Proliferation Of MHCC97H Cells Research Articles

Pharmacological Inhibition or Silencing of TREM1 Restrains HCC Cell Metastasis by Inactivating TLR/PI3K/AKT Signaling.

Hepatocellular carcinoma (HCC), a widely prevalent malignancy strongly linked to inflammation, remains a significant public health concern. Triggering receptor expressed on myeloid cells 1 (TREM1), a modulator of inflammatory responses identified in recent years, has emerged as a crucial facilitator in cancer progression. Despite its significance, the precise regulatory mechanism of TREM1 in HCC metastasis remains unanswered. In the present investigation, we observed aberrant upregulation of TREM1 in HCC tissues, which was significantly linked to poorer overall survival. Inhibition of TREM1 expression resulted in a significant reduction in HCC Huh-7 and MHCC-97H cell proliferation, invasion, and epithelial-mesenchymal transition (EMT) process. Furthermore, inhibiting TREM1 decreased protein expressions of toll-like receptor 2/4 (TLR2/4) and major myeloid differentiation response gene 88 (MyD88), leading to the inactivation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) in HCC cells. Notably, these effects were reversed by treatment with TLR2-specific agonist (CU-T12-9), indicating a potential crosstalk between TREM1 and TLR2/4. Mechanistic studies revealed a direct interaction between TREM1 and both TLR2 and TLR4. In vivo studies demonstrated that inhibition of TREM1 suppressed the growth of HCC cells in the orthotopic implant model and its metastatic potential in the experimental lung metastasis model. Overall, our findings underscore the role of TREM1 inhibition in regulating EMT and metastasis of HCC cells by inactivating the TLR/PI3K/AKT signaling pathway, thereby providing deeper mechanistic insights into how TREM1 regulates metastasis during HCC progression.

Interaction and binding mechanism of anticancer echinacoside phenylethanoid glycoside against hepatocellular carcinoma with subdomains of human serum albumin

A phenylethanoid glycoside known as echinacoside has demonstrated promising anticancer properties against numerous cancer cell types. However, its pharmacokinetics or anticancer mechanism has remained unclear. Herein, the interaction of the echinacoside with human serum albumin (HSA) was explored by intrinsic/extrinsic fluorescence spectroscopy, circular dichroism (CD) spectroscopy as well as molecular docking simulation. Also, the anticancer effects and possible mechanism of action of the echinacoside against hepatocellular carcinoma (HCC) cells, MHCC-97-H with high tumorgenicity and metastasis, were assessed by cell viability, flow cytometry, qRT-PCR, and western blot assays. The results showed a static quenching mechanism in the formation of echinacoside-HSA complex, one binding site on HSA for echinacoside, contribution of hydrophobic interactions (PHE157, GLU188 and PRO447), and slight conformational changes of HSA in the complex form. Cellular assays disclosed that treatment with echinacoside concentration-dependently mitigated the proliferation of MHCC97-H cells in vitro with an IC50 concentration of around 30 µM. Also, echinacoside triggered apoptosis through the regulation of caspase-3 at mRNA and protein levels. Moreover, echinacoside reduced the expression of astrocyte elevated gene-1 (AEG-1)and N-cadherin, while enhancing the expression of E-cadherin, as the main hallmarks of epithelial–mesenchymal transition (EMT) in tumorigenicity and metastasis. Therefore, these data indicated that echinacoside with a promising binding affinity with HSA in a mimicking physiological condition may inhibit the proliferation and metastatic activity of MHCC97-H cells mediated by manipulation of the AEG-1/EMT pathway.

