Cell Line MHCC97-H Research Articles

Eradication of Hepatocellular Carcinoma by NKG2D-Based CAR-T Cells.

Despite the great success of chimeric antigen receptor T (CAR-T)-cell therapy in the treatment of hematologic malignancies, CAR-T-cell therapy is limited in solid tumors, including hepatocellular carcinoma (HCC). NK group 2 member D (NKG2D) ligands (NKG2DL) are generally absent on the surface of normal cells but are overexpressed on malignant cells, offering good targets for CAR-T therapy. Indeed, analysis of The Cancer Genome Atlas and HCC tumor samples showed that the expression of most NKG2DLs was elevated in tumors compared with normal tissues. Thus, we designed a novel NKG2D-based CAR comprising the extracellular domain of human NKG2D, 4-1BB, and CD3ζ signaling domains (BBz). NKG2D-BBz CAR-T cells efficiently killed the HCC cell lines SMMC-7721 and MHCC97H in vitro, which express high levels of NKG2DLs, whereas they less efficiently killed NKG2DL-silenced SMMC-7721 cells or NKG2DL-negative Hep3B cells. Overexpression of MICA or ULBP2 in Hep3B improved the killing capacity of NKG2D-BBz CAR-T cells. T cells expressing the NKG2D-BBz CAR effectively eradicated SMMC-7721 HCC xenografts. Collectively, these results suggested that NKG2D-BBz CAR-T cells could potently eliminate NKG2DL-high HCC cells both in vitro and in vivo, thereby providing a promising therapeutic intervention for patients with NKG2DL-positive HCC.

Myricetin inhibits migration and invasion of hepatocellular carcinoma MHCC97H cell line by inhibiting the EMT process.

The recurrence and metastasis of hepatocellular carcinoma (HCC) are a major concern in current research. Epithelial-mesenchymal transition (EMT) is the leading cause underlying the high mobility and invasiveness of tumor cells. Myricetin is a natural flavonol with various pharmacological activities. The effects of myricetin on the migration and invasion of HCC MHCC97H cells were evaluated in the present study. Wound healing, Transwell migration and invasion assays were used to examine cell migration and invasion. Western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to examine the expression of epithelial (E)-cadherin, neural (N)-cadherin and vimentin. The present study aimed to investigate the effects of myricetin on the migration and invasion of HCC MHCC97H cells. It was indicated that myricetin decreased the viability of MHCC97H cells in a concentration and time-dependent manner, and inhibited MHCC97H cells migration and invasion. As the concentration of myricetin increased, filopodia and lamellipodia in cells weakened and cells were arranged more closely. RT-qPCR and western blotting revealed that myricetin upregulated E-cadherin expression and downregulated N-cadherin. Collectively, the results of the present study demonstrate that myricetin may inhibit the migration and invasion of HCC MHCC97H cells by inhibiting the EMT process.

Dual roles of glutathione S-transferase mu 1 in the development and metastasis of hepatocellular carcinoma

PurposeReactive oxygen species (ROS) are implicated in carcinogenesis, and cellular antioxidant systems are important for detoxifying ROS and reversing oxidant-mediated modifications. Glutathione S-transferase mu (GSTM) belongs to a family of phase II detoxification enzymes that catalyze the conjugation of reduced glutathione (GSH) to a wide range of endogenous and exogenous electrophilic compounds. The genotype of GSTM1 was associated with the risk and prognosis of cancer in several meta-analyses. This study explored the function of GSTM1 in hepatocellular carcinoma (HCC). MethodsPolymerase chain reaction (PCR) and western blotting (WB) were used to detect the levels of gene and protein expression. MTS assays, Transwell assays, and flow cytometry were used to explore the function of GSTM1 in vitro. The xenograft assay and tail vein injection model were used to explore the function of GSTM1 in vivo. ResultsThe mRNA and protein expression of GSTM1 was downregulated in HCC, but the expression levels of GSTM1 were not correlated with patient survival time. In vitro, Transwell and doxorubicin (DOX)-induced apoptosis assays revealed that GSTM1 showed opposite functions in different HCC cell lines with varied TP53 genotype statuses. The overexpression of GSTM1 in the above cell lines led to a significant decrease in ROS and an increase in GSH concentration and TP53 levels, suggesting that the controversial role of GSTM1 resulted from the TP53 genotype of HCC cells. The overexpression of GSTM1 promoted cell migration and inhibited apoptosis in the MHCC-97H cell line (TP53, R249S), but inhibited cell migration and increased apoptosis in the SMMC-7721 cell line (TP53 wildtype). ConclusionGSTM1 down-regulation may partially account for ROS-mediated oxidative damage and HCC carcinogenesis. GSTM1 also regulates tumor progression by disrupting the ROS-TP53 axis in HCC cells with different genetic backgrounds.

