Effect Of Down-regulation Of Pin1 By RNA Inference On The Proliferation And Apoptosis Of Colorectal Carcinoma SW620 Cell

Backgroud:Colorectal cancer (CRC) is one of digestive system cancer that seriously threatens to the human's survival. Its morbidity ranks the second among the cancer in western countries, and ranks the first four in china. The clinical study found that the 5-year survival rate of colorectal cancer is lower. So, Inchoatly discovery, diagnosis and treatment are the key to improve survival of colorectal cancer patient. But because of the bad of traditional treatments in specificity and sensitivity, which lead to the inefficiency of treatment. In recent years, with the rapid development of molecular biology technology,the molecular mechanism of colorectal cancer occurrence and development has made a breakthrough research too. However, the clinical study suggests that cell proliferation is important reasons causesing tumor development.Pin1 (peptidy-prolyl cis/trans isomerase PPIase) which a highly conservative and specific cis-trans isomerase is one of the members of the family peptidyl-prolyl cis-trans isomerases, can specifically bind to the protein phosphorylated at serine or threonine-lymphocy ammonia acylating (Ser/Thr Pro base sequence). Following, Pin1 catalyze the protein from cis to trans, which regulate substrate protein function, the action between protein and protein and the subcellular positioning. Research shows that Pin1 is overexpressed in human many malignancies and concerned with tumor proliferation and angiogenesis, invasive and metastasis, so called oncogenesis and catalytic molecules. Further study found that Pin1 is overexpressed in colorect al cancer, and the phenomenon has related to malignant significantly of colorectal cancer. This study used a technique called RNA interference to further explore the influence and mechanism of Pin1 to proliferation and apoptosis ability of colorectal cancer SW620 cell.Objective:1. Construction RNA inference expression vectors targeting to Pin1 gene: pGenesil-1-Pin1,ab -breviation P-shRNA);2. Observation inhibitory effect of pGenesil-1-Pin1 to colorectal cancer cells SW620 Pin1 gene expression;3. To investigate the effect of decresing of Pin1 expression on SW620 cell proliferation and apoptosis ability, observate related gene expression changes and preliminarily analyze the possible molecular mechanism.Methods:1. Constructing interference expression vector of Pin1 genes which have short hairpin structure : pGenesil-1-Pin1(abbreviation P-shRNA) and controlled expression vector: pGenesil-1-CON (abbreviations P-CON), the plasmid was detected by the double enzyme cuts and DNA sequencing , finally the plasmid was obedivested by agarose gel electrophoresis;2. To transfection plasmid p-shRNA and p-CON using liposome into colorectal cancer SW620 cell lines respectively and extraction total protein; detecting Pin1 gene protein expression level by Western blot after transfected plasmid.3. After transfected with p-shRNA and p-CON, draw the growth curve by count cell; cell proliferation and apoptosis ability were tested by MTT and FCM.4. After transfected with pGenesil-1-Pin1, detecting nuclear positioning of NF-κB/p65 by cellular immune fluorescence; Western blot was used to test NF -κB/p65 nuclear protein quantification and apoptosis related factor Bcl-2 expression.Results:1. The RNA inference expression vertor of Pin1 gene was successfully constructed. The plasmid was detected by the double enzyme cuts and DNA sequencing, finally the plasmid was obedivested by agarose gel electrophoresi.The construction was right.2. After transfected with pGenesil-1-Pin1 by liposome 2000, the protein expression of Pin1 wa -s suppressed in the p-shRNA/SW620 group.The relatively expression of Pin1 protein in p-shRNA/SW620 group and p-CON/SW620 group was 0.065±0.005, 0.34 0.027.and its inhibition ratio at 48h was 79.3% ; tatistical analysis showed that the two group difference has a statistical significance. The result point out that transfection Pin1 interference plasmid can effectively restrain Pin1 gene expression in SW620 cells.3. The result of MTT, growth curve, FCM shown that the cell proliferation ability was restrained, apoptosis ability was promoted. Apoptosis rate of SW620 cell was 12.48% in experiment group, apoptosis rate was 0.65% in control group. Statistical analysis showed that the two group difference has a statistical significance.4. The result of immune fluorescence shown that after transfection reference plasmid, the nuclear Fluorescence expression of NF-κB/p65 was lower. The protein quantification of NF-κB/p65 was decresed by Western blot, and the protein expression of antiapoptotic gene Bcl-2 was lower. The protein quantification of p65 in p-shRNA/SW620 group,p-CON/sw620 group and sw620 group was respectively 0.50±0.05,0.96±0.02,0.93±0.02; the Bcl-2 was 0.13±0.016,0.39±0.023,0.38±0.026.Conclusion:1. The RNA interference expression vector of Pin1 is successfully constructeded, the experiment proved that the interference plasmid can inhibite expression of Pin1 in colorectal cancer SW620 cell.2. The decresing of Pin1 gene expression can inhibite the proliferation of colorectal cancer SW620 cell and promoto apoptosis. The probable mechanisms had related with down-regulating the nuclear diverting of NF-κB/p65 and effect the function with the down-stream. It may provide a new therapeutic approach for color cancer therapy.