N-octylpyridine hydrogen sulphate ionic liquid for multifunctional fluorescent response in different solvents.

Ionic liquids (ILs) have attracted considerable interest in the last two decades owing to their unique fluorescent properties. Herein, N-octylpyridine hydrogen sulphate ([OP]HSO4) was synthesised and characterised using 1H NMR and infrared spectroscopies. In addition, the fluorescence spectra of [OP]HSO4 in water, methanol, ethanol and acetonitrile were studied. In a single solvent, as the concentration of the solvent (methanol, ethanol or acetonitrile) increases, the fluorescence intensity of the IL first increases and then decreases. A similar trend was observed in their mixed solvents with water. Moreover, the fluorescence intensity of [OP]HSO4 decreases with increasing temperature. A fluorescence intensity reduction of only 4.46% for [OP]HSO4 after continuous scanning for 40 cycles under the maximum excitation state was analysed. The lack of photobleaching observed in [OP]HSO4 indicates its good photobleaching resistance.

Modified Sijunzi Granules Exhibit Hemostatic Effect by Activating Akt and Erk Signal Pathways via Regulating 5-HT and Its Receptors Levels.

OBJECTIVE: To investigate the hemostatic effect of modified Sijunzi Granules (MSG) in primary immune thrombocytopenia (ITP) zebrafish model and explore the potential mechanism. METHODS: AB strain wild type zebrafish were treated with simvastatin (6 µmol/L) for 24 h to establish the hemorrhage model (model control group). The zebrafish were treated with MSG at different doses (55.6, 167, and 500 µg/mL), respectively. The hemostatic effect was assessed by examining the intestinal bleeding and hemostatic rate. 5-Hydroxytryptamine (5-HT) content was determined using enzyme-linked immunosorbent assay (ELISA) assay. The expressions of 5-HT2aR, 5-HT2bR, and SERT genes were detected by quantitative real-time polymerase chain reaction(PCR). The protein expressions of protein kinase B (Akt), p-Akt, extracellular regulated protein kinases (Erk), and p-Erk were examined using Western blot analysis. RESULTS: The intestinal bleeding rate was 37%, 40%, and 80% in the 55.6, 167, and 500 µg/mL dose of MSG, respectively, in which 55.6 and 167 µg/mL MSG dose groups were associated with significantly decreased intestinal bleeding rate when compared with the model control group (70%, P<0.05). Significantly higher hemostatic rates were also observed in the 55.6 (54%) and 167 (52%) µg/mL MSG dose groups (P<0.05). MSG increased the 5-HT content and mRNA expression levels of 5-HT2aR, 5-HT2bR, and SERT (P<0.05). In addition, caspase3/7 activity was inhibited (P<0.05). Significant increase in p-Akt and p-Erk was also detected after treatment with MSG (P<0.05). CONCLUSIONS: MSG could reduce the incidence and severity of intestinal bleeding in zebrafish by activating MAPK/Erk and PI3K/Akt signal pathways through regulating the levels of 5-HT and its receptors, which may provide evidence for the treatment of ITP.

The Development Trend of Medical Animals in the Last Ten Years: A Review.

Background: There are many kinds of medicinal animal resources, which are an important part of traditional Chinese medicine resources (TCM). However, the use of medicinal animals in TCM, especially wild animals, has become a sensitive problem at home and abroad. Systematic analysis on the research status and direction of medical animals in the last 10 years which for promoting the sustainable development of Chinese medicine. Methods: PubMed, Web of Science, Embase, CINAHL, CNKI, VIP database and WanFang Database were selected, and SPSS 25.0 software was used to analyze annual publications, journals, global distribution, authors, coauthors and co-authors rate, author institutions and high-frequency keywords. Results: Chinese Journal of modern Chinese medicine occupies the majority articles with a high co-authorship rate, but low impact factors. The development of medical animals around the world is not balanced. The top three countries are China, United Kingdom and United States. However, these countries have less exchanges and cooperation with each other. The Institute of TCM of Chinese Academy owns the most research achievements. At present, the hot spots involve the identification and quality of medical animals, applied basic research. Conclusion: The identification, quality and applied basic research of medical animals are still worthy of increasing research investment. In addition, it is necessary to strengthen exchanges and international cooperation among different countries in TCM, and promote the high-quality development in medical animals.

Hepatitis B virus X protein induces p16 gene promoter methylation through upregulation of DNA methylation transferases DNMT1 and DNMT3A.

