【Abstract】ObjectiveTo explore the effects of RECK gene on the biological behaviors of hepatocellular (HepG2). MethodsThe RECK cDNA was transfected to HepG2 with lipofectamine 2000. Detected its protein expressions with Western blot before and after transfection, analyzed the effects of RECK on MMP-9 activity using gelatin zymography, observed the effects on proliferation ability by MTT assay and plate clone formation assay, compare the changes of invasion ability by cell adhesion assay and in vitro invasion experiment. Results RECK protein was expressed steadily in transfected HepG2 cells and the amount of activated MMP-9 were decreased significantly. Their proliferation abilities weren’t different before and after transfection but their invasion abilities decreased sharply. ConclusionRECK gene can transfect HepG2 cells by liposome efficiently. It can inhibit the activity of MMP-9 and the invasion ability of HepG2.
【Abstract】ObjectiveTo investigate the proliferation rate of HepG2 cell after multiple thermotherapy and the possible reasons related to it. MethodsAfter HepG2 cell were treaded by ten repeated cycles of heat exposure at 43 ℃ for 80 minutes twice a day, the doubling time of cell was analyzed， and the cell cycle, bcl2 mRNA and bax mRNA were detected. ResultsThe proliferation rate of HepG2 cell which treated with heat speeded up, the percentage of G2 and S in cell cycle increased, the expression of bcl2 mRNA strengthened and the rate of bcl2/bax increased. ConclusionThe speeded proliferation of HepG2 cell after multiple thermotherapy is related to its high percentage of DNA duplicated and dividing cell, strengthened expression of bcl2 mRNA and increased rate of bcl2/bax.
【Abstract】Objective To investigate whether SUMO-1 enhances the apoptosis induced by wild-type p53 plasmid transfection in HepG2 cells. Methods The HepG2 cells were transfected respectively or simultaneouly with the following expressional plasmids as pcDNA3-wtp53（pwtp53，including human wild-type p53 gene），pCMV-HDM1B（pMDM2，including HDM2 gene， homologous gene as murine double minute gene 2），pcDNA3-His6-SUMO-1（pSUMO-1 ，including small ubiquitin-like modifier1 gene）and plasmid pcDNA3.The proteins expressed in cells were detected by means of Western blotting and the apoptosis rates of cells were measured by flow cytometry. Results The protein bands of p53 and MDM2 could be seen in cells transfected with pwtp53 and pMDM2. Meanwhile，the relative larger molecular weight bands were also seen in cells transfected with pSUMO-1 which represented the p53 and MDM2 protein modification by SUMO-1. Merely the trace of p53 protein was detected in cells not transfected with any plasmid or only transfected with empty plasmid and pSUMO-1. In cells transfected with pwtp53 and pwtp53+pSUMO-1，the apoptosis rates were (16.79±1.62)%and (18.15±1.36)%. When transfected with pwtp53+pMDM2，the rate decreased to (5.17±1.23)%. The apoptosis rate would come up again to (14.06±1.84)% after transfected with pwtp53+pMDM2+pSUMO-1 and the difference of rates were significant compared to the cells transfected with pwtp53+pMDM2 (PH＜0.01）. The apoptosis rates in other cells were less than 2% and had no significant difference. Conclusion SUMO-1 could increase the apoptosis induced by wild-type p53 plasmid transfection in HepG2 cells through combining to p53 protein or its post-translational modification and inhibiting p53 degradation by MDM2.
ObjectiveTo construct the human small interfering RNA (siRNA) lentiviral vector who targeting inhibitor of differentiation-1 (Id1) gene, and to detect its efficiency of gene silence for the HepG2 cells. MethodsThe most effective RNA interference sequences was screened from 4 kinds of siRNA vectors targeting Id1 gene (included pCGSIL-GFP-Id1-1, pCGSIL-GFP-Id1-2, pCGSIL-GFP-Id1-3, and pCGSIL-GFP-Id1-4), who was transfected to 293T cells. The selected siRNA vector was used to build lentiviral vector (Id1-RNAi-LV) and then infected human HepG2 cells. Then the expression levels of Id1 mRNA and its protein were detected by the real time PCR and Western blot method respectively. ResultsExpression level of Id1 protein in pCGSIL-GFP-Id1-4 group was lower than those of pCGSIL-GFP-Id1-1 group, pCGSIL-GFP-Id1-2 group, and pCGSIL-GFP-Id1-3 group (P < 0.05), who had the best efficiency of gene silence. The Id1-siRNA lentiviral vector (Id1-RNAi-LV) was successfully constructed by using pCGSIL-GFP-Id1-4. The titer of lentiviral was 2.0×109 TU/mL.results of real time-PCR and Western blot showed that, the expression levels of Id1 mRNA and its protein in HepG2 cells of Id1-RNAi-LV group were lower than those of blank control group and negative control group (P < 0.05). ConclusionsThe specific lentiviral can constantly down-regulate the expression of Id1 gene.
