Construction Of RGPR₁₅₈₆₈ Anti-sense RNA Eukaryotic Expression Vector And Study On Its Functions

Prostate cancer is the most common diseases in old men, it's usually in hormone -dependence manner in early stage,the general therapuic methods are operation, radiation treatment, and castration, etc. due to lacking of clinical symptom in early stage, most prostate cancer are diagnosed on its late stage, there are no effective way to cure the disease so far; gene therapy is a promising method to cure the disease by induce tumor cells to apoptosis and inhibit the tumor cells differentiation. recent studies showed that RGPR15868 is related to prostate cancer development which imply that RGRP15868 might be a promising target for diagnose and treatment of the disease.Our previous study showed that the first ten N -terminal amino acids of RGPR15868 are matched with the fragment of Leucine Zipper Transcription Regulator 2(LZTR2, reeducacion gene promotor region related protein, RGPR-p117, NP₁49118).Moderately strong immunohistochemical staining for LZTR2 was noted in the cytoplasm of an endometrioid adenocarcinoma, as compared to adjacent stroma and benign endometrial gland. In PSA staining negative cases of PCa patients whose PSA level were lower than 4.0ng/mL were moderately strong immunohistochemical staining of RGPR.This paper is based on the originality of the laboratory results. RGPR15868 full-length cDNA is get from the prostate cancer cell lines By RT-PCR. Confirmed fully consistent with the sequence Genebank by sequencing. Construction of antisense RNA recombinant plasmid using its full-length cDNA. The eukaryotic expression vector recombinant plasmid transfect to Prostate cancer cell line-DU145. The cell proliferation effect of RGRP15868 were detected by MTT experiment. The ability of prostate cancer cells Campaign tested by Scratch test. Polyclonal antibody Preparation through RGPR15868 protein fragment.The protein positioning in the prostate cancer cell lines. To further study the function to lay a solid theoretical foundation. The expression of Apoptosis gene mRNA in tumor cell detected by RT-PCR;The expression of cyclin D1 detected by Western blot.Objective:Construction of the eukaryotic expression vector antisense RNA expression in prostate cancer cells DU145. The RGPR15868 protein expression reduct in prostate cancer cells.Revealing RGPR15868 protein function. It may provide a new way for the treatment of prostate cancer.Methods: 1.Construc -tion of the eukaryotic expression vector antisense RNA.2. Cell culture and recombinant plasmid transfected to the DU145 cells.3. The expression of RGPR15868 protein in Transfected cells were detected by Western blot.4.The Survival of cell were detected by MTT experiment.5.The apoptosis-related cases were detected by RT-PCR.6. Scratch experiment tested cell metastasis.7. Western blot detected the cyclinD1 in tranfection cells.Results : 1.Successfully construct pcDNA3.1-RGPR15868(as) the recombinant plasmid, Digestion and sequencing prove it is correct.2. Recombinant plasmid was transfected into DU145 cells by liposome,and transfected DU145 cells successfully.β-actin as a reference,The protein expression of RGPR15868 groups are lower than control group.3.Grow curve and MTT chromatometry indicated that the proliferation ability of the Experimental group were significantly higher than those of untransfected group and empty plasmid group(p<0.01).4.β-actin as a reference, RT-PCR results show that the expression level related to apoptotic factors decreased significantly.5. Scratching experiment results show that recombinant plasmid promote cell movement.6. Western blot shows the experiment group cyclinD1 protein express higher than other three groups.Conclusions:1.The decreased of RGPR15868 protein expression promote the human DU-145 prostate cancer cell growth,and inhibit its apoptosis.2.RGPR15868 induce apotosis correlate with P53,P21,cyclinD1 express regulation.3. RGPR15868 protein can be induce cancer cells apoptosis and inhibition of tumor cell migration.