The Studys On Relationships Between The Expression Of MiR-221/222, MiR-92b And P57^kip2 In Human Heptatocellular Carcinoma

Objective:In order to explore the relation between dysregulation of p57kip2 and hepatocarcinogenesis,the expression of three microRNAs associating with p57kip2 were detected. And then the relationship among the expression of mRNA,protein of p57kip2 and three miRNAs were analyzed in hepatocellular carcinoma (HCC).Methods:The expression levels of miR-221/222,miR-92b,and p57ip2 mRNA were detected in 20 pairs of HCC and adjacent precancerous cirrhosis as well as 5 additional normal livers specimens using quantitative real-time PCR(qRT-PCR). The PCR products of three miRNAs were confirmed using sequencing.Furthermore, the p57kip2 mRNA and protein were performed in the same samples using in situ hybridization and immunohistochemistry staining.The relationship of expression pattern were analyzed among miR-221/222,miR-92b and p57kip2 mRNA,protein.Result:1. qPCR:The expression levels of miR-221 was upregulated more than o ne-fold in 85%(17/20) of HCC and 80%(16/20) of adjacent precancerous cirrho sis than normal liver tissue.Similarly,the counterpart of miR-222 were 90%(18/2 0) in HCC and 70%(14/20)in adjacent tissue,miR-92b were 90%(18/20)in HCC and 60%(12/20)in adjacent tissue more than one-fold for normal tissue. The e xpression levels of miR-221/222 in HCC were significantly upregulated than ad jacent precancerous cirrhosis (miR-221:t=2.728, p=0.014; miR-222:t=2.613, p =0.018),but miR-92b no showed statistical significance between HCC and adj acent precancerous cirrhosis (t=0.027, p=0.987>0.05).Futhermore,the expression levels of miR-221/222 were upregulated in poorly differentiated HCC than in well differentiated HCC (miR-221:z=-2.675, p=0.004<0.017; miR-222:z=-2. 548, p=0.011＜0.017),howere,miR-92b no showed statistical significance in dif ferent grading HCC (z=-1.567, p=0.311>0.017).In contrast, the rate of expre ssion of p57kip2 mRNA were 70%(14/20) in HCC,90%(18/20) in adjacent prec ancerous cirrhosis and 60%(3/5) in normal livers. Expression levels of p57kip2 mRNA were lower in 85.7%(12/14) of positive HCC and 44.4%(8/18) of positi ve precancerous cirrhosis than normal tissue.The expression levels of p57kip2 m RNA in HCC were lower than adjacent precancerous cirrhosis.Moreover, the le vels of p57kip2 mRNA were reduced in poorly differentiated HCC than in well differentiated HCC.The analysis of correlation showed no statistical significance between the expression of miR-221/222,miR-92b and p57kip2 mRNA(miR-221: HCC,r=0.068,P=0.068>0.05,adjacent tissuer=-0.266,P=0.258>0.05.miR-222: HCC,r=0.327,P=0.159>0.05;adjacent tissue,r=-0.437,P=0.054>0.05;miR-92b:HCC, r=0.229,P=0.332>0.05;adjacent tissue,r=-0.485,P=0.030<0.05).2. In suit hybridization:There were no expression in normal livers,the rate of expression of p57kip2 mRNA in HCC and adjacent precancerous cirrhosis were 55% and 90% respectively.The rate of expression of p57kip2 mRNA in different grading HCC group were 75%(3/4) in well-differentiated group,42%(5/12) in moderately-differentiated group,0%(0/4) in poorly-differentiated group. The expression percentage of p57kip2 mRNA in HCC lower than adjacent precancerous cirrhosis and normal livers (p=0.048<0.05).There were significant positive correlation between the result of in suit hybridization and levels of p57kip2 mRNA using qRT-PCR(r=0.495,p=0.001).3. Expression of P57kip2 protein:There were no expression in normal liver tissue. The rate of P57kip2 protein staining were 45%(9/20) in HCC and 80%(16/20) in adjacent precancerous cirrhosis respectively.There were statistically significance in the expression of P57kip2 protein between HCC and adjacent precancerous cirrhosis (p=0.048<0.05), the normal liver and HCC (p=0.046<0.05).Furthermore, there were statistically significance in expression of p57kip2 protein in different grading of HCC (X2=6.195,p=0.045<0.05),the expression of poorly differentiated group were lower than well-differentiated group (p=0.047<0.05).4.Analysis of correlation:There were no statistical correlation between expression of p57kip2 mRNA and protein in HCC(r=-0.168,P=0.478>0.05) and adjacent precancerous cirrhosis(r=-0.033, P=0.892>0.05). In contrast, there were significant inverse correlation between the level of miR-221/222 and the expression of p57kip2 protein in HCC(miR-221:r=-0.584, P=0.007<0.01; miR-222:r=-0.497, P=0.026 <0.05),howere,there were no significant correlation between miR-92b and p57kip2 protein(r=-0.331, P=0.153>0.05) as well as in adjacent precancerous cirrhosis group.Conclusion:1. There were aberrant expression pattern of p57kip2 mRNA and protein in HCC, which their distributing pattern were similar functions of tumor suppressor genes.2. The expression level of miR-221/222 were upregulated siginificantly in HCC, which negatively regulated expression of P57kip2 protein but there were no exhibited regulated function in adjacent precancerous cirrhosis,their biological implication remains further to be elucidated.3. Neither of miR221/222 exhibited function that upregulated or downregulated level of p57kip2 mRNA.4. The expression level of miR-92b were increased siginificantly both in HCC and adjacent precancerous cirrhosis. But miR-92b no show function that upregulated or downregulated p57kip2,which indicated that p57kip2 might not be target gene of miR-92b in HCC.