MiR-942 promotes proliferation and metastasis of hepatocellular carcinoma cells by inhibiting RRM2B.

BackgroundHepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death. MicroRNA-942 (miR-942) plays a critical role in promoting proliferation and metastasis of cancer cells and is associated with poor prognosis in some types of cancers. However, the prognostic value of miR-942 and its functional role in HCC remain unclear.Materials and methodsReal-time PCR (RT-PCR) was used to detect the expression of miR-942 in HCC tissues and adjacent normal liver tissues. Next, the correlations between miR-942 expression and clinicopathological parameters including the survival rate were analyzed. Interaction between miR-942 and ribonucleotide reductase regulatory TP53 inducible subunit M2B (RRM2B) was determined by RT-PCR, Western blot and luciferase assay. The biological influence of miR-942 on HCC cell lines was studied using CCK-8 assay, colony formation assay and transwell assay in vitro. Western blot and RT-PCR were used to analyze the change of downstream genes after miR-942 mimics transfection.ResultsmiR-942 was significantly up-regulated in HCC. Its high expression was associated with serum alanine transaminase level (P=0.0350), tumor size (P=0.0195), T stage (P=0.0045) and lymphatic metastasis (P=0.0013). High expression of miR-942 was associated with shorter overall survival and disease-free survival time of HCC patients. RRM2B was validated as a target gene of miR-942. miR-942 mimics markedly promoted the malignant phenotypes of Huh7 and MHCC97H cell lines, while its inhibitor had the opposite effect. miR-942 can regulate the downstream genes of RRM2B including Egr-1 and PTEN, markers of epithelial-mesenchymal transition and matrix metalloproteinases.ConclusionmiR-942 may serve as a potential biomarker for HCC and its inhibitor may be a therapeutic agent for the treatment of this deadly disease.

HOXA11-AS promotes the migration and invasion of hepatocellular carcinoma cells by inhibiting miR-124 expression by binding to EZH2.

The objective of this study was to examine the function of the long non-coding RNA (lncRNA) HOXA11-AS in hepatocellular carcinoma (HCC). In total, samples from liver tumor and surrounding normal liver tissues were collected from 66 cases of HCC patients. Normal liver cell line HL-7702 and HCC cell lines HepG2, Hep3B, MHCC-97H and BEL7402 were used. Cells were transfected with different small interference RNAs or vectors. Then, transwell assay, qRT-PCR, CHIP, RIP and Western blot experiments were performed. We found that the HOXA11-AS expression level was higher in HCC samples than surrounding normal liver tissues. And the higher expression level of HOXA11-AS in HCC patients indicated a lower 5-year survival rate. Knockdown of HOXA11-AS in HepG2 and Hep3B cells caused impaired cell invasion and migration abilities. Otherwise, upregulation of HOXA11-AS in MHCC-97H and BEL7402 cells displayed higher invasion and migration capabilities. We also demonstrated that HOXA11-AS could inhibit miR-124 expression by binding to EZH2. Furthermore, overexpression of miR-124 or knockdown EZH2 expression could reverse the HOXA11-AS-induced migration and invasion effects in HCC cells. In summary, the high HOXA11-AS expression in HCC patients is associated with the poor outcome. HOXA11-AS could inhibit miR-124 expression by binding to EZH2 and thus promoted the migration and invasion of HCC cells.

Chelidonine enhances the antitumor effect of lenvatinib on hepatocellular carcinoma cells.