BACKGROUND: As a tumor suppressor, p16 can competitively block the cyclin D1-CDK4/6 complex to arrest the cell cycle in the G1 phase. Lack of p16 gene expression can lead to infinite cell proliferation and malignant transformation. OBJECTIVES: To determine whether the hepatitis B virus X protein (HBx) regulates the methylation and expression of the p16 gene. MATERIAL AND METHODS: We constructed a eukaryotic expression vector carrying the HBx gene and green fluorescent protein (GFP), and transfected it into HepG2 cells to build cell lines stably expressing GFP and GFP-HBx. The p16 protein level and p16 gene methylation were measured in these cell lines. We further detected the mRNA expression of DNA methyltransferases (DNMTs) 1, 3A and 3B. The DNMT1, DNMT3A and DNMT3B gene promoter sequences were inserted into a reporter vector (pGL3-Basic) to build recombinant vectors, which were then transiently transfected into different cell lines. After 48 h, the luciferase activity was measured. RESULTS: The level of p16 protein in GFP-HBx/HepG2 cells was significantly lower than in HepG2 and GFP/HepG2 cells. The CpG methylation was present in the p16 gene promoter region of GFP-HBx/HepG2 cells. The DNMT1 and DNMT3A mRNA levels in GFP-HBx/HepG2 cells were significantly elevated compared to that in the HepG2 cells (p = 0.0495). The luciferase activity in GFP-HBx/HepG2 cells transfected with the pGL3-DNMT1/3A pro plasmid was significantly higher than in HepG2 and GFP/HepG2 cells (both p < 0.05). CONCLUSIONS: The HBx can induce p16 gene promoter methylation and inhibit the expression of p16 in HepG2 cells. This occurs due to HBx activation of DNMT1 and DNMT3A promoters and the induction of p16 promoter methylation, which downregulates the expression of p16 protein.

Biochar promotes soil organic carbon sequestration and reduces net global warming potential in apple orchard: A two-year study in the Loess Plateau of China.

The Loess Plateau is China's primary apple-growing area, and the orchard is a significant source of greenhouse gases (GHGs) emissions due to high nitrogen fertilizer input. Thus, a two-year field study was carried out to investigate the effects of apple wood derived biochar on GHGs emissions during apple orchard production, including soil organic carbon sequestration (SOCSR) and net global warming potential (NGWP) assessments. There are four treatments in this study: 20 t ha-1 biochar in a non-fertilized plot (B); no biochar in a fertilized plot (F); 20 t ha-1 biochar in a fertilized plot (FB); no biochar in a non-fertilized plot (CK). Results showed that the combined application of biochar and fertilizer stimulated CO2 emissions by 9.25% and 8.39% than either biochar or fertilizer alone. Meanwhile, biochar in fertilized plot increased annual N2O emissions by 32.6% as compared to fertilized plot without biochar amendment. Compared with CK, biochar had no significant effect on GHG emissions in unfertilized plot. The N2O emission factor of FB and F were 0.91% and 0.45% respectively in 2017-2018 and they were both 0.34% in 2018-2019. Moreover, compared with CK, the FB and B treatments increased the SOCSR by 316.52% and 354.78%, while, decreased the NGWP by 368.93% and 480.91%, respectively. Thus, biochar application may help reduce the impact of apple production on climate change by sequestering more soil organic carbon and decreasing the NGWP.

Experiment investigation of injection parameters effects on injection stability of a high pressure common rail system.

High pressure common rail system can precisely control the injection time and injection pressure to improve the thermal efficiency of the engine. The injection characteristics of the system can affect the combustion and emission process of the engine. Injection stability is defined as variation characteristics of injection. This could have an unignored influence on combustion and engine output, while few study has been published. The primary objective is to establish evaluation systems and methods for injection stability. Then to study injection stability of the high pressure common rail system. Firstly, several parameters that have an important influence on the emission and combustion are identified. Based on the characteristics data obtained, it is verified that the stability parameters are in accordance with the normal distribution. Then, the five characteristic stability parameters of close speed of injection, cycle injection mass, injection delay, maximum injection rate and open speed of injection were quantitatively analyzed by using range, relative range, kurtosis and variance. It is found that the close speed of injection is greatly depend on injection pressure, the range of close speed varies from 1.8 to 5.1 mg/ms when rail pressure varies from 60 MPa to 160 MPa at 0.6 ms energizing time. The stability of injection mass depends on energizing time, the relative range in short energizing time can be four times than long energizing time. The maximum injection rate has similar characteristics with injection mass, it is also depends on energizing time. The range of maximum injection rate is reduced from the average of 0.6-0.7 mg/ms to 0.3 or even 0.2 mg/ms with the increase of energizing time. The injection delay and open speed of injection seems to be greatly affected by pressure fluctuation, since the observed data present complex rules. Pressure fluctuation in common rail can affect injection stability a lot.

