The Study Of Effect Of STAT3 And C-myc In Hepatocellular Carcinoma

Objective (1) To investigate the expression of STAT3 and c-myc proteins in SMMC-7721cell line and human Hepatocellular carcinoma.(2) To evaluate the effects on the cell proliferation and apoptosis by the inhibition of antibodies and to explore the correlation of the two genes and the effects on the development of Hepacellular carcinoma.Methods (1) STAT3 and c-myc protein and mRNA levels in SMMC-7721 cell weredetected using immunocytochemistry and semi-quantity RT-PCR.(2) The expression of STAT3 and c-myc proteins in human Hepacellularcarcinoma were detected by immnohistochemistrial staining.(3 ) The cell line was treated with anti-STAT3 antibody or anti-c-myc antibody or the combination of anti-stat3 and anti-c-myc.antibodies respectively The cell proliferation and apoptosis were detected by MTT and Flow cytometry. RT-PCR was applied to investigate the expression changes of STAT3 and c-myc mRNA.Results (1) Immunocytochemical analysis showed that both the protein of STAT3 andc-myc protein was mainly localized in the cytoplasm.(2) The proliferation of SMMC-7721 cell was inhibited in a time-and dose dependent manner by using anti-STAT3 antibody. The inhibition rate of 20μg/mL groups and 40μg/mL groups had a significent difference comparing with that of 10μg/mL groups (P<0.05). No statistic difference of inhibition rate was found between 20μg/mL groups and 40μg/mL groups (P>0.05). ( 3 ) The growth ability of SMMC-7721 cell was inhibited in a time-and dose-dependent manner by using anti-c-myc antibody. The inhibition rate of 20μg/mL groups and 40μg/mL groups had a significent difference comparing with that of 10μg/mL groups (P<0.01). No statistic difference of inhibition rate was found between 20μg/mL groups and 40μg/mL groups(P>0.05).(4) MTT showed that the mixed treatment of anti-c-myc antibody and anti-STAT3 antibody had an inhibition effect on SMMC-7721 cell time and dose dependency.The groups of combined anti-STAT3 and anti-c-myc antibodies had a higher inhibition rate than the single treatment groups of anti-c-myc antibody or anti-STAT3 antibody on SMMC-7721 cell in 24h and 48h (P<0.05). The inhibition rate of combined antibodies was no statistic difference comparing with using a single kind of antibody in 72h (P>0.05).(5) The cell line was treated with anti-STAT3 or anti-c-myc or the combination of anti-STAT3 and anti-c-myc,the early apoptosis rate increased from 0.88% in the control cells to 7.12%,10.6% and 15.5% respectively. the percentage of late apoptosis cells increased from 1.00% in the controls to either 3.12%, 5.32% and 7.14%.The apoptosis rates blocking by combined anti-STAT3 and anti-c-myc antibodies were higher than that of using a single kind of antibody.(6) The mixed treatment of anti-STAT3 and anti-c-myc antibodies in 48h had a more remarkable decline in the mRNA level of c-myc than the single treatment of anti-STAT3 antibody in SMMC-7721 cell line by RT-PCR analysis.(7) RT-PCR analysis also showed that the STAT3 mRNA level blocking by combined anti-STAT3 and anti-c-myc antibodies in 48h decreased as comparing with the control's in SMMC-7721 cell line.(8) STAT3 proteins were detected in 33 cases of Hepatocellular carcinoma, c-myc proteins were detected in 32 cases of Hepatocellular carcinoma. The expression of STAT3 and c-myc proteins showed a statistic significance with the differentiated degree of Hepatocellular carcinoma (P<0.05).Conclusions (1) STAT3 and c-myc proteins are all activated in SMMC-7721 cell line.(2) Blocking STAT3 protein or c-myc protein or inhibiting STAT3 protein and c-myc protein combinedly can inhibit the proliferation and induce the apoptosis of SMMC-7721 cell,suggesting that STAT3 protein and c-myc protein play a crucial role in initiation and development of Hepatocellular carcinoma.(3) The expression of STAT3 and c-myc proteins all can be detected in Hepatocellular carcinoma, and the expression of STAT3 and c-myc proteins is positive correlation with the differentiated degree of Hepatocellular carcinoma,suggesting that STAT3 and c-myc proteins are related with the progression of Hepatoceilular carcinoma.(4) To block STAT3 gene can inhibit c-myc gene expression in SMMC-7721 cell, suggesting that STAT3 may up-regulate c-myc gene to inhibit SMMC-7721 cells apoptosis and to facilitate their proliferation.