MTBP enhances the activation of transcription factor ETS-1 and promotes the proliferation of hepatocellular carcinoma cells

Increasing evidence indicates that the oncoprotein murine double minute (MDM2) binding protein (MTBP) can be considered a pro-oncogene of human malignancies; however, its function and mechanisms in hepatocellular carcinoma (HCC) are still not clear. In the present work, our results demonstrate that MTBP could function as a co-activator of transcription factor E26 transformation-specific sequence (ETS-1), which plays an important role in HCC cell proliferation and/or metastasis and promotes proliferation of HCC cells. Using luciferase and real-time polymerase chain reaction (qPCR) assays, MTBP was found to enhance the transcription factor activation of ETS-1. The results from chromatin co-immunoprecipitation showed that MTBP enhanced the recruitment of ETS-1 to its downstream gene’s (mmp1’s) promoter region with ETS-1 binding sites. In cellular and nude mice models, overexpression of MTBP was shown to promote the proliferation of MHCC97-L cells with low endogenous MTBP levels, whereas the knockdown of MTBP led to inhibition of the proliferation of MHCC97-H cells that possessed high endogenous levels of MTBP. The effect of MTBP on ETS-1 was confirmed in the clinical specimens; the expression of MTBP was positively correlated with the downstream genes of ETS-1, mmp3, mmp9, and uPA. Therefore, by establishing the role of MTBP as a novel co-activator of ETS-1, this work expands our knowledge of MTBP or ETS-1 and helps to provide new ideas concerning HCC-related research.

Metabolomics and integrated network pharmacology analysis reveal SNKAF decoction suppresses cell proliferation and induced cell apoptisis in hepatocellular carcinoma via PI3K/Akt/P53/FoxO signaling axis

BackgroundThere is no comprehensive treatment method for hepatocellular carcinoma (HCC); hence, research and development are still focused on systemic therapies, including drugs. Sinikangai fang (SNKAF) decoction, a classic Chinese herbal prescription, has been widely used to treat liver cancer. However, there is no research on its core active component and target.MethodsMouse models were established to measure the anticancer effect of SNKAF decoction on HCC. Further, we investigated the effect of SNKAF decoction on inhibition of hepatoma cells proliferation using cell viability, cloning and invasion assays in vitro. The components of SNKAF were collected from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and TCM@Taiwan database. Metabolomic analysis was used to identify the potential genes and pathways in HCC treated with SNKAF decoction. Then, the expression of phosphoinositide 3-kinase (PI3K), Akt, P53, FoxO proteins of the potential signal pathways were detected using Western blot.ResultsThe animal experiments showed that SNKAF decoction inhibited tumor growth (P < 0.05) and induced no weight loss in the mice. In vitro data showed that HCCLM3 and MHCC97H cell proliferation was inhibited by SNKAF serum in a time- and concentration dependent manner. Further combined analysis network pharmacology with metabonomics showed that 217 target genes overlapped. The core target genes included BCL2, MCL1, Myc, PTEN, gsk3b, CASP9, CREB1, MDM2, pt53 and CCND1. Cancer-associated pathways were largely involved in SNKAF mechanisms, including P53, FoxO, and PI3K/Akt signaling pathways, which are closely related to induced-tumor cell apoptosis. In addition, Western bolt verified that 10% SNKAF serum significantly affected the main proteins of PI3K/Akt/P53/FoxO signaling pathway in both cell lines.ConclusionSNKAF decoction-containing serum inhibited HCCLM3 and MHCC97H cell proliferation, migration, invasion, and induced-tumor cell apoptosis in-vivo. We confirmed that SNKAF decoction is a promising alternative treatments for HCC patients.

