Migration Ability Of HepG2 Cells Research Articles

Hydroalcoholic Extract of Psoralea drupacea Inhibits Proliferation and Migration of Hepatocellular Carcinoma Cells and Decreases Angiogenesis in Chick Chorioallantoic Membrane

Objective: Hepatocellular carcinoma is one of the leading causes of cancer-related death worldwide. Experimental studies reported that some plants in the genus of Psoralea (Fabaceae family) show anticancer potential. The present study aimed to evaluate the effects of Psoralea drupacea extract (PDE) on HepG2 liver cancer cells. Methods: The proliferation, cell cycle, and migration of HepG2 cells were determined by thiazolyl blue tetrazolium bromide test, propidium iodide staining, and scratch assay, respectively. The effects of PDE on the activity of matrix metalloproteinases (MMPs) and angiogenesis were evaluated by the gelatin zymography method and chicken chorioallantoic membrane model, respectively. Results: The culture of HepG2 cells in the presence of PDE (24 hr and 48 hr) significantly reduced their viability (at a concentration of ≥ 50 µg/mL) and increased the percentage of cells in the sub-G1 stage. PDE also increased the antiproliferative and proapoptotic activities of doxorubicin (3 and 6 µg/mL). The extract significantly decreased the generation of reactive oxygen species and lipid peroxidation in the cells. Moreover, PDE (25 and 50 µg/mL) significantly suppressed the migration ability of HepG2 cells, which was associated with inhibition in the activity of MMP2 and MMP9 (50 µg/mL). Furthermore, treatment with PDE significantly reduced the number and diameter of vessels in the chick chorioallantoic membrane. Conclusion: PDE decreased the survival and cell cycle progression of liver cancer cells through a mechanism other than oxidative stress. This extract also showed an anti-angiogenesis effect and diminished the migration ability of HepG2 cells by inhibiting MMP activity

Construction of an M2 macrophage-related prognostic model in hepatocellular carcinoma.

M2 macrophages play a crucial role in promoting tumor angiogenesis and proliferation, as well as contributing to chemotherapy resistance and metastasis. However, their specific role in the tumor progression of hepatocellular carcinoma (HCC) and their impact on the clinical prognosis remain to be further elucidated. M2 macrophage-related genes were screened using CIBERSORT and weighted gene co-expression network analysis (WGCNA), while subtype identification was performed using unsupervised clustering. Prognostic models were constructed using univariate analysis/least absolute shrinkage selector operator (LASSO) Cox regression. In addition, Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG), gene set enrichment analysis (GSEA), gene set variation analysis (GSVA), and mutation analysis were used for further analysis. The relationship between the risk score and tumor mutation burden (TMB), microsatellite instability (MSI), the efficacy of transcatheter arterial chemoembolization (TACE), immunotype, and the molecular subtypes were also investigated. Moreover, the potential role of the risk score was explored using the ESTIMATE and TIDE (tumor immune dysfunction and exclusion) algorithms and stemness indices, such as the mRNA expression-based stemness index (mRNAsi) and the DNA methylation-based index (mDNAsi). In addition, the R package "pRRophetic" was used to examine the correlation between the risk score and the chemotherapeutic response. Finally, the role of TMCC1 in HepG2 cells was investigated using various techniques, including Western blotting, RT-PCR and Transwell and wound healing assays. This study identified 158 M2 macrophage-related genes enriched in small molecule catabolic processes and fatty acid metabolic processes in HCC. Two M2 macrophage-related subtypes were found and a four-gene prognostic model was developed, revealing a positive correlation between the risk score and advanced stage/grade. The high-risk group exhibited higher proliferation and invasion capacity, MSI, and degree of stemness. The risk score was identified as a promising prognostic marker for TACE response, and the high-risk subgroup showed higher sensitivity to chemotherapeutic drugs (e.g., sorafenib, doxorubicin, cisplatin, and mitomycin) and immune checkpoint inhibitor (ICI) treatments. The expression levels of four genes related to the macrophage-related risk score were investigated, with SLC2A2 and ECM2 showing low expression and SLC16A11 and TMCC1 exhibiting high expression in HCC. In vitro experiments showed that TMCC1 may enhance the migration ability of HepG2 cells by activating the Wnt signaling pathway. We identified 158 HCC-related M2 macrophage genes and constructed an M2 macrophage-related prognostic model. This study advances the understanding of the role of M2 macrophages in HCC and proposes new prognostic markers and therapeutic targets.

