ABSTRACT
@#ObjectiveTo investigate the effect of UHRF1 gene silencing on the progression of pancreatic adenocarcinoma (PAAD) cells. MethodsA total of 30 patients with PAAD who underwent pancreaticoduodenectomy in Department of Hepatobiliary Surgery, Leshan People’s Hospital, from July to December, 2018, were enrolled, and the non-necrotic PAAD tissue and the adjacent tissue were resected as samples. Semi-quantitative RT-PCR and Western blot were used to measure the mRNA and protein expression of UHRF1 in these samples. After the short-hairpin RNA (shRNA) plasmid vectors were constructed for the targeted silencing of the UHRF1 gene and were transfected into PAAD cells by the lentivirus infection method, MTT assay was used to evaluate cell proliferation, and Western blot was used to measure the expression of UHRF1, Bax, and Bcl-2. The independent samples t-test was used for comparison of continuous data between two groups; a one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsThe expression of UHRF1 in PAAD tissue was significantly higher than that in adjacent tissue (t=18.131, P＜0.001). Compared with the UHRF1 group, the UHRF1-shRNA group had a significant reduction in the protein expression of UHRF1 in PAAD cells (t=3.882, P=0.023), with inhibited cell proliferation at 12 and 36 hours after transfection (t=4.365 and 19042, P=0.005 and P＜0.001). Compared with the adjacent tissue group and the UHRF1 group, the UHRF1-shRNA group had a significant increase in the expression of the pro-apoptotic protein Bax (F=862.366, P＜0.001) and a significant reduction in the expression of the anti-apoptotic protein Bcl-2 (F=170.167, P＜0.001), suggesting that UHRF1 gene silencing had a pro-apoptotic effect. ConclusionThe expression of the UHRF1 gene is significantly upregulated in PAAD tissue, and UHRF1 gene silencing can inhibit the growth of PAAD tissue, possibly by inducing tumor cell apoptosis. UHRF1 may be a new molecular target for the treatment of PAAD.
ABSTRACT
Objective To study the effect of UHRF1 expression inhibition by RNA interference on the radiosensitivity of esophageal cancer cell line TE-1 and its mechanism.Methods Short hairpin RNA (shRNA) targeting UHRF1 gene was introduced into TE-1 cells by lentivector-mediated transfer.The cells were divided into three groups:non-transfected group,negative control (NC)-shRNA-transfected group,and UHRF1-shRNA-transfected group.The mRNA and protein expression levels of UHRF1 in TE-1 cells were measured by RT-PCR and Western blot before and after transfection.After transfection and X-ray radiation,the radiosensitivity of TE-1 cells was evaluated by colony formation assay; the cell cycle and cell apoptosis were determined by flow cytometry; the γ-H2AX (as a marker of DNA damage) level was measured by Western blot.Results After transfection with UHRF1-shRNA,the mRNA and protein expression levels of UHRF1 were significantly decreased in TE-1 cells,as compared with those in the NC-shRNA-transfected group and non-transfected group (0.11 vs 0.96 and 0.98,F =124.21,P =0.000;0.10 vs 0.89 and 0.94,F =125.25,P =0.000).The UHRF1-shRNA-transfected group had sensitization enhancement ratios of 1.53 (D0 ratio) and 1.95 (Dq ratio).X-ray radiation could cause G2/M arrest and increase apoptotic rate and γ-H2AX expression in TE-1 cells.Compared with the two control groups,the UHRF1-shRNA-transfected group showed significantly less G2/M arrest (F =500.15,P =0.000),a significantly higher apoptotic rate (F =100.10,P =0.000),and significantly higher residual γ-H2AX expression (F =61.00,P =0.000) at 24 hours after X-ray radiation.Conclusions RNA interference can effectively inhibit the UHRF1 expression and enhance the radiosensitivity of TE-1 cells.The mechanism may be related to cell cycle regulation,cell apoptosis,and DNA damage repair.