Exportin-5 SUMOylation promotes hepatocellular carcinoma progression

Increasing evidence has shown that abnormal expression of XPO5 is found in many human cancers and acts as an oncoprotein in certain cancers. However, its functional role in hepatocellular carcinoma (HCC) remains unexplored. In our study, we found that XPO5 was highly expressed in HCC, which was associated with SUMO modification. Moreover, we found that XPO5 was SUMOylated by SUMO2 at K125. Functional experiments revealed that XPO5 SUMOylation could promote MHCC97H cell proliferation, migration and invasion. In addition, we found that the nuclear export of pre-miR-3184 was suppressed by SUMOylated XPO5. Moreover, PLCB1 was identified as the common target of miR-3184-5p and miR-3184-3p. The suppressed phenotype induced by miR-3184-5p and miR-3184-3p could be rescued by overexpression of PLCB1. Bioinformatics analysis showed that PLCB1 expression had a negative relationship with HCC patient survival. The inhibitory effects of MHCC97H cells resulted from abnormal XPO5 SUMO modification could be blocked by miR-3184 inhibitor or PLCB1 overexpression. In conclusion, our findings demonstrate a novel mechanism of XPO5 in HCC, that is, the SUMOylated XPO5 acts as an “oncogenic” role in MHCC97H cells proliferation, migration and invasion by controlling the nuclear-cytoplasm transportation of miR-3184, thus up-regulating PLCB1 expression.

LncRNA SNHG14 aggravates invasion and migration as ceRNA via regulating miR-656-3p/SIRT5 pathway in hepatocellular carcinoma.

Recurrence and adverse events after hepatocellular carcinoma (HCC) treatment occur frequently even treated with the most efficient therapy for HCC, liver transplantation. Therefore, better understanding of HCC progression is required to advance the therapeutic strategy of HCC. This study aims to explore the effect and mechanism of small nucleolar RNA host gene 14 (SNHG14) on HCC cell invasion and migration. SNHG14 and miR-656-3p expression in HCC tissues and cells were examined by qRT-PCR. After co-transfection with sh-SNHG14, miR-656-3p inhibitor, miR-656-3p mimic, si-SIRT5, pcDNA3.1-SIRT5 and corresponding negative controls, HepG2 and MHCC97H cell proliferation, invasion and migration were detected. Then the expression levels of SNHG14, miR-656-3p and SIRT5 were measured by qRT-PCR and Western blot. Luciferases reporter gene assay and RNA pull down identified the relation between SNHG14 and miR-656-3p and between miR-656-3p and SIRT5. SNHG14 was upregulated and miR-656-3p was downregulated in HCC cells. Inhibition of SNHG14 could inhibit HepG2 and MHCC97H cell proliferation, invasion and migration. Upregulation of miR-656-3p or knockdown of SIRT5 significantly suppressed the biological process of HepG2 and MHCC97H cells. SNHG14 directly acted on miR-656-3p and SIRT5 was a target gene of miR-656-3p. miR-656-3p inhibitor or pcDNA3.1-SIRT5 could reverse the inhibition of sh-SNHG14 on cell proliferation, invasion and migration of HCC cells. SNHG14 promotes HCC cell invasion and migration through regulating miR-656-3p/SIRT5 axis.

Hepatitis B virus X protein boosts hepatocellular carcinoma progression by downregulating microRNA-137

BackgroundHepatocellular carcinoma (HCC) is a frequent diagnosed malignancy. microRNAs (miRs) are involved in various cellular processes during cancer development. This study attempted to probe the miR-based mechanism in hepatitis B virus X protein (HBx) small interfering RNA (siRNA)-treated HCC cells. MethodsHBx expression in hepatocyte and HCC cells was detected, and cells with highest HBx expression were screened out and transfected with HBx-siRNAs. Then the effect of HBx on HCC cell proliferation was detected. miRs differentially expressed in HBx-siRNA-transfected MHCC97H cells were analyzed and verified. miR-137 methylation was analyzed by bioinformatics, and miR-137 restoration was detected after Aza treatment. Furthermore, miR-137 methylation in MHCC97H cells with HBx knockdown or HBx overexpression was detected by methylation specific PCR. The targeting relationship between miR-137 and Notch1 was verified. Then the gain-and-loss functions of miR-137 or/and Notch1 were performed to estimate their roles in HCC cell proliferation. The effects of HBx-siRNA and overexpressed miR-137 in vivo were observed by tumor xenograft in nude mice and immunohistochemistry. ResultsHBx-siRNA weakened MHCC97H cell proliferation and tumor growth. miR-137 was highly expressed in HBx-siRNA-treated HCC cells and targeted Notch1. HBx knockdown decreased miR-137 methylation and restored miR-137 expression. miR-137 overexpression prevented HCC cell proliferation and tumor growth, while miR-137 downregulation reversed the repressing effects of HBx-siRNA on HCC cell proliferation. Inhibition of Notch1 reversed HCC cell proliferation induced by miR-137 downregulation. ConclusionOverexpression of miR-137 blocks HCC cell proliferation in HBx-siRNA-treated MHCC97H cells by targeting Notch1. This study may offer novel target for HCC treatment.

