Shikonin induces hepatocellular carcinoma cell apoptosis by suppressing PKM2/PHD3/HIF-1α signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the mechanism of shikonin-induced death of human hepatocellular carcinoma SMMC-7721 cells.@*METHODS@#Cultured SMMC-7721 cells and normal hepatocytes (L-02 cells) were treated with 4, 8, or 16 μmol/L shikonin, and the changes in cell viability was assessed using MTT assay. The levels of ATP and lactic acid in the cell cultures were detected using commercial kits. Co-immunoprecipitation and immunofluorescence staining were used to determine the relationship among pyruvate kinase M2 (PKM2), prolyl hydroxylase 3 (PHD3), and hypoxia-inducible factor-1α (HIF-1α). The expressions of PHD3, PKM2, HIF-1α, Bax, cleaved caspase-3, and Bcl-2 in SMMC-7721 cells were detected with Western blotting, and cell apoptosis was analyzed with annexin V-FITC/PI staining. The effects of RNA interference of PKM2 on PHD3 and HIF-1α expressions in SMMC-7721 cells were detected using Western blotting.@*RESULTS@#The IC50 of shikonin against SMMC-7721 and L-02 cells was 8.041 μmol/L and 31.75 μmol/L, respectively. Treatment with shikonin significantly inhibited the protein expressions of PKM2, HIF-1α and PHD3 and nuclear translocation of PKM2 and HIF-1α in SMMC-7721 cells. Coimmunoprecipitation and immunofluorescence staining confirmed that shikonin inhibited the formation of PKM2/PHD3/HIF-1α complex and significantly reduced the contents of lactic acid and ATP in SMMC-7721 cells (P < 0.05). The expressions of PHD3 and HIF-1α decreased significantly after PKM2 knockdown (P < 0.05). Shikonin treatment significantly increased the apoptosis rate, enhanced the expressions of Bax and cleaved caspase-3, and decreased Bcl-2 expression in SMMC-7721 cells (P < 0.05).@*CONCLUSIONS@#Shikonin induces apoptosis of SMMC-7721 cells possibly by inhibiting aerobic glycolysis through the PKM2/PHD3/HIF-1α signaling pathway to cause energy supply dysfunction in the cells.

Is mild erectile dysfunction associated with severe psychological symptoms in Chinese patients with moderate-to-severe chronic prostatitis/chronic pelvic pain syndrome? / 亚洲男科学杂志(英文版)

This study aimed to assess the association between psychological disorders and erectile dysfunction (ED) in patients with different degrees of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). This was a retrospective study conducted from June 2017 to October 2019 and included 182 outpatients. Patients were interviewed using the Structured Interview on Erectile Dysfunction (SIEDY) for pathogenic quantification. The National Institutes of Health-Chronic Prostatitis Symptom Index (NIH-CPSI) and the International Index of Erectile Function-5 (IIEF-5) were used for the evaluation of CP/CPPS and ED. The Generalized Anxiety Disorder-7 (GAD-7) and Patient Health Questionnaire-9 (PHQ-9) were used to assess anxiety symptoms and depressive symptoms. The number of patients with mild CP/CPPS and mild ED, mild CP/CPPS and moderate-to-severe ED, moderate-to-severe CP/CPPS and mild ED, and moderate-to-severe CP/CPPS and moderate-to-severe ED was 69 (37.9%), 36 (19.8%), 35 (19.2%), and 42 (23.1%), respectively. The corresponding PHQ-9 scores of the four groups were 6.22, 7.19, 10.69, and 7.71, respectively. The corresponding GAD-7 scores of the four groups were 5.26, 6.31, 8.77, and 6.36, respectively. Among patients with moderate-to-severe CP/CPPS, the PHQ-9 and GAD-7 scores of the moderate-to-severe ED group were significantly lower than those of the mild ED group (P = 0.007 and P = 0.010, respectively). The prevalence of ED and premature ejaculation (PE) in patients with moderate-to-severe CP/CPPS was significantly higher than that in patients with mild CP/CPPS (P = 0.001 and P = 0.024, respectively). Our findings proved that the severity of ED was negatively associated with psychological symptoms in outpatients with moderate-to-severe CP/CPPS.

