Overexpression of microRNA-30a-5p inhibits liver cancer cell proliferation and induces apoptosis by targeting MTDH/PTEN/AKT pathway.

Increasing results suggest microRNAs (miRNAs) could function important roles in malignant tumor progression. miR-30a-5p is downregulated in variety of cancers and acts as a cancer suppressing gene. The functions and molecular mechanisms of miRNA-30a-5p in hepatocellular carcinoma (HCC) remain unclear. In the present study, quantitative real-time PCR (qRT-PCR) was used to detect miR-30a-5p expression in 16 pairs of HCC and their adjacent non-cancerous tissues and HCC cell lines. By overexpression of miRNA-30a-5p, CCK-8 and colon formation assay were used to evaluate cell growth and flow cytometry to evaluate cell apoptosis. Western blot was used to test protein expression. And potential mechanisms were analyzed with luciferase activity assay. In vivo HepG2 tumor growth was observed with nude mice. Our results showed that miR-30a-5p expression in HCC tissues was significantly lower compared to adjacent non-cancerous liver tissues, and lower miR-30a-5p expression was also observed in HCC cell lines compared to normal liver cell. Luciferase assay showed that metadherin (MTDH) mRNA was a direct target of miR-30a-5p. A significant reverse correlation between miR-30a-5p and MTDH in liver cancer tissues was observed. miR-30a-5p overexpression in HCC cells significantly inhibited cell proliferation, colon formation, and induced apoptosis while MTDH overexpression reversed growth inhibition and apoptosis induction of miRNA-30a-5p in HCC cells. miRNA-30a-5p upregulated phosphatase and tensin homolog (PTEN) protein expression and thus inhibited AKT activating by targeting MTDH. miRNA-30a-5p also significantly inhibited HepG2 tumor growth in vivo. Our results suggest that underexpression of miR-30a-5p might function as a tumor suppressing miRNA by directly targeting MTDH in HCC and is therefore a potential candidate biomarker for HCC targeting therapy.

Shear Wave Elastography of the Spleen for Monitoring Transjugular Intrahepatic Portosystemic Shunt Function: A Pilot Study.

OBJECTIVES: To assess the feasibility of splenic shear wave elastography in monitoring transjugular intrahepatic portosystemic shunt (TIPS) function. METHODS: We measured splenic shear wave velocity (SWV), main portal vein velocity (PVV), and splenic vein velocity (SVV) in 33 patients 1 day before and 3 days to 12 months after TIPS placement. We also measured PVV, SVV, and SWV in 10 of 33 patients with TIPS dysfunction 1 day before and 3 to 6 days after TIPS revision. Analyses included differences in portosystemic pressure gradient (PPG), PVV, SVV, and mean SWV before and after TIPS procedures; comparison of median SWV before and after TIPS procedures; differences in PVV, SVV, and SWV before and at different times up to 12 months after TIPS placement; accuracy of PVV, SVV, and SWV in determining TIPS dysfunction; and correlation between PPG and SWV. RESULTS: During 12 months of follow-up, 23 of 33 patients had functioning TIPS, and 10 had TIPS dysfunction. The median SWV was significantly different before and after primary TIPS placement (3.60 versus 3.05 m/s; P = .005), as well as before and after revision (3.73 versus 3.06 m/s; P = .003). The PPG, PVV, and SVV were also significantly different before and after TIPS placement and revision (P < .001). The PPG and SWV decreased, whereas PVV and SVV increased, after successful TIPS procedures. A positive correlation was observed between PPG and SWV (r = 0.70; P < .001), and a negative correlation was observed between PPG and PVV and SVV (r = -0.65; P < .001). The areas under the receiver operating characteristic curve for PVV, SVV, and SWV in determining TIPS dysfunction were 0.82, 0.84, and 0.81, respectively. CONCLUSIONS: Splenic SWV is compatible with splenoportal venous velocity in quantitatively monitoring TIPS function and determining TIPS dysfunction.

[Effect of inflammatory stress on hepatic cholesterol accumulation and hepatic fibrosis in C57BL/6J mice].