Cinobufagininhibitsproliferation and induces apoptosis of hepatocarcinoma cellsby activating apoptosis, AKT, and ERK pathways.

Cinobufagin is one of the pharmaceutically active ingredients in the parotoid glands of the Chinese toad Bufo bufo gargarizansCantor. This study was conducted to investigate the effect of cinobufagin on viability, migration, and apoptosis of hepatocellular carcinoma (HCC) cells and its mechanisms. Human HCC cells (HepG2) were treated with cinobufagin and assessed for viability, apoptosis, and migration using CCK-8 assay, flow cytometry, and wound healing assay. The expression of genes related to the p53, AKT, and ERK pathways was detected using RT-PCR and Western blot analysis. Cell viability assays showed that cinobufagin reduced the viability of HepG2 cells in a concentration- and time-dependent manner. Significantly increased apoptosis was detected in cinobufagin-treated cells as compared with non-treated cells. The migration ability of HepG2 cells was significantly reduced after they were exposed to cinobufagin as compared with control. RT-PCR and Western blot analyses showed that the expression levels of p53, caspase-3, and Bax were significantly upregulated, and the expression levels of AKT and ERK were significantly downregulated after cinobufagin treatment. Our data demonstrated that cinobufagin reduces the viability and induces apoptosis of HepG2 cells. The cytotoxicity is likely achieved by upregulating the p53 pathway and downregulating the Akt and ERK pathways.

Molecular characterization, expression and anti-tumor function analysis of yak IFITM2 gene

IFITM2 is interferon-induced transmembrane protein 2, which plays an extremely key role in anti-tumor and anti-virus diseases. In this study, the 602 bp cDNA sequence of the yak (Bos grunniens) IFITM2 (BgIFITM2) gene was obtained. Moreover, the prokaryotic expression vector of BgIFITM2 protein was constructed and expressed successfully, with a molecular weight of 33.680 kDa. The proliferation activities and migration abilities of HepG2 cells were significantly inhibited after treatment with BgIFITM2 protein (0.1 and 1 μg/mL) (P < 0.05). The expressions of B cell lymphoma-2 (BCL2)/BCL2-associated X (BAX) and molecular target of rapamycin (mTOR) genes were significantly decreased, but the expressions of BAX gene were significantly increased after treatment with BgIFITM2 protein (0.1 and 1 μg/mL) (P < 0.05). The expression of BAX protein was also significantly increased after treatment with 1 μg/mL BgIFITM2 protein (P < 0.05). Finally, the addition of BgIFITM2 protein attenuated the formation of tumor lesions in mice, and the pathological damage of the lung was less than that in the model group. The expression of Ki67 protein in the model group was significantly higher than that in the control group (P < 0.05), but the expression of Ki67 protein in the BgIFITM2 group was significantly lower than that in the model group (P < 0.05). Taken together, BgIFITM2 protein could inhibit the proliferative activity of HepG2 cells by regulating apoptosis-related genes, and reduce the invasiveness of HepG2 cells in mice lung tissue. These results facilitate further studies on the function of BgIFITM2 protein.

Identification and Verification of Ubiquitin D as a Gene Associated with Hepatitis C Virus-Induced Hepatocellular Carcinoma.

Accumulated studies have suggested that hepatitis C virus (HCV) infection is one of the leading causes for hepatocellular carcinoma (HCC). However, the mechanisms underlying the effect of HCV on the occurrence of HCC are still poorly understood. HCV infection datasets (GSE82177 and GSE17856) and HCC datasets (The Cancer Genome Atlas Liver Hepatocellular Carcinoma and GSE89377) were downloaded from Gene Expression Omnibus or TCGA for analysis. The common differentially expressed genes in the above four datasets were identified by R software. The expression of ubiquitin D (UBD) in HCV-infected HepG2 cells was detected by RT-qPCR and Western blot, respectively. The interaction between NS3 and p53 was detected by co-immunoprecipitation. The influence of UBD on the proliferation and migration ability of HepG2 cells was evaluated by CCK-8 and wound healing assay, respectively. UBD was upregulated in both HCV-infected samples and HCC samples. HCV NS3 interacted with p53 and inhibited its expression. HCV NS3-induced UBD promoted the proliferation and migration of HepG2 cells. Our results suggest that HCV NS3-induced UBD is positively correlated with the development of HCV-related HCC during HCV infection. Targeting UBD could be a potential strategy for preventing and treating HCV-induced HCC.