BackgroundLenvatinib is a newly approved molecular targeted drug for the treatment of advanced hepatocellular carcinoma (HCC). However, the high cost associated with this treatment poses a huge financial burden on patients and the entire public health system. Therefore, there is an urgent need to develop novel strategies that enhance the antitumor effect of lenvatinib.MethodsThe antitumor effects of chelidonine or/and lenvatinib on HCC cell lines MHCC97-H and LM-3 were examined using the 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2- H-tetrazolium bromide (MTT) assay. For the in-vivo investigation, the effect on subcutaneous or intrahepatic tumor growth in nude mice was also determined. The mRNA levels of epithelial mesenchymal transition (EMT)-related factors were examined through quantitative polymerase chain reaction or Western blot.ResultsIn the present study, we found that treatment with chelidonine enhanced the apoptotic effect of lenvatinib on HCC cells and the in-vivo growth of HCC tumors in nude mice. Mechanistically, treatment with chelidonine increased the expression of epithelial indicator E-cadherin, whereas it decreased the expression of mesenchymal indicators N-cadherin and Vimentin. These findings suggest that chelidonine restricted the EMT in HCC cells.ConclusionChelidonine inhibits the process of EMT and enhances the antitumor effect of lenvatinib on HCC cells.

Overlapping and unique roles played by ROCK1 and 2 in the modulation of coding and long noncoding RNA expression

BackgroundOur previous study described the crucial role of Rho-associated coiled-coil containing-kinases (ROCK) in hepatocellular carcinoma (HCC). However, the potential significance of long noncoding RNA downstream of ROCK is largely unknown. Here, a comprehensive comparative bioinformatics analysis of a microarray of an MHCC-97H cell line overexpressing ROCK1 or ROCK2 was performed.ResultsNumerous lncRNAs and mRNAs were deregulated by Rho-associated coiled-coil containing kinases 1 and 2. These results were consistent with the qRT-PCR results. Compared with MHCC-97H-Con, which was transfected with a null vector, the GO analysis revealed differentially expressed mRNAs (DEmRNAs) in MHCC-97H-ROCK1 (ROCK1 was overexpressed) enriched in apoptotic cell clearance, the cyclooxygenase pathway and bone trabecula morphogenesis; the DEmRNAs in MHCC-97H-ROCK2 (ROCK2 was overexpressed) were enriched in VEGF production, chemokine-associated signaling pathways, acute inflammatory response and vasoconstriction. Compared with MHCC-97H-ROCK2, the DEmRNAs in MHCC-97H-ROCK1 were involved in the JAK-STAT cascade, the Akt signaling pathway and the activity of several different peptidases. The pathway analysis of ROCK1 and ROCK2 revealed an overlap in the VEGF signaling pathway, ECM-receptor interaction, and adhesion and differences in the PPAR signaling pathway and mismatch repair. The predicted targets of the differentially expressed lncRNA (DElncRNAs) were enriched in the p53 signaling pathway, Jak-STAT signaling pathway, etc.Several hub DElncRNAs were identified.ConclusionsROCK1 and 2 modulate the expression of numerous mRNAs and lncRNAs and may participate in several signaling pathways in HCC. Several hub molecules were identified in the lncRNA-mRNA networks. Our results provide baseline data for ROCK1 and 2 regulation in HCC that might have implications for further research.

RETRACTED ARTICLE: Long non-coding RNA TPTEP1 inhibits hepatocellular carcinoma progression by suppressing STAT3 phosphorylation

BackgroundHepatocellular carcinoma (HCC) is still the most common cause of tumor-related death worldwide and accumulating studies report that long non-coding RNAs (LncRNAs) are closely related with HCC development, metastasis and prognosis. Cisplatinum, a well-known chemotherapeutic drug, has been widely used for treatment of numerous human cancers including HCC. This study aimed to investigate the differential expressions of LncRNAs in HCC cells treated with cisplatinum and its underlying mechanism.MethodsThe differential expressions of LncRNAs in HCC cells treated with cisplatinum were determined by RNA-seq. The roles of TPTEP1 in HCC development by applying gene function gain and loss analysis in MHCC97H and QYG-7703 cell lines were detected by quantitative real-time polymerase chain reaction (qRT-PCR), cell proliferation, colony formation, cell invasion and flow cytometry assays. The underlying mechanism of TPTEP1 sensitizing hepatocellular carcinoma cells to cisplatinum was examined by RNA-pull down, western blotting, subcellular fractionation, RNA immunoprecipitation and dual luciferase reporter assays. The effect of TPTEP1 on tumorigenesis in vivo was performed with a subcutaneous xenograft mouse model of HCC. In addition, TPTEP1 expression was detected in clinical tumor tissue samples by qRT-PCR.ResultsLncRNA TPTEP1 was highly expressed in cisplatinum-treated HCC cells, which sensitizes hepatocellular carcinoma cell to cisplatinum-induced apoptosis. TPTEP1 overexpression inhibited, while TPTEP1 knockdown promoted HCC cell proliferation, tumorigenicity and invasion. Furthermore, TPTEP1 exerted its tumor suppressing activities by interacting with signal transducer and activator of transcription 3 (STAT3) to inhibit its phosphorylation, homodimerization, nuclear translocation and down-stream genes transcription. Moreover, TPTEP1 overexpression obviously inhibits tumor masses in vivo in a subcutaneous xenograft mouse model of HCC and TPTEP1 is frequently downregulated in HCC tissues, compared to its corresponding pre-tumor tissues.ConclusionLncRNA TPTEP1 inhibits hepatocellular carcinoma cells progression by affecting IL-6/STAT3 signaling. Taken together, our findings suggest a tumor suppressing role of TPTEP1 in HCC progression and provide a novel understanding of TPTEP1 during the chemotherapy for HCC.