Characterization and Cytotoxicity of Polyprenol Lipid and Vitamin E-TPGS Hybrid Nanoparticles for Betulinic Acid and Low-Substituted Hydroxyl Fullerenol in MHCC97H and L02 Cells.

BackgroundThis study demonstrated an innovative formulation including the polyprenol (GBP) lipid and vitamin E-TPGS hybrid nanoparticles (NPs) which was aimed to control the transfer of betulinic acid (BA) and low-substituted hydroxyl fullerenol (C60(OH)n). Additionally, it developed BA-C60(OH)n-GBP-TPGS-NPs delivery system and researched the anti-hepatocellular carcinoma (HCC) effects.Materials and MethodsThe NPs were prepared by nanoprecipitation with ultrasonic-assisted emulsification (UAE) method. It was characterized by scanning electronic microscopy (SEM), transmission electron microscopy (TEM), FTIR spectrum, size distribution and zeta potential. Physical and chemical properties were evaluated through measurement of drug release, stability studies, drug loading efficiency (DE) and encapsulation efficiency (EE). Biological activities were evaluated through measurement of MTT assay, lactate dehydrogenase leakage assay (LDH), cell proliferation assays, cell apoptosis analysis, comet assay, wound healing assay, cell invasion and Western blot analysis.Results and ConclusionsThe NPs exhibited clear distribution characteristics, improved solubility and stability. BA and C60(OH)n for the NPs displayed a biphasic release pattern with sustained drug release properties. The mixture of C60(OH)n with different hydroxyl groups may have a certain effect on the stability of the NPs system itself. The NPs could effectively inhibit MHCC97H cell proliferation, migration and invasion in vitro. Combined use of C60(OH)n and BA in GBP lipids may improve the inhibit effect of C60(OH)n or BA against HCC cells and reduce cytotoxicity and genotoxicity of C60(OH)n for normal cells. We concluded that one of the important mechanisms of BA-C60(OH)n-GBP-TPGS-NPs inhibiting MHCC97H cells is achieved by up-regulating the expression of Caspase-3, Caspase-8 and Caspase-9.

Yin Yang Gong Ji pill is an ancient formula with antitumor activity against hepatoma cells.

Yin Yang Gong Ji pill (YYGJ) is a formula that was used in the Ming Dynasty. This study investigated the effects of YYGJ on HepG2 and MHCC97H hepatoma cells. The effects of YYGJ drug-containing rat serum (YYGJ serum) on cell proliferation and the cell cycle were investigated by a tetrazolium dye-based MTS assay and flow cytometry. Apoptosis was assayed by TUNEL and flow cytometry. E-cadherin, vimentin, c-Myc, Smad4, and MMP2 expression were assayed by real-time quantitative PCR and Western blot assays. The effects on cell invasiveness and migration were evaluated by wound healing and transwell assays. The antitumor activity of 10% YYGJ serum was compared to that of blank control, 10% rat serum control and 5-fluorouracil(FU). HepG2 and MHCC97H cell proliferation was inhibited by YYGJ serum in a time- and concentration-dependent manner. Cells accumulated in G0/G1 and apoptosis was increased in both cell lines by 10% YYGJ serum. The effects of apoptosis in 10% YYGJ serum were weaker than those in response to 5-FU. E-cadherin and Smad4 expression were upregulated by 10% YYGJ serum, but c-Myc, vimentin and MMP2 expression were downregulated in both hepatoma cell lines. The protein expression of Smad4 in HepG2, and mRNA expression of MMP2 and E-cadherin in both cell lines had no difference between 10% YYGJ serum and 5-FU treated groups. Cell invasion and migration were decreased by 10%YYGJ serum while cell cytotoxicity was shown in 5-FU treated group. YYGJ drug-containing serum inhibited HepG2 and MHCC97H cell proliferation, induced apoptosis, and regulated the expression of tumor-related genes and proteins. It reduced tumor cell invasion and migration. Further study to investigate the antitumor activity of YYGJ is warranted.

