| Objective:Hepatocellular carcinoma (HCC) is the most frequent primary neoplasia of the liver. Its incidence and mortality has risen globally and has become one of the world’s top six malignant tumors. Therefore, strengthening the basic research of HCC is extremely important. Previous found that retinoic acid/interferon in the induction of apoptosis related gene (gene associated with retinoid-interferon-induced mortality19, GRIM19) is a tumor suppressor gene, and its expression is closely associated with cell death and cell growth inhibition. In this experiment we mainly study was designed by detection of GRIM-19mRNA in hepatocellular carcinoma and expression analysis of its clinical and pathological staging, and further through the construction of GRIM-19kd and SCR cell lines HL-7701and Huh-7study of GRIM-19on HCC cell migration.Methods:(1) Application of real-time quantitative polymerase chain reaction technique to study the effect of GRIM-19mRNA in83cases of he patocellular carcinoma and its adjacent nontumorous liver tissues and8cases of normal tissues expression, and analysis of its clinical and pathological relationship.(2) Design of GRIM-19knockdown and the control sequence, by Trans1-T1Phage Resistant chemical competent bacteria, LA solid culture medium screening selection of positive recombinants. A good selection of positive recombinants by liquid LA medium containing GRIM-19to obtain a large plasmid bacterial plasmid extraction, through small kit to obtain GRIM-19kd and scr plasmide.(3) Culture of low invasiveness cell lines HL-7701, MHCC97-L and high invasiveness cell lines MHCC97-H, Huh-7, and through Western Blot detect the expression of GRIM-19protein in these cell lines. Through the cell transfection technology trnfect the GRIM-19kd and scr plasmid into the HL-7701and Huh-7cell lines. Use G-418screen this cell lines in order to construct GRIM-19kd and scr cell lines, through Western Blot detect is constructed successfully.(4) Detect the ability of migration in HL-7701kd and scr、Huh-7kd and scr by Transwell cell, and hematoxylin staining method for the detection of cell number, thereby understanding the migration of ability of GRIM-19in HCC cell.Result:(1) Real time quantitative polymerase chain reaction technique the results showed: hepatocellular carcinoma GRIM-19mRNA expression is lower than the corresponding noncancerous liver tissue expression of a total of52cases, the positive rate was62.65%, the difference has statistical significance (P<0.01). Hepatocellular carcinoma GRIM-19mRNA expression compared with normal liver tissue, liver cancer cells GRIM-19mRNA average expression levels, significantly lower than that in GRIM-19mRNA in normal liver tissues, has significant difference (P<0.005).(2) GRIM-19mRNA expression in hepatocellular carcinoma and clinical pathologic correlation study shows:GRIM-19mRNA and hepatocellular carcinoma clinical TMN staging (P=0.011), capsular invasion (P=0.047), microvascular invasion (P=0.030) closely related. Thus prompting GRIM-19with the HCC cell migration must be in contact.(3) Western Blot showed normal liver cell line HL-7701, hepatocellular carcinoma cell lines MHCC97-L, MHCC-97H and Huh-7expression of GRIM-19protein and low invasiveness, HL-7701and MHCC97-L GRIM-19protein expression was higher than that of MHCC97-H and Huh-7.(4) Western Blot results showed that the expression of GRIM-19protein in HL-7701kd cells than SCR; Huh-7kd cell expression of protein GRIM-19in less than SCR, thereby displaying the GRIM-19knockout cell lines for building successful.(5) Transwell cell the results show that compared to scr, HL-7701kd cell number increased, whereas Huh-7kd cell number increased, difference has obvious difference (P<0.05).Conclusion:1. GRIM-19mRNA in hepatocellular carcinoma tissue expression was significantly lower in the corresponding noncancerous liver tissues and normal tissues, GRIM-19mRNA in hepatocellular carcinoma by GRIM-19mRNA expression levels, and low expression of hepatocellular carcinoma clinical TMN staging, capsular invasion and vascular invasion are closely related.2. GRIM-19and HCC cell migration ability is closely related to, the low expression of HCC cells promote migration, which may lead to tumor metastasis and proliferation.3GRIM-19and HCC of the occurrence and development of distant metastases are closely linked, the regulation and mechanism research for cancer diagnosis and therapy to provide new ideas and therapeutic targets. Key words:GRIM-191iver tumor migration ability... |
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