Preliminary Study On The Functions And Molecular Mechanisms Of Long Noncoding RNA PCA3 In Prostate Cancer

Part one:Construction of knock down vector pGLV3/H1-GFP-shRNA and over expression vector pCDH-GFP-PCA3Objective PCA3 has been found to be specific and overexpressed in prostate cancer, suggesting that PCA3 may contribute to the development of prostate cancer. The function and mechanism of PCA3 in prostate cancer is not clear yet. To investigate its role in prostate cancer and filter out the steady transfer lines, provi ding the basis for the subsequent furnctional studies in vitro, we constructed the knockdown vector and over-expression vector.Methods Short hairpin RNAs (shRNAs) against PCA3, including pGLV3/H1-sh2456, pGLV3/H1-sh2618, pGLV3/H1-sh2702, pGLV3/H1-sh2913 and pGLV3/H1-control, were designined to decrease its expression level in LNCaP cell. After packaging the knock down vector into virus, we used it to infect LNCaP cell and the silencing effect was examined by quantitative RT-PCR. LNCaP-2618 was proved to be the highest cell line. The total RNA of PCA3 was extracted from LNCaP cell line, and then it was reversely transcribed into cDNA. Three pairs of primers were designed to amplify the corresponding fragments using the template cDNA, we got the full length of PCA3 through overlapping real time PCR. After PCA3 and plasmid pCDH-CMV-MCS-EF1-copGFP were double digested and recombined, we transformed them into DH5a. Positive clones were selected to sequence and varied by PCR and double digestion. After packaging the over expression vector into virus, we used it to infect DU145 and PC3 cells, and the expression effect was examined by quantitative RT-PCR. The pCDH-copGFP-control and pCDH-copGFP-PCA3 plasmids were obtained successfully.Results After sequencing and real time PCR validation, the pGLV3/H1-shRNA-PCA3 knock down vector and pCDH-GFP-PCA3 over expression vector was successfully constructed. And the stablely transfected cell lines were finaly obtained.Conclusions The successful construction of knock down vector and over expression vector laid a good foundation for in-depth study of the role of PCA3 in prostate cancer and provided a new way to explore the treatment of prostate cancer.Part two:A preliminary study on the function of interference and overexpression of PCA3 in prostate cancer and its moleculer mechanismsObjective To investate the biological function of interference and overexpression of PC A3 in prostate cancer on the aspects of cell growth, cell cycle, cell migrateon and cell invation.Methods The established stably transfected cell lines on the first part were applied to study. The biological functions of PCA3 on cells were investigated by examining the cell proliferation by cck8 assay (or EdU assay) and cell colony assay. Cell cycle was examined by flow cytometricass assay. Transwell assay was used to study cell migration and invation. The knock down cell line was sent to sequence on transcriptome, to explore the differences of the expression in two groups at the transcriptional level.Results Downregulation of PCA3 by shRNA inhibited the growth in the experimental group compared with the control group, and the migration and invation abilities of prostate cancer also greatly decreased, the difference was statistically significant. While after over expression in DU145 cell and PC3 cell, no significant difference was found in the experimental group compared with the control group in the cell growth, cell migration and invasion. Transcriptome sequencing results showed that after knock down PCA3, the gene expression involving in cell adhesion signaling pathways had a significant decrease in experimental group, and gene expression involving in TGF-beta signaling pathway increased significantly comcompared with control group.Conclusions We demonstrated that PCA3 might play a significant role in the development of prostate cancer. Knocking down of PCA3 extremely inhibits cell growth, migration and metastasis, indicating that PCA3 may be a promising therapeutic target in prostate cancer. Transcriptome sequencing results suggest that PCA3 molecular probably influenced cell growth, migration and invasion by affecting the expression level of cell adhesion molecule and TGS-beta.