Analysis of adverse reactions risk signal of Xinyuan Capsules based on national ADR monitoring spontaneous reporting system / 中国中药杂志

In recent years, the safety problems and events of traditional Chinese medicine represented by liver injury have occurred frequently. In particular, Polygonum multiflorum has been widely used and considered as a "non-toxic" tonic traditional Chinese medicine for thousands of years. However, in recent years, frequent reports of liver injury events have attracted widespread attention at home and abroad, which has made unfavorable impacts on traditional Chinese medicine and its international development. Some scho-lars have found that susceptible genes of P. multiflorum on liver injury lay a scientific foundation for formulating rational comprehensive prevention and control measures for liver injury risk of P. multiflorum and its relevant preparations. But what are the risk signals of adverse reactions of P. multiflorum in clinical application? Spontaneous reporting system is an important way to monitor and find adverse drug reaction(ADR) signals after the drug is launched in the market. It can find the ADR signals in time and effectively, and then effectively prevent and avoid the occurrence of adverse drug events. At present, the data mining technique has gradually become the main method of ADR/adverse event(AE) report analysis and evaluation at home and abroad. Specifically, Bayesian confidence propagation neural network in Bayesian method is a commonly used risk signal early warning analysis method. In this paper, BCPNN method was used to excavate the risk signals of adverse reactions of Xinyuan Capsules, a traditional Chinese medicine preparation containing P. multiflorum, such as nausea, diarrhea, rash, dizziness, vomiting, abdominal pain, headache, liver cell damage, so as to provide evidence-based evidence for clinical safe and rational use of drugs.

Tumor-associated macrophages attenuate apoptosis-inducing effect of sorafenib in hepatoma cells by increasing autophagy / 南方医科大学学报

OBJECTIVE@#To explore the molecular mechanism of sorafenib resistance in hepatoma cells and identify for new targets to reverse drug resistance.@*METHODS@#THP-1 cells were induced into M2 tumor-associated macrophages (M2-TAMs) in vitro and identified by immunofluorescence. SMMC-7721 cells were co-cultured with M2-TAMs with or without sorafenib treatment. CCK-8 assay was used to observe the inhibitory effect of sorafenib on the cell proliferation. Annexin V/PI double staining and protein immunoblotting were used to assess the effect of sorafenib on the proliferation, apoptosis and the expressions of apoptosis-related proteins and autophagy-related protein in SMMC-7721 cells co-cultured with M2-TAMs in the presence or absence of the autophagy inhibitor chloroquine (CQ).@*RESULTS@#The IC of sorafenib at 48 h was 2.25 μmol/L in SMMC-7721 cells cultured alone, and increased to 4.72 μmol/L in the cells co-cultured with M2-TAMs. Compared with the cells cultured alone, the co-cultured SMMC-7721 cells showed significantly reduced apoptosis rate in response to sorafenib ( < 0.01) and significantly increased expression of Bcl-2 and Bcl-2/Bax ratio ( < 0.05) with also increased LC3-II/LC3-I ratio ( < 0.001) and lowered expression of p62 ( < 0.05), suggesting a significantly enhanced level of autophagy. CQ treatment significantly inhibited the proliferation of the co-cultured SMMC-7721 cells ( < 0.05), increased the cell apoptosis ( < 0.05) and reduced the Bcl-2/Bax ratio ( < 0.01).@*CONCLUSIONS@#M2-TAMs can attenuate the inhibitory effect of sorafenib on the proliferation of hepatoma cells by increasing the level of autophagy, suggesting a new strategy for reversing sorafenib resistance induced by the tumor microenvironment by inhibiting autophagy.

