Effectiveness of Mindful Self-Compassion Training Supported by Online Peer Groups in China: A Pilot Study.

Context: Self-compassion training involves the cultivation of feelings of warmth and safety, presence, and interconnectedness. Mindful Self-Compassion (MSC) training in a group setting has been found to increase self-compassion, mindfulness, and emotional well-being. Objective: The current study intended to examine the outcomes of live, online, videoconference-based MSC training with online peer-support for nonclinical populations in different cities in China. Design: The research team designed a pre-post pilot study. Setting: The study took place at Renmin University in Beijing, China. Participants: Participants were 253 Chinese individuals who were recruited from different regions in China through online advertisements. Intervention: Participants took part in online MSC training in a two-hour, group class each week for eight weeks and received support from online peer groups and through a half-day in-person retreat. Outcome Measures: Self-report outcomes were obtained at baseline and postintervention, using the Self Compassion Scale (SCS) and the Compassion for Others Scale (CS) for primary outcomes, and the Depression, Anxiety, and Stress Scale (DASS-21), the Fear of Compassion Scale (FOCS), the Satisfaction with Life Scale (SWLS), the Subjective Happiness Scale (SHS), and the Cognitive and Affective Mindfulness Scale (CAMS-R), for secondary outcomes. A fixed effects model was used to test for within-group changes in the scales. Results: The online MSC program yielded a high retention rate. Of the 206 first-time participants, 179 (86.9%) attended six or more of the eight MSC sessions, and 183 (88.8%) completed the assessments at both baseline and postintervention. Of the 183 retained participants, 97.8% were female, with an average age of 37.8 ± 7.9; 94% had college or higher education. For all scales, the within-person changes occurred in the expected direction; positive attributes and experiences increased, while negative attributes and experiences decreased. Conclusions: The study showed that first-time participants in China in an online MSC training that was supported by online peer groups had high attendance rates, high assessment completion, and favorable results. These preliminary outcomes suggest that future studies with more rigorous designs are warranted to further investigate online training with peer support as an effective and efficient approach to disseminate MSC training in China.

[Expression of microRNA-126 in myocardial tissue of rats in the early stage of severe burn injury and its relation with myocardial damage].

OBJECTIVE: To observe the changes in the expressions of microRNA-126 in myocardial tissue and cardiac troponin I (cTnI) in serum of rats in the early stage of severe burn injury with analysis of their relationship, and to validate the relationship between microRNA-126 and myocardial damage in cellular level. METHODS: (1) Forty-eight SD rats were divided into sham injury group (n=8, without fluid therapy after sham injury) and burn injury group (n=40, inflicted with 30% TBSA full-thickness scald on the back, hereinafter referred to as burn, and received intraperitoneally injection of lactic acid Ringer's solution) according to the random number table. Blood was collected from abdominal aorta of rats in sham injury group at post injury hour (PIH) 1, and then these 8 rats were sacrificed for obtaining left ventricular tissue. Blood was respectively collected from abdominal aorta of 8 rats in burn injury group at PIH 3, 6, 12, 24, and 48, and then they were sacrificed and the left ventricular tissue was obtained at each time point. The expression of microRNA-126 in myocardial tissue was assessed by real-time fluorescent quantitative RT-PCR. Serum level of cTnI was assessed by ELISA. (2) Rat myocardial cell line H9C2 was divided into normal control group (NC, routinely cultured), stimulation group (S), negative transfection+stimulation group (NT+S), and transfection+stimulation group (T+S) according to the random number table. Cells in S group were treated with hypoxia for 24 h after being cultured with DMEM containing 10% burn serum obtained from rats in burn injury group at PIH 6 in experiment (1). Cells in NT+S group and T+S group were respectively transfected with the negative control of microRNA mimics and microRNA-126 mimics for 24 h, and then were given the same treatment as that of S group. The expression of microRNA-126 in myocardial cells was determined by real-time fluorescent quantitative RT-PCR (with the sample number of 3). Cell counting kit 8 was used to examine the vitality of myocardial cell (with the sample number of 4, denoted as absorbance value). Apoptotic rate of myocardial cells was determined by flow cytometer (with the sample number of 3). Data were processed with one-way analysis of variance and LSD-t test. The relationship between microRNA-126 expression in myocardial tissue and serum level of cTnI of rats was assessed by linear correlation analysis. RESULTS: (1) Compared with that of sham injury group at PIH 1, the expression levels of microRNA-126 in myocardial tissue of rats in burn injury group at PIH 3, 6, 12, 24, and 48 were significantly decreased (with t values from 5.68 to 9.79, P values below 0.01), reaching its nadir at PIH 24 (0.40 ± 0.08). Compared with that of sham injury group at PIH 1, the serum levels of cTnI of rats in burn injury group at PIH 3, 6, 12, 24, and 48 were significantly increased (with t values from 6.68 to 12.79, P values below 0.01), peaking at PIH 12 [(1 035 ± 177) pg/mL]. A significant negative correlation between the expression level of microRNA-126 in myocardial tissue and serum level of cTnI was observed in rats of burn injury group at each time point (r=-0.797, P<0.001). (2) Compared with those of NC group, the microRNA-126 expression levels in myocardial cells of S group and T+S group were respectively decreased and increased (with t values respectively 4.57 and 5.73, P<0.05 or P<0.01), the cell vitality levels were obviously decreased (with t values respectively 14.88 and 6.48, P values below 0.01), and the apoptotic rates were significantly increased (with t values respectively 13.82 and 6.96, P values below 0.01). Compared with that in NT+S group, the microRNA-126 expression level in myocardial cells of T+S group was significantly increased (t=6.77, P<0.01), the cell vitality level was obviously increased (t=8.23, P<0.001), and the apoptotic rate was significantly decreased (t=6.14, P<0.001). CONCLUSIONS: Expression level of microRNA-126 in myocardial tissue of rat was decreased in the early stage of severe burn injury. It may participate in regulating myocardial damage and play a protective role.

