No abstract

The human transmembrane 4 superfamily member 4 or intestinal and liver tetraspan membrane protein (TM4SF4/il-TMP) was originally cloned as an intestinal and liver tetraspan membrane protein and mediates density-dependent cell proliferation. The rat homolog of TM4SF4 was found to be up-regulated in regenerating liver after two-thirds hepatectomy and overexpression of TM4SF4 could enhance liver injury induced by CCl 4 . However, the expression and significance of TM4SF4/il-TMP in liver cancer remain unknown. Here, we report that TM4SF4/il-TMP is frequently and significantly overexpressed in hepatocellular carcinoma (HCC). Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis showed that TM4SF4/il-TMP mRNA and protein levels were upregulated in ∼80% of HCC tissues. Immunohistochemical analysis of a 75 paired HCC tissue microarray revealed that TM4SF4/il-TMP was significantly overexpressed in HCC tissues (P < 0.001), and high immunointensity of TM4SF4/ il-TMP tended to be in well-to-moderately differentiated HCC compared with poorly differentiated tumors. Functional studies showed that overexpression of TM4SF4/ il-TMP in QGY-7701 and BEL-7404 HCC cell lines through stable transfection of TM4SF4 expression plasmid significantly promoted both cell growth and colony formation of HCC cells. Reduction of TM4SF4/il-TMP expression in QGY-7701 and BEL-7404 cells by stably transfecting TM4SF4 antisense plasmid caused great inhibition of cell proliferation. Our findings suggest that TM4SF4/il-TMP has the potential to be biomarker in HCC and plays a crucial role in promotion of cancer cell proliferation.

Gene targeting using short interfering RNA (siRNA) has become a common strategy to explore gene function because of its prominent efficacy and specificity. The human transmembrane 4 superfamily member 4 (TM4SF4) was originally identified in intestine and liver as a cell proliferation-related gene. Recently, it showed an increased expression in the hepatocellular carcinoma (HCC) tissues. In this study, we developed an adenoviral vector harboring an effective siRNA targeting TM4SF4 (AdSiTM4SF4) and identified its function in suppression of tumor cell growth. It was confirmed that TM4SF4 was overexpressed in HCC tissues compared with its paired non-tumor tissues by western blot analysis and immunohistochemistry. Remarkably, it was more abundant on the cell surface of HCC cells. The signals of ectopically expressed TM4SF4 in four cell lines dramatically localized in the plasma membrane, slightly in the cytoplasm, and absent in the nucleus, demonstrating that TM4SF4 is a membrane protein. Targeting TM4SF4 by AdSiTM4SF4 successfully exerted a gene knockdown effect. The QGY-7701 and SMMC-7721 HCC cells infected with AdSiTM4SF4 displayed remarkably attenuated growth potential.Moreover, intratumoral injection of AdSiTM4SF4 significantly suppressed tumor growth in a xenograft mouse model using SMMC-7721 hepatoma cells. Our results indicated that targeting TM4SF4 might be a promising modality for inhibition of HCC.