The Preliminary Study Of Correlation Between Skp2 And Human Pancreatic Carcinoma

Backgroud and Objective: S-phase kinase associated protein 2 (Skp2) constituting the sbustrate-recognition subunit of the SCFskp2 ubiquitin ligase complex,targets the cyclin-dependent kinase inhibitor p27kip1.Skp2 protein was overexpressed in many tumors,and was associated witn tumor progression and prognosis.Some researches showed that down-regulation of Skp2 can inhibit tumor growth.The relationship between Skp2 expression and pancreatic carcinoma is less studied,especially the effect of skp2 down-regulation on pancreatic cell has mot been reported yet.In this study,we detect the expression of Skp2 and its related proteins in pancreatic carcinoma and study their interrelationship; Then we investigate the effect of Skp2 down-regulation by skp2siRNA on pancreatic cancer cell line and provide experimental basis for the next possible research of Skp2 being a new therapeutic target in pancreatic carcinoma.Method: (1)Study of the expression of Skp2,p27kip1 and PTEN in pancreatic carcinoma:P-V method of immunohistochemistry was employed to detect the expression of Skp2,p27kip1 and PTEN in 32 cases of pancreatic carcinoma tissue and 13 tissues adjacent to pancreatic carcinoma.(2) Study of the effect of Skp2 down-regulation by skp2 siRNA on pancreatic cancer cell line:①RT-PCR was used to detect the expression of Skp2 in two pancreatic cancer cell lines SW1990 and Panc-1;②Cationic liposome transfection method was employed to transfect siRNA into pancreatic cancer cell line, transfection efficiency was determined by immunofluorescence method, and optimization of transfection efficiency was detected by using different dose ratio of siRNA and liposome and different time points after tranfection.③The cells was divided randomly into some groups: A.blank control group: nontreatment cultured cell line; B.liposome group: cultured cell line+liposome; C.negative control group: cultured cell line+liposome+ Non-silencing siRNA; D.positive control group: cultured cell line+liposome+positive siRNA; E.expenrimental group: cultured cell line+liposome+Skp2 siRNA.④RT-PCR was used to detect Skp2 expression in cells of the aboved groups;⑤The cell cycle in cells of the aboved groups were detected by the method of PI-single staining;⑥MTT method was employed to detect cell proliferation in cells of the aboved groups.Results: (1)The expression of Skp2,p27kip1 and PTEN in pancreatic carcinoma andtheir interrelationship :①The expression rate of Skp2 in pancreatic carcinoma (56.2%,18/32)was significantly higher than that (23.1%,3/13)in tissues adjacent to pancreatic carcinoma (P<0.05).Positive rate of Skp2 protein in pancreatic carcinoma became higher when tumor differentiation went poorer(r=-0.483,P=0.005).②The expression rate of p27kip1 in pancreatic carcinoma (25.0%,8/32)) was significantly lower than that (76.9%,10/13)in tissues adjacent to pancreatic carcinoma (P<0.05).Positive rate of p27kip1 protein in pancreatic carcinoma became lower when tumor differentiation went poore(rr=0.444,P =0.011).③The expression rate of PTEN in pancreatic carcinoma (31.8%,10/32) was significantly lower than that (69.2%,9/13)in tissues adjacent to pancreatic carcinoma (P<0.05).Positive rate of PTEN protein in pancreatic carcinoma became lower when tumor differentiation went poorer(r=0.446,P=0.010).④Spearman's rank correlation test showed that Skp2 expression in pancreatic carcinoma had negative correlation with p27kip1 expression.(r=-0.437,P=0.012).but there seemed no correlation between Skp2 expression and PTEN expression(r=-0.159,P=0.384), p27kip1 expression and PTEN expression(r=0.276,P=0.126).(2) The effect of Skp2 down-regulation by skp2 siRNA on pancreatic cancer cell line:①The expression of Skp2 was detected but without significant difference in pancreatic cancer cell lines SW1990 and Panc-1 (P>0.05).②The highest transfection efficiency could be obtained when the dose ratio of liposome to RNA was 1:2, and the tansfection time was 9 hours. But the efficiency of SW1990 was relatively lower than that of Hela cell line.③Skp2 mRNA level of Skp2siRNA group did not change comparing with that of Skp2-nonsilencing group (P>0.05).④The S phase in cells of expenrimental group did not change comparing with that of Skp2-nonsilencing group(P > 0.05).;⑤The growth of cells of experimental group did not change comparing with that of Skp2-nonsilencing group(P>0.05)..Conclusions: (1).Protein of Skp2 was highly expressed in pancreatic carcinoma,and Skp2 expression in pancreatic carcinoma became higher when tumor differentiation went poorer ,it suggested Skp2 may had role in promoting tumor occurrence and development. (2). p27kip1 and PTEN was poorly expressed in pancreatic carcinoma, and their expression in pancreatic carcinoma became lower when tumor differentiation went poorer, it meaned that their low expression or deletion may involved in tumor occurrence and development.(3). Skp2 expression in pancreatic carcinoma had negative correlation with p27kip1 expression.but there seemed on correlation between PTEN expression and Skp2 expression, PTEN expression and p27kip1 expression.It may suggested that although PTEN could affect p27kip1 through Skp2,PTEN's role in the occurrence and progression of pancreatic carcinoma may not related to Skp2 and p27kip1.(4).Expression of Skp2 was detected in both pancreatic cancer cell lines SW1990 and Panc -1,then Skp2 may becomes a new potential therapeutic target in pancreatic carcinoma.(5).The low transfection efficiency and the own defects of cationic liposome transient transfection method were possibly the main reasons of the unsuccessful RNAi experiment.