A new isoflavone glycoside from Abrus cantoniensis

A new isoflavone glycoside named as 8-O-methylrelusin-7-O-β-D-apifuranosyl-(1→2)-β-D-glucopyranoside (1), together with two known compounds, 8-O-methylrelusin-7-O-β-D-glucopyranoside (2) and isobiflorin (3), were isolated from Abrus cantoniensis. The structure of the new compound was elucidated on the basis of spectroscopic methods including extensive 1D NMR, 2D NMR, and HRESIMS. This is the first report of isoflavone from Abrus cantoniensis. Moreover, all isolated compounds were evaluated for their cytotoxicity against SMMC-7721 and MHCC97-H cell lines.

Inhibition of NET-1 suppresses proliferation and promotes apoptosis of hepatocellular carcinoma cells by activating the PI3K/AKT signaling pathway.

The present study aimed to elucidate the underlying mechanism of neuroepithelial cell transforming 1 (NET-1), a member of the Ras homolog gene family, in hepatocellular carcinoma (HCC). To determine the association between the expression of NET-1 and the proliferation and migration of MHCC97-H cells, the cells were transfected with NET-1 small interfering (si)RNA and si negative control. Following transfection with NET-1 siRNA, the proliferation rate of MHCC97-H cells decreased significantly and the percentage of apoptotic cells increased. The HCC cell line MHCC97-H was used in the present study as it exhibited an increased expression level of NET-1 compared with the MHCC97-L cell line. Expression levels of apoptosis-associated proteins including apoptosis regulator Bax (Bax), cyclinD1, apoptosis regulator Bcl-2 (Bcl-2) and caspase-3 were determined. Expression levels of phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) and their phosphorylated forms were also measured by western blotting. Following NET-1 knockdown, the expression of Bax and cyclinD1 decreased, the expression of Bcl-2 and caspase-3 increased, and the PI3K/AKT signaling pathway was inhibited. The results of the present study suggest that inhibition of NET-1 can suppress the progression of HCC by targeting the PI3K/AKT signaling pathway. NET-1 expression level in HCC cells increased compared with normal liver cells.

Effect of liver kinase B1 on biological function of hepatocellular carcinoma cells and its relationship with postoperative prognosis

Objective To investigate the effect of liver kinase B1 (LKB1) on the biological function of hepatocellular carcinoma cells and its relationship with prognosis. Methods LKB1 overexpression lentiviral vector was constructed by molecular biology technology, and the MHCC97H cells with high metastasis were infected by lentiviruses. LKB1 control MHCC97H cell lines (control group) and LKB1 overexpression MHCC97H cell lines (observation group) were established. The proliferation ability of cells was analyzed by cell counting kit-8 (CCK-8) assay in control group and observation group. The invasion ability of cells was analyzed by Transwell in control group and observation group. 142 patients with liver cancer from June 2012 to June 2018 in our hospital were selected as subjects. The expression of LKB1 was analyzed by immunohistochemistry and the recurrence rate of patients was analyzed in LKB1 high expression and low expression groups. Results The LKB1 overexpression MHCC97H cell line was successfully constructed in this study. As compared with control group, the proliferative capacity of MHCC97H cells in observation group was significantly decreased (P<0.05). As compared with control group, the invasive ability of cells in the observation group [(28.32±7.32) cells] was significantly decreased (P<0.05). Immunohistochemical analysis showed there were 98 cases (69.01%) in the LKB1 low expression group and 44 cases (30.99%) in the LKB1 high expression group. The 2- and 3-year recurrence rate was 27.27% and 47.62% in the high-expression group, and 40.82% and 68.37% in the low-expression group, respectively. There was significant difference between the two groups (Log-rank=2.013, P<0.05). Conclusion LKB1 is related to the proliferation and migration of hepatocellular carcinoma cells, and the recurrence rate of patients with high LKB1 expression is significantly increased after 5 years. Key words: Liver kinase B1; Carcinoma, hepatocellular; Proliferation; Invasion; Recurrence

