The Effects Of Silencing CHAF1b On The Biological Behavior Of Human Hepatoma Cell Line SMMC-7721

Objective: 1、To examine the expression of the subunit p60 of Chromatin assembly factor 1(CHAF1b) in human hepatocellular carcinomas(HCCs), and analyze the relationship between its expression and HCCs clinical pathological features(gender, age, tumor size, portal invasion, liver cirrhosis, degree of differentiation, TNM stage). 2、To examine the effects on cell apoptosis、proliferation and cell cycle of human hepatoma cell line SMMC-7721, transfected by recombinant lentiviruses for RNA interference(RNAi) of CHAF1 b gene. Initially clarify the role of CHAF1 b on cell proliferation、 apoptosis of HCCs.Methods: 1、Using immunohistochemical method to detect CHAF1 b expression in the three kinds of tissues,which are collected by 20 cases of normal liver tissue, surgical removal of the 110 cases of liver cancer tissue which have been diagnosed pathologically hepatocellular carcinoma and the corresponding adjacent tissue(at least lcm away from the tumor) of our hosptial. According to the test results,comparativily analyze the relationship of the different expression of CHAF1 b with clinical pathological features(gender, age, tumor size, degree of differentiation, portal invasion, liver cirrhosis, TNM stage). 2、The expression of CHAF1 b m RNA in different hepatoma cell lines(SMMC-7721 Hep G2、Hep3B、Huh-7) were observed by Real-time fluorescent quantitative PCR. 3、A lentiviral expression vector for RNA interference(RNAi) of CHAF1 b gene was constructed,then the lentivirus vector which contained RNAi cassette was used to transduct into SMMC-7721, which were divided into experimental group and negative control group. 4、CHAF1b-targeting RNAi lentivims inhibited the expression of CHAF1 b gene in SMMC-7721 was observed by Real-time fluorescent quantitative PCR.And its silencing effect on cell proliferation、apoptosis、cell cycle and colony formation in SMMC-7721 was observed by flow cytometry analysis and MTT assay.Results: 1 、 Using immunohistochemical method to detect CHAF1 b expression in normal liver tissue、 liver cancer tissue and the corresponding adjacent tissue,only cells with a definite brown nuclear staining were judged positive for the CHAF1 b antibody. There are 70 CHAF1b-positive in HCCs, 4 in the corresponding adjacent tissue and 0 in normal liver tissue, the positive rates were 63.6%(70/110)、3.6%(4/110)、0.0%(0/20)respectively, the difference was statistically significant(P<0.001),the CHAF1 b positive rate of HCCs was significantly higher than the corresponding adjacent tissue and normal liver tissue, the difference was statistically significant(P<0.001). The expression of CHAF1 b in HCCs was not related with patient’s gender, age, portal invasion, liver cirrhosis, degree of differentiation and TNM stage( P>0.05), but the tumor size(P<0.05). 2、CHAF1b m RNA was expressed in human hepatoma cell line SMMC-7721. 3、The lentiviral vector for RNA interference of CHAF1 b gene was successfully constructed, and the sequence and correct site of ds oligos inserted were confirmed by PCR and sequencing assay. 4、The results of Real-time fluorescent quantitative PCR showed that the expression of CHAF1 b in SMMC-7721 could be inhibited efficiently by RNAi(P= 0.006). The test results showed that the growth of the experimental group cells significantly slower than the negative control group(P<0. 05); the G1 phase cells of the experimental group was significantly reduced(P<0. 05), G2 / M phase and S phase cells of the experimental group were significantly increased(P < 0. 05); the apoptotic cells of the experimental group was significantly higher than the negative control group(P<0. 05); the proliferation multiple of the experimental group was lower than the negative control group(P<0. 05).Conclusion: 1 、 The expression of CHAF1 b in HCCs was obviously higher than the corresponding adjacent tissue and normal liver tissue, the expression of CHAF1 b in HCCs was not related with patient’s gender, age, portal invasion, liver cirrhosis, degree of differentiation and TNM stage,but the tumor size. 2、CHAF1b gene was expressed in human hepatoma cell line SMMC-7721. 3、The CHAF1b-targeting RNAi lentiviral vector was an important tool that can inhibit the expression of CHAF1 b gene efficiently. Inhibiting the expression of CHAF1 b could inhibit the proliferation of SMMC-7721, promote its apoptosis, influence its cell cycle.