OBJECTIVE: To investigate whether inflammatory stress exacerbates hepatic cholesterol accumulation and liver fibrosis using a C57BL/6J mouse model of chronic inflammation. METHODS: Twelve male C57BL/6J mice were given a high-fat diet (15.0% fat, 1.25% cholesterol, 0.5% cholic acid) and randomly assigned to the normal control group (n=6; subcutaneously injected with 0.5 mL of isotonic saline, every other day for 14 weeks) or the chronic inflammation model group (n=6; subcutaneously injected with of 0.5 mL of 10% casein, every other day for 14 weeks). At the end of week 14, the animals were sacrificed and blood was collected from the left ventricle for serological analysis of inflammatory markers and lipid profile, including serum amyloid A (SAA), interleukin-6 (IL-6), total cholesterol (TC) and free cholesterol (FC), low-density lipoprotein (LDL), and high-density lipoprotein (HDL)). Extracted liver tissues were divided for use in histological analysis (lipid accumulation and fibrosis evaluated by Oil Red O, Sirius red and Masson's trichrome staining) and quantitative fluorescence real-time PCR (to measure b-actin normalized expression of TNF-a MCP1, SREBP-2, LDLr, HMGCoA-r, ATF-6, GRP78, BMP-7, TGF-b, and collagens type I and type IV). Comparisons between groups were made by the two-samples t-test or Satterthwaite t-approximation test, collagen type I and type IV. RESULTS: Compared to the normal control group, the inflammation model group showed elevated serum IL-6 (12.55+/-4.75 vs. 32.41+/-7.42 pg/mL, P less than 0.01), reduced serum TC (14.82+/-1.56 vs. 10.62+/-0.48 mmol/L, P less than 0.01), up-regulated hepatic TNF-a mRNA expression (1.05+/-0.35 vs. 2.12+/-0.72, P less than 0.01), and elevated hepatic TC (12.10+/-2.57 vs. 23.21+/-8.75 mmol/L, P less than 0.05). In addition, the inflammation group showed abnormal lipid deposition, and increased and thickened reticular fibers. The livers of the inflammation group also showed up-regulated mRNA expression of SREBP-2 (normal control: 1.01+/-0.19 vs. 2.63+/-0.13, P less than 0.05), GRP78 (1.07+/-0.47 vs. 2.21+/-0.99, P less than 0.05), TGF-b (1.01+/-0.14 vs. 1.38+/-0.28, P less than 0.05), and collagen type I (1.02+/-0.27 vs. 1.71+/-0.51, P less than 0.05) and down-regulation of BMP-7 (1.01+/-0.15 vs. 0.55+/-0.25, P less than 0.01). CONCLUSION: Activation of the inflammatory system exacerbates hepatic cholesterol accumulation and hepatic fibrosis in C57BL/6J mice.

[Sorafenib and octreotide combination therapy can inhibit proliferation of and induce apoptosis in human hepatoma cells].

To investigate the effects of sorafenib and octreotide combination treatment on cellular proliferation and explore the underlying molecular mechanisms by using an in vitro cell culture system with the human hepatoma cell line, HepG2. HepG2 cells were treated with different concentrations of sorafenib and octreotide alone or in combination. Untreated HepG2 cells were used as controls. Treatment-induced cytotoxicity was determined with the cell counting kit-8 by Sigma-Aldrich, and rate of apoptosis was detected by flow cytometry. Fluorescent microscopy was used to observe rates of cell growth under the various treatments. Treatment-induced changes in protein expressions were detected by enzyme-linked immunosorbent assay (for vascular endothelial growth factor (VEGF)) and Western blotting (for the Mcl-1 apoptosis mediator and the ERK1/2 and PERK1/2 kinases). Sorafenib and octreotide, used alone or in combination, inhibited proliferation and induced apoptosis in HepG2 cells. Combination treatment was more effective than either mono-treatment (F = 200.398, P less than 0.05). Fluorescent microscopy showed that combination treatment stimulated phosphatidylserine, the marker of early apoptosis, better than either mono-treatment. VEGF expression in cultures exposed to combination treatment was also significantly lower than in mono-treatment or untreated control cultures (F = 1019.725, P less than 0.05). Western blotting showed that octreotide mono-treatment had no effect on Mcl-1 expression (vs. control group; P more than 0.05) and that combination treatment significantly lowered Mcl-1 expression (vs. mono-treatment and control groups; P less than 0.05). None of the treatments affected ERK1/2 expression (all, P more than 0.05), while all treatments significantly lowered PERK1/2 expression (vs. control group; F = 2.401, P less than 0.05) and the combination treatment lowered PERK1/2 significantly more than either mono-treatment (P less than 0.05). Sorafenib and octreotide can inhibit proliferation and induce apoptosis in the human hepatoma cell line, HepG2. Combination treatment is significantly more efficacious (P less than 0.05) and produced synergistic effects. The mechanism underlying this phenomenon may depend on synergistic inhibition of VEGF, the anti-apoptotic protein Mcl-1, and the proliferation-inducing PERK1/2.

