[Inhibitory effect of blocking expression of human augmenter of liver regeneration (hALR) on proliferation of hepatocellular carcinoma cell line HepG2].
OBJECTIVE
Human augmenter of liver regeneration (hALR) is highly expressed in hepatocellular carcinoma (HCC), but rarely expressed in normal liver cells. ALR could stimulate the proliferation of HCC cell line in vitro, but has no effect on normal hepatocytes. This study was to investigate the inhibitory effect of blocking the expression of hALR on the proliferation of HCC cell line HepG2 using small interfering RNA (siRNA) targeting ALR and anti-ALR monoclonal antibody (McAb).


METHODS
RNA interference (RNAi) plasmid pSIALR-A targeting hALR cDNA and control plasmid pSIALR-B were constructed and transfected into HepG2 cells, respectively. After transfection, the expression of green fluorescent protein was observed under a fluorescent microscope to calculate transfection efficiency, the protein level of hALR was measured by immunocytochemistry, meanwhile, the expression of hALR mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). (3)H-TdR incorporation approach was performed to detect the proliferation of HepG2 cells after transfection and after neutralization with anti-hALR McAb.


RESULTS
hALR was expressed in HepG2 cells. Plasmids pSIALR-A and pSIALR-B were successfully constructed. Both immunocytochemistry and RT-PCR showed that pSIALR-A inhibited the expression of hALR in HepG2 cells significantly by 83% as compared with pSIALR-B. pSIALR-A specifically inhibited the growth of HepG2 cells after transfection. (3)H-TdR incorporation of pSIALR-A/HepG2 cells was significantly lower than that of pSIALR-B/HepG2 cells (67 687+/-6 548 vs. 104 807+/-5 713, P<0.05). Anti-hALR McAb markedly inhibited the autonomous growth of HepG2 cells (P<0.05).


CONCLUSIONS
hALR is highly expressed in HepG2 cells. The short interfering RNA targeting hALR gene could specifically suppress the expression of hALR, and subsequently inhibit the growth of HepG2 cells. Anti-hALR McAb could partially inhibit the autonomous growth of HepG2 cells.
Tang
Lin
L
Laboratory of Molecular Biology for Infectious Disease, Institute for Viral Hepatitis, Chongqing University of Medical Sciences, Chongqing, 400010, P. R. China.
Sun
Hang
H
Zhang
Lin
L
Guo
Hui
H
Zhang
Ling
L
Liu
Qi
Q
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology*; D010957:Plasmids; D011506:Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D050862:Oxidoreductases Acting on Sulfur Group Donors
D000911:Antibodies, Monoclonal; D049452:Green Fluorescent Proteins; D011506:Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C088825:GFER protein, rat; D050862:Oxidoreductases Acting on Sulfur Group Donors