Blockade of ARHGAP11A reverses malignant progress via inactivating Rac1B in hepatocellular carcinoma

BackgroundThe molecular signaling events involving in high malignancy and poor prognosis of hepatocellular carcinoma (HCC) are extremely complicated. Blockade of currently known targets has not yet led to successful clinical outcome. More understanding about the regulatory mechanisms in HCC is necessary for developing new effective therapeutic strategies for HCC patients.MethodsThe expression of Rho GTPase-activating protein 11A (ARHGAP11A) was examined in human normal liver and HCC tissues. The correlations between ARHGAP11A expression and clinicopathological stage or prognosis in HCC patients were analyzed. ARHGAP11A was downregulated to determine its role in the proliferation, invasion, migration, epithelial-to-mesenchymal transition (EMT) development, and regulatory signaling of HCC cells in vitro and in vivo.ResultsARHGAP11A exhibited high expression in HCC, and was significantly correlated with clinicopathological stage and prognosis in HCC patients. Moreover, ARHGAP11A facilitated Hep3B and MHCC97-H cell proliferation, invasion, migration and EMT development in vitro. ARHGAP11A knockdown significantly inhibited the in vivo growth and metastasis of HCC cells. Furthermore, ARHGAP11A directly interacted with Rac1B independent of Rho GTPase- activating activity. Rac1B blockade effectively interrupted ARHGAP11A-elicited HCC malignant phenotype. Meanwhile, upregulation of Rac1B reversed ARHGAP11A knockdown mediated mesenchymal-to-epithelial transition (MET) development in HCC cells.ConclusionARHGAP11A facilitates malignant progression in HCC patients via ARHGAP11A-Rac1B interaction. The ARHGAP11A/Rac1B signaling could be a potential therapeutic target in the clinical treatment of HCC.

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.

Diltiazem inhibits proliferation and motility of hepatocellular cells in vitro by downregulating calcium-activated chloride channel TMEM16A

To assess the inhibitory effect of diltiazem, a calcium channel inhibitor, on the proliferation and mobility of human hepatocellular carcinoma cells in vitro and explore the possible mechanism. Two human hepatocellular carcinoma cell lines, MHCC97H and 7402, were treated with different concentrations (0-400 μmol/L) of diltiazem for 12, 24, or 48 h, and the changes in the cell proliferation and mobility were observed with MTT assay and wound healing assay, respectively. The changes in the expressions of calcium-activated chloride channel TMEM16A at mRNA and protein levels in the treated cells were detected using RT-PCR and immunocytochemistry. Treatment with diltiazem obviously inhibited the proliferation and suppressed the mobility of MHCC97H and 7402 cells in a time- and concentration-dependent manner (P < 0.05). Treatment with 100 μmol/L diltiazem for 24 h significantly inhibited the proliferation of MHCC97H cells and down-regulated the mRNA and protein levels of TMEM16A. In 7402 cells, diltiazem treatment at 50 μmol/L for 48 h resulted in the most significant inhibitory effect on the cell proliferation and TMEM16A expressions. Diltiazem can transiently inhibit the invasion of hepatocellular carcinoma cells in vitro possibly by down-regulating the expression of TMEM16A at both the mRNA and protein levels.

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.

Triclosan treatment decreased the antitumor effect of sorafenib on hepatocellular carcinoma cells.