[Inhibitory effect of pyrrolidine dithiocarbamate combined with matrine on the growth of human hepatocellular carcinoma xenografts].

OBJECTIVE: To investigate the relationship between activation of nuclear factor-K-gene binding (NF-κB) and apoptosis induced by matrine(MT) in transplanted tumor of human hepatocellular carcinoma in nude mouse. METHODS: Tumors were established by injection of hepatocellular carcinoma cell line HepG2 into the back of nude mice. The mice were divided randomly into four groups: Control group, MT group (35 mg/kg), PDTC group (120 mg/kg) and Combination group: PDTC + MT group (120 mg/kg + 35 mg/kg), the reagents were injected peritoneally. The tumor growth curve of nude mice bearing transplanted tumor were observed and the inhibition ratios were evaluated. Apoptosis of carcinoma cells was analyzed by TUNEL. The DNA-binding activity of NF-κB was determined by electrophoretic mobility shift assay (EMSA). Expression of bcl-2 and bax in carcinoma tissue were detected by immunohistochemical method. NF-κB mRNA, bcl-2 mRNA and bax mRNA in carcinoma tissue were detected by RT-PCR. RESULTS: Pyrrolidine dithiocarbamate (PDTC) could enhance the inhibition of matrine on carcinoma proliferation (P < 0.05). The apoptosis and activation of NF-κB in carcinoma cells could be induced by matrine. PDTC significantly suppressed NF-κB activation induced by matrine in carcinoma cells from 93.64 ± 2.95 to 65.78 ± 5.65 (F = 124.754, P < 0.01). Meanwhile, PDTC increased the apoptosis induced by matrine from 55.9% ± 2.8% to 74.3% ± 4.8% (P < 0.05).A positive correlation observed between the expressions of NF-κB and of bcl-2 (Pearson correlation coefficient = 0.983, P < 0.01). CONCLUSIONS: Matrine could induce apoptosis and activation of NF-κB in transplanted tumor. PDTC could increase apoptosis in hepatocellular carcinoma cells might be due to the suppression of NF-κB activation and the enhancement of bcl-2 expression.

[Intercellular transfer of P-glycoprotein in hepatocellular cell line HepG2].

OBJECTIVE: To investigate whether there is intercellular transfer of functional P-glycoprotein(P-gp) from P-gp-positive cells to P-gp-negative cells in vitro. METHODS: HepG2/GFP cells, a HepG2 cell line stably expressing GFP, were co-cultured with HepG2/ADM cells, an adriamycin-resistant cell line derived from HepG2 cells. The distribution of P-gp in hepatocellular carcinoma cell was observed under laser scanning confocal microscope (LSCM). Immunomagnetic beads were used to separate HepG2/GFP cells from the mixed culture. The abundance of P-gp was analyzed by western blot, and the expression of mdr1 mRNA was detected by qRT-PCR. RESULTS: Yellow fluorescence was detected in HepG2/aqMDR cells, green fluorescence was detected in HepG2/GFP cells, red fluorescence was detected in HepG2/ADM cells by LSCM. The level of P-gp protein in HepG2/aqMDR cells was lower than that in HepG2/ADM cells, but higher than that in HepG2/GFP cells (q = 35.07, P < 0.05) and HepG2 cells (q = 36.87, P < 0.05). The expression of mdr1 mRNA in HepG2/ADM cells was higher than that in HepG2/aqMDR, HepG2 and HepG2/GFP cells, but there was no significant difference in mdr1 mRNA among HepG2/aqMDR, HepG2 and HepG2/GFP cells (F = 2.30, P > 0.05). CONCLUSIONS: P-gp can transfer from drug resistant hepatocellular cells to sensitive hepatocellular carcinoma cells. This study suggests a novel mechanism of multidrug resistance in hepatocellular carcinoma.