Change of the thickness of macular inner and outer retina in patients with chronic primary angle-closure glaucoma / 眼科新进展

Objective To measure the thickness of macular inner and outer retina of chronic primary angle-closure glaucoma (CPACG) and observe its features.Methods Together 58 patients (58 eyes) including 14 patients with early CPACG,23 patients with moderate CPACG and 21 patients with severe CPACG were recruited as early CPACG group,moderate CPACG and severe CPACG group,respectively;additional 23 healthy volunteers were chosen as controls.The thickness of macular inner and outer retina at all parts were measured by RTVue-100 SD-OCT.Results The macular inner retinal thicknesses in fovea were (135.62 ± 2.96) μm,(124.21 ± 6.47) μm,(119.74 ± 10.67) μm and (94.95 ± 11.24) μm in the control group,early CPACG,moderate CPACG and severe CPACG group,respectively.The thicknesses of macular inner retina of fovea were significantly decreased in the early CPACG group,moderate CPACG group and severe CPACG group when compared with the control group (all P ＜ 0.05).There were also significant differences between early CPACG group and moderate CPACG group,severe CPACG group and moderate CPACG group and early CPACG group and severe CPACG group (all P ＜ 0.05).The thicknesses of macular outer retina of fovea,parafovea and perifovea were significantly decreased in the severe CPACG group compared with the control group (all P ＜ 0.05).Conclusion The thickness of macular inner and outer retina are decreased at all parts in patients with the severe CPACG.The macular inner retinal thickness only in the fovea is thinner in the patients with the early CPACG.

2-deoxyglucose inhibits angiogenesis of rheumatoid arthritis via activating AMPK pathway / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effects of 2-deoxyglucose inhibiting synovial pannus of adjuvant arthritis rats and to explore its potential mechanism of inhibiting angiogenesis by investigating proliferation, migration and matrigel tube formation assay .</p><p><b>METHODS</b>The effect of 2-DG on synovial pannus was evaluated by histopathology of HE staining; HUVEC proliferation was determined by CCK-8 method; migration of FLS were determined by transwell; matrigel tube formation assay was made for assessing tube number of HUVEC; p-AMPK and Bcl-2 were detected by Western blot assay; AMPK signaling pathway in HUVEC was inhibited by compound C, which is an inhibitor of AMPK activation.</p><p><b>RESULTS</b>2-DG (200 mg/kg) obviously decreased appearance of synovial pannus ( < 0.01); , 2-DG (0.5 mmol/L and/or 5 mmol/L) obviously inhibited proliferation, migration and tube number of HUVEC ( < 0.01 or < 0.001), and its effects on HUVEC were reversed by using AMPK antagonist (Compound C); Western blot showed that 2-DG (5 mmol/L) increased expression of p-AMPK and decreased expression of Bcl-2 ( < 0.05).</p><p><b>CONCLUSIONS</b>Activating AMPK pathway and decreasing expression of Bcl-2 may the potential mechanism by which 2-DG contributes to anti-angiogenesis and effects of inhibiting proliferation, migration and tube number of HUVEC.</p>

A survey on the prevalence of overweight and obesity among city residents in Wenzhou City / 预防医学

Objective To learn the prevalence of overweight and obesity among city residents in Wenzhou city.Methods Byusing multistage stratified cluster sampling method,a total of 10 449 residents aged 18 years and above were selected forquestionnaires interview,physical examination and laboratory testing ,analyzed the prevalence and risk factors of overweightand obesity among city residents.Results The crude prevalence rates of overweight and obesity were 32.60% and7.70%,and the age -adjusted rate were 27.50% and 6.51%.The multivariate logistic regression analysis showed that age(OR =1.01,95%CI:1.01 -1.02)and drinking (OR =1.14,95%CI:1.02 -1.27)were risk factors of overweight andobesity,while high degree of education(OR =0.87,95%CI:0.84 -0.91)and active physical exercise (OR =0.75,95%CI:0.57 -0.98)were protective factors of overweight and obesity.The prevalence rates of hypertension,diabetes,dyslipidemia and hyperuricemia were much higher among adults with overweight and obesity,compared with that of normalweight adults (P <0.01).Conclusion Prevalence of overweight and obesity is high in Wenzhou City.Intervention shouldbe actively carried out for the prevention and control of overweight and obesity.