Plasminogen activator inhibitor 2 (PAI2) inhibits invasive potential of hepatocellular carcinoma cells in vitro via uPA- and RB/E2F1-related mechanisms

Plasminogen activator inhibitor 2 (PAI2) has been shown to be associated with invasive phenotypes and prognosis in hepatocellular carcinoma (HCC). However, its biological roles and underlying mechanisms in invasion of HCC have not been explored. The present study aimed to address the issues. First, sub-lines in that PAI2 was stably overexpressed and silenced were established based on MHCC97H and BEL7402 cell lines, respectively. Wound-healing and transwell assays were applied to evaluate cell migration and invasion. Urokinase-type plasminogen activator (uPA) activity was measured using an ELISA kit. Real-time RT-PCR and western blotting were used to show gene expression at mRNA and protein levels. E2F1 expression in human specimens was determined by tissue microarray-based immunohistochemical staining. The sub-lines, MHCC97H-PAI2 and BEL7402-siPAI2, were successfully established. The two sub-lines carried much lower and higher migration and invasion powers, respectively, in contrast to the controls. In MHCC97H-PAI2 sub-line, intra-medium uPA activity was significantly decreased, while RB expression was obviously elevated, compared with the controls. The BEL7402-siPAI2 sub-line presented the opposite trend. To identify the role of RB/E2F1 pathway, we transiently overexpressed E2F1 in MHCC97H-PAI2 sub-line, and largely reversed the inhibitory effects of PAI2 on cell migration and invasion, through regulating multiple matrix metalloproteinases and epithelial-mesenchymal transition. In HCC specimens, E2F1 expression was much higher in tumor than in non-tumor tissues, and was significantly related to Edmondson-Steiner grade, overall as well as tumor-free survival. Our data suggest that PAI2 inhibits invasive potential of HCC cells via uPA- and RB/E2F1-related mechanisms.

Overexpressions of sentrin-specific protease-1 is associated with the invasion and metastasis of human hepatocellular carcinoma

Objective To investigate the expressions of sentrin specific protease-1 (SENP-1) in hepatocellular carcinoma (HCC), and explore its correlations with tumor metastasis. Methods The expression of SENP-1 and hypoxia inducible factor-1α (HIF-1α) were assessed by polymerase chain reaction (PCR)and immunohistochemical staining in human hepatocellular carcinoma, edge tissues and non-cancer tissues, microvascular density (MVD)was determined. The expressions of SENP-1 in HCC cell lines MHCC97H, MHCC97L and HepG2 were assessed by reverse transcriptase-PCR (RT-PCR), quantitative real-time quantitative PCR (Real-time PCR) and Western blotting. The plasmid SENP1-small interfering RNA (siRNA) conducted was used to transfected into MHCC97H cells. Western blotting analysis was identified for the effect of transfection. The expression of cysteinyl aspartate specific proteinase-8 (Caspase-8), B-cell lymphoma-2 (bcl-2), HIF-1α and vascular endothelial growth factor (VEGF) protein in hepatoma cells before and after transfection was detected by Western blotting. The invasion and metastasis of hepatoma cells were detected by wound assay and Transwell invasion assay. Results Compared with the hepatocellular carcinoma tissues and non-cancer tissues, the expression of SENP-1 and HIF-1α in tumor-surrounding tissues were much higher. Compared with immortalized liver cell line, the HCC cell lines showed higher expression of SENP1 mRNA and protein, the expressions of SENP-1 was positive correlation with the metastasis ability of HCC cell lines. After transfection of SENP1-siRNA plasmid into MHCC97H cells, the expression of apoptosis promoting protein Caspase-8 was increased, and the expression of apoptotic protein bcl-2 was inhibited. Scratch healing assay revealed that the scuffing distance of hepatoma cells after transfection for 24 h was significantly increased compared with non-treatment group. Transwel1 assay showed that the number of hepatoma cells that migrated through the matrigel after transfection for 24 h was significantly decreased compared with non-treatment group (P=0.000). Conclusion The high expressions of SENP-1 in HCC might play an important role in tumor metastasis. The mechanism may be related to the over expression of SENP-1, which promotes liver cancer cells to adapt to hypoxic microenvironment and inhibit cell apoptosis. Key words: Sentrin specific peptidase 1; Hepatocellular carcinoma; Metastasis