Effect of CD16a, the surface receptor of Kupffer cells, on the growth of hepatocellular carcinoma cells.

FcγRIIIa (CD16) is a low-affinity Fc receptor of IgG. As the idio-binding receptor of IgG Fc, it plays an important role in the antibody-dependent cellular cytotoxicity of natural killer cells. The aim of the present study was to investigate the distribution of Kupffer cells (KCs) and the expression of their surface receptor FcγRIIIa in hepatocellular carcinoma. Furthermore, we also aimed to observe the functional mechanism of FcγRIIIa. Immunohistochemical analysis was employed to study KCs and FcγRIIIa. In order to explore the role of FcγRIIIa in the growth of cancer cells, KCs and H22 tumor cells were co-cultured in different serum. The mRNA expression levels of tumor necrosis factor (TNF)-α and FcγRIIIa were analyzed by RT-qPCR; the TNF-α and FcγRIIIa protein expression levels were examined by enzyme­linked immunosorbent assay and western blot analysis, respectively. Our results showed that the number of Kuppfer cells in cancerous tissues (21.6±7.8) was lower than those in para-cancerous (68.8±9.1) tissues and adjacent normal hepatic tissues (62.0±1.9) (P<0.01); this decreased with the reduction in the differentiation degree of cancer (P<0.05). FcγRIIIa-positive cells were similar in morphology to KCs, and their distributive tendency was coincident (P<0.05). The increase in CD16a mRNA levels in the group treated with immune serum was 3.9-, 4.9- and 3.9-fold greater than that in the ordinary serum group at different time points, and CD16a protein expression also markedly increased (P<0.05). However, these effects were inhibited by the addition of anti-IgG Fc serum (P<0.05). The results of the present study suggested that FcγRIIIa resided in KCs, and it contributed to the inhibition of the growth of liver tumor cells.

Synthesis of endotoxin receptor CD14 protein in Kupffer cells and its role in alcohol-induced liver disease.

AIM: To observe the synthesis of endotoxin receptor CD14 protein and its mRNA expression in Kupffer cells (KCs), and evaluate the role of CD14 in the pathogenesis of liver injury in rats with alcohol-induced liver disease (ALD). METHODS: Twenty-eight Wistar rats were divided into two groups: ethanol-fed group and control group. Ethanol-fed group was fed ethanol (dose of 5g-12 g/kg/d) and control group received dextrose instead of ethanol. Two groups were sacrificed at 4 wk and 8 wk, respectively. KCs were isolated and the synthesis of CD14 protein and its mRNA expression in KCs were determined by flow cytometric analysis (FCM) or the reverse transcription polymerase chain reaction (RT-PCR) analysis. The levels of plasma endotoxin and alanine transaminase (ALT) were measured by Limulus Amebocyte Lysate assay and standard enzymatic procedures respectively, and the levels of plasma tumor necosis factor (TNF)-alpha and interleukin (IL)-6 were both determined by ELISA. The liver pathology change was observed under light and electric microscopy. RESULTS: In ethanol-fed group, the percentages of FITC-CD14 positive cells were 76.23 % and 89.42 % at 4 wk and 8 wk, respectively. Compared with control group (4.45 % and 5.38 %), the difference was significant (P<0.05). The expressions of CD14 mRNA were 7.56+/-1.02 and 8.74+/-1.37 at 4 wk and 8 wk, respectively, which were significantly higher compared with the control group (1.77+/-0.21 and 1.98+/-0.23) (P<0.05). Plasma endotoxin levels at 4 wk and 8 wk increased significantly in ethanol-fed group (129+/-21 ng/L and 187+/-35 ng/L) than those in control rats (48+/-9 ng/L and 53+/-11 ng/L)(P<0.05). Mean values of plasma ALT levels increased dramatically in ethanol-fed rats (112+/-15 IU/L and 147+/-22 IU/L) than those in the control animals (31+/-12 IU/L and 33+/-9 IU/L) (P<0.05). In ethanol-fed rats, the levels of TNF-alpha were 326+/-42 ng/L and 402+/-51 ng/L at 4 wk and 8 wk, respectively which were significantly higher than those in control group (86+/-12 ng/L and 97+/-13 ng/L) (P<0.05). The levels of IL-6 were 387+/-46 ng/L and 413+/-51 ng/L, which were also higher than control group (78+/-11 ng/Land 73+/-10 ng/L) (P<0.05). In liver section from ethanol-fed rats, there were marked pathological changes including steatosis, cell infiltration and necrosis. No marked pathological changes were seen in control group. CONCLUSION: Ethanol administration led to a significant synthesis of endotoxin receptor CD14 protein and its gene expression in KCs, which maybe result in the pathological changes of liver tissue and hepatic functional damages.

