miR-762在胰腺癌中的表达及对胰腺癌细胞生物学行为的影响

背景与目的：miR-762在多种恶性肿瘤中存在表达异常，参与肿瘤的增殖、凋亡及侵袭转移。观察miR-762在胰腺癌组织和细胞系中的表达及对胰腺癌细胞增殖、侵袭转移的影响。方法：采用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）技术检测于河北医科大学第四医院行胰腺癌根治术的胰腺癌组织和细胞株中miR-762的表达。通过Lipofectamine ^TM 2000将miR-762模拟物（mimics）、miR-762抑制物（inhibitors）及其阴性对照序列（scramble序列）分别转染胰腺癌PANC-1细胞。采用细胞计数试剂盒（cell counting kit-8，CCK-8）实验检测细胞增殖；采用流式细胞术检测细胞凋亡；采用划痕实验和Transwell侵袭实验检测细胞侵袭转移能力；采用蛋白质印迹法（Western blot）检测上皮-间质转化（epithelial-mesenchymal transformation，EMT）相关分子标志物表达。结果：胰腺癌组织中miR-762 mRNA表达量显著高于癌旁组织（P<0.01）。胰腺癌细胞株BxPC-3、PANC-1、AsPC-1、SW-1990中miR-762 mRNA的表达量也显著高于正常胰腺上皮细胞HPDE（P<0.01）。转染miR-762 mimics后PANC-1细胞miR-762 mRNA表达量显著增加，而转染miR-762 inhibitors后PANC-1细胞miR-762 mRNA表达量显著降低（P<0.01）。同时miR-762 mimics组450 nm处的吸光度（D ₄₅₀ ）值、细胞迁移距离和穿膜细胞数及间质表型细胞标志物N-钙黏蛋白（N-cadherin）、波形蛋白（vimentin）表达量显著增加，细胞凋亡率及上皮细胞标志物E-钙黏蛋白（E-cadherin）表达量显著降低；而miR-762inhibitors组D ₄₅₀ 、细胞迁移距离和穿膜细胞数及间质表型细胞标志物N-cadherin、vimentin表达量显著降低，细胞凋亡率及上皮细胞标志物E-cadherin表达量显著增加（P<0.05）。结论：miR-762在胰腺癌组织和细胞株中高表达，上调miR-762表达可能通过调控N-cadherin、vimentin、E-cadherin表达促进EMT进程，从而增强PANC-1细胞的增殖和侵袭转移能力。

Expression of miR-762 in pancreatic cancer and its effect on biological behavior of pancreatic cancer cells

Background and purpose: The abnormally expressed miR-762 in various malignant tumors is involved in the proliferation, apoptosis, invasion and metastasis of tumors. Aim of the present study was to study the expression of miR-762 in pancreatic cancer tissues and cell lines and its effect on the proliferation, metastasis and invasion of pancreatic cancer cells. Methods: Real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) was used to detect the expression of miR-762 mRNA in pancreatic cancer tissues who received radical resection of pancreatic cancer in the Fourth Hospital of Hebei Medical University and cell lines. The miR-762 mimics, miR-762 inhibitors and scramble sequences were transfected into PANC-1 cells with Lipofectamine ^TM 2000. The proliferation, apoptosis and migratory and invasive abilities of PANC-1 cells were detected with cell counting kit-8 (CCK-8) assay, flow cytometry, wound healing assay and Transwell assay. The expression of epithelial-mesenchymal transition (EMT) related markers were determined by Western blot. Results: The miR-762 expression was significantly up-regulated in pancreatic cancer tissues compared with paracancerous tissues (P<0.01). Similarly, miR-762 expressions of the four pancreaticcancer cell lines (BxPC-3, PANC-1, AsPC-1 and SW-1990) were significantly higher than those in the normal pancreatic ductal epithelial cells HPDE (P<0.01). miR-762 mRNA expression in PANC-1 cell line was obviously increased after transfection with miR-762 mimics, but markedly decreased after transfection with miR-762 inhibitors (P<0.01). Meanwhile, D ₄₅₀ , the healing rate, invasive cells and expressions of N-cadherin and vimentin proteins in miR-762 mimics group were significantly higher than those in negative control group after transfection, and apoptotic rate and E-cadherin protein expression were significantly lower than those in negative control group after transfection. D ₄₅₀ , the healing rate, invasive cells and expressions of N-cadherin and vimentin in miR-762 inhibitors group were significantly lower than those in negative control group after transfection, while apoptotic rate and E-cadherin expression were significantly higher than those in negative control group after transfection (P<0.05). Conclusion: The over-expression of miR-762 can effectively enhance the proliferation, metastasis and invasion ability in PANC-1 cells, in which EMT related markers including N-cadherin, vimentin and E-cadherin may play a role.