Effect of low-molecular-weight heparin combined with doxorubicin on hepatocellular cancer cell migration in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the anti-cancer effect of low-molecular-weight heparin (LMWH) combined with doxorubicin and explore the mechanism.</p><p><b>METHODS</b>Hepatocellular cancer HepG2 cells exposed to LMWH, doxorubicin, or both were evaluated for cell viability with MTT assay and for changes in their migration ability using wound healing assay and Transwell migration assay. The changes in cellular expressions of matrix metalloproteinase-9 (MMP-9) and MMP-2 mRNA and proteins were analyzed with quantitative real-time PCR (qRT-PCR) and Western blotting, and ELISA was used to determine heparanase (HPA) concentration in the cell culture medium.</p><p><b>RESULTS</b>HepG2 cells exhibited suppressed proliferation in response to LMWH and doxorubicin treatments. The combined treatment caused a significantly higher inhibition rate of cell migration than LMWH and doxorubicin alone. LMWH enhanced doxorubicin-induced down-regulation of MMP-9, MMP-2 and HPA in the cells.</p><p><b>CONCLUSIONS</b>LMWH can enhance the inhibitory effect of doxorubicin on the migration of HepG2 cells, the mechanism of which may involve the down-regulation of MMP-9, MMP-2 and HPA expressions.</p>

Simple osteotomy for correct hallux valgus with increased I, II intermetatarsal angle / 中国骨伤

<p><b>OBJECTIVE</b>To investigate efficacy of simple osteotomy for correct hallux valgus,and explore its scope and condition.</p><p><b>METHODS</b>From December 2009 to April 2011, 20 patients (32 feet) with hallux valgus were treated by simple osteotomy. There were 1 male (1 foot), 19 females (31 feet) with an average age of 40 years (ranged, 22 to 64 years). The course of disease ranged from 2 to 31 years(mean 12 years). Among them, 6 feet were mild, 20 feet were moderate, 6 feet were serious. Patients had symptoms of metatarsophalangeal joint pain, but tensity of lateral soft tissue were normal. Hallux Valgus Angle (HVA) and Intermetatarsal Angle (IMA) were examined before and after treatment. The criteria of hallux valgus was used to evaluate the effects from valgus deformity, activity of metatarsophalangeal joint, satisfaction of patients.</p><p><b>RESULTS</b>Twenty patients were followed up from 6 to 18 months with an average of 8.5 months. The wounds were healed well, no infection and metatarsal head necrosis occurred, 95.1% of patients were satisfied with the efficacy. Average AOFAS score increased from preoperative (53.1 +/- 7.5) points to the final follow-up (93.1 +/- l.9) points (P<0.05), the average correct HVA increased from preoperative (33.4 +/- 7.8) degrees to postoperative (11.9 +/- 3.6) degrees (P<0.05), the average IMA were decreased from preoperative (12.3 +/- 3.0) degrees to postoperative (6.3 +/- 1.9) degrees (P<0.05), tibial sesamoid position improved from 1.9 to 0.9 (P<0.05).</p><p><b>CONCLUSION</b>the main pathological changes of hallux valgus patients with normal tension of lateral soft tissue is metatarsal adduction, simple osteotomy can get satisfactory results, no need to cut adductor muscle.</p>

Influence of fast inhibition to virus replication on immune rejection and prognosis of acute-on-chron-ic livre failur e patients / 中国医师杂志