Effect and possible mechanism of monocyte-derived VEGF on monocyte-endothelial cellular adhesion after electrical burns.

OBJECTIVE: One of the major obstacles in the treatment of severe electrical burns is properly handling the resulting uncontrolled inflammation. Such inflammation often causes secondary injury and necrosis, thus complicating patient outcomes. Vascular endothelial grow factor (VEGF) has emerged as an important mediator for the recruitment of monocytes to the site inflammation. This study was designed to explore the effects and possible mechanism of VEGF on monocyte-endothelial cellular adhesion. To do so, we used a cultured human monocytic cell line (THP-1) that was stimulated with serum derived from rats that had received electrical burns. METHODS: Serum was obtained from rats that had received electrical burns. Both the VEGF and soluble flt-1 (sflt-1) concentrations of the serum were determined by double-antibody sandwich ELISA. The concentrations of VEGF, sflt-1, and TNF-α obtained from the cell-free cultured supernatant of THP-1 cells that had been exposed to the serum were then determined by double-antibody sandwich ELISA. Serum-stimulated THP-1 cells were added to wells with a monolayer of endothelial cells to detect the level of monocyte-endothelial cells adhesion. Finally, the state of phosphorylation of AKT was determined by Western blotting. RESULTS: Both in vivo and in vitro studies showed that compared to controls, the levels of VEGF were significantly increased after electrical burns. This increased was accompanied by a reduction of sflt-1 levels. Furthermore, the serum of rats that had received electrical burns was able to both activate monocytes to secrete TNF-α and enhance monocyte-endothelial cell adhesion. Treatment with the serum also resulted in an up-regulation of the phosphorylation of AKT, but had no effect on the total levels of AKT. Phosphatidylinositide 3-kinases (PI3K) inhibition decreased the number of THP-1 cells that were adhered to endothelial cells. Finally, sequestering VEGF with sflt-1 was able to reduce the effect on monocyte-endothelial cells adhesion by blocking the PI3K signaling pathway. CONCLUSION: Our results indicate that VEGF is implicated in the pathogenesis of inflammation after electrical burns. Inhibition of VEGF activity could attenuate monocyte-endothelial cells adhesion by suppressing the state of phosphorylation of AKT, which is downstream of the PI3K signaling pathway.

[Expression of microRNA-21 in myocardial tissue of rats in the early stage of severe scald injury and its mechanism].