Overexpression of Rac GTPase Activating Protein 1 Contributes to Proliferation of Cancer Cells by Reducing Hippo Signaling to Promote Cytokinesis

Agents designed to block or alter cytokinesis can kill or stop proliferation of cancer cells. We aimed to identify cytokinesis-related proteins that are overexpressed in hepatocellular carcinoma (HCC) cells and might be targeted to slow liver tumor growth. Using the Oncomine database, we compared the gene expression patterns in 16 cancer microarray datasets and assessed gene enrichment sets using gene ontology. We performed immunohistochemical analysis of an HCC tissue microarray and identified changes in protein levels that are associated with patient survival times. Candidate genes were overexpressed or knocked down with small hairpin RNAs in SMMC7721, MHCC97H, or HCCLM3 cell lines; we analyzed their proliferation, viability, and clone-formation ability and their growth as subcutaneous or orthotopic xenograft tumors in mice. We performed microarray analyses to identify alterations in signaling pathways and immunoblot and immunofluorescence assays to detect and localize proteins in tissues. Yeast 2-hybrid screens and massspectrometry combined with co-immunoprecipitation experiments were used to identify binding proteins. Protein interactions were validated with co-immunoprecipitation and proximity ligation assays. Chromatin immunoprecipitation, promoter luciferase activity, and quantitative real-time polymerase chain reaction analyses were used to identify factors that regulate transcription of specific genes. The genes that were most frequently overexpressed in different types of cancer cells were involved in cell division processes. We identified 3 cytokinesis-regulatory proteins among the 10genes most frequently overexpressed by all cancer cell types. Rac GTPase activating protein 1 (RACGAP1) was the cytokinesis-regulatory protein that was most highly overexpressed in multiple cancers. Increased expression of RACGAP1 in tumor tissues was associated with shorter survival times of patients with cancer. Knockdown of RACGAP1 in HCC cells induced cytokinesis failure and cell apoptosis. In microarray analyses, we found knockdown of RACGAP1 in SMMC7721 cells to reduce expression of genes regulated by yes-associated protein (YAP) and WW domain containing transcription regulator 1 (WWTR1 or TAZ). RACGAP1 reduced activation of the Hippo pathway in HCC cells by increasing activity of RhoA and polymerization of filamentous actin. Knockdown of YAP reduced phosphorylation of RACGAP1 and redistribution at the anaphase central spindle. We found transcription of the translocated promoter region, nuclear basket protein (TPR) to be regulated by YAP and coordinately expressed with RACGAP1 to promote proliferation of HCC cells. TPR redistributed upon nuclear envelope breakdown and formed complexes with RACGAP1 during mitosis. Knockdown of TPR in HCC cells reduced phosphorylation of RACGAP1 by aurora kinase B and impaired their redistribution at the central spindle during cytokinesis. STAT3 activated transcription of RACGAP in HCCcells. In an analysis of gene expression patterns of multiple tumor types, we found RACGAP1 to be frequently overexpressed, which is associated with shorter survival times of patients. RACGAP1 promotes proliferation ofHCC cells by reducing activation of the Hippo and YAPpathways and promoting cytokinesis in coordination withTPR.

MicroRNA-23a induces apoptosis of hepatocarcinoma cell line MHCC97H via down-regulating KIAP: a mechanism study.

