Influence Of Overexpression Of DPT And SPON1 Gene On Proliferation,Invasion And Migration Of Human Hepatocellular Carcinoma Sk-Hep-1 And HepG2 Cells

ObjectiveTo study the influence of overexpression of DPT and SPON1 gene on proliferation,invasion and migration of human hepatocellular carcinoma cells.To explore the role of DPT and SPON1 gene in the proliferation of hepatocellular carcinoma,and to provide a new biological indicator for invasion and metastasis of HCC and target for gene therapy of HCC.Methods1.The detection of gene and protein expression of DPT and SPON1 in hepatocellular carcinoma SK-Hep-1,HepG2 cells and normal human hepatocyte L-02: To culture three kinds of cells and extract the total RNA,then to reverse transcriptional cDNA,and use qPCR to detect the background expression of DPT and SPON1 genes.The total protein from three cell samples was extracted,and the background expression of DPT and SPON1 protein was detected by Western blot.2.The packaging of DPT and SPON1 gene lentivirus plasmid carrier and transfection of hepatoma cells: pLenti6.3-DPT-IRES-EGFP and pLenti6.3-SPON1-IRES-EGFP of DPT,SPON1 gene Lentivirus Expression Vector and package plasmid were co transfected to 293 T cells respectively to carry out the slow virus package of the target gene overexpression.After the packing was determined,the culture solution of 293 T cells expressing lentivirus-containing DPT and SPON1 and the lentivirus negative control virus(lentivirus negative control)were infected with SK-Hep-1 and HepG2 of the target cells respectively,respectively.The experiment set blank control group,empty plasmid negative control group(NC group)and target gene overexpression group.QPCR was used to detect the expression of DPT and SPON1 gene in transfected cells.WB was used to detect the expression of DPT and SPON1 protein in transfected cells.3.Detection of the transfected liver cancer cell proliferation,migration and invasion ability: CCK-8 test detected cell proliferation,transfection of hepatoma cells at different time points(1D,2D,3D,4D,5D)use enzyme labeled instrument to read the absorption value(OD value)at 450 nm,and calculate the cell growth activity according to the results of OD.Transwell test was used to detect the cell migration and invasion ability.In the experiment,the cells were fixed and stained after 24 hours in the Transwell compartment,and the OD values were read by the enzyme labeling instrument,and the cell migration ability was compared.The cells were transferred into the Transwell chamber with the matrix glue for 48 hours,then fixed and stained.The OD value was read out by the enzyme labelling instrument,and the invasion ability of the cells was compared.Results1.The expression of DPT and SPON1 gene in SK-Hep-1,HepG2 and normal human liver cell line L-02 was detected by qPCR.The results showed that the expression of DPT gene and protein in SK-Hep-1 and HepG2 of liver cancer cells were lower than that of normal human hepatocyte strain L-02,and the lowest was found in SK-Hep-1.A follow-up experiment was performed to express the proliferation and migration ability of hepatoma cells.The expression of SPON1 gene and protein in SK-Hep-1 and HepG2 of hepatoma cells was lower than that of normal human liver cell line L-02,which was the lowest in HepG2.Therefore,HepG2 cells were selected as the follow-up experiments of the overexpression of the SPON1 gene.2.The morphological observation of lentivirus transfected cells: DPT and SPON1 overexpressed lentivirus transfected with fluorescent labeling were transfected to SK-Hep-1,HepG2 cells and 48 h respectively.The fluorescence microscopy showed that the green fluorescence signal was obvious and the cell growth was good,which indicated that the transfection was successful.3.The expression of DPT and SPON1 genes in transfected cells: the expression of DPT gene in SK-Hep-1 cell and SPON1 gene in HepG2 cell was detected by qPCR.The results showed that compared with the blank group and the NC group in the SK-Hep-1 cell experiment,the expression of DPT gene in the experimental group(over expression group,SK-Hep-1-DPT)cell was significantly up(P<0.01),and the SPON1 gene expression in the experimental group(over expressed group,HepG2-SPON1)cells was also significant compared with the blank group and the NC group in the HepG2 cell experiment.Up regulation(P<0.01)indicated that two kinds of over expressing lentivirus packaging were successful.4.The expression of DPT and SPON1 protein in transfected cells: lentivirus mediated target gene transfected to the target cells to extract the total protein.WB detection showed that the expression of DPT protein in the overexpressed group(SK-Hep-1-DPT)cells was significantly higher than that in the blank group and NC group(P<0.05),and the expression of SPON1 protein in the overexpressed group(HepG2-SPON1)cells was also higher than that of the protein group(P<0.05).The blank group and the NC group(P<0.01).It is further proved that two kinds of over expression lentivirus package is successful.5.CCK-8 assay was used to detect the proliferation of transfected hepatoma cells: the proliferation of three groups of cells at different time points was detected by CCK-8 method.The results showed that the proliferation activity of DPT group(SK-Hep-1-DPT)was significantly inhibited(P<0.05)compared with the blank group and the NC group.Over expression of SPON1 group(HepG2-SPON1)cell proliferation activity was also inhibited(P<0.05).Overexpression of DPT and SPON1 significantly inhibited the proliferation of hepatoma cells.6.The migration and invasion of transfected hepatoma cells were detected by Transwell experiment.The results showed that the migration and invasion ability of overexpressed DPT group(SK-Hep-1-DPT)cells were significantly inhibited compared with the blank group and the NC group(P<0.05).The migration and invasion ability of cells overexpressing SPON1(HepG2-SPON1)was also significantly inhibited(P<0.01).Overexpression of DPT and SPON1 significantly inhibited the metastasis of HCC cells.Conclusions1.The expression of DPT gene and protein is the lowest in SK-Hep-1 cells,and SK-Hep-1 cells are selected as DPT overexpression and subsequent experiments that affect the proliferation and migration of hepatoma cells.The expression of SPON1 gene and protein is the lowest in HepG2 cells,and HepG2 cells are selected as a follow-up experiment for the overexpression of SPON1 and the ability to influence the proliferation and migration of liver cancer cells.2.The expression of DPT and SPON1 gene and protein expression was significantly up-regulated after two target gene overexpression lentivirus vectors were transfected to two hepatoma cells respectively,indicating that the overexpression of lentivirus was successfully packaged.3.The results of CCK-8 test showed that overexpression of DPT gene significantly inhibited the proliferation of SK-Hep-1 cells.Overexpression of SPON1 gene significantly inhibited the proliferation of HepG2 cells.Overexpression of DPT and SPON1 significantly inhibited the proliferation of hepatoma cells.4.The results of Transwell test showed that overexpression of DPT gene significantly inhibited the migration and invasion of SK-Hep-1 cells,and overexpression of SPON1 gene significantly inhibited the migration and invasion of HepG2 cells.Overexpression of DPT and SPON1 significantly inhibited the metastasis of HCC cells.