody Preparation and Basic Research of LRP16 Gene in theDifferent Tissues of HumanObjective: To prepare the anti-LRP16 polyclonal antibody and investigate thesubcellular localization of LRP16 protein.Methods: The full length LRP16 gene was obtained by RT-PCR. The predicatedantigen epitope was cloned into protein expression plasmid pRSET-C,transformed into E.coli BL21(DE3) cells for expressing recombinant pRSET-C-LRP16 protein induced by IPTG. The purified pRSET-C-LRP16 protein wasemployed to immunize rabbit for preparing the polyclonal antibody. Theexpression of LRP16 was analyzed by Western blot and immunohistochemistry incancer such as: breast cancer and ovarian serous cystadenoma.Results: Specific anti- LRP16 rabbit polyclonal antibody was obtained, and bothWestern blot and immunohistochemistry showed that LRP16 was expressed in thecancer with high-expressing of ER, localized in the cellular nucleus.Conclusion: LRP16 protein was a nuclear protein located in cellular nucleus. Theanti LRP16 polyclonal antibody could be used for studying the physiologicalfunction of LRP16 and its pathology.(Chapter 3) LRP16 Suppresses the Transcriptional Activity of E-cadherinthrough Estrogen Receptor a Mediation in Ishikawa cellsObjective: It has been previously demonstrated that the ectopic expression of LRP16 gene in Ishikawa human endometrial cancer cells promoted the invasive ability of the cells through down-regulation of E-cahderin. In this study, the involved molecular pathway was investigated.Methods: The expression of E-cadherin protein in LRP16-overexpressed Ishikawa cells was measured by immunohistological staining. Cotransfection and luciferase assays were used to determine the effect of LRP16 on the transcriptional activity of E-cadherin promoter. The recruitment of both estrogen receptor a (ERa) and LRP16 proteins was detected by chromatin immunoprecipitation (ChIP) assay.Results: E-cadherin was significantly down-regulated in Ishikawa cells with LRP16 overexpression. LRP16 inhibited the transactivation of E-cadherin promoter in an estrogen dependent manner. The recruitment of ERα at the E-cadherin promoter was inhibited by LRP16.Conclusion: LRP16 suppressed the transcriptional activity of E-cadherin in Ishikawa cells through blocking the recruitment of ERa to its promoter region.