MicroRNA Expression Profiles and MiR-10a Target in Anti-benzo[a] pyrene-7,8-diol-9,10-epoxide-transformed Human 16HBE Cells

Objective To screen miRNA profiles of malignantly transformed human bronchial epithelial cells, 16HBE-T, induced by anti-benzo[a]pyrene-trans-7,8-diol-9,10-epoxide （anti-BPDE）, and to analyze putative miR-10a targets in 16HBE-T. Methods A novel microarray platform was employed to screen miRNA profiles of 16HBE-T cells transformed by anti-BPDE. Microarray data for miR-10a and miR-320 were validated using quantitative real time polymerase chain reaction （QRT-PCR）. The expression of a putative target for miR-10a, HOXA1, was analyzed by reverse transcription polymerase chain reaction （RT-PCR） and QRT-PCR. Results In comparison with the vehicle-treated cells （16HBE-N）, 16HBE-T exhibited differential expression of 54 miRNAs, in which, 45 were over-expressed and 9 were down-regulated. The five most highly expressed miRNAs were miR-494, miR-320, miR-498, miR-129, and miR-106a. The lowest expressed miRNAs were miR-10a, miR-493-5p, and miR-363＊. Three members of miR-17-92 cluster, miR-17-5p, miR-20a, and miR-92, showed significantly higher abundance in 16BHE-T as miR-21, miR-141, miR-27a, miR-27b, miR-16 and miRNAs of the let-7 family. The putative target for miR-10a, HOXA1 mRNA was up-regulated 3-9-fold in 16HBE-T, as compared with 16HBE-N. Conclusion The findings of the study provide information on differentially expressed miRNA in malignant 16HBE-T, and also suggest a potential role of these miRNAs in cell transformation induced by anti-BPDE. HOXA1 is similarly up-regulated, suggesting that miR-10a is associated with the process of HOXA 1-mediated transformation.

Overexpression of pim-3 and protective role in lipopolysaccharide-stimulated hepatic stellate cells

AIM: To investigate pim-3 expression in hepatic stellate cells(HSCs) stimulated by lipopolysaccharide(LPS), and its protective effect on HSCs. METHODS: Rat HSC-T6 cells were stimulated by LPS. The effect of LPS on proliferation and apoptosis of HSC-T6 cells was investigated by methyl thiazoyltetrazolium(MTT) assay and flow cytometry after annexin V-fluorescein isothiocyanate/propidium iodide double staining. pim-3 m RNA and protein were detected by reverse transcriptase polymerase chain reaction and Western blotting at 48 h when HSC-T6 cells were stimulated with 1 μg/m L LPS for 0, 3, 6, 12, 24 and 48 h. The cells without stimulation served as controls. To study the effect of pim-3 kinase on HSC-T6 cells, si-pim3(si RNA against pim-3) was transfected into HSC-T6 cells. HSC-T6 cells were subjected to different treatments, including LPS, si-pim3, or si-pim3 plus LPS, and control cells were untreated. Protein expression of pim-3 was detected at 48 h after treatment, and cell proliferation at 24 and 48 h by MTT assay. Apoptosis was detected by flow cytometry, and confirmed with caspase-3 activity assay.RESULTS: LPS promoted HSC-T6 cell proliferation and protected against apoptosis. Significantly delayed upregulation of pim-3 expression induced by LPSoccurred at 24 and 48 h for m RNA expression(pim-3/β-actin RNA, 24 or 48 h vs 0 h, 0.81 ± 0.20 or 0.78 ± 0.21 vs 0.42 ± 0.13, P < 0.05), and occurred at 12 h and peaked at 24 and 48 h for protein expression(pim-3/GAPDH protein, 12, or 24 or 48 h vs 0 h, 0.68 ± 0.12, 1.47 ± 0.25 or 1.51 ± 0.23 vs 0.34 ± 0.04, P < 0.01). pim-3 protein was ablated by si-pim3 and upregulated by LPS in HSC-T6 cells at 48 h after treatment(pim-3/GAPDH: si-pim3, si-pim3 plus LPS or LPS vs control, 0.11 ± 0.05, 0.12 ± 0.05 or 1.08 ± 0.02 vs 0.39 ± 0.03, P < 0.01). Ablation of pim-3 by si-pim3 in HSC-T6 cells partly abolished proliferation(OD at 24 h, si-pim3 group or si-pim3 plus LPS vs control, 0.2987 ± 0.050 or 0.4063 ± 0.051 vs 0.5267 ± 0.030, P < 0.01; at 48 h 0.4634 ± 0.056 or 0.5433 ± 0.031 vs 0.8435 ± 0.028, P < 0.01; si-pim3 group vs si-pim3 plus LPS, P < 0.01 at 24 h and P < 0.05 at 48 h), and overexpression of pim-3 in the LPS group increased cell proliferation(OD: LPS vs control, at 24 h, 0.7435 ± 0.028 vs 0.5267 ± 0.030, P < 0.01; at 48 h, 1.2136 ± 0.048 vs 0.8435 ± 0.028, P < 0.01). Ablation of pim3 with si-pim3 in HSC-T6 cells aggravated apoptosis(si-pim3 or si-pim3 plus LPS vs control, 42.3% ±1.1% or 40.6% ± 1.3% vs 16.8% ± 3.3%, P < 0.01; si-pim3 vs si-pim3 plus LPS, P > 0.05), and overexpression of pim-3 in the LPS group attenuated apoptosis(LPS vs control, 7.32% ± 2.1% vs 16.8% ± 3.3%, P < 0.05). These results were confirmed by caspase-3 activity assay.CONCLUSION: Overexpression of pim-3 plays a protective role in LPS-stimulated HSC-T6 cells.

阿是穴针刺放血治疗蛇伤局部肿痛临床疗效观察

Objective:To evaluate the clinical therapeutic effect of acupuncture bloodletting therapy on local swelling and pain of snakebite in the patients bitten by snakes.Methods:A total of 106 patients with trimeresurus stejnegeri bite were randomly divided into two groups,a conventional treatment group and a conventional treatment combined with bloodletting therapy group,53 cases in each one.In the conventional treatment group,the convention treatment of western medicine was adopted.In the conventional treatment combined with bloodletting therapy group,on the base of the conventional treatment,bloodletting therapy was applied at Ashi points selected at the tender points around the wound.The intervention and observation were performed not less than 7 days in two groups.Before and after treatment,swelling and pain degrees were measured and remission time of both the limb swelling and pain were recorded in the patients.Results:Compared with the values on day 1 of treatment,swelling degree and visual analogue scale(VAS) score of the upper and lower limbs were all lower on day 3 and day 7 of treatment in the patients of the two groups(all P <0.05).Compared with the conventional treatment group,swelling degree and VAS score of the upper and lower limbs were all lower in the values of the conventional treatment combined with bloodletting therapy group on day 3 and day 7 of treatment respectively(all P <0.05).The remission time of either limb swelling or pain in the patients of the conventional treatment combined with bloodletting therapy group was shorter than the conventional treatment group respectively(both P <0.05).Conclusion:Acupuncture bloodletting therapy can effectively relieve the local swelling and pain caused by snakebite,promote the recovery of limb function,shorten the treatment course and improve the clinical therapeutic effect.