Experimental Study of Rhein Inhibits the Invasion and Migration of HepG2 Cells by ERK Signaling Pathway

Objective: To investigate the effectiveness of Rhein on the proliferation, invasion and migration of human hepatoma cell line HepG2 and its possible mechanism.Methods: Human hepatoma cell line HepG2 was treated with different concentrations of Rhein (Rhein treatment group) and culture in culture medium alone (control group).The proliferation activity of the cells was determined by methyl Thiazolyl Tetrazolium (MTT) colorimetry.Transwell assay detected the invasion and migration of cells in each group.Cell scratch test was used to detect the migration ability of cells in each group.Excella-phospho-excellar signal-regulated kinase (P-ERK) activity was determined by ELISA after treatment with 50μ mol/L Rhein at different times.Western blot was used to detect ERK protein expression in HepG2 cells treated with 50 μmol/L Rhein.Results: Compared with the control group, the proliferation activity, invasion and migration ability of HepG2 cells in the Rhein treatment group were all decreased (P&lt; 0.05), and the p-ERK relative activity of HepG2 cells treated with Rhein was decreased (P &lt; 0.05).Conclusion: Rhein inhibits the invasion and migration of HCC cells, possibly by inhibiting the ERK pathway

Effect of small interfering RNA silencing of the AFP gene on the migration and invasion of hepatocellular carcinoma HepG2 cells

Objective To investigate the effect of alpha-fetoprotein (AFP) on the migration and invasion abilities of hepatocellular carcinoma (HCC) HepG2 cells. Methods HCC HepG2 cells were transfected with synthesized small interference RNA (siRNA) for targeted silencing of AFP, and then HepG2 cells were divided into blank control group, negative control group, and AFP siRNA group. After 48 hours of intervention, quantitative real-time PCR and ELISA were used to measure the efficiency of silencing after transfection; Transwell assay was used to analyze the invasion and migration abilities of HepG2 cells after silencing of the AFP gene; Western blot was used to investigate the effect of AFP silencing on the expression of epithelial-mesenchymal transition (EMT)-related proteins (N-cadherin, vimentin, and E-cadherin), AKT, and p-AKT. A one-way analysis of variance was used for comparison of continuous data between groups, and the LSD-t-test was used for further comparison between two groups. Results After transfection and silencing, compared with the blank control group, the AFP siRNA group had a significant reduction in the relative mRNA expression of AFP (P Conclusion Silencing AFP can inhibit the migration of HepG2 cells, possibly by blocking the PI3K/AKT signaling pathway and inhibiting EMT based on the mechanism of action of HepG2 cell line.

Molecular characterization, expression and functional analysis of yak IFITM3 gene

IFITM3 is interferon-induced transmembrane 3, which plays an extremely key role in anti-proliferation, anti-virus and anti-tumor diseases.In this study, the yak (Bos grunniens) IFITM3 (BgIFITM3) gene contained a 5′-untranslated region (UTR) (25 bp), a coding region (441 bp), and a 3′-UTR (115 bp). The expression of BgIFITM3 gene in liver was significantly higher than that in heart, spleen, lung and kidney (P < 0.01). BgIFITM3 protein was localized on the yak hepatocyte plasma membrane, and its expression was significantly different between 1 day and 15 months of age (P < 0.05). Moreover, the prokaryotic expression vector of BgIFITM3 protein was constructed and expressed successfully, with a molecular weight of 19.5 kDa. The activities of yak hepatocyte were significantly inhibited after treating with BgIFITM3 protein (10 and 20 μg/mL) (P < 0.01). The expression levels of ERBB-2, IRS-1, PI3KR-1, AKT-1 and MAPK-3 were significantly lower after treating with 20 μg/mL BgIFITM3 protein (P < 0.05). Besides, the activities of HepG2 cells were significantly inhibited after treating with BgIFITM3 protein (1, 10 and 20 μg/mL) (P < 0.05). While, the cloning ability and migration ability of HepG2 cells were significantly inhibited after treating with 10 μg/mL BgIFITM3 protein (P < 0.05). Finally, the mitochondria of HepG2 cells was concentrated, cristae widened, and the double film density of mitochondria was increased after treating with 10 μg/mL BgIFITM3 protein. After 10 μg/mL BgIFITM3 protein treating, the expression levels of VDAC-2, VDAC-3 and p53 genes were significantly increased, but the expression level of GPX-4 gene was significantly decreased (P < 0.01).Taken together, the BgIFITM3 protein could inhibit the proliferations of yak hepatocyte and HepG2 cells by regulating the PI3K/Akt pathway or ferroptosis-related genes, respectively. These results benefit for further study of the function of BgIFITM3 protein.

