Expression Of Rab25 In PCA Tissue And Effect On Biological Behavior Of LNCaP Cells And Molecular Mechanism Of Rab25 Regulating Apoptosis

Prostate cancer(PCA)was a common urological carcinoma,many patients had bone or lymph node metastasis,When they were diagnosed with prostate cancer.Even in patients with early-stage,half of the treatment would progress to androgen-independent prostate cancer(HRPC)and metastasis.The majority of patients had a short survival and a poor prognosis.There was no effective clinical treatment.Therefore,to explore the pathogenesis of prostate cancer,to seek early diagnosis methods and effective treatment of these aspects in prostate cancer was a hot and difficult point.The rat sarcoma(RAS)oncoprotein small GTPase superfamily contains over 170 members,divided into five subfamilies-RAS,RHO,RAB,RAN and ARF.Among these small G proteins,the Ras subfamily is the most studied,primarily because of its critical roles in human oncogenesis Members of the RAB superfamily play important roles in regulating signal transduction and,subsequently,a diverse range of cellular processes,including differentiation,proliferation,vesicle transport,nuclear assembly and cytoskeleton formation.It regulated vesicle transport in almost all eukaryotic cells,thus affecting cell growth,proliferation,survival and other aspects.In tumor development,Rab GTPase are known to play potential roles such as regulation of cell proliferation,migration,invasion,communication,and drug resistance in multiple tumors.However,the correlation between Rabs expression and the occurrence,development,and metastasis of tumor remains unclear.RAB25,a member of the rat sarcoma(RAS)family of small GTPase,has been implicated in the pathophysiology of ovarian,breast and other cancers.Its role in endosomal transport and recycling of cell-surface receptors and signaling proteins presents a novel paradigm for the disruption of cellular pathways and promotion of tumor development and aggressiveness.We will determine the expression level of Rab25 in prostate cancer tissues and benign prostatic hyperplasia tissues and evaluate its relationship with clinicopathological significance.Exploreing its effect on biological behavior of LNCa P cells,to find a new target gene associated with the development and progression of prostate cancer,and to provide experimental basis for the clinical application of prostate cancer gene therapy.This study has there parts.Part I Expression of Rab25 in prostate cancer and its clinical significanceObjective: To investigate the differential expression of the Rab25 in prostate cancer and prostate hyperplasia tissue,reveal the clinical significance Rab25 in prostate cancer.Methods: The expression of Rab25 is detected by immunohistochemistry S-P method in 100 cases of prostate cancer and 60 cases of hyperplasia of prostate tissue,The study was foucsed on the different expression of Rab25 in prostate cancer and prostate hyperplasia tissues and its relationship with clinical pathological features,and serum PSA.Result: The positive rate of expression of Rab25 in 100 cases of prostate cancer tissue was 81.0%,The positive rate of expression of Rab25 in 60 cases of hyperplasia of prostate tissue was 21.7 % the difference has statistically significant(X2 = 54.48, P<0.01).There was no statistical significance(P>0.05)between the expression and the tumor size or the difference of patient age,and it showed positive correlation with prostate cancer Gleason score(rs =0.369,P<0.01)and serun PSA (rs= 0.538,P<0.05).Conclusion: 1.Rab25 expression was overexpressed in prostate cancer..2.The expression of Rab25 in prostate cancer was not relevant to the size of the tumor or the age.3.Rab25's expression was positively correlated with pathological grades and serum PSA level,suggesting that Rab25 may have a certain relationship with the malignant degree of prostate cancer.4?Rab25's expression was positively correlated with the overall survival and the biochemical recurrence after surgery.Part II: Influence of knocking down Rab25 gene with siRNA on the biological behavior of human prostate cancer LNCAP cellObjective To investigate influence of knocking down Rab25 gene with specific small interference RNAs(siRNAs)selected on biological behavior of human prostate cancer LNCa P cell,including cell migration force,apoptosis,invasiveness,expression of Rab25 proliferation.The research can provide not only a new and effective target of RNA interference on Rab25 gene,but also provide a new approach for gene therapy of prostate cancer.Methods 1.Three pairs of siRNAs targeting Rab25 and a pair of FAM-siRNA as control were designed and synthesized in vitro.FAM-siRNA is transfected into LNCAP cells by using OligofectamineTM2000 reagent.The transfecting efficiency was calculated by fluorescent microscopy.The expression of Rab25 mRNA was identified by using RT-PCR in different transfectants treated with different Rab25-siRNAs.The most effective siRNA was selected;2.The most effective siRNA was transfected into LNCAP.LNCAP cell was divided into three groups: LNCAP group,NC+ siRNA group and LNCAP +Rab25 siRNA group;The protein expression of Rab25 was detected by using Western-blot,and calculated inhibition ratio.Transwell test was used to observe invasiveness.CCK-8 test was used to investigate the proliferation of LNCAP cell.Annexin V-PI test was used to investigate the prophase and advanced stage apoptosis of LNCAP cells.Results The result showed that the transfection efficiency was over 75%,which detected by fluorescent microscopy.The expression of Rab25 mRNA decreased after three siRNA groups transfected into LNCAP cells by 100 n M.The siRNA-3 group had the strongest inhibitory effect compared to the other groups(P<0.05),which was choosed as the most effective siRNA.The most effective siRNA was transfected into LNCa P cells.The expression of Rab25 protein decreased to(59.82±4.16)% by using western-blot and better than the other groups(P<0.05).Cell proliferation after transfection was estimated by CCK-8 test.Comparing the three groups of cells survival rate,according to the measured OD values of cells,CCK-8 test showed,at 24 h,48h,and72 h after transfection,the proliferation rate of siRNA group was different to the others(P<0.05),and the interference effect at 72 h was the most efficient of all.The total prophase and advanced stage apoptosis on LNCAP cells could be counted to 15.6±2.1 and 21.30±3.30 by using Annexin V-PI test.Its inhibition was better than other groups(P<0.05).Transwell method was used to measure the invasion and migration after transfection.Results showed that: the cell number of siRNA group going through the Transwell chamber were significantly less than the number of other groups(P<0.05).Conclusion The special siRNA targeting Rab25 in prostate cancer LNCAP cells can down-regulate the expression of Rab25 both in the level of mRNA and protein obviously;Down-regulation of Rab25 expression can significantly inhibit proliferation,migration and invasion of LNCAP cell Line in vitro.It could also induce the apoptosis in LNCAP cells;The optimal Rab25 siRNA sequence segment may provide a valid target for treating prostate cancer.Part III: Molecular Mechanism of Rab25 Regulating ApoptosisObjective Exploring the role of Caspase-3 and Caspase-9 in inhibiting the apoptosis of LNCa P cells induced by Rab25,provide experimental evidence for further study on the specific mechanism of apoptosis induction.Methods The Rab25-siRNA was transfected into LNCAP.LNCAP cell was divided into three groups: LNCAP group,NC+ siRNA group and LNCAP +Rab25 siRNA group;The mRNA expression of Caspase-3 and Caspase-9 was detected by using RT-PCR.The protein expression of Caspase-3 and Caspase-9 was detected by using Western-blot.Results The expression of Caspase-3 mRNA and protein was higher than that of NC group and blank control group,the difference was statistically significant.The expression of Caspase-9 mRNA and protein was higher than that of NC group and blank control group,the difference was statistically significant.Conclusion Rab25-siRNA could induce the apoptosis in LNCa P cell through Caspase-3,Caspase-9 apoptotic signaling pathway...