ABSTRACT
Objective:To study the effect of miR-539-5p on apalutamide (ARN-509) sensitivity and malignant phenotype of androgen independent prostate cancer cell line C4-2B and related mechanisms.Methods:Castrated resistant prostate cancer, castrated sensitive prostate cancer and benign prostate tissue were obtained. C4-2B cell lines were divided into blank group, transfection group (miR-539-5p plasmid) and control group (control plasmid). qPCR was used to detect the expression of miR-539-5p, androgen receptor (AR) and HSBP1 in the tissues and 3 group of cells. The protein expressions of AR and HSBP1 were detected by western blot. Transwell assay was used to detect the invasion and migration ability of three groups of cells. CCK-8 assay was used to detect the proliferation ability and semi-inhibitory concentration (IC50) of AR antagonist ARN-509. The colony forming ability of the three groups of cells was detected by plate cloning experiment.Results:Tissue-qPCR indicated that, in the benign prostate tissue, tumor tissue of castration sensitive patients and tumor tissue of castration resistant patients, the expressions of miR-539-5p were 0.29 ± 0.04, 0.17 ± 0.02 and 0.07 ± 0.01, the expressions of AR were 0.13 ± 0.02, 0.28 ± 0.04 and 0.79 ± 0.11, and the expressions of HSBP1 were 0.20 ± 0.03, 0.38 ± 0.04 and 0.72 ± 0.11, respectively. Compared with benign prostate tissue and prostate cancer tissue, the expression of AR and HSBP1 gene was higher in prostate cancer tissues with castration resistance, and the expression of miR-539-5p was lower. Cell-qPCR demonstrated that the expressions of miR-539-5p in blank group, control group and transfection group were 1.00±0.09, 1.07±0.11 and 7.19±0.51, the expressions of AR were 1.00±0.10, 1.03±0.14 and 0.51±0.08, and the expressions of HSBP1 were 1.00±0.10, 0.96±0.12 and 0.97±0.11. The expression of miR-539-5p in the transfection cells was significantly higher than that in the control group and the blank group, the expression of AR gene was significantly lower than that in the control group and the blank group, and there was no significant difference in the expression of HSBP1. Western blot showed that, in blank group, control group and transfection group, the protein expressions of AR were 1.00±0.10, 1.12±0.22 and 0.72±0.16, and the expressions of HSBP1 were 1.00±0.10, 0.94±0.18 and 0.48±0.11. The protein expression of AR and HSBP1 in the transfection group was significantly lower than that in the control group and the blank group. Transwell experiment showed that the invasion and migration of cells in the transfection group were significantly lower than that in the control group and the blank group. CCK-8 assay and plate cloning experiment showed that the proliferative capacity and the number of clone formation in the transfection group were significantly lower than those in the control group and the blank group, and the expression of AR and HSBP1 in the transfection group was significantly lower than that in the control group and blank group. Compared with the control group and blank group, the IC50 value of ARN-509 decreased significantly in the transfection group.Conclusion:miR-539-5p may inhibit the malignant phenotype and castration resistance of cells via interfering with the translation level of HSBP1.
ABSTRACT
Objective:To explore the effect of low expression of human epidermal growth factor-like domain protein 6 (EGFL6) gene in human bladder cancer cell 5637 on its proliferation ability in vitro and in vivo.Methods:Human bladder cancer cells 5637 were divided into experimental group and control group. The experimental group cells targeted human EGFL6 gene with small interfering RNA (siRNA) , and the control group cells were transfected with Mock-siRNA. The cells in the experimental group and the control group were detected by real-time quantitative PCR. The content of EGFL6 mRNA in the medium. CCK8 was used to detect the proliferation ability of cells. Nude mice were injected subcutaneously with human bladder cancer cells 5637 in the experimental and control groups respectively, and the proliferation ability of the cells in vivo was detected by subcutaneous transplantation tumor assay in nude mice. The expression of EGFL6, p-P13K, and p-AKT was detected by western blotting.Results:The expression of EGFL6 was 0.19±0.03 and 0.91±0.11 in the experimental and control groups, respectively. siRNA-EGFL6 decreased the protein expression of EGFL6 in human bladder cancer 5637 cells in the experimental group. CCK8 results showed that the absorbance of the experimental group and the control group were 1.558±0.152 and 2.287±0.182, respectively. The results of subcutaneous tumor transplantation in nude mice showed that the volume of tumor in experimental group and control group was (1192.07±250.9) μm 3 and (2280.5±600.1) μm 3, respectively. The mass were (0.66±0.31) g and (1.52±0.48) g, respectively. The tumor volume and mass of the experimental group decreased after 4 weeks. The results of protein immunoblotting experiments revealed that the expression of p-P13K was 0.79±0.14 and 0.33±0.09 in the control and experimental groups, respectively, and the expression of p-AKT was 0.93±0.13 and 0.28±0.06, respectively, confirming that the expression of p-P13K and p-AKT were decreased in the experimental group of cells compared with the control group. Conclusion:The low expression of EGFL6 can inhibit the proliferation of human bladder cancer cell 5637 in vivo and in vitro through the P13K-AKT signaling pathway.
ABSTRACT
Objective:To establish the alcoholic fatty liver(AFL) animal models,and to explore the protective effect of Zhi Zi Da Huang Tang (ZZDHT) on the rats with AFL and its dosage.Methods:A total of 54 SD rats were divided into normal control group (n=10) and model control group (n=44).The rats in model control group were given alcohol by lavage (50% ethanol solution 6.0 mL·kg-1) combined with high-fat feed to establish the rat models of AFL.After 4 weeks,the rats in model control group were randomly divided into model group(treated with water),simvastatin group (10 mg·kg-1),low and high doses (5.0 and 10.0 g·kg-1) of ZZDHT groups,and there were 10 rats in each group.Every 2 weeks during the process,the body weights and levels of serum total cholesterol (TC),triglyceride (TG),aspertate aminotransferase (AST),and alanine aminotransferase (ALT) were detected.After 4 weeks,the body weights and liver weights of the rats were detected;the levels of TC,TG,AST,ALT,alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in serum and liver tissue of the rats of were detected;the levels of tumor necrosis factor-alpha (TNF-α),interleukin-6 (IL-6) and interleukin-1β(IL-1β) in serum of the rats were detected;the morphology of liver tissue was observed by HE staining and the pathological examination was performed.Results:Compared with normal control group,the levels of serum TC,AST,ALT,TG,TNF-α,IL-6,and IL-1β of the rats in model group 4 weeks after administration were significantly increased (P<0.05 or P<0.01).Compared with model group,the levels of serum TC,TG,AST,and ALT of the rats in low and high doses of ZZDHT groups 4 weeks after administration were decreased (P<0.05 or P<0.01);the levels of serum TNF-α and IL-6,IL-1β of the rats in high dose of ZZDHT group were decreased (P<0.01);the levels of serum TNF-α and IL-1β of the rats in low dose of ZZDHT group were decreased (P<0.01);the levels of serum ADH and ALDH in liver tissue of the rats in low and high doses of ZZDHT groups and simvastatin group were decreased(P<0.05 or P<0.01).The HE staining result showed that compared with model group,the pathological conditions of the liver tissue of the AFL rats in ZZDHT groups were significantly improved.Conclusion:ZZDHT can significantly improve the liver injury caused by high fat diet combined with alcohol and fat liver lesions.