Transmembrane 4 Superfamily Member 4 In Acute Liver Injury And Hepatocellular Carcinoma

Transmembrane 4 superfamily is a group of hydrophobic proteins with four transmembrane domains and two extracellular loops, both with conserved residues. They are expressed in a wide variety of cell types and have functional roles in processes, such as cellular adhesion, migration, proliferation and tumour invasion. TM4SF4 (transmembrane 4 superfamily member 4, also known as intestine and liver tetraspan membrane protein, il-TMP) is a distant member of the transmembrane 4 superfamily that was originally identified as a tetraspan membrane glycoprotein produced in the human intestinal eptithelium and in non-dividing hepatocytes that can mediate the cell density-associated inhibition of proliferation. Our lab firstly cloned the rat homolog of TM4SF4 gene. Rat TM4SF4 was identified as a gene up-regulated in liver regeneration and supposed to be a proliferation-associated gene. However, the in vivo functions of TM4SF4 and the possible molecular mechanism for TM4SF4 action are largely unknown.Firstly, we investigated the in vivo function of TM4SF4 in a rat carbon tetrachloride (CCl4)-mediated liver injury model. Expression of TM4SF4 was analyzed by RT-PCR and Western blot in normal and CCl4-injured rats. Our study shows that TM4SF4 gene is overexpressed in acutely injured liver. Overexpression or reduced expression of TM4SF4 in the liver was achieved by injection of sense or antisense TM4SF4 expression plasmids. Assessment of liver injury (histology, serum ALT and AST levels), apoptosis by TUNEL assay were performed. TM4SF4 overexpression is found to be associated with more elevated ALT/AST, increased necrosis, and enhanced apoptosis. Decreased TM4SF4 gene expression showed minimal liver necrosis and depressed ALT/AST levels. Expression of injury-related genes was analyzed by quantitative real-time PCR. Increased expression of TM4SF4 affected the expression levels of growth factors and receptors, such as TNF-α, TNFR1 and c-met. Furthermore, pro-apoptotic and anti-apoptotic gene expression was altered after TM4SF4 administration. Our findings suggest that TM4SF4 plays an important role in accelerating CCl4 liver injury, which may be mediated by the TNF-αand HGF/c-met signaling pathways.Secondly, we investigated the relation between TM4SF4 expression and hepatocellular carcinoma. Western blot and immunohistological analysis showed that the TM4SF4 expression was most abundant in HCC tissues and significantly reduced or undetected in corresponding noncancerous tissues. After introduction of the sense and antisense cDNA of TM4SF4 into HCC cell line QGY-7701 and BEL-7404, we observed that the overexpression of TM4SF4 enhanced both colony formation and cell proliferation in vitro. Using the transgenic short hairpin RNA (shRNA) to knock down the expression of TM4SF4 gene in BEL-7404 HCC cell lines resulted in the cell growth greatly inhibited. Further immunofluresence analysis explored that TM4SF4 was localized on the cellular membrane and could translocate from membrane to cytoplasma with EGFR under the induction of EGF. Moreover, reduction of the TM4SF4 gene expression could also inhibit the phosphorylation of JAK and STAT3. Overexpression of TM4SF4 gene enhances transcriptional activity of STAT3-regulated promoter, which is inhibited by an inhibitor of Janus tyrosine kinase activity. Therefore, TM4SF4 might be involved in EGFR/STAT3 signaling pathway.In conclusion, TM4SF4 gene is up-regulated in acute liver injury and hepatocellular carcinoma. Overexpression of TM4SF4 accelerates CCl4-induced acute liver injury, which might be mediated by the TNF-αand HGF/c-met signaling pathways. In addition, overexpressing TM4SF4 could enhance cell proliferation and tumor invasion in HCC, which might be associated with EGFR/JAK/STAT3 pathway. The further functional analysis and the transcriptional regulation of this gene are being performed now.