The Role And Mechanism Of LncRNA DLX6-AS1 In The Development Of Prostate Cancer

Objectives:This topic is based on TCGA database analysis to screen out long-chain non-coding RNA associated with prostate cancer,and to study its role in regulating prostate cancer proliferation and invasion and metastasis,to explore the therapeutic target of prostate cancer,and to search for the biological diagnosis of prostate cancer with minimally invasive body fluids and markers provided references for individualized knitting of prostate cancer.Therefore,it provides a reliable theoretical basis for the development of new types of prostate cancer targeted drugs and the optimization of clinical treatment plans.Methods:1.Using TCGA database to perform cluster analysis on 52 normal prostate cancer tissues and 499 prostate cancer tissues to screen for tumor-associated lncRNA molecules with significantly different expressions;2.Culture 4 prostate cancer cell lines PC3 and DU 145 in vitro,22Rv1 and LnCaP and human normal prostate epithelial cells RWPE-2,after extracting RNA respectively,using real-time fluorescence quantitative method to detect the mRNA expression of lncRNA DLX6-AS1 in these five cells;3.Detection of lncRNA DLX6-by nuclear-plasma separation test Localization of AS1 in prostate cancer cells 4.After lentivirus-transfected prostate cancer cell lines PC3,DU145,22Rv1 and LnCaP downregulated lncRNA DLX6-AS1 by expressing shRNA,CCK8 cell proliferation activity kit detected lncRNA DLX6-AS1 knockdown for prostate The effect of cancer cell proliferation ability,clone formation experiment to detect the impact of lncRNA DLX6-AS1 knockdown on the survival ability of prostate cancer cells,Transwell laboratory test to detect the effect of lncRNA DLX6-AS1 knockdown on the migration and invasion ability of prostate cancer cells,apoptosis reagents detection of the effect of lncRNA DLX6-AS1 knockdown on the anti-apoptotic ability of prostate cancer cells;5.qRT-PCR and protein immunoassay Western blot to detect the effect of lncRNA DLX6-AS1 knockdown on prostate cancer cells on EMT-related markers;6.RNA pull-down and RIP experiments capture and verify functional proteins that specifically bind to lncRNA DLX6-AS1.Results:1.After TCGA analysis,lncRNA DLX6-AS1 was identified as the target gene in the experient;2.The mRNA expression levels of PC3 cells(10.36±0.32),DU145 cells(4.59±0.09),22Rv1 cells(2.47±0.13),and LnCaP cells(2.70±0.20)were all higher than that of normal prostatic epithelial cells RWPE-2(1.00±0.16)(p<0.05).3.The results of nuclear and cytoplasmic separation test suggested that lncRNA DLX6-AS1 was mainly enriched in the cytoplasm of prostate cancer cells.4.After lentivirus infected prostate cancer cells,CCK8 experiment results show that the absorbance of experimental group in 450 nm is lower than the control group(p<0.05);The cell clone formation experiment showed that the experimental group of the DLX6-AS1 knockdown decreased the cell colony numbers compared with the control group(p<0.05);Transwell invasive and migration experiments showed that the experimental group of the DLX6-AS1 knockdown the invasion and migration ability is weaker than the control group(p<0.05);Annexin V-FITC/PI apoptosis experiment results showed that the anti-apoptotic ability of down-regulated DLX6-AS1 group was lower than that of the control group(p<0.05).5.qRT-PCR and Western blot experiments showed that the mRNA and protein expressions of snail,twist and Vimentin in the down-regulated DLX6-AS1 group were lower than those in the control group(p<0.05),while the mRNA and protein expressions of e-cadherin,Claudinl and zo-1 were higher than those in the control group(p<0.05);6.The results of RNA pull-down and RIP experiments showed that plectin protein could specifically bind to lncRNA DLX6-AS1.Conclusions:1.lncRNA DLX6-AS1 is highly expressed in prostate cancer cell lines,and may play a role as a pro-oncogene in prostate cancer;2.lncRNA DLX6-AS1 can promote the proliferation,invasion and metastasis of prostate cancer cells and inhibit apoptosis;3.lncRNA DLX6-AS1 promotes the invasion and metastasis of prostate cancer by promoting the EMT process;4.lncRNA DLX6-AS1 may promote the occurrence and development of prostate cancer by combining with plectin,and is expected to become a potential target for clinical diagnosis and active treatment.