Effect of miR-449a on radiosensitivity of pancreatic cancer cells / 中华放射肿瘤学杂志

Objective The purpose of this study is to investigate the effect of miR-449a on pancreatic cancer cells and the molecular mechanism. Methods The expression levels of miR-449a in pancreatic cancer cells treated or untreated with radiation was detected by qRT-PCR.High expression of miR-449a was achieved by transfecting miR-449a mimics into SW1990 cells. The cell growth,apoptosis and colony formation ability was assessed by MTT assay,flow cytometry and colony formation assay,respectively. The relationship of miR-449a and Cyclin D1 was determined by the TargetScan and dual luciferase reporter. Immunohistochemistry was used to examine protein levels of Cyclin D1 in pancreatic cancer and normal pancreas tissues. Si-Cyclin D1 was used to detecte the effect of Cyclin D1 on radiosensitivity of pancreatic cancer cells. Results The expression levels of miR-449a in pancreatic cancer cells with radiation treatment were decreased significantly. Mir-449a mimics increased the cell proliferation rates and apoptosis rates obviously,and decreased the colony formation ability in SW1990 cells treated with radiation. Results from the TargetScan and dual luciferase reporter showed that Cyclin D1 was the target of miR-449a. The positive staining rates of Cyclin D1 in pancreatic cancer tissue(85.7%,30/35)was higher than those in normal pancreas tissue(20%,2/10).Knockdown of Cyclin D1 enhanced the radiosensitivity of pancreatic cancer cells.Conclusion MiR-449a enhances the radiosensitivity of pancreatic cancer cells by targeting Cyclin D1.

Construction of pEGFP-C1-HSP27 recombinant eukaryotic expression vector and screening of human pancreatic cancer SW1990 cell line stably expressing HSP27 / 中华胰腺病杂志

Objective To construct pEGFP-C1-HSP27 recombinant eukaryotic expression vector and establish human pancreatic cancer SW1990 cell line stably expressing HSP27.Methods RT-PCR was applied to amplify human HSP27 cDNA from human pancreatic cancer SW1990 cells with a pair of specific primers carrying a restriction enzyme site BamH Ⅰ or Hind Ⅲ on each 5' end.HSP27 cDNA was inserted into pEGFP-C1 vector and then identified by restriction enzyme digestion and sequencing.Successful constructed pEGFP-C1-HSP27 or empty vector was transfected into SW1990 cells by lipofectamine 2000,respectively.The location of HSP72 was determined by fluoroscopy,RT-PCR and Western blot was used to detect the expression of HSP27 in transfected cell.Results The DNA sequence of pEGFP-C1-HSP27 recombinant plasmid was completely correct,and it was successfully transfected into SW1990 cell lines and stably transfected SW1990 cell lines were obtained,which were confirmed by restriction enzyme and sequencing.The expression of EGFP was distributed in cytoplasm,the HSP27mRNA expression was significantly increased (1.458 ± 0.160vs0.897 ±0.051,P ＜0.05).In addition,it was showed that EGFP-HSP27 fusion protein was expressed.Conclusions The eukaryotic expression vector pEGFP-C1-HSP27 was constructed successfully and stably transfected SW1990 cell line expressing HSP27 was obtained.