ObjectiveTo explore the effects of hypoxia inducible factor-1 alpha (HIF-1α) on the reverse differentiation of hepatocellular carcinoma cells into liver cancer stem cells, and the maintenance of malignant biological behavior in hypoxic environment.MethodsCD133-negative cells in HepG2 cells were separated by immunomagnetic beads and divided into two groups. The cells of siRNA group were transfected with siRNA-HIF-1α to silence the expression of HIF-1α gene, while cells of the blank control group did not transfect any siRNA fragments. The two groups of cells were cultured under normal and hypoxic conditions respectively. MTT, cloning and Transwell chamber experiments were used to detect the proliferation and invasion ability of cells. Western blot and real-time PCR (RT-PCR) were used to detect the expressions of HIF-1α, CD133, CD90, and CD44 protein and mRNA in cells.ResultsMTT results showed that the cell proliferation rate increased with the prolongation of hypoxia in four groups. Compared with the blank control group at 24, 32, 40, and 48 hours, the cell proliferation rate decreased significantly after siRNA-HIF-1a transfection, on both two kinds of cultured conditions (P<0.05). The results of plate cloning experiment showed that the number of cell-forming clones increased significantly after hypoxic culture (there were significant differences between the transfected normoxic group and transfected hypoxic group, blank control normoxic group and blank control hypoxic group, P<0.05); and the formation of transfected hypoxic condition group at the same time of hypoxia was also significant (P<0.05). The number of clones were significantly less than that of the blank control group at the hypoxic condition (P<0.05). Transwell lab experiment showed that after hypoxic culture, the number of cells migrated to the inferior chamber in the transfection group was significantly reduced compared with that of the blank control group (P<0.05). Western blot and RT-PCR results showed that the expression levels of HIF-1α protein and tumor stem cell markers (CD133, CD90, and CD44 protein) in the blank control hypoxic condition group were significantly higher than those in the other three groups (P<0.05); after siRNA-HIF-1a transfection, HIF-1α mRNA and tumor stem cell markers mRNA (CD133, CD90, and CD44 mRNA) in the transfected hypoxic condition group were significantly lower than those in the transfected normal condition group and the blank control normal condition group (P<0.05).ConclusionsIn hypoxia environment, HIF-1α can promote hepatocellular carcinoma cells to differentiate into liver cancer stem cells and enhance their malignant biological behavior.
The article aims to explore the optimal concentration of arsenic trioxide (As2O3) on HepG2 of liver cancer cells, and the effect of As2O3 on the migration, invasion and apoptosis of HepG2 cells. In this study, the activity of HepG2 cells treated with 0, 1, 2, 4, 8, 16, 32 μmol/L As2O3 was tested by CCK-8 method, the semi-inhibitory concentration (IC50) was calculated, and the morphological changes of HepG2 cells were observed after the action of As2O3 at IC50 concentration for 12, 24, 48 h. The effect of As2O3 on cell migration and invasion ability was verified by wound healing experiment and Transwell invasion experiment. Western blot and qRT-PCR were used to detect the effects of As2O3 on the gene and protein expression levels related to cell migration, invasion and apoptosis. The results showed that, compared with the control group, the activity of HepG2 cells decreased with the increase of the concentration of As2O3 treatment, showing a dose-dependent effect, and its IC50 was 7.3 μmol/L. After 24 hours’ treatment with 8 μmol/L As2O3, HepG2 cells underwent significant apoptosis, and its migration and invasion abilities were significantly reduced. In addition, the protein expression levels of RhoA, Cdc42, Rac1 and matrix metalloproteinase-9 (MMP-9) were down-regulated, the protein and mRNA expression levels of anti-apoptotic gene Bcl-2 were significantly down-regulated, and the protein and mRNA expression levels of pro-apoptotic genes Bax and Caspase-3 were significantly up-regulated. The above results indicate that certain concentration of As2O3 can inhibit the migration and invasion of hepatocellular carcinoma cells and promote the apoptosis of hepatocellular carcinoma cells.