Objective To analyze the result of treatment for acute-on-chronic liver failure patients with fast high efficiency Nucleoside and to explore the relations among inhibition to virus replication , liver failure development and immune rejection .Methods Sixty-two cases of acute-on-chronic liver failure pa-tients with HBV DNA(+) were divided into study group (treated with a kind of fast and nucleoside , n =30) and control group( n =32).HBV DNA,CD4 +T,CD8 +T, C3,C4 TBIL,PTA were observed at treat-ment 0w,2w and 4w.Results All of the study and control group patients , serum HBV DNA were positive before treated.And the levels of CD4+,CD8 +,C3,C 4,TBIL,PTA of study group were not significantly compared with control group .At treatment 2w , the rate of HBV DNA diverted negative in study group 90.0%(27/30), was significantly more then control group (9.4%, 3/32)(χ2=37.14 , P <0.01).But the CD4 +,CD8 +,C3,C4,TBIL,PTA levels were not significantly however between study and control group . At treatment 4w ,the rate of HBV DNA diverted negative in study group (96.7%, 29/30), was significant-ly more then control group(12.5%,4/32) (χ2 =40.74, P <0.01).CD4 +, CD8 +,C3,C4,TBIL,PTA levels of the study group were significantly more compared with the control group .The CD4 +level of study group (495.33 ±89.91)cells/ml, was higher significantly then control group (270.34 ±97.74)cells/ml( t=9.42, P <0.01),the CD8 +level (571.03 ±120.15 ) cells/ml, was higher significantly then control group(224.88 ±79.68)cells/ml( t =13.45, P <0.01).The C3 level of the study group (0.28 ±0.11) g/L, was lower significantly then control group ( 0.68 ±0.13 ) g/L ( t =13.13 , P <0.01 ) , the C4 level (0.12 ±0.06)g/L, was lower significantly then control group (0.23 ±0.10)g/L( t =4.92, P <0.01). The TBIL level of study group ( 653.93 ±131.02 )μmol/L, was higher significantly then control group (285.63 ±154.63)μmol/L( t =10.09, P <0.01),the PTA level (17.13 ±7.07)%, was lower signifi-cantly then control group(50.94 ±13.68)%( t =12.10, P <0.01).The death rate of the study group( 57.9%) was higher significantly compared with the control group (28.1%)(χ2 =6.39, P <0.05).Con-clusion Treatment of chronic severe hepatitis with fast and high efficiency nucleoside may arise the T cell subset level and make the immune rejection strength , as a result the liver failure maybe far away from cure .

Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100 percent efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.

The effect of telbivudine on peripheral blood CD4+CD25+ regulatory T cells and its significance in patients with chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of telbivudine on peripheral blood regulatory T cells and its significance in patients with chronic hepatitis B (CHB).</p><p><b>METHODS</b>Thirty-six HbeAg positive chronic hepatitis B patients were recruited and received telbivudine treatment for 9 months. Before and during the 3, 6, 9 months of treatment, flow cytometry was used to detect the proportion of peripheral blood Tregs; real-time PCR was used to detect the levels of HBV DNA in the serum. Markers of hepatitis B virus infection were detected by ELISA assay and levels of alanine aminotransferase in the serum were measured.</p><p><b>RESULTS</b>The proportion of peripheral blood Tregs in patients with CHB was significantly higher than that in healthy controls and decreased over 6 or 9 months of telbivudine treatment to a level comparable to that of the healthy controls. After 3 months of telbivudine treatment, the rate of undetectable HBV DNA in patients whose proportion of peripheral blood Tregs was decreased was higher than those whose Tregs had been reduced, but the difference was not statistically significant (P more than 0.05). Three, 6 or 9 months of telbivudine treatment resulted in HbeAg negativity in 4 (11.1%) patients, 7 (19.4%) patients or 9 (25.0%) patients respectively. In 7 (19.4%) patients who had seroconversion from HBeAg to anti-HBeAg, after 3 or 6 months of telbivudine treatment, their proportion of peripheral blood Tregs had decreased to a level comparable to that of the healthy controls.</p><p><b>CONCLUSION</b>Telbivudine treatment reduces HBV replication and the proportion of peripheral blood Tregs. In addition, patients who have their proportion of peripheral blood Tregs decreased quickly at the early phase of telbivudine treatment are prone to have HBeAg to anti-HBeAg seroconversion.</p>

Effect of KLF6 and its splice variant KLF6V on proliferation and differentiation of human hepatocellular carcinoma HepG2 cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the roles of Kruppel-like factor 6 (KLF6) and its splice variant KLF6V on suppressing growth and inducing differentiation of human hepatocellular carcinoma hepG2 cells.</p><p><b>METHOD</b>KLF6V cDNA was amplificated by RT-PCR from human hepatocellular carcinoma (HCC) tissue and then sequenced. The recombinant vectors expressing KLF6 variant (KLF6V) were constructed using molecular clone technology based on established plasmid pcDNA3.1A(-)/wtKLF6. KLF6V or KLF6-transfected HepG2 cells were established after being screened with G418. Growth activity of HepG2/KLF6 or HepG2/KLF6V cells was detected by in vitro MTT assay. Expression of p21WAF1 or cyclin D1 protein was detected by Western blot, and expressions of AFP or ALB protein were measured by radioimmunoassay.</p><p><b>RESULTS</b>A novel alternatively spliced transcript of the human KLF6 gene was found and its sequencing revealed that the variant form of KLF6 lacked 126nt and its encoded protein products had a deletion of 42 aa near the COOH-terminal amino acid in comparison with full-length KLF6. Although KLF6 alternative splicing was present in both normal and cancerous tissues, expression of the KLF6 splice variants seemed to be up-regulated in HCCs tissues. The isoform of KLF6 proteins antagonized the ability of wild-type KLF6 to up-regulate p21 expression or down-regulate cyclin D1 expression and suppress HepG2 cell proliferation. KLF6 gene increased albumin production and decreased alpha fetoprotein production of the cells.</p><p><b>CONCLUSION</b>The isoform of KLF6 protein, present in HCC tissue, antagonizes the ability of wild-type KLF6 to suppress cell proliferation and induce cellular differentiation.</p>