OBJECTIVE: To explore the molecular mechanism of microRNA-21 in myocardial damage of rats in the early stage of severe scald injury by observing the expression of microRNA-21 and programmed cell death 4 (PDCD4) in myocardial tissue of rat and to validate the relationship between them in cell model. METHODS: (1) Forty SD rats were divided into sham injury group (n =8, sham injured) and scald injury group (n =32, inflicted with 30% TBSA full-thickness scald on the back) according to the random number table. The left ventricular tissue was collected from rats in sham injury group at post injury hour 1 without any fluid infusion. Rats in scald injury group were given an intraperitoneal injection of lactic acid Ringer's solution and 8 rats were respectively sacrificed at post injury hour 3, 6, 12, 24 to harvest left ventricular tissue. The expression of microRNA-21 in myocardial tissue was assessed by real-time fluorescent quantitative RT-PCR. The protein expression of PDCD4 in myocardial tissue was assessed by Western blotting. (2) Rat myocardial cell line H9C2 was divided into microRNA-21 inhibitor group (cells were transfected with microRNA-21 inhibitor) and negative transfection control group (cells were transfected with negative control of microRNA inhibitor) according to the random number table. At post transfection hour 48, real-time fluorescent quantitative RT-PCR and Western blotting were performed respectively to determine the mRNA and protein expression levels of PDCD4 in cells. Data were processed with one-way analysis of variance, LSD-t and two independent samples t test. The relationship between microRNA-21 expression and PDCD4 protein level in myocardial tissue of rats was assessed by linear correlation analysis. RESULTS: (1) The expression levels of microRNA-21 in myocardial tissue of rats in sham injury group at post injury hour 1 and in scald injury group at post injury hour 3, 6, 12, 24 were respectively 0. 96 ± 0. 13, 0. 44 ± 0. 08, 0. 42 ± 0. 10, 0.33 +0.07, and 0.61 0.10 (F = 27.331, P <0.001). Compared with that in myocardial tissue of rats in sham injury group at post injury hour 1, expression level of microRNA-21 was significantly decreased in scald injury group at post injury hour 3, 6, 12, 24 (with t values from 4. 558 to 9.410, P values below 0.01). The protein expression levels of PDCD4 in myocardial tissue of rats in sham injury group at post injury hour 1 and in scald injury group at post injury hour 3, 6, 12, 24 were respectively 0.44 ± 0.05, 0.60 ± 0.09, 0.92 ± 0. 15, 0. 86 ± 0.11, and 0.57 ± 0. 10 (F =8.622, P =0.003). Compared with that in sham injury group at post injury hour 1, protein expression level of PDCD4 was significantly increased in scald injury group at post injury hour 6 and 12 (with t values respectively 4. 968 and 4. 122, P values below 0.01). A significant negative correlation between the expression of microRNA-21 and PDCD4 protein in myocardial tissue of rats of scald injury group was observed at each time point (r = -0. 572, P = 0. 026). (2) The mRNA and protein expression levels of PDCD4 of myocardial cells in microRNA-21 inhibitor group were respectively 1.73 ± 0. 29 and 0. 38 ± 0. 08, which were significantly higher than those in negative transfection control group (0.95 ± 0.14 and 0.23 ± 0.03, with t values respectively 4. 857 and 3.356, P <0.05 or P <0.01). CONCLUSIONS: Expression of microRNA-21 was decreased, while expression of PDCD4 was increased, in myocardial tissue of rats in the early stage of severe scald injury. MicroRNA-21 might participate in myocardial damage in the early stage of scald injury by negatively regulating expression of PDCD4.

[Role of phosphoinositide 3 kinase/protein kinase B signal pathway in monocyte-endothelial adhesion induced by serum of rats with electrical burn].