ObjectiveThis study aims to study the effects and mechanism of resveratrol on hepatocellular carcinoma (HCC) cells through phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) axis.MethodsHepG2 cells at logarithmic growth stage were treated with different concentrations (0, 12.5, 25.0, and 50.0 μmol/L) of resveratrol, respectively. Then the proliferation of HepG2 cells was detected by the CCK8 method and real time cell anaIysis (RTCA) system, the expressions of signal molecules associated with PI3K/Akt axis was detected by the Western blot method, including PI3K p58, phosphorylation protein kinase B (p-Akt), total protein kinase B (t-Akt), and CyclinA2 protein.ResultsResveratrol had a significant inhibitory effect on the growth of HepG2 cells in a time and dosage dependent manner. After 48 h treatment of resveratrol to HepG2 cells, 50.0 μmol/L resveratrol inhibited the growth of HepG2 cells most significantly. Further, the RTCA system studies also found that resveratrol had a time and concentration dependent effect on the reduction of normalized cell index (NCI) in HepG2 cells. Flow cytometry results showed that, apoptosis rates of 12.5, 25.0, and 50.0 μmol/L group were higher than that of 0 μmol/L group. Compard with 0 μmol/L group, the expressions of PI3K p85, p-Akt, and CyclinA2 protein in HepG2 cells of 12.5, 25.0, and 50.0 μmol/L resveratrol group was significantly higher (P<0.05), although there was no significant effect of resveratrol on the expression of t-Akt in HepG2 cells (P>0.05).ConclusionsResveratrol might have anti-proliferation effects on HepG2 cells through PI3K p85/Akt signaling axis. This study could provide a novel idea for the treatment to HCC.
ObjectiveTo study the effects of sea cucumber polysaccharide (SCPS) on the proliferation and apoptosis of hepatocellular carcinoma (HCC) cells, and to explore its effect on JAK2/STAT3/survivin pathway. MethodsThe human HCC cell lines HepG2 were placed into 24-well plates after culturing to the logarithmic phase, then dealed with different concentrations (0, 50, 100, 200 μg/mL) of SCPS. The MTT assay was used to detect the effects of different concentrations of SCPS on cell proliferation at 12 h, 24 h, and 36 h. The effect of SCPS on cell apoptosis was analyzed by flow cytometry. The mRNA and protein expression levels of JAK2, STAT3, and survivin were detected by real-time quantitative PCR (qRT-PCR) and Western blot at 36 h after treatment with SCPS, respectively. Then, the human HepG2 cells treated with different concentrations (0, 50, 100, 200 μg/mL) of SCPS were subcutaneously xenografted into nude mice to observe the effect of SCPS on the growth of tumor tissues in nude mice. At the same time, the expressions of phosphorylated JAK2, STAT3, and survivin proteins in tumor tissues were detected by immunohistochemical method. ResultsAfter treatment with different concentrations of SCPS, the proliferation inhibition rate of HepG2 cells increased over time and increased SCPS concentrations (P<0.05). After 36 h cultivation time, the apoptosis rate of cells treated with different concentrations of SCPS was statistically significant (F=117.110, P<0.001) and increased with the increase of SCPS concentrations (P<0.05). The protein expression levels of JAK2, STAT3, survival and their phosphorylated proteins decreased gradually with the increase of SCPS concentrations (P<0.05), but there was no significant difference in the mRNA expression level (P>0.05). With the increase of SCPS concentration, the tumor volume of nude mice gradually reduced (P<0.05). At the same time, the results of immunohistochemical detection showed that the positive expression rates of phosphorylated JAK2, STAT3, and survivin proteins in tumor tissues decreased with the increase of SCPS concentrations (P<0.05). ConclusionFrom preliminary results of this study, SCPS could inhibit the proliferation of HCC cells and promote their apoptosis, which might be achieved by regulating the phosphorylated expressions of JAK2, STAT3, and survivin in the JAK2/STAT3/survivin pathway.