The Effect Of P57Downregulation On Growth And Invasion Of Hepatocellular Carcinoma And Its Molecular Mechanism

Background:Hepatocellular carcinoma (HCC) is one of common malignant tumors in the world, and over50%new cases and dead cases come from China. HCC is one of the serious diseases thatthreaten the health of our people. Although the diagnostic and therapeutic techniques ofHCC have made great progress, their5-year relative survival rate is not exceeding10%.Recently, molecular targeting therapy as a new method provides the hope of HCC therapy.However, the effectiveness of EGFR inhibitor, VEGF inhibitor and multi-target inhibitorremain disappointing in cilinical application. Therefore, finding a new target has become anew direction for HCC research.The CKI p57is found in1995as one of tumor suppressor. As a multifunctional protein, p57could regulate cell cycle, apoptosis, tissue and cell differention, tumor invasion andmetastasis, and angiogenesis. Numorous evidences showed p57expression decreased inmany types of human tumors and associated with malignant behaviors, suggesting that p57deletion is the important hereditary basis of an increased risk of tumors. Previous studiesshowed p57expression decreased in HCC by immunohistochemistry and p57downregulationby MicroRNAs and ubiquitin involved in hepatocarcinogenesis, indicating p57might be animportant regulator in biological behavior of HCC. It is of great importance to elucidate themechanism of HCC growth and invasion and find a new molecular target by exploring therole of p57in HCC.Objective:To detect the levels of p57mRNA and protein in HCC tissues and adjacent non-canceroustissues, to analyze the relationships between p57expression and clinicopathological factorsand prognosis, to evaluate the effect of p57downregulation on the growth and invasion ofHCC in vitro and in vivo, to elucidate the mechanism and signal pathway of p57inregulating growth and invasion, and to propose theoretical basis for finding a new target ofHCC.Methods:We examined levels of p57mRNA and protein in HCC tissues and adjacent non-cancerous tissues by immunohistochemistry, Real-time PCR and Western blot. The relationshipsbetween p57expression and clinicopathological factors and prognosis were analyzed bystatistics.p57shRNA was transfected into HCC cells with p57expression by Lipofectamine, and thetransfected cells were selected by G418to form stable transfectants.The proliferative ability of the transfected cells was detected by MTT and colony formationassay; cell cycle and cell apoptosis were analyzed by flow cytometry. Moreover, weconstructed the model of subcutaneous xenografts in vivo, obsearved tumor growthconstituly for6weeks with in vivo small animals imaging, and drew growth curve. At theend of probation, animals were killed to observe the growth of the tumor xenografts. Theexpression of G1/S phase related proteins was detected by Western blot, and kinase activitiesof cyclin/CDK complexes were performed by kinase activity assay and co-immunoprecipitation.The migration of transfected cells were performed by cell scratch assay and Transwell model,and the invasion of transfected cells were evaluated by matrigel-coated Transwell model.Furthermore, we generated the model of subrenal capsule xenografts in vivo, observed thegrowth of xenografts, and evaluated invasive depth by H/E staining. The expression ofRhoA/ROCK1/LIMK1/cofilin pathway related genes was detected by Western blot.Moreover, the transfected cells were treated with Y27632, a specific inhibitor of ROCK, andthe expression of realted genes were analyzed by Western blot. The subcellular distributionof p57and its association with cell motility were detected by imunofluorescent confocalanalysis and Western blot of nclear and cytoplasmic extracts.Results:The results of imunohistochemistry showed that p57expression decreased in the HCCtissues compared to the adjacent non-cancerous tissues. Furthermore, p57expression wasreduced in specimens with a larger tumor size, a later TNM stage, and the presence ofcapsule invasion and extrahepatic metastasis. The survival analysis indicated the mediansurvival time of the p57-negative group was shorter than that of the p57-positive group.Real-time PCR analysis showed the decreased level of p57mRNA in HCC tissues comparedto adjacent non-cancerous tissues. Western blot further deonstrated low level of p57proteinin HCC tissues compared to the adjacent non-cancerous tissues.We transfected eukaryotic expression plasmid of p57shRNA into HCC cells and formedstable transfectants, successfully. Compared to control, p57downregulation accelerated theproliferation and colony formation of HCC cells by regulating cell from G1phase to S phaserather than apoptosis. To verify the in vitro findings, we subsequently conducted the modelof subcutaneous xenografts, and the results showed that p57downregulation accelerated thegrowth of xenografts. Additionally, p57downregulation increased the expression of cyclin D1and CDK2, as well as kinase activity of CDK4/cyclin D1and CDK2/cyclin E.p57downregulation enhanced the migration and invasion of HCC cells in vitro and invasionof subrenal capsule xenografts in vivo. Furthermore, p57downregulation decreased theactivity of the LIMK1and subsequently inhibited phosphorylation of cofilin. In addtion, weobserved that p57expressed in both the nucleus and the cytoplasm. The decrease ofcytoplasmic p57was associated with the presence of capsule invasion and extrahepaticmetastasis, and p57downregualtion decreased the level of p-cofilin in the cytoplasm,suggesting that p57might regulate HCC invasion in the cytoplasm.Conclusion:The levels of p57mRNA and protein were significantly decreased in HCC tissues comparedto the adjacent non-cancerous tissues. The reduction of p57protein correlated withaggressive phenotypes and a short median survival time, suggesting that p57downregulationis an important molecular event and involves in carcinogenesis and development of HCC.p57downregulation increased cyclin D1and CDK2expression and kinase activity ofCDK4/cyclin D1and CDK2/cyclin E resulting in the acceleration of cell from G1phase intoS phase, cell proliferation and xenografts growth. These evidences indicate p57negativelyregulate the growth of HCC.p57downregulation enhanced invasion of HCC cells and subrenal capsule xenografts bydecreasing the activity of LIMK1and phosphorylation of cofilin, demonstrating p57is annegative regulator of HCC invasion and correlated with HCC progression. Additionally, p57were expressed in both the nucleus and the cytoplasm，and p57might regulate HCC invasionin the cytoplasm.