OBJECTIVE: To observe the change in phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signal pathway in monocytes as induced by serum of rats with electrical burn, and to explore the effects of PI3K/Akt pathway on monocyte-endothelial cell adhesion. METHODS: Sixty-four SD rats of clean grade were inflicted with electrical burn for the collection of serum of rats with electrical burn; another group of twenty-four SD rats were used to obtain normal serum without treatment. (1) Human monocyte line THP-1 was routinely cultured. The THP-1 cells in logarithmic phase were divided into normal serum group (resuspended in RPMI 1640 medium with 20% normal rat serum) and burn serum group (resuspended with RPMI 1640 medium with 20% serum of rats with electrical burn) according to the random number table, with 6 wells in each group. Morphology of THP-1 cells in normal serum group was observed at post culture hour (PCH) 24, and that in burn serum group at PCH 3, 6, 24. The contents of TNF-α in culture supernatant were determined by double-antibody sandwich ELISA at the corresponding time point in each group. The state of Akt activation was determined by Western blotting at PCH 3, 6, 24. (2) Another portion of THP-1 cells were divided into 4 groups according to the random number table, with 6 wells in each group. Cells in normal serum group and burn serum group were given with the same culture condition as above; cells in normal serum+inhibitor group and burn serum+inhibitor group were cultured with the same culture conditions as in the former two groups correspondingly with addition of 100 nmol/L wortmannin in the nutrient solution. At PCH 3 and 6, THP-1 cells were added into the well with a monolayer of endothelial cell line EA.hy926 to observe the monocyte-endothelial cell adhesion. Data were processed with one-way analysis of variance and LSD- t test. RESULTS: (1) In normal serum group, THP-1 cells showed growth in suspension, with uniform shape at PCH 24. In burn serum group, the cell shape became irregular though the membrane was complete at PCH 3; cellular size became irregular and cell membrane and cytoplasm were swollen at PCH 6; cell membrane was disrupted with death of cells at PCH 24. The contents of TNF-α in culture supernatant in normal serum group at PCH 24 and in burn serum group at PCH 3, 6, 24 were respectively (38.5 ± 1.4), (75.1 ± 1.5), (91.5 ± 1.8), (117.0 ± 1.4) pg/mL (F = 1 415.306, P < 0.01). The contents of TNF-α in culture supernatant in burn serum group at PCH 3, 6, 24 were all significantly higher than the content of TNF-α in normal serum group at PCH 24 (with t values respectively 29.614, 42.852, 63.485, P values below 0.01). The ratio values of phosphorylated Akt to Akt in burn serum group at PCH 3, 6, 24 were respectively 2.66, 3.69, 1.17 times of those in normal serum group at the corresponding time point. (2) In normal serum group, normal serum+inhibitor group, burn serum group, and burn serum+inhibitor group at PCH 3 and 6, the numbers of THP-1 cells adherent to endothelial cells were respectively (231 ± 45), (280 ± 47), (703 ± 169), (335 ± 85) per 100-time field; (219 ± 49), (235 ± 21), (562 ± 123), (226 ± 29) per 100-time field (with F values respectively 25.630 and 18.975, P values below 0.01). The number of THP-1 cells adhered to EA.hy926 cells was significantly more in burn serum group than in normal serum group at PCH 3 and 6 (with t values respectively 6.189 and 6.601, P values below 0.01). The number of THP-1 cells adherent to EA.hy926 cells was significantly fewer in burn serum+inhibitor group than in burn serum group at PCH 3 and 6 (with t values respectively 6.821 and 6.465, P values below 0.01). CONCLUSIONS: The serum of rats suffering from electrical burn can induce the monocytes to secrete TNF-α, thus enhancing monocyte-endothelial cell adhesion, but it can be inhibited by blocking PI3K/Akt signal pathway.

[Effects of microRNA-21 on the myocardial cell apoptosis induced by ischemia and hypoxia in rat].

OBJECTIVE: To explore the effects of microRNA-21 on apoptosis of myocardial cell of rats as induced by ischemia and hypoxia, and to analyze the underlying mechanisms. METHODS: (1) Rat myocardial cell line H9C2 was cultured in a serum-free and low glucose DMEM medium using a hypoxic incubator which was filled with 1% oxygen, 5% carbon dioxide, and 94% nitrogen to simulate ischemic environment. The expression of microRNA-21 in normal myocardial cells and cells treated with low oxygen exposure for 6 and 24 h were assessed by real-time fluorescent quantitative RT-PCR. (2) Another portion of myocardial cells were divided into 4 groups according to the random number table: normal control group (NC, ordinary culture without any treatment), ischemia/hypoxia group (IH, treated with ischemia and hypoxia for 24 h), negative transfection control+ischemia/hypoxia group (NC+IH, treated with ischemia and hypoxia for 24 h after the transfection of microRNA mimics control for 24 h), microRNA-21+ischemia/hypoxia group (M+IH, treated with ischemia and hypoxia for 24 h after the transfection of microRNA-21 mimics for 24 h). The cells in the latter three groups were examined immediately after treatment, and cells in group NC were collected and examined at the same time point. Apoptosis rate of myocardial cells was determined by flow cytometer. The mRNA and protein expression levels of programmed cell death 4 (PDCD4) in myocardial cells were determined by real-time fluorescent quantitative RT-PCR and Western blotting respectively. The sample numbers in this experiment were 6 or 3. Data were processed with one-way analysis of variance and LSD- t test. RESULTS: (1) The expression level of microRNA-21 in normal myocardial cells and cells treated with ischemia and hypoxia for 6 and 24 h were respectively 1.09 ± 0.17, 0.75 ± 0.08, and 0.67 ± 0.08 (F = 11.280, P = 0.009). Compared with expression level of microRNA-21 in normal myocardial cells, those of cells treated for 24 h (t = 4.461, P = 0.004) and 6 h (t = 3.642, P = 0.011) were both lower, and the former was more obvious. Therefore all the ischemia and hypoxia treatment time of cells in the following experiment was 24 h. (2) The apoptosis rate of myocardial cells in group NC was (3.5 ± 0.7)%. After being treated with ischemia and hypoxia for 24 h, the apoptosis rates of myocardial cells in groups IH, NC+IH, and M+IH were respectively (17.3 ± 3.2)%, (16.4 ± 3.0)%, and (7.6 ± 2.0)% (F = 15.176, P = 0.001). Compared with that of group NC, the apoptosis rate of myocardial cells of group IH was significantly increased (t = 5.641, P < 0.001), while it was significantly decreased in group M+IH as compared with group NC+IH (t = 3.588, P = 0.007). The mRNA expression level of PDCD4 in group NC was 1.06 ± 0.21. After being treated with ischemia and hypoxia for 24 h, the mRNA expression levels of PDCD4 in groups IH, NC+IH, and M+IH were respectively 3.01 ± 0.34, 3.05 ± 0.25, and 1.48 ± 0.24 (F = 44.952, P < 0.001). Compared with that of group NC, the mRNA expression level of PDCD4 in group IH was higher (t = 8.945, P < 0.001), while it was significantly lower in group M+IH as compared with group NC+IH (t = 7.253, P < 0.001). The protein expression level of PDCD4 in group NC was 0.44 ± 0.08. After being treated with ischemia and hypoxia for 24 h, the protein expression levels of PDCD4 in groups IH, NC+IH, and M+IH were respectively 0.96 ± 0.13, 1.05 ± 0.12, and 0.58 ± 0.12 (F = 18.804, P = 0.008). Compared with that of group NC, the protein expression level of PDCD4 in group IH was higher (t = 5.429, P = 0.006), while it was significantly reduced in group M+IH as compared with group NC+IH (t = 4.903, P = 0.008). CONCLUSIONS: Ischemia and hypoxia reduce the expression of microRNA-21 in myocardial cells, while increasing the expression of microRNA-21 can alleviate the ischemia/hypoxia-induced apoptosis by lowering the expression of PDCD4.