Mutations of the tumor suppressor Kruppel-like factor 6 (KLF6) gene in hepatocellular carcinoma and its effect of growth suppression on human hepatocellular carcinoma cell line HepG2 / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the mutated KLF6 gene in hepatocellular carcinoma (HCC) and to characterize its behavior in human hepatocellular carcinoma cell line HepG2.</p><p><b>METHODS</b>We analyzed the DNA isolated from 23 hepatocellular carcinoma tissues and their adjacent nontumor tissues by polymerase chain reaction (PCR). Direct sequencing was used to establish the incidence of mutation in exon2 of the KLF6 gene. Loss of growth suppressive function of the HCC-derived KLF6 mutants was characterized by in vitro analyzing alteration of cell cycle and MTT assay. Expression of p21WAF1, a possible downstream gene of KLF6, was detected in human hepatocellular carcinoma cell line HepG2 transiently transfected with KLF6 genes.</p><p><b>RESULTS</b>Mutations of KLF6 were found in 2 of the 23 (8.7%) hepatocellular carcinomas. The two mutations were located in the transactivation domain and one of them resulted in single amino acid substitution of TGG (W) by GGG (G) at codon 162. Unlike the wild-type KLF6, cancer-derived KLF6 mutants neither suppressed growth nor induced p21WAF1 following transfection into culture cells.</p><p><b>CONCLUSIONS</b>Mutations of the KLF6 gene may play a role in the pathogenesis of HCC, but are not the dominating mechanism resulting in inactivation of KLF6 functions. KLF6 suppresses hepatocellular carcinoma cell proliferation partly through upregulating expression of the p21WAF1 gene.</p>

Inactivation of the tumor suppressor Krüppel-like factor 6 (KLF6) by mutation or decreased expression in hepatocellular carcinomas / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>BACKGROUND AND AIM</b>The Krüppel-like transcription factor KLF6 is a novel tumor-suppressor gene. It was inactivated in human prostate cancer and other tumors tissue, as the result of frequent mutation and loss of heterozygosity (LOH). However, there is no data reporting the levels of KLF6 both mRNA and protein in hepatocellular carcinomas (HCCs). We therefore detected mutations and expression of KLF6 in HCC tissues and further observed the effect of it on cell growth in HCC cell lines.</p><p><b>METHODS</b>We analyzed the exon-2 of KLF6 gene by direct DNA sequencing, and detected the expression of KLF6 by RT-PCR and Western blot in 23 HCC tissues and corresponding nontumorous tissues. Loss of growth suppressive effect of the HCC-derived KLF6 mutant was characterized by in vitro growth curves plotted, flow cytometry and Western blotting.</p><p><b>RESULTS</b>KLF6 mutations were found in 2 of 23 HCC tissues and one of mutations was missense. Expression of KLF6 mRNA or protein was down-regulated in 8 (34.7%) or 9 (39.1%) of 23 HCC tissues. Wild-type KLF6 (wtKLF6) inhibited cellular proliferation and prolonged G1-S transition by inducing the expression of p21WAF1 following stable transfection into cultured HepG2 cells, but tumor-derived KLF6 mutant (mKLF6) had no effects.</p><p><b>CONCLUSION</b>Our findings suggest that KLF6 may be involved in pathogenesis of HCC.</p>

