Hepatitis B Virus Carboxyl-terminal Truncated Pres2 / S Protein-coding (mhbst) Preliminary Study Of The Sub-trans-activation Of Telomerase Reverse Transcriptase

Aim:To study the transactivated effect of MHBst on hTERT promotion, MHBst expression vector and hTERT promoter report vector were constructed, then cotransfected into different cell lines in vitro.This research provides a new insight for study the mechanism of HBV associated disease. Methods:1. Construct MHBst expression vector and study its transactivator mechanism in hepatocellular cellsPolymerase chain reaction was employed to amplify the coding sequence of two different MHBst from plasmid pUC19/3HBV-wt(adr), and the products were cloned into pcDNA3 to construct two MHBSt expression vectors, respectively named pcS2 and pcTS . HepG2 cell line was contransfected with reporter plasmid pGL3-control and pcS2/pcTS to estimate the transactivation function of MHBst by assay of luciferase activity using dual-luciferase reporter assay systems. In order to investigate the effects of pcS2/pcTS on hepatocellular cells, pcS2/pcTS was transfected into HepG2 cells and G418 resistant stable transfected clones were selected, respectively named HepG2-pcS2 and HepG2-pcTS.The cell growth curve and clone-forming experiment were used to assay the direct effect of MHBst on hepatocellular cells.2. Construct two different report plasmids containing human telomerase reverse transcriptase gene promoterFull-length hTERT promoter sequence(TRTP) and deleted hTERT promoter sequence which contain HBV homology sequence(del445) were respectively cloned into pGL3-basic multiple cloning sites to construct report plasmids pGL3-TRTP and pGL3-del445.To detect the transcriptional activity of hTERT promoter in the plasmids, transient transfection was performed in different cells- HELF,L02, Bel7402 cells. pRL-tk was used to normalize the transfection efficiency. Detect the luciferase activityby Dual-luciferase reporter assay.3. Investigate MHBst transactivator function on hTERT promoterMHBst expression vector pcS2 and hTERT promoter report vector pGL3~TRTP were cotranfected into different cell lines- L02, HepG2 and Bel7402 cells to detect the luciferase relative activity. The S-modified antisense oligodeoxyribonucleotide(asON) was synthesized to complement to preS2 region. By RT-PCR select the suitable concentration of asON. asON, pcS2 and pGL3~TRTP were cotranfected into different cells to study the specific transactivator function of MHBst on hTERT promoter. Results1. MHBst expression vector was successfully constructed and its transact ivator effects in hepatocel/lular cells was assayedTwo MHBSt coding sequence were cloned and sequenced. After being cotransfected into HepG2 cell line, the luciferase activity of pGL3~control was augmented by the pcS2/pcTS compared to pcDNA3. They were respectively 2.092 and 1.534 fold higher than pcDNA3. It indicated that both pcS2 and pcTS could transactivate the activity of SV40 promoter. Compared with pcTS, pcS2 showed more powerful transactivator function (p<0.01).In vitro study showed that after stable transfection of pcS2/pcTS, hepatocellular cells got more rapid growth speed and higher ability of colony forming ability. Growth curve showed that at sixth day, the cell number of HepG2-pcS2 and HepG2-pcTS were 1. 621 fold and 1. 548 fold higher compared with HepG2-pcDNA3 which was stable transfected with pcDNA3 as control. The ability of form soft agar colonies of HepG2-pcS2 and HepG2-pcTS were respectively 1.266 and 1.286 higher compared with HepG2-pcDNA3 control.2. Two different report pi asm ids containing human telomerase reverse transcriptase gene promoter were successfully constructedThe sequencing data of report vectors pGL3~TRTP and pGL3~del445were correspondence with the design. The results of transient transfection showed that the two report plasmids could highly expressed in immortal cells L02 and carcinoma cells Bel7402. pGL3-del445 was respectively 15.381,13.481 fold higher than pGL3-basic. pGL3-TRTP was respectively 15. 351, 23. 726 fold higher than pGL3-basic. While luciferase was not expressed in mortal cells HELF.3. MHBst transactivate the activation of hTERT promoter significantly Dual-luciferase report assay indicated that MHBst expression vector pcS2 might transactivate hTERT promoter in different cells(p<0. 01). The luciferase expression in cells treated with pcS2 was higher than that of pcDNA3.They were respectively 28. 16%, 17.55% and 19.41% higher in L02, HepG2^ Bel7402 cells. Antisense results showed the luciferase expresion can be lowed by asON against preS2. The luciferase expression of the asON group were lower than random group (p<0. 05). They were respectively 18. 81%, 21.21%, 22.15% lower in L02x HepG2, Bel7402 cells. While there was no significance between random and non-random treated group. These indicated MHBst might play specific transactivator on hTERT promoter. Cone I us i on1. MHBst expression vectors were constructed successfully which could play transactivator role. Compared with pcTS, pcS2 showed more powerful transactivator function.2. The stable expression MHBst cell lines were selected. Growth curve and colon-forming assay indicated that MHBst could improve both the growth speed and the tumorigenesis of hepatoma cells.3. hTERT promoter report vector with the ability of selectively expression in hTERT positive cells was successfully constructed.4. MHBst show specific transactivator function on hTERT promoter by cotransfection and antisense assay in vitro.