Influence of cytotoxic T lymphocyte-associated antigen 4 polymorphisms on the outcomes of hepatitis B virus infection.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) regulates T-cell activation and Th1/Th2 cytokine production and is involved in the immune response against Hepatitis B virus (HBV) infection. To detect the association of the CTLA-4 gene polymorphisms with susceptibility to HBV infection a hospital-based case-control study was conducted. A total of 1,119 unrelated individuals were recruited. The CTLA-4 variants rs5742909, rs231775 and rs3087243 were genotyped via the TaqMan method in this cohort. A comparison with a chronic active hepatitis B group revealed that the SNP rs231775 exhibited significant susceptibility to HBV progression, with the highest odds ratio (OR) reaching 1.659 and P=0.009-0.049. Although an HBV clearance group was used as a control, results of the present study demonstrated an association of rs5742909 with viral persistence [OR=1.694, 95% confidence intervals (CI)=1.124-2.553 and P=0.012]. Subsequent analyses revealed risk haplotypes (C-A-A and T-A-G, for which the highest OR reached 1.865) compared with the protective haplotype C-G-G. Therefore, SNPs in the CTLA-4 gene may be associated with HBV progression and viral persistence which is consistent with its emerging role in the T regulatory cells in the pathogenesis of disease.

[Analysis of diagnosis and management of 21 patients with Marjolin's ulcers].

