Effect Of HCCR-2 Antisense-nucleic Acids And RNA Interference On Biological Behaviours Of Liver Cancer Cell Line HepG2

Objective HCCR gene was screened and identified from cervical cancer tissues by differential display RT-PCR ,and HCCR is associated with many human tumors. The HCCR gene is classified into two species, HCCR-1 and HCCR-2 accroding to their molecular characteristics.Immunohistochemical staining analyses showed that HCCR was overexpressed in the HCC and liver cirrhosis revealed weak staining.In normal liver, however,there was no expression of HCCR. The role of HCCR-2 in tumorigenesis may be reside as a negative regulator of the p53 tumor suppressor gene .To investigate the role of HCCR-2 we observed the changes of biological behaviour,the expression of Bax,Bcl-2 and mutation of p53 in HepG2 cells through inhibiting the expression of HCCR by antisense-nucleic acids and RNAi, and this may provide a new way for liver cancer gene therapy.Methods 1,Human cervical cancer oncogene HCCR-2 was cloned by touch down RT-PCR and constructed its sense and antisense expression vector. We selected three pieces of interference sequence targeting HCCR-2 through on-line design tool in web site according to the principle of RNAi design.We further established HCCR-2 RNAi plasmids including interference sequences and negative control sequence. 2,The antisense expression vector and the siRNA eukaryotic expression vectors of HCCR-2 were transfected into hepatocellular carcinoma cell line HepG2 through lipofectamine 2000 . After G418 selection, positive clones were picked and further established their stable transfected cell line. 3,The change of HCCR-2 in mRNA level was detected by semi-determinalion RT- PCR and real-time quantitive PCR. 4,The cell morphology change of HepG2 cells was observed by HE staining,transmission electron microscopy and interactive laser cytometer respectively.5,The capability of cell proliferation,survival and migration was detected by MTT, plate clone formation experiment and cell migration experiment. 6,Cell cycle and cell apoptosis were analysed by FCM. 7,The expression of P53 in mRNA level was determined by semi-determinalion RT- PCR. 8,The protein expression of Bax and Bcl-2 was detected by western blot method.Results 1,A 1kb fragment of HCCR-2 was successfully amplified by touch down RT-PCR, sequencing suggested that the recombinant antisense and RNAi eukaryotic expression vectors targeting HCCR-2 possesse correct read frame and nucleotide sequence 2,Cells containing stable transformants were selected by the ability of resistance to G418 and isolated with a limited dilution. Green fluorescene of the stable transfected HepG2 cell lines could be observed under inverted fluorescence microscope.3,The mRNA level of HCCR-2 was down–regulated in the cells transfected with HCCR-2 antisense plasmid.Of three HCCR-2 siRNA vectors ,two could effectively supprress the HCCR-2 mRNA level expression.4,(1)The transected cell morphology was transformed into fusiform and katyoplasmic ratio decreased to some extent. Cellular nucleus contracted and became round,cell kytoplasm condensed in the transfected cells. Chromatin margination and crimple also could observed under transmission electron in the transfected cells.(2) MTT results showed that cell proliferation ability of HepG2/ HCCR-2(-), HepG2/H1 and HepG2/H3 cells was significantly lower than that of HepG2,HepG2/P and HepG2/ N cells.(3) Plate clone formation experiment demonstrated that single cell clone formation capacity of HepG2/ HCCR-2(-), HepG2/H1 and HepG2/H3 cells was obviously lower than that of HepG2 and HepG2/ N cells. (4)Cell migration experiment showed that the migration capacity of HepG2/ HCCR-2(-), HepG2/H1 and HepG2/H3 cells was also decreased compared with that of HepG2 and HepG2/ N cells.5,Flow cytometry suggested that the apoptosis of cells was increased by antisense and siRNA HCCR-2 gene eukaryotic expression vectors. And the growth of HepG2 transfected by antisense and siRNA HCCR-2 plasmids was retarded, which was blocked in G0/G1 stage.6,Semi-determinalion RT- PCR result manifested that there was no significant change in P53 mRNA level in the transfected HepG2 cells.7,Western blot showed that Bax was diminished and Bcl-2 was augmented in HepG2/ HCCR-2(-), HepG2/H1 and HepG2/H3 cells.Conclusion 1,We successfully constructed antisense-nucleic acids and siRNA vectors of HCCR-2 and established their stably transfected cell lines.2,Two RNAi sequence specific targeting HCCR-2 were identified.3,With the expression of HCCR-2 in hepatocellular carcinoma cell line HepG2 was inhibited by antisense-nucleic acids and siRNA, biological behaviours including cell growth , plating efficiency, cell migration were affected . 4,HCCR-2 can participate in the regulation of cell cycle in HepG2 and may be related to cell proliferation and apotosis. 5,The expression change of HCCR-2 could not influence the mRNA level of P53 in HepG2 cells. 6,Bax and Bcl-2 were involved in apoptosis which was induced by antisense-nucleic acids and siRNA in HepG2 cells.