Identification Of The Functional Fragment Of ZHX2 And Its Repression Effects On Cell Growth

Objective:To identify the functional fragment of ZHX2 repressing AFP expression and explore its inhibition effects on cell growth.Method:PartⅠ.Identification of repressional fragment of ZHX2 on AFP1.Cloning of ZHX2 truncated fragments(1) Construction of ZHX2 truncated fragments expression plasmidsAccording to the sequence of ZHX2 cDNA in PubMed,specific primers for the ZHX2 different truncated fragments using Primer Premier5.0 software.HA tag were included in the reverse primers at its 5 'end.PCR reaction were used to amplify ZHX2 truncated fragments 1-501AA,242-338AA and 242-501AA.The PCR products were then cloned into the vector pcDNA3.0 to construct the expression plasmids:p1ZHX2(1-501AA)-HA,p2ZHX2 (242-338)-HA and p3ZHX2(242-501AA)-HA.The recombinants were transformed into JM109,and the positive clones were identified by restrictive enzymes assay and DNA sequencing.(2) Western blot analysis for ZHX2 truncated protein expressionIn order to assay the expression of ZHX2 truncated fragments in vitro,p1ZHX2 (1-501AA)-HA,p2ZHX2(242-338AA)-HA and p3ZHX2(242-501AA)-HA were transfected into HepG2,HepG2.2.15and COS-7 cells respectively.Cells were collected 48 h later for western blot.2.The repression effect of ZHX2 truncated fragments on AFP (1) Overexpression of ZHX2 truncated fragments in hepatma cell lines HepG2 and HepG2.2.15Three vectors containing ZHX2 different truncated fragments were transfected into HepG2 and HepG2.2.15 cell lines respectively.48 h later,the medium and the cells were collected seperately to detect the expression of AFP and ZHX2 truncated fragments AFP were analyzed by ELISA and ZHX2 were analyzed by western blot using anti-HA monoclonal antibody as first antibody and anti-mouse IgG antibody labeled with HRP as second antibody.(2) The repression of ZHX2 truncated fragments on AFP core promoter0.6μg phAF269 plasmid was cotransfected with different truncated fragments of ZHX2 plasmids(0.3μg) into HepG2 and HepG2.2.15 cells,and 20ng of pRL-TK plasmid DNA was also involved in each group to normalize transfection efficiency.Cells were harvested 48 h for luciferase assay.(3) Dual-Luciferase reporter assaysFollowing the Promega protocol,dual-Luciferase reporter assays were performed in triplicate,using 20μl of cell extract,100μl of Luciferase Assay ReagentⅡReagent and 100μl of Stop&Glo Reagent by using luminoskan TL plus(Labsystems,Fr ankfurt,Germany).The firefly luciferase activity was normalized with the renilla reniformis luciferase activity of the cotransfected pRL-TK to control for variations in transferction efficiency.All the data shown in this study were obtained from at least three independent experiments,and each experiment was established double-wells.(4)Statistical analysisThe software GraphPad Prism 4 was used in the statistical analysis.Results were expressed as mean value±standard deviation(X~-±1s ).T test was used to evaluate the significance,where a value less than 0.05(p＜0.05) denoted the statistical significance.PartⅡ.The research of ZHX2 and its truncated fragments on liver cancer cells grow in vitro.1.The effect of ZHX2 and its truncated fragments on liver cancer cell grow were detected by CCK-8.HepG2 and HepG2.2.15 cells were inoculated by 1×10~4 cells per well in 96-well plates.The next day the recombinant plasmids of ZHX2 and ZHX2 truncated fragments were transfected into HepG2.2.15 and HepG2 cells respectively.The cell grow were continued detected for five days by CCK-8 every 24h after transfection.The growth curve were drawed to observe the effect of ZHX2 and ZHX2 functional fragment on cell growth.2The colony formation experimentsHepG2 cell were inoculated at 1×10~4 cells per well in 96-well plates.The next day the recombinant plasmids of ZHX2 and its truncated fragments were transfected into HepG2 cell.The cells were digested by trypsin after transfected 24h,the cell were inoculated in the six-well plates at appropriate density,after 2-3 weeks the gentian violet dyed the cells and counted the number of cell clones,calculated cloning formation rate.Result:1 The expression of ZHX2 truncated fragments in vitro(1)The successfully construction of ZHX2 truncated fragment-expression plasmidsEnzymes digestion,PCR and DNA sequencing confirmed that three combinant vectors, p1ZHX2(1-501AA)-HA,p2ZHX2(242-338AA)-HA and p3ZHX2(242-501AA)-HA were successfully constructed.(2) Expression of ZHX2 different truncated fragments in vitroWestern blot results showed that ZHX2 different truncated fragments proteins could all express in HepG2,HepG2.2.15 and COS-7 cells.2.Repression of ZHX2 truncated fragments on AFP in human hepatoma cell lines(1) Overexpression of human ZHX2 truncated fragments repressed AFP expression in hepatoma cell linesELISA results showed that AFP was suppressed by all three ZHX2 truncated fragments, while p3ZHX2(242-501AA)-HA has the stronggest repression function with the inhibition ratio 31.7%±2%(AFP in HepG2 and HepG2.2.15 cells transfected with p3ZHX2(242- 501 AA)-HA was 1386.7±193.6ng/ml and 1726.2±111.7ng/ml;compared with ZHX2-HA: 1897.4±73.1 ng/ml,1896.1±81.1 ng/ml).(2)The repression of ZHX2 truncated fragments on AFP core promoterResults of dual-Luciferase reporter assay showed that all ZHX2 truncated fragments could suppress the activity of AFP core promoter,while p3ZHX2(242-501AA) -HA has the most powful repression with the inhibition rate of 49.9%±0.9%. 3 ZHX2 and its truncated fragments can repress the cancer cell growth in vitro(1) ZHX2 and its truncated fragments can repress the cancer cell growthThe expression plasmids of ZHX2 and its truncated fragments were transfected into HepG2 and HepG2.2.15 cells.The cell growth were continued monitoring by CCK-8.The result shows ZHX2 and ZHX2 truncated fragments can repress the cancer cell growth.,while p3ZHX2(242-501AA) -HA has the most powful repression effects with the inhibition rate of 15%.(2) ZHX2 and its functional fragment can repress the colony forming ability of hepatoma cells in vitroThe expression plasmids of ZHX2 and its functional fragments were transfected into HepG2 cell.The cell colonies were counted by hexamethylpararosaniline dyeing 2 weeks later.The result showed ZHX2 and its functional fragments can repress the colony forming ability of hepatoma cells in vitro,while p3ZHX2(242-501AA) -HA has the most powful repression with the inhibition rate of 83%.Conclusions:1.We successfully constructed three fusion expression vectors p1ZHX2(1-501AA) -HA, p2ZHX2(242-338AA)-HA and p3ZHX2(242-501AA)-HA,which could express ZHX2 truncated fragments fusion proteins in vitro efficiently.This can provide requirement for further study of ZHX2 function.2.This study confirmed that p3ZHX2(242-501AA)-HA is the minimum function fragment owning the repression function on AFP and it has the most powful repression effects.This study provided necessary experimental data for further research on the structure and function of ZHX2.3.This study found that both ZHX2 and its truncated fragments could suppress the cell growth and the colony forming ability in vitro which may have role in HCC therapy.