OBJECTIVE: To investigate the clinical manifestation, diagnosis, and treatment of patients with Marjolin's ulcers. METHODS: The clinical materials of 21 patients with Marjolin's ulcers hospitalized from January 2007 to January 2013 were retrospectively analyzed, including age, gender, injury causes, duration time of primary disease in developing Marjolin's ulcer, duration of ulcer, lesion site, ulcer area, symptoms and signs of ulcer region, bacterial culture results before operation, histopathological type, grade of carcinoma cell differentiation, depth of invasion, treatment, and outcome. RESULTS: (1) The age of 21 patients at the time of diagnosis of Marjolin's ulcers was 19-74 (47 ± 13) years, and the ratio of male to female was nearly 0.9:1.0. (2) The main primary lesions were flame burns and high temperature liquid scald, respectively occurred in 12 cases (57.1%) and 7 cases (33.3%). The time for development of Marjolin's ulcers from primary injury was 10-56 (40 ± 14) years. (3) Ulceration on top of scar lasted for longer than one year in 12 patients (57.1%). (4) Lesion site was mainly located in the limbs in 13 patients (61.9%), and on head and face in 6 patients (28.6%), respectively. (5) Ulcer area ranged 0.25-74.25 (39 ± 25) cm(2). Foul excretion, bleeding, intensified pain, and gradual enlargement of ulceration were observed in the lesion of most patients. (6) Bacterial culture of wound excretion before operation showed positive results in 16 patients (76.2%). RESULTS: of bacterial culture of blood were negative in all patients. (7) Pathological examination revealed squamous cell carcinoma in 20 cases and basal cell carcinoma in 1 case, and mostly of high or medium differentiation. Cancer cells in nearly 40% patients had invaded the subcutaneous tissue or deeper area. (8) All patients were treated by surgery, among them autologous skin grafting was done after excision of lesion in 11 patients, and in 5 patients the defects were closed with skin flaps after excision of lesion, and in 5 patients limbs harboring the lesion were amputated. Twelve patients (57.1%) received postoperative rehabilitation treatment. Two patients with pulmonary metastasis received chemotherapy. (9) Most of the flaps and skin grafts survived well after surgery, and a few cases with failure of skin grafting or transplantation of flaps underwent skin grafts again. Patients were followed up for 6 months to 5 years, in 4 patients recurrence occurred after surgery, and 2 of them died. The other patients survived without recurrence. CONCLUSIONS: Squamous cell carcinoma was the most common pathological type of Marjolin's ulcer admitted to our unit. A recurrent ulcer with long course should be considered as Marjolin's ulcer, and it should be scrutinized pathologically. Currently, surgery remains the optimal treatment for Marjolin's ulcer. Regular follow-up should be carried out after resection of the lesion to detect carcinoma recurrence and metastasis.

[WISP-1: a novel mediator of hepatitis B virus-related hepatocellular carcinoma].

OBJECTIVE: To explore the effect of hepatitis B virus (HBV) X protein (HBX) on expression of the host gene Wnt induced secreted protein-1 (WISP-1) that is related to the pathogenic process of hepatocellular carcinoma (HCC). METHODS: Tumor and paratumor tissues were collected from HCC patients, and normal liver tissues were collected from healthy controls. Immunohistochemistry was used to evaluate the in vivo presence and expression levels of HBX and WISP-1 in the three tissue types. HepG2 cells stably transfected with pc-DNA3.1(+)-HBX or with pc-DNA3.1(+) only (G0, control) were generated and used to examine in vitro the HBX-induced changes in WISP-1 expression at the mRNA and protein levels by reverse transcription polymerase chain reaction and western blotting, respectively. RESULTS: The HCC tissues showed significantly higher rates of positivity for WISP-1 expression than the non-tumor controls (76.6% vs. paratumor: 23.4% or normal tissues: 0%, x2= 35.967, P less than 0.01). HBX increased WISP-1 expression in HepG2 cells at both the mRNA (1170.33 +/- 41.26 vs. G0: 265.34 +/- 27.47, t = 31.63, P less than 0.01) and protein (240.33 +/- 11.37 vs. G0: 40.33 +/- 7.09, F = 600.57, P less than 0.01) levels. CONCLUSION: HBV may up-regulate expression of the host gene WISP-1 through its X protein and thus promote the development of HCC.

Association of a single-nucleotide polymorphism within the miR-146a gene with susceptibility for acute-on-chronic hepatitis B liver failure.

Excessive activation of innate immune response contributes to the pathogenesis of acute-on-chronic hepatitis B liver failure (ACLF-HBV). miR-146a was recently found to be implicated in the regulation of innate immunity. In this study, we explored the biological significance of a single-nucleotide polymorphism (rs2910164) within the miR-146a gene in the risk of acquiring ACLF-HBV. We completed a hospital-based case-control study including 717 cases of HBV-infected patients--251 cases of ACLF-HBV and 466 cases of chronic hepatitis B. Whole blood samples were collected for isolation of DNA and peripheral blood mononuclear cells (PBMCs). The association between genotypes and risk of ACLF-HBV was analyzed by multivariate unconditional logistic regression, with adjustment for sex and age. Our results showed that the GG homozygote was a protective genotype in terms of susceptibility to ACLF-HBV, with odds ratio = 0.496, 95 % confidence interval = 0.309-0.797, P = 0.004 compared with CC+GC genotypes. The amount of mature miR-146a in PBMCs was significantly higher in the GG homozygote group than those in the CC and CG genotype groups of ACLF-HBV patients. The GG genotype group also represented lower serum level of TNF-α and higher survival rate (follow-up period = 4 months). In conclusion, The GG genotype within the pre-miR-146a is reversely associated with susceptibility of ACLF-HBV in the studied Chinese population. This may be partially explained by the relatively higher amount of mature miR-146a and the lower serum level of TNF-α in this genotype group.