Hepatocarcinoma is a great threat to global health. MicroRNA-23a was suggested to regulate growth and apoptosis in certain cell lines. Our study was focused on growth, proliferation, and apoptosis of hepatocarcinoma cell line MHCC97H under the influence of microRNA-23a, and explored the mechanism of pro-apoptosis microRNA-23a. MicroRNA-23a and control microRNA (scramble miRNA, for short as miRNA) were synthesized with the routine protocol. Lipofection transfection was performed in hepatocarcinoma cell line MHCC97H. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, caspase-3 activity detection, and flow cytometry were performed to examine growth, proliferation, and apoptosis of hepatocarcinoma cell line MHCC97H, respectively. Kidney inhibitor of apoptosis protein (KIAP) and small interfere RNA (siRNA) was synthesized for inhibition of KIAP. KIAP plasmid was established for activation of KIAP. Western blot was performed to examine the protein expression of KIAP and caspase protein family after transfection of KIAP siRNA or KIAP plasmid. Compared with miRNA transfection, microRNA-23a transfection significantly reduced the growth of MHCC97H cells, and decreased the expression of KIAP (p < 0.05). Enhanced translocation of phosphatidylserine and activation of caspase-3 were observed in microRNA-23a transfection cells. Moreover, inhibition of KIAP enhanced the pro-apoptosis effect of microRNA-23a, while activation of KIAP abrogated pro-apoptosis effect of microRNA-23a. MicroRNA-23a inhibits growth and proliferation of MHCC97H cells, and induces apoptosis of MHCC97H cells via down-regulating KIAP. KIAP could be a potential therapeutic target for hepatocarcinoma treatment.

Lentiviral-mediated microRNA-26b up-regulation inhibits proliferation and migration of hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is a frequently occurred malignancy worldwide with a high mortality. The treatment for HCC is still controversial. Emerging evidences have demonstrated that microRNAs (miRs) play a role in HCC. This study aims to investigate the effects of lentiviral-mediated miRNA-26b (miR-26b) on the proliferation and metastasis of HCC cells. The normal hepatic cell line HL-7702 and HCC cell lines HepG2 (without metastatic potential), SMMC-7721 (with low metastatic potential) and MHCC97H (with high metastatic potential) were purchased for our experiment. The lentiviral-mediated miR-26b overexpression (miR-26b-LV) and low expression (sh-miR-26b) were constructed to transfect the cells. The miR-26b expression and expressions of Karyopherin α-2 (KPNA2), matrix metalloproteinase 1 (MMP-1), MMP-7 and MMP-14 were determined by RT-qPCR and western blot analysis. The proliferation and metastasis of transfected HCC cells were detected by MTT and Transwell assay respectively. The miR-26b expressions were decreased significantly in MHCC97H cells. With lentiviral-mediated miR-26b overexpression, the proliferation and migration of HepG2, MHCC97H and SMMC-7721 cells were decreased significantly. The RT-qPCR and western blot analysis results revealed that the mRNA and protein expressions of KPNA2, MMP-1, MMP-7 and MMP-14 were decreased by lentiviral-mediated miR-26b overexpression. All the above indexes in the HepG2, MHCC97H and SMMC-7721 cells treated by sh-miR-26b exhibited opposite trends. These results show that overexpressed miR-26b could inhibit the proliferation and metastasis of HCC cells significantly, which provides a novel target and theoretical foundation for the treatment of HCC.

Effect of PAK1 gene silencing on proliferation and apoptosis in hepatocellular carcinoma cell lines MHCC97-H and HepG2 and cells in xenograft tumor.

This study intends to explore the effect of the PAK1 gene silencing on apoptosis and proliferation of hepatocellular carcinoma (HCC) MHCC97-H and HepG2 cells and cells in xenograft tumor. MHCC97-H and HepG2 cells and mice with xenograft tumor in vivo were randomly divided into control, empty vector and PAK1 shRNA groups. Morphology and the expression of green fluorescent protein of MHCC97-H and HepG2 cells and cells in xenograft tumor were observed. MTT assay and flow cytometry were used to detect proliferation, cell cycle and apoptosis of MHCC97-H and HepG2 cells and cells in xenograft tumor. The expressions of PAK1, PCNA, Ki67, Cyclin E, CDK2, p21, p53, Bax and Bcl-2 were measured using the quantitative reverse transcription polymerase chain reaction and western blotting. Compared with the control and empty vector groups, number of adherent cells of MHCC97-H and HepG2 cells and cells in xenograft tumor was reduced, and green fluorescent cells became round and reduced in the PAK1 shRNA group. Cell proliferation, the cells at S phase, the mRNA and protein expressions of PAK1, PCNA, Ki67, Cyclin E, CDK2 and Bcl-2 of MHCC97-H and HepG2 cells and cells in xenograft tumor were decreased, while the cells at G1 phase, apoptosis rate, the mRNA and protein expressions of p21, p53 and Bax of MHCC97-H and HepG2 cells and cells in xenograft tumor were increased in the PAK1 shRNA group. PAK1 gene silencing decreases proliferation of MHCC97-H cells, HepG2 cells and cells in xenograft tumor through the p53/p21 pathway.