Effect of atractylone on the viability and apoptosis of hepatoma HepG2 cells and related mechanism

Objective To investigate the effect of atractylone on the viability and apoptosis of hepatoma HepG2 cells and its mechanism of action. Methods Hepatoma HepG2 cells were selected and divided into low-, middle-, and high-dose atractylone groups (5, 10, and 20 μmol/L), and the cells in the control group were added with an equal volume of DMSO. MTT colorimetry was used to measure the viability of HepG2 cells after treatment with different concentrations of atractylone; flow cytometry was used to measure the apoptosis rate and mitochondrial membrane potential of HepG2 cells; the DCFH-DA fluorescent probe labeling method was used to measure the level of reactive oxygen species (ROS) in HepG2 cells; Transwell assay was used to evaluate the effect of atractylone on the migration ability of HepG2 cells; Western blot was used to measure the protein expression levels of Bcl-2, Bax, and cleaved caspase-3. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for comparison between two groups. Results After 24 and 48 hours of treatment with atractylone, compared with the control group, the low-, middle-, and high-dose atractylone groups had a tendency of reduction in cell viability (all P Conclusion Atractylone can induce the apoptosis and inhibit the migration of HepG2 cells, which provides an experimental basis for further development and utilization of atractylone.

Down-regulation of miRNA-196b expression inhibits the proliferation, migration and invasiveness of HepG2 cells while promoting their apoptosis via the PI3K/Akt signaling pathway

The purpose of this study was to investigate the effects of microRNA-196b (miRNA-196b) on proliferation, migration, invasiveness and apoptosis of hepatocellular carcinoma cell line (HepG2), and the mechanism involved. MiRNA-196b inhibitor or negative control were transfected into HepG2 cells, while empty liposome vector was used as normal control. The results of transfection were assessed using real-time quantitative polymerase chain reaction (qRT-PCR). Cell proliferation, migration, invasiveness and apoptosis were determined using cell counting kit 8 (CCK-8), scratch test, Transwell invasion assay, and flow cytometric analysis, respectively. The expressions of PIK3, Akt and p-Akt proteins were determined using Western blotting. The HepG2 cells were also treated with PI3K/Akt signaling pathway inhibitor LY294002, and its effect on cell proliferation, migration, invasion, and apoptosis, and expressions of PIK3, Akt, and p-Akt proteins were determined. The results of RT-PCR showed that the relative expression of miRNA-196b in the inhibitor group (0.42 ± 0.13) was significantly lower than that in the blank control group (0.96 ± 0.10) and the negative control group (1.01 ± 0.32) (p &lt; 0.05). The miRNA-196b inhibitor significantly and time-dependently reduced the invasiveness, proliferation migration abilities of HepG2 cells, while promoting their apoptosis (p &lt; 0.05). The expressions of PIK3 and p-Akt proteins were significantly down-regulated in the inhibitor group, when compared with normal and negative control groups (p &lt; 0.05). However, there were no significant differences in the expression of Akt protein among the groups (p &gt; 0.05). After treatment of HepG2 cells with PI3K/Akt signaling pathway inhibitor LY294002, the proliferative, migratory and invasive abilities of cells in the treatment group were significantly enhanced, while cell apoptosis was significantly reduced (p &lt; 0.05). Similarly, the protein expressions of PIK3 and p-Akt in the non-treatment group was significantly upregulated, relative to the treatment group (p &lt; 0.05), but there was no significant difference in the expression of Akt protein between the two groups (p &gt; 0.05). Downregulation of miRNA-196b expression inhibits the proliferation, migration and invasiveness of HepG2 cells, while promoting their apoptosis via a mechanism involving the PI3K/Akt signaling pathway.