BackgroundTriclosan is a widely applied antimicrobial agent which affects the endocrine system and homeostasis; it may also promote the cirrhosis and hepatocellular carcinoma (HCC) growth in a mice model. The exact roles of triclosan in regulating human hepatocellular carcinoma development and treatment remain unknown.MethodsMHCC97-H, a highly aggressive HCC cell line, was treated with indicated concentration of triclosan or sorafenib. The expression of drug-resistance genes was examined by qPCR. The clearance or metabolism of sorafenib was determined by liquid chromatograph-mass spectrometer/mass spectrometer (LC-MS/MS). MTT assay was used to examine the MHCC97-H cell proliferation. Nude mice were used to exam the anti-tumor effect of sorafenib on subcutaneous and intrahepatic growth of MHCC97-H cells.ResultsIn the present study, triclosan could induce the expression of drug-resistance genes in MHCC97-H cells (a highly aggressive HCC cell line), accelerate the clearance of sorafenib, and attenuate the anti-proliferation effect of this molecular targeted agent in MHCC97-H cells. Triclosan decreased the antitumor effect of sorafenib on subcutaneous and intrahepatic growth of MHCC97-H in nude mice.ConclusionBy discovering the fact that triclosan treatment enhances sorafenib resistance in HCC cells, this work suggests exposure of triclosan is detrimental to HCC patients during chemotherapy.

Canopy Homolog 2 Expression Predicts Poor Prognosis in Hepatocellular Carcinoma with Tumor Hemorrhage

Background/Aims: Canopy homolog 2 (CNPY2) is a signature gene highly associated with tumor progression, including hepatocellular carcinoma (HCC). The presence of tumor hemorrhage (TH) implies a fast-growing and worse tumor microenvironment. We examined a possible association between CNPY2 levels and TH and evaluated their prognostic values in patients with HCC. Methods: CNPY2 mRNA and protein levels were respectively determined in two independent cohorts of HCC specimens using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry of tissue microarrays. Kaplan-Meier survival and Cox regression analyses were executed to evaluate the prognosis of HCC. CNPY2 knockout HCC cell lines were established by the CRISPR/Cas9 gene editing system, and the functional role of CNPY2 in HCC cell proliferation and growth was examined in vitro and in vivo. Results: qRT-PCR showed that CNPY2 expression was significantly higher in HCC tumor tissue than in adjacent non-tumor tissue. Immunohistochemistry of HCC tissue microarrays demonstrated that CNPY2 expression was significantly correlated with TH and clinicopathological features indicating worse HCC progression. The prognostic value of CNPY2 expression and TH was validated by Cox proportional hazards analyses. Furthermore, CNPY2 knockout resulted in the significant suppression of MHCC97H cell proliferation, tumor growth, and hemorrhage. Bioinformatics analysis revealed that CNPY2 was closely associated with the expression levels of 6 positive impact genes in HCC, namely, ROMO1, BOLA2, HSF1, ATG4B, ATF4, and DENR, which are implicated in the regulation of the tumor microenvironment. Conclusion: CNPY2 is an oncogene that plays a critical role in the progression of HCC with TH. CNPY2 could be exploited as a novel prognostic marker and potential target for therapeutic intervention in HCC.

MiR-564 inhibits hepatocellular carcinoma cell proliferation and invasion by targeting the GRB2-ERK1/2-AKT axis.

Recent studies have shown that miR-564 is closely related to the development of various tumors, including breast cancer, lung cancer and glioma. However, few studies have examined miR-564 in hepatocellular carcinoma (HCC). Here, we demonstrated that miR-564 expression in HCC tissues was lower than that in adjacent noncancerous tissues and that miR-564 expression was associated with tumor size, tumor number and vein invasion. Bioinformatics analyses showed that low levels of miR-564 were correlated with poor prognosis. Furthermore, upregulation of miR-564 impaired SMCC7721 and MHCC97H cell proliferation, migration and invasion in vitro and reduced tumorigenesis in vivo. Next, we found that GRB2 was a direct target gene of miR-564 in the HCC cell lines. GRB2 was highly expressed in HCC tissues and negatively correlated with miR-564 expression levels. When GRB2 was downregulated by GRB2-siRNA, HCC cell proliferation, invasion and metastasis were impaired, and restoring GRB2 expression partially reversed the inhibitory effects of miR-564. Western blot analysis showed that miR-564 overexpression reduced GRB2 expression in HCC cell lines and inhibited ERK1/2 and AKT phosphorylation. miR-564 overexpression also upregulated the epithelial-like cell marker E-cadherin and downregulated the interstitial cell-like markers N-cadherin and vimentin. These results suggest that miR-564 inhibits the malignant phenotype of HCC cells by targeting the GRB2-ERK1/2-AKT axis. Consequently, miR-564 may be used as a prognostic marker and therapeutic target for HCC.

