Experimental Study Of Suppressing Effect Of SiRNA And ShRNA Targeting PCNA Gene On Growth Of Bladder Cancer In Vitro And In Vive

Bladder cancer is the most common type of urinary system malignant tumours.There is a higher recurrence rate after surgical treatment.At the present,how to treat it and prevent recurrence effectually is also the difficult issue.The RNA interference was discovered in 1998,but the internal source small interference RNA(siRNA) has not been found in mammals as yet.Some chemosynthesis siRNAs were used to study on oncotherapy.The intra-cellular action time of siRNAs is transient,it refer to be studied for short-term effect,and not suitable for long-term alleosis of cell biological function after RNA interference(RNAi).The eukaryotic expression plasmid can express short hairpin RNA(shRNA),it may overcome the demerit of siRNA.Investigators transfer the plasmid into tumour cells by liposomes or other assistant transfection methods,and in the cytoplasm,the plasmid express shRNAs continually,then shRNAs are cutted by Dicer enzyme and changed to siRNAs,and siRNAs develop the RNAi.For the past few years, RNAi has provided firenew research path for bladder cancer.Malignant proliferation is one of the key feature of cancer cells.Ever since a long time ago,the proliferating cell nuclear antigen(PCNA) is only a malignant proliferation extent and worse prognostic marker,there are few of investigators connect PCNA directly to treatment of malignant neoplasm,taking PCNA as a target gene for bladder cancer treatment has not been seen in domestic and abroad literatures.The thread of this topic: First of all,we designed and obtained three chemical synthesis siRNAs,and transferred it into bladder cancer cells by liposomes to study the short-term effects and the effects on biological behaviour of bladder cancer cells in vitro.Again,on the basis of siRNAs,we constructed eukaryotic expression plasmid to study the long-term effects in vitro and the effects on growth of bladder cancer in vive.To explore the effective methods of treatment and prevention for bladder cance is the goals of this study.This study has four parts. PartⅠScreening of effective siRNA targeting PCNA geneObjective:To design and select effective siRNA targeting PCNA gene for subsequent research.Methods:Three chemical synthesis siRNAs targeting human PCNA-mRNA (NM-002592) were designed by ourselves.100nM was choosed as the best final concentration for siRNAs.The quantitative RT-PCR(RTq-PCR) was used to detect the expression of PCNA-mRNA in T24 cells at 24,48,72,and 96 hours after transfection.The western-blotting was used to measure the expression of PCNA protein at 72 hour post transfection.Results:At 48 hours after transfection,three siRNAs had their best interfering results and PCNA-mRNA inhibition ratio were all over 80%.The PCNA-siRNA3 had the highest inhibition ratio which was 90.1±1.7%.The expression trend of PCNA protein and PCNA-mRNA was at equal pace roughly.Conclusions:PCNA-siRNA1(target mRNA site location:291-309),PCNA-siRNA2 (target mRNA site location:550-568) and PCNA-siRNA3(target mRNA site location: 679-697) were all utility siRNAs and The PCNA-siRNA3 was the most effective one.PartⅡExperimental study of suppressing effect of siRNA targeting PCNA gene on growth of bladder cancer in vitroObjective:To investigate biological behaviour changes of T24 cells after PCNA gene was inhibited transiently by PCNA-siRNA.Methods:The most effective PCNA-siRNA3 was seleced to inhibit expression of PCNA gene in T24 cells.After inhibition,the cell growth inhibition ratio was measured with MTT,apoptosis was detected with DAPI staining,the cell cycle and apoptosis were checked out with FCM and the invasive ability in vitro was measured by the matrigel invasion assays. Results:At day 1,2,3,4and 5 post transfection,The T24 cell growth inhibition ratio of interfering groups were 23.7±2.8%,70.8±0.7%,69.8±0.4%,48.8±0.3%,31.7±0.2%,and it of negative control groups were 2.4±0.9%,3.9±0.1%,5.8±0.6%,12.2±0.4%,8.1±0.1%.the inhibition ratio of interfering group were obviously higher than it of negative control group,and at day 2,3 the inhibitory effects were the best(p＜0.05).At day 3 post inhibition,in most visual fields of inversion fluorescence microscope there were few apoptosis cells in interfering groups,but there was no apoptosis cell in negative control groups and blank control groups.The apoptosis ratio of interfering group was 7.4±0.6%,it was obviously higher than negative control group and blank control group(p＜0.05).There were more cells were blocked on s and G1 phase in interfering group(p＜0.05).At day 3 post inhibition,the permeating cell numbers were 