ABSTRACT
Objective To investigate the effect of miR-29c on radiosensitivity of hepatoma HepG2 cells by targeting AKT2 gene.Methods The expression of miR-29c in human normal hepatocytes THLE-3 and hepatoma cell HepG2 was detected by RT-PCR.The relationship between miR-29c and AKT2 were predicted by predicted by informative analysis and verified by dual luciferase reporter gene test and Western blot.miR-29c mimic/AKT2 gene recombinant plasmid and miR-29c inhibitor/ lentivirus vector AKT2 shRNA were transfected into HepG2 cells by Liposome 2000.The cells were irradiated with different doses (0,2,4,6 and 8 Gy) of X-rays,and the effects of miR-29/AKT2 on the survival and cell viability of HepG2 cells were detected by cloning and MTT assays.Results Compared with THLE-3 cells,the expression of miR-29c in HepG2 cells was significantly lower (t=17.816,P＜0.05).After 2,4,6 and 8 Gy X-ray irradiation,the survival of HepG2 cells was significantly lower than that of THLE-3 cells (t =4.541,6.823,7.218,9.363,P＜0.05),and the expression of miR-29c in HepG2 cells was significantly decreased (t =5.599,9.262,10.470,10.873,P＜0.05).The survival and viability of HepG2 cells were decreased by miR-29c overexpression (tsurvival rate =4.307,7.668,7.668,6.894,P＜0.05;tcell viability =3.443,8.116,13.434,P ＜ 0.05) but they were increased by miR-29c inhibition (tsurvival rate =4.003,6.713,7.141,P＜0.05;tcell viability =4.282,5.113,P＜0.05).Double luciferase reporter gene experiments showed that AKT2 was the target gene of miR-29c since the expression of AKT2 was negatively regulated by miR-29c.After the silence of AKT2 or overexpression of AKT2,the survival and viability of HepG2 cells were consistent with the overexpression of miR-29c or the inhibition of miR-29c,respectively.Conclusions MiR-29c increases the radiosensitivity of hepatoma cell HepG2 by targeting AKT2.
ABSTRACT
Objective: To investigate the predictive effect of major adverse cardiac events [MACE] in malignant obstructive jaundice [OJ] patients using plasma brain natriuretic peptide [BNP] level and surgical Apgar scoring [SAS] system
Methods:Forty one malignant OJ patients undergoing surgical treatments were studied at a single center. Pre-and postoperative plasma BNP level, total bilirubin [TBil] and data of cardiac function [HR, CVP, CI, LVEF%] were detected, the SAS was calculated during the surgery, the relationship of both plasma BNP level and SAS with MACE after surgery was analyzed
Results:Thirteen patients out of 41 [31.71%] experienced MACE without cardiac death. OJ patients had a higher plasma BNP level than baseline before operation [191.61+/-105.76 pg/ml VS 175 pg/ml, P<0.05], the cardiac function data was improved [CVP: t=4.761, p=0.000; CI: t=3.539, p=0.001; LVEF%: t=3.632, p=0.001] after the operation. Patients with lower SAS had increasing incidence of MACE after surgery
Conclusion:Malignant OJ patients with higher preoperative BNP level and lower surgical Apgar score were identified at high risk of MACE after surgery
ABSTRACT
Objective To investigate the anti-tumor efficiency of cytotoxic T-lymphocyte (CTL) induced by activated B lymphocyte after hepatocellular carcinoma (HCC) alpha fetoprotein (AFP) mRNA transfection.Methods B lymphocytes were fractionated,purified and activated by recombinant human soluble sCD40L.PGEM4Z/AFP/A64-EGFP plasmid was established in vitro,mixed with polymerase T7RNA,and then transcribed into AFP mRNA with Poly (A) sequence.B lymphocytes electrotransfected with AFP mRNA were in the experimental group,B lymphocytes electrotransfected with GAPDH mRNA were in the negative control group,and untreated B lymphocytes were in the blank control group.The expressions of antigen-presenting cell (APC)markers (CD19,CD20,CD21,CD40,CD80,CD83) and major histocompatibility complex (MHC) of the 3 groups were detected.B lymphocytes of the 3 groups were cultured with T lymphocytes at ratios of 1∶40,1 ∶ 20,1∶10 and 1∶5 to induce and ampify CTL,and then the absorbance values were detected to evaluate the proliferation ability of T lymphocytes.The killing activity of CTL was investigated with HCC cell line SMMC7721 as the target cells.All data were analyzed using the paired t test,one-way analysis of variance or Tamhane's T2 test.Results The expressions of CD19,CD20,CD21,CD40,CD80 and CD83 of the experimental group were 74 ± 11,78 ±8,80 ± 10,90 ± 11,82 ± 6,56 ± 5,which were significantly higher than 51 ± 5,60 ± 7,53 ± 5,73 ± 8,50 ± 5 and 49 ± 6 of the negative control group,and 46 ± 3,54 ± 5,41 ± 3,56 ± 5,52 ± 6 and 21 ± 4 of the blank control group (t =5.302,4.812,7.627,5.932,9.142,7.813; 11.581,7.036,13.592,12.873,9.235,14.619,P ＜ 0.01).The proliferation of CTL of the experimental group was significantly higher than that in the negative control group and blank control group (t =18.203,23.714,15.062,9.417 ; 16.833,19.392,13.871,6.592,P ＜0.01).When the T lymphocytes were mixed with the HCC cell line SMMC7721 at the ratios of 40∶ 1,20∶1and 10∶1,the killing rates of HCC cells by CTL of the exprimental group were 43％ 4％,32％ ± 4％ and 22％ ±3％,which were significantly higher than 15％ ± 5％,7％ ± 3％ and 6％ ±2％ of the negative control group,and 7％±3％,8％±3％ and 9％±4％ of the blank control group (t =9.141,13.272,11.901; 14.372,12.835,9.507,P ＜ 0.01).Conclusion Activated B lymphocytes after HCC AFP mRNA transfection may effectively induce CTL to kill HCC cells.
ABSTRACT
Objective To investigate AXIN2 mRNA expression level in hepatocellular carcinoma (HCC) , and to analyze the effect of AXIN2 gene methylation status on its mRNA expression and HCC genesis and development. Methods Fifty-three surgical excised HCC specimens and paired adjacent non-cancerous specimens, seven normal liver specimens and five HCC cell lines were collected. The expression of AXIN2 at mRNA level and the methylation status of AXIN2 gene promoter were determined by quantitative PCR. Results The expression of AXIN2 mRNA was lower in HCC tissues (0.1629 + 0.0679) than that in adjacent non-cancerous tissues (0. 4155 + 0. 2330), and there was significant difference (Z= -2. 567, P = 0. 010). The methylation level of AXIN2 gene in HCC and adjacent non-cancerous tissues (39. 77% ±3. 89%, and 36. 92% ±2. 81%) was significantly higher than that in normal liver tissues (7. 38% ±2. 40% , t=-3. 663 ,P = 0. 009;t= -4. 591 ,P = 0. 007).AXIN2 gene was hypermethylated in all five HCC cell lines. There was a negative correlation between AXIN2 mRNA expression level and the degree of methylation ( r = -0. 458, P = 0. 032). The methylation level was higher in TNM Ⅲ patients of HCC than that in TNM Ⅰ and Ⅱ patients (P =0.008). Conclusion The down-regulation of AXIN2 gene mRNA expression is correlated with its hypermethylation status. The low expression of AXIN2 mRNA and the abnormal methylation of promoter may be one of the important mechanism of HCC genesis and development.