Inhibition of CD9 expression reduces the metastatic capacity of human hepatocellular carcinoma cell line MHCC97-H.

Metastasis is a characteristic of malignant tumors and may be a fatal clinical factor for many patients with cancer. Hepatocellular carcinoma (HCC) cells are highly metastatic; the mechanism of metastasis is complicated and may be influenced by a number of factors. Membrane proteins may block receptors or inhibit important enzymes, thus inhibiting tumor progression, and may be potential therapeutic targets for tumor prognosis and treatment. The present study aimed to use proteomics to analyze the dynamic changes of membrane proteins in HCC cells, to improve our understanding of membrane protein functions and to clarify the important components of the mechanisms of HCC metastasis. The present study used the highly metastatic MHCC97-H and the lowly metastatic MHCC97-L HCC cell lines, and the isobaric tags for relative and absolute quantitation (iTRAQ) approach was used for high-throughput screening of metastasis-related membrane proteins. A total of 22 membrane proteins were identified as differentially expressed between the MHCC97-H and MHCC97-L cell lines; these results were verified by reverse transcription-quantitative polymerase chain reaction and western blotting. A number of the identified proteins were revealed to be related to tumor metastasis, including the tetraspan in transmembrane protein CD9. CD9 was demonstrated to be highly expressed in MHCC97-H cells compared with MHCC97-L cells. The functional role of CD9 was characterized by inhibiting its expression using a small interfering RNAs, which demonstrated that reduced CD9 expression inhibited cell migration and metastasis, as determined by wound-healing and invasion assays. Results from the present study demonstrated that CD9 was highly expressed in the highly metastatic HCC cells and promoted HCC cell migration. This protein may be a novel target for regulating the invasive phenotype in HCC.

Two new anthraquinone derivatives and one new triarylbenzophenone analog from Selaginella tamariscina

Two new anthraquinone derivatives, selaginones A (1) and B (2), and one new triarylbenzophenone analog, selagibenzophenone B (3), were isolated from Selaginella tamariscina (Beauv.) Spring. Their structures were established by 1D-, 2D-NMR and HR-ESI-MS data. Compounds 1 and 2 represent the uncommon examples of aryl substituted anthraquinone derivatives. Especially, compound 2 is a unique anthranone with exceptional structural feature, in which a p-hydroxyphenyl moiety is attached to the C-10 position. Compound 3 is the second naturally occurring triarylbenzophenone and showed moderate activity against SMCC-7721 and MHCC97-H cell lines with IC50 values of 39.8, 51.5 μM respectively.

Effect of Naringenin on apoptosis and epithelial mesenchymal transition of human high-invasive hepatoma cell line MHCC97-H

Objective To investigate the effect of Naringenin (Nar) on apoptosis, epithelial mesenchymal transition (EMT), of human high-invasive hepatoma cell line MHCC97-H and related molecular mechanisms. Methods MHCC97-H cells were routinely cultured. MTT assay was used to test the effect of Nar on viability of MHCC97-H cells treated with different doses of 0, 40, 80, 160 and 320 μmol/L. Flow cytometry (FCM) was applied to assess the apoptosis rate of MHCC97-H cells treated with Nar. Wound assay and Transwell invasive assay were utilized to assess the EMT of MHCC97-H cells treated with Nar. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting were applied to detect the expression of associated mRNA and proteins with apoptosis and EMT. Results Results of MTT assay showed that decreased cell activity was induced by Nar in a dose-dependent manner. After treatment with 0, 40, 80, 160, 320 μmol/L Nar for 24 h, cell activity was (96.49±13.28)%, (73.65±8.93)%, (60.75±15.75)%, (37.31±7.77)%, and (33.20±10.00)% respectively (F=31.028, P=0.000). After treatment with Nar, apoptosis rate of MHCC97-H cells was increased significantly (t=8.368, P=0.000). Migration and invasion of MHCC97-H cells were decreased after Nar treatment (t=2.964, P=0.014; t=8.899, P=0.000). Results of qPCR and Western blotting demonstrated that associated mRNA and proteins varied after Nar treatment. Conclusion Nar could induce apoptosis and inhibit EMT of MHCC97-H cells. Key words: Liver cancer; Naringenin; Apoptosis; Epithelial mesenchymal transition