| Part 1:Expression of macrophage migration inhibitor factor and CyclinD1 in hepatocellular carcinomaObjective To explore the possible relationship between the exression of macrophage migration inhibitor factor (MIF), CyclinD1, cyclin-dependent kinase4 (CDK4), phosphorylated- retinoblastoma susceptibility gene product Rb protein (phospho-Rb) and the development of hepatocellular carcinoma (HCC). Method The paraffined HCC tissues in 93 patients and the normal liver tissues in 5 non-liver disease patients were used to investigate the expression of MIF, CyclinD1, CDK4 and phospho-Rb by tissue microarray and immunohistochemistry methods. Using quantitative real-time PCR and Western blotting detect the mRNA and protein expression of MIF and Cyclin D1 in 5 fresh HCC cancer tissues and their adjacent non-tumor tissues. Results The expression rates of MIF,CyclinD1,CDK4,phospho-Rb in the HCC tissues were 71%,41%,82% and 14% respectively,and in the normal liver tissues,they were 0%,0%,80% and 20% respectively.The expression rates of MIF and CyclinD1 were statistically significant different between the tumor and the normal liver tissues and the expression rates of CDK4 and phospho-Rb were not significantly different between the tumor and the normal liver tissues.The rate difference (79% versus 48%) of MIF expression between the large tumors (≧3.5cm) and the smaller tumors(<3.5cm) was of statistcal significance (P<0.01).The expression rate(62%) of CyclinD1 in the tumors with metastasis was significantly higher than the expression rate(35%) in the tumors without metastasis (P<0.05).MIF expression was positively correlated with CyclinD1 expression in the tumor tissues(P<0.01) .CDK4 and phospho-Rb expressions were not significantly associated with the tumor sizes and metastasis status. MIF and Cyclin D1 protein and mRNA were overexpressed in the HCC tumor tissues. Quantitatively, the expression of MIF and CyclinD1 proteins in cancer tissues were 3.6±0.8 and 2.3±0.1 folds of those in the tumor adjacent tissues (FMIF=15.5 ,P<0.01;FCyclinD1=87.5,P<0.01), and the expression of MIF and CyclinD1 mRNA in cancer tissues were 7.31±1.85 and 4.27±1.05 folds of those in the tumor adjacent tissues (FMIF=111.4,P<0.01;FCyclinD1=58.4,P<0.01). Conclusion:Our results indicate that MIF and cyclinD1 mRNA and protein were overexpressin in HCC tissue.MIF and cyclinD1 were related with tumor cell growth and metastasis in HCC.Part 2:Inhibition on the proliferation, migration and growth of HCC by MIF siRNAObjective To investigate the interfering effects of specific small interfering RNA(siRNA)targeting MIF gene on hepatocyte carcinoma cell. Methods Double strain siRNAs which were sythesised by chemical methods and then transfected into PLC and HepG2 cell with liposome method.the MIF mRNA and protein expression levels were detected by quantitative RT-PCR , western blot and immunofluorescent staining.Tumor cell growth were tested by MTT method and cell migration were measured by counting the cell number of trans-well.Results Expression of MIF mRNA decreased 81.3% and 89.1% and MIF protein levels decreased 69.50% and 72.31% respectively in PLC and HepG2 cells when compared with those in the control groups 24h after 100nM MIF siRNA transfection ( p<0.01 ) . MIF immunostaining was obsviously reduced in MIF siRNA treated cells. Cell growth rates decreased 16.79% and 47.14% respectively when compared with those in the control groups( both p <0.05);The cell numbers of migration were 51±11.27 and 18.56±4.72 per well after 48h trans-well growth,which were significantly decreased compared with those in control groups (both p <0.05).Conclusion:Our results indicate that specific MIF small RNA can down-regulating MIF gene expression and inhibiting of tumor cell growth and migration.Part 3:The Possible inhibition mechanism of MIF siRNA on the growth of HCCObjective To investigate the interfering effects of specific small interfering RNA(siRNA)targeting MIF gene on PLC and HepG2 cell line and the changes of tumor cell growth and migration.explore the possible mechanism how MIF inhibit the tumor cell growth or migration.Methods Double strain siRNAs which were sythesised by chemical methods and then transfected into PLC and HepG2 cell with liposome method.MIF and CyclinD1 mRNA were tested by EvaGreen real-time PCR. Western blot were used to detect the expression of p- ERK, ERK,p- AKT, AKT,p- GSK3β, GSK3β,CyclinD1,CDK4,CDK6. Results MIF and CyclinD1 mRNA levels were significantly decreased after transfection of MIFsiRNA .The phospholated levels of p- ERK and p- AKT and expression of CyclinD1 were significantly decreased. phospholated of GSK3βwas up-regulated. Total ERK ,AKT cdk4 and cdk6 had no change between MIF siRNA treated groups and control siRNA treated groups.Conclusion: Our results indicate that down-regulating MIF gene expression with specific siRNA inhibit tumor cell growth and migration which may be related with activation of ERK , AKT , GSK3βsignal phathway and regulation of cyclinD1and MMP2 expression.
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