HMGA1 participates in MHCC97H cell proliferation and invasion through the ILK/Akt/GSK3β signaling pathway.

Hepatocellular carcinoma (HCC) is one of the major causes of cancer-related mortality, and the prognosis of HCC patients is unsatisfactory. It is known that the occurrence and development of HCC involves numerous genes, as well as various steps and stages in the pathological process. High mobility group AT-hook 1 (HMGA1) and integrin-linked kinase (ILK) may be overexpressed in HCC and may serve important roles in the development of cancer; however, the relationship between HMGA1 and ILK in HCC has not been examined. The present study demonstrated that inhibition of HMGA1 expression significantly decreased the levels of expression of ILK and the downstream elements phosphorylated (p)-Akt, p-glycogen synthase kinase 3β (GSK3β), matrix metalloproteinase (MMP)2, MMP9, CyclinD1 and c-Myc. Transfection with an ILK expression vector was able to recover the decreased expression of these downstream genes, and affected cell proliferation and apoptosis. In addition, results from Transwell and wound-healing experiments indicated that HMGA1 participates cell invasion and migration through the ILK/Akt/GSK3β pathway. The present study aimed to improve our understanding about the regulatory pathway involved in HCC and provides the basis for exploring HMGA1 inhibition as a therapy for patients with HCC and a new treatment strategy to prevent the development of HCC.

The biological mechanism of homeo box A13 regulating hepatoma carcinoma cells

Objective To filtrate high expression of homeo box A13 (HOXA13) in hepatoma carcinoma cells and investigate the effects of HOXA13 (liver-intestine cadherin) RNA interference (RNAi) in high hepatoma expression carcinoma cells on biological effects. Methods The expression levels of HOXA13 mRNA and protein in hepatoma carcinoma cells were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting to filtrate high expression of HOXA13 in hepatoma carcinoma cells, with Lipofectamine and plasmid to transfect high expression of HOXA13 in hepatoma carcinoma cells. The expression levels of HOXA13 mRNA and protein in cells transfected with HOXA13-RNAi were detected by RT-PCR and Western blotting, respectively. The proliferative activity of hepatoma carcinoma cells was measured by cell counting kit-8 (CCK-8) method. The migration and invasive capabilities of hepatoma carcinoma cells were examined by scratch-wound migration assay and Transwell invasion assay respectively. Results The results of RT-PCR and Western blotting revealed that the expression levels of HOXA13 mRNA and protein in MHCC-97H cells (14.010±0.431, and 0.702±0.062) were signifeicantly higher than in HepG2, Huh-7, SMMC-7721 cells (12.292±0.821, 0.653±0.087; 11.750±1.271, 0.527±0.069; 11.242±2.031, 0.420±0.075, P<0.05). The results of RT-PCR and Western blotting revealed that the expression levels of HOXA13 mRNA and protein in MHCC-97H cells transfected with HOXA13-RNAi (3.215±0.022, and 0.117±0.016) were lower than in control group (9.426±1.006, 0.364±0.025; 10.517±1.412, 0.426±0.047, P<0.05). The proliferation ability in HOXA13-RNAi group (0.368±0.115) was significantly lower than in the negative control group and blank control group (0.688±0.352, and 0.684±0.322, P<0.01). The proliferation of HOXA13-RNAi MHCC-97H cells was decreased significantly due to HOXA13 knockdown (P < 0.001). The invasive and migration capabilities of MHCC-97H cells transfected with HOXA13-RNAi [(47.302±5.410) cells] were also significantly reduced as compared with the negative control group and blank control group [(82.221±11.102) cells, and (84.322±12.520) cells, P<0.01]. Conclusion Knockdown of HOXA13 may play an important role in the proliferation, invasion and migration of human hepatoma carcinoma cells and it can promote the oncogenesis and progression of human hepatoma carcinoma. Key words: Homeo box A13; RNA interference; Carcinoma, hepatocellular