[The effect of hepatitis B virus X protein on the expression of CtIP in HepG2 Cells].

To investigate the effect of hepatitis B virus X protein(HBx) on CtBP-interacting protein(CtIP) which is an important repair factor of DNA double strand break damage in HepG2 cells induced by bleomycin. A HBx stably expressing HepG2 cell line and a control HepG2 cell line with empty vector transfected were established. After the double strand break (DSB) damage occurred, the mRNA and protein levels of CtIP were detected by Real-time PCR and Western blot assay respectively, cell cycle profiles and apoptotic cell death were determined by a flow cytometry, and the position of CtIP in cells was observed by confocal laser scanning microscopy. It showed that HepG2 cells transfected with hepatitis B virus X gene could stably express HBx protein. After being induced by bleomycin, the percentage of apoptotic cell was 16.90%+/-0.89% in HBx stably expressing HepG2 cell line and 15.30%+/-0.86% in control cell line, respectively (q = 2.074, P is more than to 0.05). While the percentage of death cell was 8.71%+/-0.74% in HBx stably expressing HepG2 cell line and 4.90%+/-0.46% in control cell line, respectively (q = 7.126, P is less than to 0.01). The two cell lines manifested the increase of G2/M arrest and significant difference existed between the two cell lines. HBx down regulated the expression levels of CtIP and its mRNA. The CtIP level was 0.66+/-0.04 in HepG2-HBx cell and 0.73+/-0.05 in HepG2-vec cell, respectively (t = 2.314, P is less than to 0.05). The relative mRNA level was 1.00+/-0.06 in HepG2-HBx cell and 1.23+/-0.08 in HepG2-vec cell, respectively (t = 2. 732, P is less than to 0.05). We also found that CtIP was concentrated in the cell nucleus. The research suggests that HBx may affect DNA-repair pathways by disrupting the expression of CtIP.

MiR-192 inhibits nucleotide excision repair by targeting ERCC3 and ERCC4 in HepG2.2.15 cells.

Deficient DNA repair capacity is associated with genetic lesions accumulation and susceptibility to carcinogenesis. MicroRNAs (miRNAs) are small non-coding RNAs that regulate various cellular pathways including DNA repair. Here we hypothesized that the existence of HBV products may interfere with cellular nucleotide excision repair (NER) through microRNA-mediated gene regulation. We found that NER was impaired in HepG2.2.15 cells, a stable HBV-expressing cell line, compared with its parental cell line HepG2. Altered miRNA expression profile, in particular the significant upregulation of miR-192, was observed in HepG2.2.15 cells. Additionally, ERCC3 and ERCC4, two key factors implicated in NER, were identified as targets of miR-192 and over-expressing miR-192 significantly inhibited cellular NER. These results indicated that persistent HBV infection might trigger NER impairment in part through upregulation of miR-192, which suppressed the levels of ERCC3 and ERCC4. It provides new insight into the effect of chronic HBV infection on NER and genetic instability in cancer.

[HBx gene down-regulates miR-192 expression and inhibits apoptosis of human hepatoma cell line HepG2].

OBJECTIVE: To explore the mechanism by which HBV X gene(HBx) inhibits apoptosis of human hepatoma cell line HepG2 in terms of miRNA. METHODS: Three cell lines were prepared: HepG2 cells stably transfected with HBx (HepG2/HBx), HepG2 cells stably transfected with pcDNA3.1 (HepG2/pcDNA3.1) and HepG2 cells. Flow cytometry was adopted to measure the apoptosis of these three cells and Taqman fluorescence quantitative PCR was used to examine miR-192 expression. After HepG2 cells was transfected with miR-192, the apoptosis was analyzed by flow cytometry and the expressions of p53 and PUMA at mRNA and protein levels were evaluated by SYBR Green quantitative PCR and Western blot, respectively. RESULTS: Compared with HepG2/pcDNA3.1 cells (11.46% ± 0.69%) and HepG2 cells (12.5% ± 0.66%), the apoptosis rate of HepG2/HBx cells (2.37% ± 0.35%) was significantly reduced (F = 171.722, P < 0.01). The level of miR-192 was 49.1% ± 5.9% in HepG2 cells, which was dramatically down-regulated (F = 14.319, P = 0.019) as compared to the other two groups (HepG2/pcDNA3.1: 98.0% ± 8.9%; HepG2: 100%). Compared with HepG2 cells transfected with miR-NC (10.74% ± 1.15%), transfection of miR-192 into HepG2 cells led to increased apoptosis (15.74% ± 1.17%) (F = 18.415, P = 0.013) and higher p53 and PUMA expressions at mRNA (p53: 1.68 ± 0.12 vs 0.90 ± 0.09, F = 43.115, P = 0.003, PUMA: 1.66 ± 0.10 vs 0.98 ± 0.06, F = 22.541, P = 0.009) and protein (p53: 3.07 vs 1, PUMA: 2.13 vs 1) levels. CONCLUSION: HBx could inhibit apoptosis of HepG2 cells through down-regulation of miR-192 which induces apoptosis of HepG2 cells.