[Study on ligustrazine in reversing multidrug resistance of HepG2/ADM cell in vitro].

OBJECTIVE: To study the reverse effect of ligustrazine (TMP) on HepG2/ADM, a herd of hepatocellular carcinoma cell, multidrug resistance (MDR) and the influence of P-gp170 expression. METHOD: The reverse effect of ligustrazine on HepG2/ADM cell was observed, with the methods of cell culture, MTT's analyze, RT-PCR and Flow cytometric, etc. RESULT: Ligustrazine could make MDR of cell line of HepG2/ADM reduce the expression of P-gp170, enhance the density of adriamycin in cell and increase the adriamycin's cytotoxicity. With the Flow cytometric, the results of RT-PCR showed the transcriptional activity of the MDR1 decreased. CONCLUSION: Ligustrazine can reverse MDR of HCC cell line of HepG2/ADM and has prospect in clinical use.

Role of high-intensity focused ultrasound in treatment of hepatocellular carcinoma.

About 70 per cent of patients with hepatocellular carcinoma are diagnosed at intermediate or advanced stages, and most of them are technically unresectable. As a novel, emerging therapeutic modality, high intensity focused ultrasound (HIFU) has a great potential for tumor treatment. In this review, principle of HIFU technique is introduced, and an overview of clinical applications and limitations of HIFU for HCC treatment, as well as prospects for future development, is provided. Consequently, HIFU has been considered a safe and feasible procedure for HCC treatment.

Down-regulation of Dicer in hepatocellular carcinoma.

Dicer, the key enzyme in the RNAi pathway, is misregulated in tumor tissues. The altered expression of Dicer is associated with clinical characteristics in patients with cancer. Liver carcinoma and adjacent non-neoplastic tissues were obtained from 36 patients with hepatocellular carcinoma (HCC) undergoing surgery. Expressions of Dicer mRNA were evaluated using the Real-time reverse transcription-PCR in 36 liver carcinoma tissues and 36 adjacent histologically non-cancerous liver tissues. Dicer mRNA levels were evaluated in relation to age, sex, tumor number, tumor size, tumor stage, and distant metastasis. Dicer mRNA level was significantly lower in malignant tissues than in the corresponding non-neoplastic tissues in 34 of the 36 patients with HCC (94.4%). The Dicer expression level was not associated with clinical characteristics, including age, sex, tumor number, tumor size, tumor stage, or distant metastasis in HCC cases. These results demonstrate that Dicer is significantly down-regulated in HCC, suggesting that reduced expression of Dicer may play an important role during the process of hepatocarcinogenesis.

Knockdown of damage-specific DNA binding protein 1 (DDB1) enhances the HBx-siRNA-mediated inhibition of HBV replication.

Recent studies have demonstrated that the effect of inhibition of HBV replication can be achieved by RNA interference (RNAi) at both the cellular and organismal levels. However, HBV replication cannot be completely inhibited by this method. To completely inhibit HBV replication, new strategies for improving the inhibition efficacy of HBV-specific siRNAs are needed. In this study, we demonstrated that knockdown of damage-specific DNA binding protein 1(DDB1), a protein involved in nucleotide-excision repair and HBV replication, significantly enhanced the HBx-siRNA-mediated inhibition of HBV replication. Although knockdown of DDB1 may be toxic to normal liver cells, our results indeed suggest a new direction to enhance the efficacy of HBV-siRNA-mediated inhibition of HBV replication.