Application of PCR-based tRNAval Pol III-shRNA expression cassettes in screening of efficient siRNA for inhibiting hepatitis B virus / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To screen efficient siRNA for inhibiting hepatitis B virus using the technique of PCR-based tRNA(val) Pol III-shRNA expression cassettes (SECs).</p><p><b>METHODS</b>Based on core gene sequence of HBV, five target sites of siRNA were designed. tRNAval Pol III-shRNA expression cassettes produced by one-step overlapping extension PCR strategy were co-transfected with HBV C gene and pC-EGFP plasmid into AD293 cells respectively. Forty-eight hours after transfection, fluorescence of HBVC-GFP protein was detected by fluorescence-activated cell sorting (FACS); HBV C mRNA was detected by semi-quantitative RT-PCR. HBV-producing HepG2. 2. 15 cells were transfected with selected SECs for 72 h, HBsAg and HBeAg in the cell culture medium were detected by radioimmunoassay assay (RIA). HBV pgRNA from cell total RNA was detected by semi-quantitative PCR.</p><p><b>RESULT</b>Co-transfection with pC-GFP plasmid and SECs into AD293 cells resulted in inhibition expression of HBV C gene and decrease of EGFP fluorescence intensity. SEC-492i showed most significant inhibition effect on HBV C-EGFP expression compared with other SECs. Selected SEC-492i or SEC-282i targeting core gene could efficiently decrease expression of HBeAg and the level of HBV pgRNA in a dose-dependent manner. SEC-492i inhibited HBV replication and antigen expression in a more efficient way than SEC-282i at the same final concentration.</p><p><b>CONCLUSION</b>The expressed shRNA, which targets sites on HBV C mRNA in 492i, is to have having most efficient RNAi effect. tRNAval Pol III-shRNA expression cassettes produced by one-step overlapping extension PCR strategy should be useful for identification of optimal siRNA.</p>

Inhibition of HBs-GFP fusion gene expression by RNA interference / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To develop an effective report gene system to test the effect of small interfering RNA (siRNA).</p><p><b>METHODS</b>HBV S gene was fused with enhanced green fluorescent protein (EGFP) gene to form HBs-GFP and the plasmid containing HBs-GFP was constructed. A vector expressing small hairpin RNA (shRNA) pAVU6 + 4sh357 was also constructed. Two plasmids were co-transfected into HepG2 cells transiently. The fluorescence of HBs-GFP was detected by fluorescence-activated cell sorting (FACS). The mRNA expression in HepG2 cells was detected by conventional RT-PCR and real-time PCR.</p><p><b>RESULTS</b>siRNA inhibited the expression of HBs-GFP 72 hours post transfection. The fluorescence of HBs-GFP in HepG2 cells treated with pAVU6+4sh357 was reduced by 55.4% compared with that of controls. The HBs-GFP expression in HepG2 cells treated with pAVU6+4sh357 was reduced by 76.3% and 90% as measured with conventional RT-PCR and real-time PCR, respectively.</p><p><b>CONCLUSION</b>This investigation demonstrated siRNA derived from shRNA expression vectors can inhibit the expression of HBs-GFP in HepG2 cells.</p>

Establish a screening system for selection of mRNA target sites for HBsAg to construct siRNA with shRNA / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To find some effective short interfering RNA's sites targeting HBV surface gene sequence using shRNA expression vectors.</p><p><b>METHODS</b>Four shRNA expression vectors targeting HBV surface gene sequence were constructed based on pAVU6 + 27 vector, and cotransfected into AD293 cells with HBs-EGFP fusion gene plasmid. The changes of HBs-EGFP image were detected by FACS and microscopy. The HBs-EGFP mRNA expression was evaluated by RT-PCR.</p><p><b>RESULTS</b>Four shRNA expression vectors and HBs-EGFP fusion gene plasmid were successfully constructed. pAVU6 + 4sh579 vector inhibited the HBs-EGFP expression by 69.8% in AD293 and suppressed the HBs-EGFP mRNA expression by 74.6%.</p><p><b>CONCLUSIONS</b>The results showed that the 579 site of HBV surface gene sequence was an effective target and pAVU6 + 4sh579 vector could suppress the HBs-EGFP expression in AD293 cells</p>