Trihexyphenidyl induced malignant hyperthermia in a patient with Parkinson's disease complicated with pneumonia: A case report.

INTRODUCTION: Drug-induced fever is easy to overlook in respiratory departments. High fever is a rare side effect of trihexyphenidyl, which can be used clinically to treat Parkinson's disease. Syndrome of inappropriate antidiuretic hormone secretion (SIADH) is a group of clinical syndromes caused by various diseases, resulting in water retention and refractory hyponatremia. However, pneumonia combined with malignant hyperthermia and SIADH has rarely been reported. We describe an unusual case of malignant hyperthermia and refractory hyponatremia due to trihexyphenidyl adverse reaction. PATIENT CONCERNS: Fifty-five-year-old male with pneumonia presented with malignant hyperthermia and refractory hyponatremia has a history of Parkinson's disease. DIAGNOSIS: Early considerations related the described hyperthermia findings to the manifestations of pneumonia. However, the last findings were due to trihexyphenidyl adverse reaction. INTERVENTIONS: Broad-spectrum antibiotics, oral and intravenous supplement of concentrated sodium chloride, drug, and physical cooling. OUTCOMES: The patient survived. During the 3-month follow up, the patient was no recurrence of fever or hyponatremia. CONCLUSION: High fever and SIADH can be a rare adverse reaction to trihexyphenidyl. Therefore, possible drug factors should be considered in the case. Consideration of other possible causes can improve early diagnosis and treatment of patients with fever of unknown origins.

miR-497 may enhance the sensitivity of non-small cell lung cancer cells to gefitinib through targeting the insulin-like growth factor-1 receptor.

BACKGROUND: Recently, studies have demonstrated that microRNA-497 (miR-497) plays an important role in modulating tumor cell sensitivity to chemotherapeutic drugs; however, its role in cellular resistance to the effects of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in treatment of non-small cell lung cancer (NSCLC) is not fully understood. In this study, we explored the potential of miR-497 in targeting the insulin-like growth factor-1 receptor (IGF-1R) signaling pathways to overcome gefitinib resistance. METHODS: A gefitinib resistant human lung adenocarcinoma A549 cell line (A549/GR) was established by the method of gefitinib mutagenesis culture. Next, the A549/GR cells were transfected with miR-497 mimics to establish an miR-497 overexpression model, designated A549/GR-miR497-mimic. MTT assay was used to assess cell resistance to gefitinib, and western blot assay was employed to evaluate alterations of IGF-1R and the AKT1 signaling pathway. RESULTS: We found that A549/GR-miR497-mimic cells (IC50 =33.76±0.97 µmol/L) were more sensitive to gefitinib than the control group (P<0.01). In addition, the expression levels of IGF-1R and phosphorylated AKT1 (p-AKT1) in A549/GR-miR497-mimic cells were reduced. CONCLUSIONS: We demonstrated that miR-497 may have the effect of reversing gefitinib resistance and increasing the sensitivity of NSCLC cells to EGFR-TKIs by inhibiting the expression of IGF-1R and reducing activation of the downstream AKT signaling pathway. Thus, miR-497 plays a vital role in the acquired resistance to EGFR-TKIs, and it may represent a potential therapeutic strategy to treat NSCLC exhibiting resistance to EGFR-TKIs.

Effects of PON1 Gene Promoter DNA Methylation and Genetic Variations on the Clinical Outcomes of Dual Antiplatelet Therapy for Patients Undergoing Percutaneous Coronary Intervention.

