Establishment Of Stable ANGPTL4-expressing HCC Cell Lines And Their Characteristics

ANGPTL4(angiopoietin-like 4),one of the angiopoietin-like gene family,is associated with angiogenesis and the growth,migration and invasion of tumor cells. Previous studies of our lab on this gene indicated that ANGPTL4 can evidently inhibit the colony formation of two HCC cell lines BEL-7402 and SMMC-7721 in vitro.It can also inhibit the xenograft tumor formation of transfected BEL-7402 and SMMC-7721 cells in Balb/c nude mice.The mRNA expression of ANGPTL4 was detected in 9 cell lines(7 HCC cell lines and two immorted hepatic cell lines) by semi-Q RT-PCR:low expression of ANGPTL4 was found in Huh-7,MHCC-LM3 cell lines and Chang's liver,middle expression in MHCC-97L cell line,but high expression in Hep3B cell line. Furthermore,Huh-7,MHCC-97L and MHCC-LM3 cells were transfected with ANGPTL4 by lipofectAMINE 2000.These transfected cells were screened with G418, and sorted by FACS.The Huh-7,MHCC-97L and MHCC-LM3 cell lines stably overexpressing ANGPTL4 were established.Moreover,ectopic tumor formation assay was carried out,which indicated that the cell growth was significantly inhibited in Huh-7 cells(P=0.001),but was slightly promoted in MHCC-97L(P=0.019) and MHCC-LM3 cells(P=0.058) by ANGPTL4 gene.In addition,cDNA microarray analysis was performed to detect the altered expression profiles of ANGPTL4 transfection in Huh-7,MHCC-97L and MHCC-LM3 cells,which were confirmed by fluorescent quantitative RT-PCR.Moreover,the present study observed the effect of mutated ANGPTL4(named as ANGPTL4-mut),a point mutation on the 646^th site of its cDNA sequence and the Cystine mutated into Arginine,on the growth of MHCC-97L cells.Three deletion mutations from the ANGPTL4-mut designed by our lab before,were also used to be transfected to compare the effects on the growth of MHCC-97L cells with normal ANGPTL4 full-length cDNA.The results showed that the normal ANGPTL4 full-length cDNA could distinctly promote the xenogrft tumor formation of MHCC-97L cells(P=0.019).ANGPTL4-mut protein mainly located in the cytoplasm of MHCC-97L cells,which is consistent with the previous results of our lab.However, the ANGPTL4-mut and its deletion mutations had no distinct effect on xenograft tumor formation of MHCC-97L cells(P＞0.05).