| Objective: Liver cancer is one of most common advanced tumors in the world,and hepatocellular carcinoma(HCC)is the most frequent phenotype,accounting for 90% approximately.Liver cancer in men is the fifth most frequently diagnosed cancer worldwide but the second most frequent cause of cancer death,and the rates of hepatocellular carcinoma cases and deaths are remaining high.According to the global cancer statistics,an estimated 782500 new liver cancer cases and 745500 deaths occurred worldwide during 2012,with China alone accounting for about 50% of the total number of cases and deaths.Due to the improvement of medical condition,the liver cancer death rate of China both in men and women tend to decrease(2000-2011).However,hepatocellular carcinoma is still the fourth most frequently diagnosed cancer and the second most common cause of death in whole China owning to the big population base.Liver cancer,characterized by the insidious condition and poor prognosis,was most common and mortal malignant tumor which was much more in men than in women under the age of sixty.Thus,it is not only theoretical but clinical valuable to explore the liver tumor cell's ability of proliferation,apoptosis,migration and invasion.The Wnt7 a protein is a member of the 19 Wnt proteins family that play an irreplaceable role in the development of nerves and muscles and the maintenance of organ function.Compared to other Wnt proteins,the WNT7 A gene has only one transcript and contains four exons.In addition,it also contains a CpG island and two CpG-rich sequences,and the first exon is located on the CpG island which is very likely methylated.And the methylation is closely related to the occurrence of tumors.The expression level of Wnt7 a in various tumor tissues or cells is also very different.Some studies have found that low expression of Wnt7 a in tumors was caused by WNT7 A methylation.However,the roles of this protein in hepatocellular carcinoma remain unclear.Our study aims to explore the expression of Wnt7 a in hepatocellular carcinoma tissues and cell lines,and to analyze its clinical significance.Then,further investigate the roles of Wnt7 a in the progression of hepatocellular carcinoma and its related molecular mechanisms to illustrate the application value of Wnt7 a in the diagnosis,treatment and prognosis of liver cancer.Methods:1 Specimen collection33 hepatocellular carcinoma tissues from January 2013 to January 2015 were used to this research,and the HCC tissues contain both of cancer tissue and adjacent tissues.Frozen hepatocellular carcinoma cell lines including HepG2?SMMC-7721?Hep3B?Huh-7 are all from lab.2 Database analysis of WNT7 A mRNA expression in HCC and its clinical prognosisThe Oncomine database was used to analyze the expression of WNT7 A mRNA in normal liver tissues and HCC tissues.The relationship between WNT7 A mRNA expression and survival was analyzed using www.kmplot.com.3 Detection the expression of Wnt7 a in HCC tissues and cell lines(1)Immunohistochemistry was used to detect the expression of Wnt7 a protein in 33 cases of HCC tissues and their corresponding adjacent tissues.(2)Western blotting was used to detect the expression levels of Wnt7 a protein in hepatocellular carcinoma cell lines HepG2,SMMC-7721,Hep3 B,Huh-7,and the corresponding cell lines were used for subsequent experiments.4 Relationship between Wnt7 a expression and biological characteristics such as proliferation,apoptosis and invasion of hepatocellular carcinoma cells.Hep3B with low expression of Wnt7 a was overexpressed with WNT7 A or treated with rWnt7 a.Then,MTT assay,transwell assay and flow cytometry were used to detect the effects of proliferation,migration and apoptosis,respectively.5 Mechanism researches of effects of Wnt7 a on biological characteristics in HCCThe expression levels of Caspase-3,E-cadherin,Vimentin,and SKP2/P21 were detected by Western Blot and Immunofluorescence after overexpressed with WNT7 A.Results:1 The expression level of WNT7 A mRNA in liver cancer tissues was lower than that in normal liver tissues.HCC patients with low expression of WNT7 A had higher survival rates.The expression level of Wnt7 a protein in hepatocellular carcinoma tissues was significantly lower than that in adjacent tissues.Wnt7 a protein hardly expressed in Hep3 B cells,but weakly expressed in the other three cells(SMMC-7721,HepG2,Huh-7).2 Overexpressed with WNT7 A,the proliferation,migration and invasion of Hep3 B cells were significantly decreased,but apoptosis was increased compared to the control group.3 Overexpressed with WNT7 A,cleaved-caspase-3 and E-cadherin expression were increased but N-cadherin expression was decreased.Wnt7 a inhibits HCC not through the Wnt/?-catenin pathway,but through inactivating SKP2 leading to upregulation of P21.Conclusion:The Wnt7 a was weakly expressed in HCC tissues and cell lines,and its abnormal expression may be related to the invasion and metastasis of HCC.Wnt7a inhibited the EMT to inhibit migration and invasion of HCC cells and promote apoptosis by up-regulating caspase-3 activity.In addition,Wnt7 a inactivated SKP2 leading to upregulation of P21. |
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