LASP2 is downregulated in human liver cancer and contributes to hepatoblastoma cell malignant phenotypes through MAPK/ERK pathway

LASP2 was recently demonstrated to serve as multifaceted roles in several types of cancers. However, its underlying mechanism in the progression of human liver cancer has not been explored. The aims of the current study were to detect LASP2 expression in a liver tissue microarray, and to determine whether LASP2 contributes to malignant phenotypes of HepG2 human hepatoblastoma cells. Our results revealed that LASP2 expression was downregulated in liver cancer tissues relative to normal non-cancerous tissues, and its downregulated expression was closely correlated with malignant process of liver cancer. In vitro, upregulation of LASP2 expression by transfection with LASP2 vector significantly suppressed HepG2 cells viability, colony formation and migration activities. Conversely, the viability, colony formation and migration abilities of HepG2 cells were increased when downregulating LASP2 expression by transfection with small interfering RNA targeting LASP2. Interaction study showed that silencing of LASP2 in HepG2 cells triggered high expression of Cyclin D1, ERK and p-ERK, and low expression of Bax, respectively. In addition, LASP2 silencing-induced malignant phenotypes were further attenuated after HepG2 cells treatment with ERK1/2 blocker PD98059. Collectively, our data suggest a link between LASP2 and MAPK/ERK axis in the development of hepatoblastoma and LASP2 may be a potential marker for assessment of liver cancer prognosis and staging.

Effects of knockout of lincRNA‑p21 on the proliferation, migration and invasion ability of HepG2 liver cancer cells

Effects of long intergenic non-coding RNA (lincRNA)-p21 on the proliferation, migration and invasion ability of HepG2 liver cancer cells were assessed to explore the underlying mechanism. The lincRNA-p21 small interfering RNA (siRNA) lentivirus vector was constructed, transfected and screened to obtain a stable cell line, which constituted the experimental group. At the same time, the empty virus vector was transfected as the control group. The messenger RNA (mRNA) expression of lincRNA-p21 in cells was detected via reverse transcription-polymerase chain reaction (RT-PCR). The proliferation ability of cells was detected via Cell Counting kit-8 (CCK-8) assay. Transwell chamber experiment was used to observe cell migration and invasion ability. Compared with that in the control group, the mRNA expression level of lincRNA-p21 in cells in the experimental group was obviously decreased (p<0.05). Results of CCK-8 showed that the proliferation ability of liver cancer cells was remarkably higher than that in the control group after knockout of lincRNA-p21 (p<0.05). Results of the Transwell chamber experiment revealed that the invasion and migration ability of HepG2 cells in experimental group was markedly higher than that in control group (p<0.05). When lincRNA-p21 was inhibited, the proliferation, invasion and migration ability of HepG2 cells were significantly enhanced, and the apoptosis rate was significantly decreased. Thus, lincRNA-p21 on the surface may play an inhibitory role in the occurrence, development and metastasis of liver cancer.

Effects of curcumin combined with cisplatin on cell proliferation, apoptosis, migration and invasion in hepatocarcinoma HepG2 cell

Objective To study the synergistic effects of curcumin and Cisplatin (DDP) on the proliferation, apoptosis, migration and invasion of hepatocellular carcinoma cells HepG2. Methods HepG2 cells were randomly divided into multiple groups, including control [dmethyl sulfoxide (DMSO)], curcumin group (5, 10, 20 μmol/L), DDP group (1, 5, 10 μmol/L) and combination treatment (Curcumin + DDP) group (5 μmol/L Curcumin + 5 μmol/L DDP, 10 μmol/L Curcumin+ 5 μmol/L DDP). The proliferation of HepG2 cells was detected by methyl thiazol tetrazolium (MTT) assay. Additionally, Hoechst 33258 assay was used to determine morphological changes of nucleus and cell apoptosis. The migration and invasion ability of HepG2 cells were detected by scratch healing assay and Transwell invasion assay, respectively. Results The viability of HepG2 cells in combination treatment group was significantly lower than that in DDP group as indicated by MTT assay [(40.27±4.80)% vs. (61.80±9.14)%, P=0.023; (19.37±5.02)% vs. (61.80±9.14)%, P=0.002]. Hoechst 33258 staining showed that combination treatment triggered (10 μmol/L Curcumin+ 5 μmol/L DDP) more severe cell apoptosis compared with DDP alone (5 μmol/L) [(18.95±1.30)% vs. (10.35±1.46)%, P=0.012]. Scratch healing assay revealed that curcumin or DDP alone induced significant inhibition of cell healing in a dose-dependent manner, while combination treatment exhibited more stronger effects on cell healing than curcumin alone [(33.81±1.63)% vs. (39.77±2.48)%, P=0.026; (24.54±4.93)% vs. (39.77±2.48)%, P=0.003]. Transwell invasion assay indicated that cell invasion was reduced under the treatment of curcumin or DDP, and combination treatment group significantly reduced the number of invasive cells more obvious (24.33±2.52 vs. 32.00±3.61, P=0.039; 15.67±3.06 vs. 32.00±3.61, P=0.004). Conclusion Combination treatment could induce marked cell apoptosis of HepG2 and significantly inhibite cell proliferation, migration and invasion. Key words: Curcumin; Cisplatin; Hepatocarcinoma; HepG2 cell; Migration; Invasion

