Effect Of SaRNA Upregulation Of Numb On AIPC Of Hedgehog Signaling Pathways On SHh,GLi And CyclinE

Objectiv Based on the Numb protein is a kind of important cell differentiation factors,which affect the cell cycle G1 / S transition and use the small activating RNA(sa RNA to activate the numb protein expression and block the prostate cancer cells in the G1 cycle.In this study we inorder to search the activating and relatively efficient sa RNA for Numb,to determine the expression level of m RNA and protein on Numb,and verify the expression level of m RNA and protein on SHh,GLi and Cyclin E,then discuss he effect of sa RNA Upregulation of Numb on AIPC of Hedgehog signaling pathways on SHh,GLi and Cyclin E.Methods 1.Design and synthesis of small double-stranded RNA(ds RNA)for Numb;2.The ds RNA molecules transfected in human prostate cancer(PC-3)cells to get stablly high expression of numb recombinant PC-3 cells on experimental groups;Empty load the virus PC-3 cells as the positive control group;and the PC-3 cell as the negative control group.3.We use the RT-q PCR to detecte m RNA expression level of Numb,SHh,GLi and Cyclin E on the three groups of cells.4.We use the Western Blot to detecte Protein expression level of Numb,SHh,GLi and Cyclin E on the three groups of cells.5.We use the Flow cytometry technology test cells apoptosis on three groups.6.Effect of sa RNA Upregulation of Numb on AIPC of Hedgehog signaling pathways on SHh,GLi and Cyclin E.Results 1.Filtered the activating specific sa RNA which can activate the high expression of Numb in AIPC.2.We use the flow cytometry technology to detect cell apoptosis,and find that there are not obvious difference between without androgen treatment group transfection cells apoptosis and the negative control group(P>0.05).3.We use the Western bolting to verify the expression of protein and find that protein expression of Hedgehog signaling pathways important factor SHh,GLi,and Cyclin E is significantly lower than control group(P<0.05).4.We use the Real-time PCR to verify the expression of m RNA and find that m RNA expression of Hedgehog signaling pathways important factor SHh,GLi,and Cyclin E is significantly lower than control group(P<0.05).5.After high expressed Numb gene on transfected group of PC-3 cells,we find that the cell signaling pathways through Hedgehog pathway to control and made cell proliferation in stagnation and the stagnation of the cell cycle at G1 phase(P<0.05).Conclusion 1.Numb-Sa RNA transfected into human prostate cancer(PC-3)cells can make numb in PC-3 cell gains stable expression;2.Numb expressed increasing in experimental group PC-3 cells,the Hh signaling pathways important factors SHh,GLi and Cyclin E expressed significantly lower than the control group;3.Numb protein may affect AIPC cell proliferate through Hh signaling pathways.