INTRODUCTION AND OBJECTIVE: The relationship between either paraoxonase 1 (PON1) gene promoter DNA methylation or genetic variations and bleeding or major adverse cardiac events after dual antiplatelet therapy has been incompletely characterized. We aimed to systematically investigate the role of genetic variations and DNA methylation of the PON1 CpG island promoter on the clinical outcomes of dual antiplatelet therapy for patients with coronary artery disease (CAD) who underwent percutaneous coronary intervention (PCI). METHODS: This study included 653 patients with CAD undergoing PCI and receiving dual antiplatelet therapy. Genomic DNAs were isolated from whole blood and were genotyped for the three single nucleotide polymorphisms (SNPs) of the PON1 gene. The DNA methylation levels in the PON1 promoter region were determined by bisulfite sequencing or pyrosequencing at five CpG sites (positions -142, -161, -163, -170, and -184 from the transcription start site). Clopidogrel and its metabolites in plasma were examined using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), and platelet function analysis was performed using the VerifyNow assay. RESULTS: Statistically significant associations between methylation levels at five PON1 CpG sites and bleeding were observed: -184 [odds ratio (OR) 0.98, 95% confidence interval (CI) 0.96-1.00, p = 0.028]; -170 (OR 0.99, 95% CI 0.97-1.00, p = 0.048); -163 (OR 0.98, 95% CI 0.96-1.00, p = 0.029); -161 (OR 0.98, 95% CI 0.97-1.00, p = 0.026); and -142 (OR 0.98, 95% CI 0.97-1.00, p = 0.042) at a false discovery rate of <5%. Statistical analysis also revealed that aspirin reaction units (ARUs) were significantly associated with PON1 methylation level at CpG site -163 (p = 0.0342). The ARUs of patients with the PON1 126 CC genotype was 527 ± 94, which was higher than the ARUs (473 ± 89) of patients with the 126 CG genotype (p = 0.0163). Multivariate logistic regression analysis indicated that the PON1 methylation level at CpG site -161 (OR 0.95, 95% CI 0.92-0.98, p = 0.002) and the use of angiotensin-converting enzyme inhibitors (OR 0.48, 95% CI 0.26-0.89, p = 0.021) were associated with a decreased risk of bleeding events. CONCLUSIONS: Hypomethylation of CpGs in the PON1 promoter may be a weak, albeit statistically significant, risk factor of bleeding after dual antiplatelet therapy. Further large-scale studies are needed to verify our results.

Halofuginone ameliorates inflammation in severe acute hepatitis B virus (HBV)-infected SD rats through AMPK activation.

The hepatitis B virus (HBV) has caused acute and chronic liver diseases in ~350 million infected people worldwide. Halofuginone (HF) is a plant alkaloid which has been demonstrated to play a crucial role in immune regulation. Our present study explored the function of HF in the immune response of HBV-infected Sprague Dawley (SD) rats. Plasmid containing pCDNA3.1-HBV1.3 was injected in SD rats for the construction of an acute HBV-infected animal model. Our data showed that HF reduced the high concentrations of serum hepatitis B e-antigen, hepatitis B surface antigen, and HBV DNA induced by HBV infection. HF also reduced the number of T helper (Th)17 cells and the expression of interleukin (IL)-17 compared with the pCDNA3.1-HBV1.3 group. Moreover, pro-inflammatory cytokine levels (IL-17, IL-23, interferon-γ, and IL-2) were downregulated and anti-inflammatory cytokine levels (IL-4 and IL-13) were upregulated by HF. Through further research we found that the expression of AMP-activated protein kinase (AMPK) and IKBA which suppressed NF-κB activation was increased while the expression of p-NF-κB P65 was decreased in pCDNA3.1-HBV1.3+HF group compared with pCDNA3.1-HBV1.3 group, indicating that HF may work through the activation of AMPK. Finally, our conjecture was further verified by using the AMPK inhibitor compound C, which counteracted the anti-inflammation effect of HF, resulting in the decreased expression of AMPK, IKBA and increased expression of p-NF-κB P65 and reduced number of Th17 cells. In our present study, HF was considered as an anti-inflammatory factor in acute HBV-infected SD rats and worked through AMPK-mediated NF-κB p65 inactivation. This study implicated HF as a potential therapeutic strategy for hepatitis B.

Identification of microRNAs involved in gefitinib resistance of non-small-cell lung cancer through the insulin-like growth factor receptor 1 signaling pathway.