Downregulation of matrix metalloproteinases contributes to the inhibition of cell migration and invasion in HepG2 cells by sodium valproate.

Histone deacetylation levels are closely associated with the genesis and development of tumors. However, the anti-tumor effect and mechanism of class I histone deacetylase inhibitor sodium valproate (VPA) on hepatocellular carcinoma cells has not been demonstrated clearly. In the present study, HepG2 hepatocellular carcinoma cells were exposed to VPA, and the effects of VPA on the proliferation, apoptosis, metastasis and invasion of the cells were evaluated. The MTT method and colony assays were used to detect cell proliferation, and cell migration and Transwell invasion were also evaluated. In addition, the protein expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 were measured by flow cytometric analysis. The results indicated that VPA was able to inhibit proliferation and reverse the malignant phenotypes of hepatocellular carcinoma cells by inducing cell apoptosis. Furthermore, the colony formation and migration abilities of HepG2 cells were downregulated by VPA. Protein expression levels of MMP-2 and MMP-9 in HepG2 cells were also downregulated following VPA treatment, which contributed to suppression of the migration and invasion ability of HepG2 cells.

Inhibition effect of sodium demethylcantharidate injection on adherence, invasion and metastasis of human hepatocarcinoma HepG2 cells

Objective To investigate the effect of sodium demethylcantharidate injection on adherence,invasion and metastasis and to investigate the related mechanism in human hepatocarcinoma cell line HepG2.Methods Adherence ability,migration and invasion of HepG2 cells inhibited by sodium demethylcantharidate injection were assessed by MTT and Transwell techniques.Expression levels of MMP-9 protein in HepG2 cells were determined by immunohistochemistry.Results The number of adhesion,migration and invasion of HepG2 cells were significantly lower in sodium demethylcantharidate injection group than those in the control group (P ＜ 0.05).HepG2 cells co-incubated with sodium demethylcantharidate injection in the concentration of 0.25 μg/ml for 30,60,90 and 120 min showed higher cell adhesion than the control group.The adhesion inhibition ratios were 48.11％,33.81％,28.97 ％ and 16.83 ％,respectively.The migration and invasion inhibition rates were 64.19 ％ and 58.19 ％.With concentration of sodium demethylcantharidate injection to increasing,expression levels of MMP-9 protein in HepG2 cells more and more lower than control group.Conclusion The adherence,migration and invasion abilities of HepG2 cells are markedly inhibited by sodium demethylcantharidate injection,the mechanisms is possible related to the expression levels of MMP-9 protein. Key words: Hepatocarcinoma HepG2 cells; Sodium demethylcantharidate injection; Metastasis; Adherence; Invasion; Matrix metalloproteinase 9

Effect of silencing RRM2 by specific small interference RNA on biological behaviors of human hepatoma cancer cell line HepG2