95±7,90±8 and 20±2 for blank control group,negative control group and interfering group.The amount of cells which had permeated filter membrane less in interfering group than in the other two groups(p＜0.05).Conclusions:PCNA-siRNA3 had inhibited the proliferation and invasive ability of T24 cell effectively,and it also promoted the apoptosis of T24 cell.There were more cells were blocked on s and G1 phase in interfering group.PartⅢConstruction,identification and screening of shRNA eukaryotic expression plasmid targeting PCNA geneObjective:To Construct effective shRNA eukaryotic expression plasmid targeting PCNA gene for research in vivoMethods:The pGPU6/GFP/Neo-PCNA-shRNA1～3 eukaryotic expression plasmids were constructed according to subsequences of PCNA-siRNA1～3,This type plasmid has U6 promoter,Neomycin resistance screening marker and green fluorescence protein gene. The identification was done by BamHⅠand PstⅠenzymes,and the sequencing was made by the shanghai Invitrogen Biotech company.The transfection ratio of T24 cell was increased with G418,and multiclone T24 cells were acquired at week 2 post screening. For selecting effective shRNA eukaryotic expression plasmid,RTq-PCR and western-blotting were made to detect expression of PCNA gene.Results:pGPU6/GFP/Neo-PCNA-shRNA1～3 were all positive recombinant vectors and its sequences were all correct.The transfection ratio of T24 cell was more than 50%. The inhibition ratio of PCNA-mRNA in T24-PCNA-shRNA3 multiclone cells was 47.0±1.9%,it was obviously higher than in T24-shNC multiclone cells which was 1.8±0.7% (p＜0.05).The expression trend of PCNA protein and PCNA-mRNA was at equal pace roughly.Conclusions:pGPU6/GFP/Neo-PCNA-shRNA1～3 eukaryotic expression plasmids had been constructed successfully,pGPU6/GFP/Neo-PCNA-shRNA3 plasmid had inhibited the expression of PCNA gene continuously and efficiently.PartⅣExperimental study of suppressing effect of shRNA targeting PCNA gene on growth of bladder cancer in viveObjective:To investigate suppressing effect of PCNA-shRNA3 eukaryotic expression plasmid on growth of bladder cancer in vive.Methods:1.BALB/c nude mice models bearing human bladder cancer on the right flank were established,and divided randomly to three groups.there were six mice in each group.The first one was blank control group(no intratumoural injection),the second one was negative control group(20ug shNC plasmids were injected into tumour of each nude mouse) and the third one was interfereing group(20ug PCNA-shRNA3 plasmids were injected into tumour of each nude mouse).Intratumoural injections at the same dose were repeated on days 8,10,13,16,19,22and25 after T24 cells were injected subcutaneously into the right flank of nude mice(day 0),and all nude mice were sacrificed humanely on day 28.2.The relative stable expression multiclone T24 cells were acquired at week 6 post screening,and it were injected subcutaneously into the right flank of nude mice,this multiclone group also had six nude mouse,and all nude mice were sacrificed humanely on day 28.The tumor volumes were measured,tissues of tumor were checked out with hematoxylin and eosin stain and immunohistostaining.Results:1.The tumor growth is more quickly in blank control group and negative control group than in interfereing group.At day 28,the average volumes of blank control group,negative control group and interfereing group were 761.3±149.9 mm~3,718.3±75.1 mm~3 and 328.1±150.1 mm~3.The mice in interfereing group had a significantly smaller tumour size compared with that in blank control group(p＜0.05) or that in negative control group(p＜0.05),no significant difference in tumour size between the blank control group and the negative control group(p＞0.05).2.At day 28,in multiclone group,there were four nude mice had tumours to be seen by naked eye,the biggest diameter was 4mm and the average volume was 6.8±1.6mm~3.This group had a significantly smaller tumour size compared with that in the other three groups(p＜0.05).3.Immunohistostaining revealed that tumours in multiclone group and interfereing group had significantly weaker expression of PCNA protein compared with tumours in blank control group(p＜0.05) or in negative control group(p＜0.05),The tumour expression of PCNA protein had no significant difference between the blank control group and the negative control group (p＞0.05).The tumours in multiclone group had the weakest expression of PCNA protein (p＜0.05).Conclusions:1.PCNA-shRNA3 eukaryotic expression plasmid had inhibited the subcutaneous proliferation ability of T24 cells efficiently and continuously.2.PCNA-shRNA3 eukaryotic expression plasmid had inhibited the growth of bladder cancer efficiently in vive.