Incarvine C suppresses proliferation and vasculogenic mimicry of hepatocellular carcinoma cells via targeting ROCK inhibition.

BackgroundStudies have described vasculogenic mimicry (VM) as an alternative circulatory system to blood vessels in multiple malignant tumor types, including hepatocellular carcinoma (HCC). In the current study, we aimed to seek novel and more efficient treatment strategies by targeting VM and explore the underlying mechanisms in HCC cells.MethodsCell counting kit-8 (CCK-8) assay and colony survival assay were performed to explore the inhibitory effect of incarvine C (IVC) on human cancer cell proliferation. Flow cytometry was performed to analyze the cell cycle distribution after DNA staining and cell apoptosis by the Annexin V-PE and 7-AAD assay. The effect of IVC on Rho-associated, coiled-coil-containing protein kinase (ROCK) was determined by western blotting and stress fiber formation assay. The inhibitory role of IVC on MHCC97H cell VM formation was determined by formation of tubular network structures on Matrigel in vitro, real time-qPCR, confocal microscopy and western blotting techniques.ResultsWe explored an anti-metastatic HCC agent, IVC, derived from traditional Chinese medicinal herbs, and found that IVC dose-dependently inhibited the growth of MHCC97H cells. IVC induced MHCC97H cell cycle arrest at G1 transition, which was associated with cyclin-dependent kinase 2 (CDK-2)/cyclin-E1 degradation and p21/p53 up-regulation. In addition, IVC induced apoptotic death of MHCC97H cells. Furthermore, IVC strongly suppressed the phosphorylation of the ROCK substrate myosin phosphatase target subunit-1 (MYPT-1) and ROCK-mediated actin fiber formation. Finally, IVC inhibited cell-dominant tube formation in vitro, which was accompanied with the down-regulation of VM-key factors as detected by real time-qPCR and immunofluorescence.ConclusionsTaken together, the effective inhibitory effect of IVC on MHCC97H cell proliferation and neovascularization was associated with ROCK inhibition, suggesting that IVC may be a new potential drug candidate for the treatment of HCC.

Compound K-induced apoptosis of human hepatocellular carcinoma MHCC97-H cells in vitro.

An intestinal bacterial metabolite of ginseng protopanaxadiol saponin, 20-O-(β-D-glucopyranosyl)-20(S)-protopanaxadiol (compound K), has been reported to induce apoptosis in a variety of cancer cells. However, the precise mechanisms induced by compound K in human hepatocellular carcinoma (HCC) cells remain unclear. In order to examine possible apoptotic mechanisms, we investigated the anticancer effect of compound K in MHCC97-H. MTT assay showed that compound K inhibited the proliferation of MHCC97-H cells with a relatively low toxicity in normal hepatoma cells. Cell cycle progression and cell staining showed an increase in apoptotic sub-G1 fraction. Treatment of MHCC97-H with compound K also induced a reduction in mitochondrial membrane potential (Δψm) and DNA damage. Further study showed that compound K upregulated Fas, FasL, Bax/Bcl-2 ratio and downregulated pro-caspase-9, pro-caspase-3 in a dose-dependent manner, and it also inhibited Akt phosphorylation. These results suggest that compound K significantly inhibits cell proliferation and induces apoptosis in MHCC97-H cells through Fas- and mitochondria-mediated caspase-dependent pathways in human HCC cells.