Objective To investigate the effect of small interference RNA (siRNA) silencing RRM2 on the biological behaviors of human hepatoma cancer cell line HepG2 and and the molecular mechanisms.Methods RRM2 expression was knocked down in human hepatoma cancer cell line HepG2 by RRM2 siRNA.The expression level of RRM2 was detected in human hepatoma cancer cell line HepG2 and human normal liver cell line HL-7702 at mRNA and protein levels by real-time polymerase chain reaction (PCR) or Western blotting.The cell proliferation was measured by cell counting Kit-8 (CCK-8).The migration was observed by using Transwell cell migration system.The expression levels of B lymphocytes/leukemia-2 (bcl-2),bax and cytochrome C (Cyt-C) proteins were detected by using Western blotting.Ten male BALB/C nude mice were subcutaneously injected with siRNA-RRM2/HepG2,siRNA-NC/HepG2 or NEG/HepG2 cells respectively to study the growth of tumor in each group.Results siRNA-RRM2 could down-regulate the expression of RRM2 in HepG2 remarkably,and siRNA-RRM2 could inhibit both the proliferation [(41.20 ± 2.13) ％ vs (91.35 ± 10.22) ％] and migration ability of HepG2 cells (21.20 ±1.28 vs 132.50 ±7.88) as compared with the negative control group.siRNA-RRM2 could down-regulate the expression of bcl-2 and up-regulate the expression of bax and Cyt-C.Additionally,the volume of xenograft tumors in siRNA-RRM2 group was decreased significantly as compared with control group.The volume of xenograft tumors in siRNA-RRM2 group was (171.13 ± 28.12) mm3 and that in siRNA-NC group was (1487.41 ± 168.20) mm3 at 8th week.Conclusion siRNA-RRM2 can down-regulate the expression of RRM2 in the HepG2 cells and may play an important role in the regulation of biological behaviors of HepG2 cells,and also inhibit the tumor growth of nude mice model. Key words: RNA interference ; RRM2 gene ; Carcinoma,hepatocellular

Hepatitis B virus X protein upregulates expression of calpain small subunit 1 via nuclear facter‐κB/p65 in hepatoma cells

Hepatitis B virus (HBV) infection is closely correlated with the development of hepatocellular carcinoma (HCC), in which hepatitis B virus X protein (HBx) plays crucial roles. HBx is believed to be a multifunctional oncoprotein. It has been reported that the calpain small subunit 1 (Capn4) is upregulated in the HCC tissues and involved in the metastasis of HCC. Therefore, we suppose that HBx may promote hepatoma cell migration through Capn4. In the present study, we investigated the effect of HBx on regulating Capn4 expression in human HCC cells. Our data showed that HBx could increase promoter activity of Capn4 and upregulate the expression of Capn4 at the levels of mRNA and protein in human hepatoma HepG2 (or H7402) cells using luciferase reporter gene assay, real-time quantitative RT-PCR assay and Western blot analysis. While, the RNA interference targeting HBx mRNA was able to abolish the upregulation. Interestingly, we found that the inhibition of nuclear factor-kappaB (NF-kappaB) mediated by siRNA targeting NF-kappaB/p65 mRNA or PDTC (an inhibitor of NF-kappaB) could attenuate the upregulation of Capn4. While, HBx failed to increase the promoter activity of Capn4 in hepatoma cells when the putative NF-kappaB binding site of the Capn4 promoter was mutant, suggesting that NF-kappaB is involved in the activation of Capn4 mediated by HBx. In function, wound healing assay showed that HBx could significantly enhance the migration ability of HepG2 cells through upregulating Capn4. Thus, we conclude that HBx upregulate Capn4 through NF-kappaB/p65 to promote migration of hepatoma cells.

Honokiol inhibits HepG2 migration via down-regulation of IQGAP1 expression discovered by a quantitative pharmaceutical proteomic analysis

Honokiol (HNK), a natural small molecular product, inhibited proliferation of HepG2 cells and exhibited anti-tumor activity in nude mice. In this article, we applied a novel sensitive stable isotope labeling with amino acids in cell culture-based quantitative proteomic method and a model of nude mice to investigate the correlation between HNK and the hotspot migration molecule Ras GTPase-activating-like protein (IQGAP1). The quantitative proteomic analysis showed that IQGAP1 was 0.53-fold down-regulated under 10 microg/mL HNK exposure for 24 h on HepG2 cells. Migration ability of HepG2 cells under HNK treatment was correlated with its expression level of IQGAP1. In addition, the biochemical validation on HepG2 cells and the tumor xenograft model further demonstrated that HNK decreased the expression level of IQGAP1 and its upstream proteins Cdc42/Rac1. These data supported that HNK can modulate cell adhesion and cell migration by acting on Cdc42/Rac1 signaling via IQGAP1 interactions with its upstream Cdc42/Rac1 proteins, which is a new molecular mechanism of HNK to exert its anti-tumor activity.