TGFβ1 Gene Leu10Pro Variation Influences TGFβ1 Secretion And Functions Of Liver Cells

Objective:Hepatocellular carcinoma (HCC) is one of the most common neoplasms worldwide and the third cause of death among those patients with malignant tumors. One of the important risk factors for HCC is chronic infection by hepatitis B or C virus, although other risk factors, such as Aflatoxin and alcoholism, have been implicated in its pathogenesis. Chronic hepatitis B is one of the most common infectious liver diseases which had high morbidity and mortality because of the development of liver fibrosis, cirrhosis and subsequently hepatocellular carcinoma. About 2 billion people are affected and more than 10% of these populations are persistent into chronic status all over the world. The mortality rate during HBV infection caused by liver failure, liver cirrhosis and HCC is approximately 1 million each year. About half a million Chinese die from HCC caused by HBV and from end stage cirrhosis each year. The patients with chronic HBV are at increased risk of progression to cirrhosis and development of HCC. Liver cirrhosis in its early stage is able to reverse, so it is important for diagnosis and treatment of liver diseases to early diagnose of liver fibrosis.Accumulating evidences indicated that Hepatic stellate cells (HSCs) play an important role in the progression of chronic liver diseases. HSCs could express the molecules necessary for antigen presentation and modulate lymphocyte proliferation, so HSCs also could play a role in the immune function of the liver.TGFβ1 acts as an important profibrogenic cytokine in liver diseases.It plays an important role in the fibroproliferative changes that follow liver tissue damage, for it can initially recruit and stimulate fibroblasts to produce ECM, enhance ECM synthesis and inhibit ECM degradation by downregulating the expression of matrix-degrading enzymes, promoting expression of matrix metalloproteinase (MMP) inhibitors and inducing tissue inhibitor of metalloproteinases-1 (TIMP-1).Until now at least 10 single nucleotide polymorphisms (SNPs) in TGFβ1 gene have been recognized. LeulOPro is located in the signal peptide of TGFβ1 precursor; leucine is encoded when the codon is CTG while proline is encoded when the codon is CCG. Base substitution causing amino acid replacement in signal peptide might influence the secretion of some related proteins. Since TGFβ1 plays such a pivotal role in liver fibrosis and liver malignancy, serial experiments were designed in this study to analyze TGFβ1 gene-509 polymorphisms of eight cell lines, to determine whether the leucine/proline substitution influences TGFβ1 secretion and thus changes the functions of liver cells.Method:TGFβ1 gene polymorphisms at positions-509 of Huh-7, HepG2, BEL-7402, SMMC-7721, PLC/PRF/5, Hep3B, QGY, L02 cells were analyzed. Two TGFβ1 plasmid CMV-Leu and CMV-Pro were constructed by cloning TGFβ1 DNA fragment (including LAP and active portion), which encoding either the Leu or Pro forms of TGFβ1, into pcDNA3.1 vectors. L02, HepG2, SMMC-7721 and LX-2 cell lines were chosen to be transfected with CMV-Leu,CMV-Pro or pcDNA3.1 empty vectors. The cell medium was collected and assayed by ELISA kit specific for TGFβ1 separately at 24 hours after the transfection. The transfected L02 and HepG2 cells were collected at 48 hours after the transfection, stained by Annexin V and PI, then analyzed by flow cytometry. All data was calculated by Student's t test. Cell proliferation assays (MTT test) of L02 and HepG2 cells were carried out at the time point of 12,24,36,48 hours after initial transfection. HepG2 cells were collected at 24 hours after the transfection, stained by CD105-FITC and analyzed by flow cytometry. LX-2 cells were collected at 24 hours after the transfection, stained by CD80-FITC, CD83-PC5, CD1α-PE and analyzed by flow cytometry. The data were analyzed with SPSS 11.0 by Student's t test.Results:The genotype of eight cell lines'TGFβ1 gene-509 and codon10 were confirmed successfully. CMV-Leu and CMV-Pro vectors were proved to be exactly correct by DNA sequencing. The amounts of TGFβ1 secreted from both CMV-Leu and CMV-Pro transfected cells were higher than pcDNA3.1-transfected cells in four cell lines. CMV-Pro-transfected cells secreted much more TGFβ1 than CMV-Leu-transfected cells, and the difference was statistically significant (P<0.05). The apoptotic rates of CMV-Leu or CMV-Pro transfected cells were greater than pcDNA3.1-transfected cells in HepG2 cells. While in L02 cells the apoptotic rates of CMV-Leu or CMV-Pro transfected cells were much lower than pcDNA3.1-transfected cells. The apoptotic rate of CMV-Pro-transfected cells was also lower than that of CMV-Leu-transfected cells in two cells. And the differences between all transfected cells were statistically significant (P<0.05). Both TGFβ1 codon 10 genotypes could cause an advancing proliferation of liver cells and the genotype of Pro 10 is more effective than Leu 10. CMV-Pro-transfected HepG2 cells expressed a higher level of CD105 than CMV-Leu-transfected cells did (P<0.05). CMV-Pro-transfected LX-2 cells expressed lower level of CD83 than CMV-Leu-transfected cells did (P<0.01), while CD 80 and CD1αexpression pattern were not affected by the genotypes.Conclusion:TGFβ1 gene Pro10 could enhance the secretion of TGFβ1 than LeulO in liver cells. Both TGFβ1 Pro10 and Leu10 could accelerate the proliferation activity of L02 cells and HepG2 cells, and Pro10 had a better acceleration effect than Leu10. LeulOPro encoding either Pro10 or Leu10 could promote the apoptosis of hepatoma cells, but prevent the apoptosis of normal liver cells. And TGFβ1 Pro10 had stronger protective ability than Leu10. TGFβ1 Pro10 also could increase the CD105 expression than Leu10 in HepG2 cells, but Leu10 increase the CD83 expression than Pro in LX-2 cells.