DNA double-strand breaks, potential targets for HBV integration.

Hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC) is one of the most frequently occurring cancers. Hepadnaviral DNA integrations are considered to be essential agents which can promote the process of the hepatocarcinogenesis. More and more researches were designed to find the relationship of the two. In this study, we investigated whether HBV DNA integration occurred at sites of DNA double-strand breaks (DSBs), one of the most detrimental DNA damage. An 18-bp I-SceI homing endonuclease recognition site was introduced into the DNA of HepG2 cell line by stable DNA transfection, then cells were incubated in patients' serum with high HBV DNA copies and at the same time, DSBs were induced by transient expression of I-SceI after transfection of an I-SceI expression vector. By using nest PCR, the viral DNA was detected at the sites of the break. It appeared that integration occurred between part of HBVxgene and the I-SceI induced breaks. The results suggested that DSBs, as the DNA damages, may serve as potential targets for hepadnaviral DNA insertion and the integrants would lead to widespread host genome changes necessarily. It provided a new site to investigate the integration.

Persistent effect of IFNAR-1 genetic polymorphism on the long-term pathogenesis of chronic HBV infection.

Genetic polymorphism of IFNAR-1 plays a large role in determining the clearance or chronicity after hepatitis B virus (HBV) exposure. However, it is not clear whether type I interferon receptor-1 (IFNAR-1) variations continuously exert their effects to influence the final outcomes following HBV chronicity, including acute-on-chronic hepatitis B liver failure (ACLF-HBV), chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC). Here we report that these four potential outcomes of chronic HBV infection are strongly associated with IFNAR-1 polymorphisms through a hospital-based case-control study of 663 cases. ACLF-HBV and HCC were each compared with CHB+LC. In comparison with the G/G genotype, the C/G and C/C genotypes at both single-nucleotide polymorphism (SNP) sites (rs1012335 and rs2257167) showed significant susceptibility to ACLF-HBV (the highest odds ratio [OR] reached 2.374; 95% CI = 1.488, 3.788; p < 0.001 for the C/G genotype at rs2257167), as well as HCC (OR = 2.475; 95% CI = 1.435, 4.426; p < 0.001 for the C/C genotype at rs1012335). The C allele at both loci was a susceptibility allele for ACLF-HBV and HCC, with the highest ORs reaching 1.653 (95% CI = 1.233, 2.216; p < 0.01 at rs1012335) in the ACLF-HBV group, and 1.659 (95% CI = 1.274, 2.159; p < 0.01 at rs1012335) in the HCC group. A strongly linked disequilibrium was also found within these two alleles (p < 0.001). Our research indicates that genetic polymorphisms of IFNAR-1 not only contribute to the determination of clearance or chronicity in the early stages of HBV exposure, but they also persistently influence pathogenesis over the long-term process of chronic HBV infection.

DNA Double-Strand Breaks, Potential Targets for HBV Integration / 华中科技大学学报（医学）（英德文版）

Hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC) is one of the most frequently occurring cancers. Hepadnaviral DNA integrations are considered to be essential agents which can promote the process of the hepatocarcinogenesis. More and more researches were designed to find the relationship of the two. In this study, we investigated whether HBV DNA integration occurred at sites of DNA double-strand breaks (DSBs), one of the most detrimental DNA damage. An 18-bp I-SceI homing endonuclease recognition site was introduced into the DNA of HepG2 cell line by stable DNA transfection, then cells were incubated in patients' serum with high HBV DNA copies and at the same time, DSBs were induced by transient expression of I-SceI after transfection of an I-SceI expression vector. By using nest PCR, the viral DNA was detected at the sites of the break. It appeared that integration occurred between part of HBV x gene and the I-Scel induced breaks. The results suggested that DSBs, as the DNA damages, may serve as potential targets for bepadnaviral DNA insertion and the integrants would lead to widespread host genome changes necessarily. It provided a new site to investigate the integration.