Effects Of Non-coding RNA MiR-9 And CDKN2B-AS On Invasion And Gemcitabine Chemosensitivity Of Bladder Cancer Cells

The first and second sections Objective:Bladder cancer is the most common tumor in urinary and germinal system,the morbility and mortality are the highest in urinary system tumor,especially in china.90 percent of bladder cancer is transitional cell carcinoma which shows quick advancement and high recurrence.If bladder cancer is carcinoma in situ,it can cure by operation.If metastasis happens,bladder cancer shows poor sensitivity for radiotherapy and chemotherapy,and the treatment will be hard.So,it is important to study the molecular mechanism of metastasis in bladder cancer.Chromobox homolog 7(CBX7)is a member of chromobox homolog family.There are the expression and function abnormity of CBX7 gene in many kinds of tumor,such as pancreatic cancer.This research is designed to detect the level of CBX7 gene in bladder cancer tissue,identify the mechanism of altered expression;the pcDNA-CBX7 was constructed and upregulated the expression of CBX gene in T24 cells,and then detect the invasion ability of T24 cells with CBX7 over-expression.Methods:Detect the level of CBX7 gene’s expression in 45 bladder cancer tissues by real-time PCR.Predict the miRNAs that can regulate the CBX7 gene by using the Miranda software.Investigate the effect of the CBX7 under the effect of miR-9’s precursor.Detect the level of miR-9 expression in 45 bladder cancer tissues by real-time PCR.Analize the possibility of combination between miR-9 and CBX7 gene by using dual-luciferase reporter system,and the inhibition of miR-9 to CBX7 gene.Bladder cancer T24 cells was transfected with pcDNA-CBX7,the expression of CBX7 gene was detected by real-time PCR and Western blotting.Observe invasion ability of T24 cell by Transwell assay.Detect the expressing level of matrix metalloproteinase(MMP2)and vascular endothelial growth factor(VEGF)by using Western blotting under the condition of CBX7 over-expression.Result:1.The expression level of CBX7 mRNA in bladder cancer tissue.Detect the CBX7 gene’s expression level in 45 bladder cancer tissue with real-time PCR.The expression level of CBX7 gene’s mRNA in bladder cancer tissue is significantly higher than that of para-cancerous tissue,and the expression level of CBX7 gene’s mRNA in invasive bladder cancer tissue is significantly higher than that of superficial bladder cancer.2.miR-9 regulated the expression of the CBX7 gene under post-transcription level.Predict the target gene of CBX7 gene by using Miranda software with the result that miR-9 is the target gene and can combine with CBX7 gene in the mRNA-3’UTR site.With the help of real-time PCR,the expression of miR-9 shows a higher level than that of para-cancerous tissue,and the correlation of miR-9 and CBX7 comes to negative.miR-9 precursor can depress the expression of the CBX7 gene with combining the site of 3’UTR and then negatively regulates CBX7 gene’s expression by using luciferase reporter gene system.3.CBX7 gene inhibited the invasion ability of T24 cell.The pcDNA-CBX7 can significantly increase the expression of the CBX7 gene by real-time PCR and Western blotting.The over-expression of CBX7 gene can significantly inhibit the invasion ability of T24 cells,down-regulated the expression of MMP-2 protein,and up-regulated the expression of VEGF protein.Conclusion: 1.The expression of CBX7 gene down-regulates in bladder cancer,and the expression level of CBX7 gene’s mRNA in invasive bladder cancer tissue is significantly higher than that of superficial bladder cancer.The CBX7 gene had a relationship with development and invasion of bladder cancer.2.The miR-9 can down-regulate the expression of CBX7 gene by targeted combining with the 3’UTR special sequence.3.The expression of miR-9 up-regulates in bladder cancer,the expressions of CBX7 and miR-9 have a negative correlation.The up-regulation of miR-9 is a cause of downregulation of CBX7 gene in bladder cancer.4.T The over-expression of CBX7 gene can significantly inhibit the invasion ability of T24 cells,down-regulated the expression of MMP-2 protein,and up-regulated the expression of VEGF protein.CBX7 gene act as a metastasis inhibitive gene.The third section Objective: To investigate the clinical significance of long noncoding RNA(lnc RNA)CDKN2B antisense RNA 1(CDKN2B-AS)gene and its effects on Gemcitabine sensitivity in BUC.Methods: The expression of CDKN2B-AS gene was examined with real-time quantitative PCR.The cell proliferation and the half maximal inhibitory concentration(IC50)of Gemcitabine were detected with enhanced CCK-8 assay.The apoptosis rate was examined using Annexin V-FITC/PI double-staining apoptosis kit.The protein expression was examined with western blotting.The activity of Wnt signaling pathway was examined with TOP/FOP luciferase assay.Results: CDKN2B-AS gene was high-expressed in BUC tissues and J82,T24 cells compared with paracancerous normal urothelial tissues and SV-HUC-1 cells.Furthermore,the high-expression of CDKN2B-AS gene was related with high pathological grade and low Gemcitabine sensitivity of BUC tissues.The expression of CDKN2B-AS gene in Gemcitabine-resistant T24/Gem cells was much higher than that in T24 cells.Knockdown of CDKN2B-AS gene sensitized T24/Gem cells to Gemcitabine,promoted Gemcitabine-induced cytotoxicity.Knockdown of CDKN2 BAS gene inactivated Wnt signaling pathway,and Wnt signaling pathway mediated the effects on Gemcitabine sensitivity induced by CDKN2B-AS knockdown in T24/Gem cells.Conclusion: Lnc RNA CDKN2B-AS is high-expressed in BUC,and related to low Gemcitabine sensitivity of BUC.CDKN2B-AS inhibited Gemcitabine sensitivity through Wnt signaling pathway in BUC.