Multiple clinical and experimental studies have suggested that epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) may be effective at treating advanced non-small cell lung cancer (NSCLC), however, the molecular basis of primary resistance to EGFR-TKIs in NSCLC remains unclear. In the current study, the insulin-like growth factor 1 receptor (IGF-1R) gene in the gefitinib-resistant human lung adenocarcinoma epithelial cell line A549 (A549/GR) was silenced using small interfering RNA (siRNA) in order to determine the role of microRNA (miRNA) in the development of resistance against epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in lung adenocarcinoma. The relative gefitinib-resistant capacity in A549 and A549/GR cells was determined using a cell counting kit 8. A549/GR cells were transfected with chemically synthesized siRNA to silence the IGF-1R gene. A total of 48 h after siRNA transfection, IGF-1R expression in A549/GR cells was evaluated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. miRNA expression in A549/GR cells and A549/GR cells with silenced IGF-1R was analyzed using a miRNA microarray. The microarray results of 10 miRNAs were then compared with the results of RT-qPCR. The results demonstrated that the gefitinib-resistance capacity of A549/GR cells was six times higher than that of A549 cells. Additionally, RT-qPCR and western blotting demonstrated that the IGF-1R gene in A549/GR cells was successfully silenced by siRNA. The highest silencing rate (72%) of the IGF-1R gene was obtained using siRNA-2. The microarray identified 72 miRNAs with significantly different expression in A549/GR cells with silenced IGF-1R compared with A549/GR cells. Of the 72 differentially expressed miRNAs, 13 miRNAs (including miR-497-3p and miR-1273c) were up-regulated and 59 miRNAs (including miR-361-3p and miR-345-3p) were down-regulated in A549/GR cells with silenced IGF-1R compared with A549/GR cells. The changes in the expression of 10 different miRNAs were confirmed by RT-qPCR. Thus, the present study successfully established an A549/GR cell line with silenced IGF-1R. The results suggest that a number of miRNAs associated with the IGF-1R signaling pathway, including miR-497-3p and miR-144-5p, were involved in the development of resistance against EGFR-TKIs in A549 cells. These miRNAs may provide novel targets to treat lung adenocarcinoma exhibiting resistance against EGFR-TKIs.

[Hepatitis B virus X promotes HepG2 cell cycle progression and growth via downregulation expression of p16 protein].

OBJECTIVE: To investigate the effects and related mechanisms of hepatitis B virus X (HBx) protein on cell cycle and growth in hepatocellular carcinoma. METHODS: A human hepatocyte HepG2 cell line stably expressing a green fluorescent protein (GFP)-tagged HBx (HepG2/GFP-HBx cells) was used for the experiment, and HepG2 parental and HepG2/GFP cells was used as the controls. Effect of HBx on cell growth was evaluated by the MTT cell proliferation assay and on cell cycle progression by flow cytometry analysis of cells with or without treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR; 5 pmol/L). Effect of HBx expression on promoter methylation status of the p16INK4A tumor-suppressor gene was detected by methylation-specific polymerase chain reaction and on p16 protein level was analyzed with western blotting. RESULTS: The HepG2/GFP-HBx cells showed significantly higher cell proliferation at 72 hrs of culture (3.225+/-0.038 A490) than either control (HepG2: 2.012+/-0.022 A490, t = -46.86, P less than 0.001; HepG2/GFP: 2.038+/-0.029 A490, t = 42.51, P less than 0.001). The HepG2/GFP-HBx cells also showed significantly lower proportion of cells in the G0/G1 phase (16.45%+/-0.45%) than either control (HepG2: 44.81%+/-1.36%, t = -34.202, P less than 0.001; HepG2/GFP: 42.76%+/-1.58%, t = -28.88, P less than 0.001). However, 5-Aza-CdR treatment did lead to a significant amount of HepG2/GFP-HBx cells being arrested in the G0/G1 phase (33.25%+/-0.79%, t = 31.85, P less than 0.001). The p16INK4A promoter was methylated in the HepG2/GFP-HBx cells, and became demethylation after treatment with 5-Aza-CdR. However, no methylation of p16INK4A promoter was observed in both HepG2 and HepG2/GFP cells. The p16 protein level was significantly lower in the HepG2/GFP-HBx (vs. HepG2 and HepG2/GFP cells) and this level increased after treatment with 5-Aza-CdR. CONCLUSION: HBx protein promotes hepatocellular carcinoma cell cycle progression and growth by shortening the G0/G1 phase, and the underlying mechanism may involve inducing p16INK4A promoter methylation and downregulating p16 protein expression.

Association of PON1 genotype and haplotype with susceptibility to coronary artery disease and clinical outcomes in dual antiplatelet-treated Han Chinese patients.

PURPOSE: The aim of this study was to evaluate the association of PON1 genetic variants with the susceptibility to coronary artery disease (CAD) and with the clinical endpoints in aspirin and clopidogrel (dual antiplatelet therapy)-treated Han Chinese patients with CAD after percutaneous coronary intervention (PCI). METHODS: A total of 538 Han Chinese patients undergoing PCI and receiving dual-antiplatelet therapy were sequentially recruited to the study and followed for up to 1 year. Healthy controls (n = 539) were enrolled during the same period. All study participants were genotyped for five genetic variants in PON1 and the cytochrome P450 2C19*2 mutation (CYP2C19*2). The effect of genetic variants on disease risk and clinical outcome of major adverse cardiac events (MACE) within 1 year or bleeding within 6 months was assessed. RESULTS: CYP2C19*2 was associated with a higher risk of MACE (adjusted P = 0.0098), but a lower risk of bleeding events (adjusted P = 0.0016). The PON1 Q192R polymorphism was significantly associated with a lower risk of bleeding events [odds ratio (OR) 0.61, 95% confidence interval (CI) 0.43-0.87, adjusted P = 0.0066). The haplotype bearing the PON1 -126C allele was associated with a higher risk to CAD (OR 1.48, 95% CI 1.04-2.09, P = 0.029) and a higher risk of bleeding events (OR 1.68, 95% CI 1.10-2.56, P = 0.017) compared to the most frequent haplotype. The transcription activity of haplotype p-162A-126C-108C in the PON1 promoter was 2.6-fold higher than that of the most frequent haplotype (p-162G-126G-108T). CONCLUSIONS: Based on these results, we suggest that the haplotype-bearing PON1 -126C allele contributes to the disease risk and the risk of bleeding events in dual antiplatelet-treated CAD patients after PCI.

[Progress in genetic and epigenetic research on in-stent restenosis after percutaneous coronary interventions].

Coronary heart disease is one of the most important causes of death in human, and consumes vast medical resources. Percutaneous coronary intervention (PCI) has been a significant breakthrough for its treatment. However, clinical application has been hampered by in-stent restenosis (ISR). Although drug eluting stent (DES) has reduced the occurrence of restenosis, incidence of ISR is still about 5% to 10%. The main reasons for restenosis after PCI are hyperplasia of vascular endothelial cells and smooth muscle cell migration. The exact mechanism of personalized differences in restenosis is not clear yet, but there may be a variety of risk factors. In addition to aging, smoking and diabetes, an increasing number of studies have found that genetic and epigenetic factors play an important role in ISR. In this article, authors have reviewed genetic and epigenetic factors on the progression of ISR, which may help to determine the genetic risk factors in patients with ISR after PCI.

[Small interfering RNA targeting to hepatitis B virus X gene and 5-aza-2'-deoxycytidineon inhibited growth of the subcutaneous implanted tumor of hepatocellular carcinoma in nude mice].

OBJECTIVE: To investigate the anti-tumor effect of small interfering RNA targeting to HBV X gene (X-siRNA) and 5-aza-2'-deoxycytidine (5-aza-dC) on HBV-related hepatocellular carcinoma. METHODS: X-siRNA and control siRNA were synthesized. HepG2/GFP-HBx cells were treated with X-siRNA, and the levels of HBV X mRNA were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Nude mice were inoculated with HepG2/GFP and HepG2/GFP-HBx cells subcutaneous respectively to establish implant models of hepatocellular carcinoma, and were treated with X-siRNA, 5-aza-dC alone or in combination, and tumor growth was observed. The methylation of p16 gene promoter was detected by methylation specific polymerase chain reaction (MSP). RESULTS: RT-PCR showed the expression of HBV X mRNA in HepG2/GFP-HBx cells was inhibited markedly by X-siRNA. The nude mice experiment showed that the gross tumor volume was much bigger in HepG2/GFP-HBx group than that in HepG2/GFP group (P < 0.05). The growth of palpable tumors in X-siRNA or 5-aza-dC treatment group notably decreased (P < 0.05). MSP analysis showed that p16 gene methylation was observed in HepG2/ GFP-HBx-caused palpable tumors, while no methylation was detected in HepG2/GFP group. However, after treatment with X-siRNA or 5-aza-dC, p16 gene methylation reduced. CONCLUSIONS: HBV X-siRNA and methylation inhibitor can inhibit the growth of hepatoma cells via reversing p16 methylation.