抑制miR-29对胰腺癌PANC1细胞生长、侵袭和转移的影响

Inhibiting miR-29 on growth, invasion and metastasis of PANC1 cells

目的观察抑制miR-29对胰腺癌PANC1细胞生长、侵袭和转移能力的影响，探讨其可能机制。方法以抑制miR-29表达的寡核苷酸（anti-miR-29）及对照寡核苷酸（miR-NC）转染PANC1细胞，构建anti-miR-29-PANC1细胞及miR-NC-PANC1细胞，并采用瞬时转染PUMA-siRNA、E-cadherin-siRNA或NC-siRNA方法构建共转染的anti-miR-29＋PUMA-siRNA-PANC1细胞及anti-miR-29＋E-cadherin-siRNA-PANC1细胞。观察各组细胞的克隆形成数，MTT法检测细胞存活率，流式细胞仪检测细胞凋亡，Transwell小室检测细胞侵袭能力，划痕试验检测癌细胞迁移能力。采用anti-miR-29-PANC1细胞皮下注射建立裸鼠移植瘤模型，静脉注射建立肺转移模型，以注射PANC1细胞作为对照。观察移植瘤的生长情况及肺转移结节数量，TUNEL法检测移植瘤的细胞凋亡，免疫组织化学法检测移植瘤的PUMA、E-cadherin表达。结果PANC1、miR-NC-PANC1、anti-miR-29-PANC1组的细胞存活率分别为100%、（96.8±2.8）%、（24.4±3.2）%；细胞克隆形成数为（213±36）、（196±28）、（37±6）个/100倍视野；穿膜细胞数为（56.3±9.6）、（49.8±7.3）、（11.2±3.4）个/400倍视野；细胞的迁移距离为（260±48）、（247±46）、（53±7）μm；细胞凋亡率分别为（1.5＋0.9）%、（2.6＋0.9）%、（22.4＋2.8）%，anti-miR-29-PANC1组细胞与其他2组差异均有统计学意义（P值均〈0.05）。anti-miR-29＋PUMA-siRNA-PANC1组的细胞存活率为（84.7±10.9）%，细胞凋亡率为（1.3±0.8）%；anti-miR-29＋E-cadherin-siRNA-PANC1的穿膜细胞数为（49.7±6.4）个/400倍视野，细胞迁移距离为（182±36）μm，均消除抑制miR-29表达对PANC1细胞所带来的影响（P值均〈0.05）。PANC1、anti-miR-29-PANC1细胞移植瘤的体积分别为（3 800±270）、（1 890±160）mm3，细胞凋亡指数为0.93±0.14、8.26±1.15，肺转移结节为（26.4±6.5）、（8.6±2.7）个，PUMA阳性表达率为（7.2±1.6）%、（43.8±7.6）%，E-cadherin阳性表达率为（8.3±3.6）%、（47.4±5.7）%。anti-miR-29-PANC1组移植瘤体积、肺转移结节较对照组显著减小或减少，而细胞凋亡、PUMA及E-cadherin表达显著增加，差异均有统计学意义（P值均〈0.05）。结论抑制miR-29表达后PANC1细胞的增殖、侵袭、转移能力下降，其机制可能与上调PUMA及E-adherin表达有关。

ObjectiveTo investigate the effects of inhibiting miR-29 on growth, invasion and metastasis of pancreatic cancer PANC1 cells, and explore the potential mechanism.MethodsOligonucleotides inhibiting miR-29 （anti miR-29） and control oligonucleotides （miR NC） were used to transfect PANC1 cells to establish anti miR-29 PANC1 cells and miR NC PANC1 cells. Transient transfection of PUMA siRNA, E-cadherin siRNA or NC siRNA was used to construct cotransfected anti miR29＋ PUMA-siRNA-PANC1 cells and anti-miR-29＋ E-cadherin-siRNA-PANC1 cells. Number of colony formations was observed, cell survival was detected by MTT, cell apoptosis was measured by flow cytometry, cell invasion was detected by transwell chamber assay, and cell migration was detected by wound healing assay. Subcutaneous injection of anti miR-29 PANC1 cells was used to establish xenograft nude mice model, and venous injection of anti miR-29 PANC1 cells was used to establish lung metastasis nude mice model, and the subcutaneous and venous injection of PANC1 cells served as control. The growth of xenograft and the number of lung metastatic nodules were observed. TUNEL method was used to detect cell apoptosis in xenograft and immunohistochemical analysis was used to detect PUMA and E-cadherin in xenograft.ResultsThe survival rate of PANC1, miR-NC-PANC1 and anti-miR-29-PANC1 cells was 100%, （96.8±2.8）% and（24.4±3.2）%. The number of colony formation was （213±36）, （196±28） and（37±6）per 100 high power field. The number of transmembrane cells was （56.3±9.6）, （49.8±7.3） and （11.2±3.4） per 400 high power field. The distance of cell migration was（260±48）, （247±46） and（53±7）μm. Cell apoptosis rate was（1.5＋ 0.9）%, （2.6＋ 0.9）% and（22.4＋ 2.8）%. There was statistically significant difference between anti miR 29 PANC1 cells and other PANC1 cells （P〈0.05）. The survival rate, apoptosis rate, transmembrane cells and migration distance of anti-miR-29＋ PUMA-siRNA-PANC1 cells was （84.7±10.9）%, （1.3±0.8）%, （49.7±6.4）per 400 high power field and（182±36）μm, indicating that the effects of miR 29 inhibition on PANC1 cells were abolished （all P〈0.05）. The volume of the xenograft of PANC1 and anti-miR-29-PANC1 cells was （3 800±270） and （1 890±160）mm3, the cell apoptosis rate was 0.93±0.14 and 8.26±1.15, the number of metastatic lung lesions was （26.4±6.5） and（8.6±2.7）, the PUMA positivity was（7.2±1.6）% and（43.8±7.6）%, E-cadherin positivity was（8.3±3.6）% and（47.4±5.7）%, respectively. The xenograft volume and the number of metastatic lung nodules of anti miR29 PANC1 cells was obviously decreased or decreased, but cell apoptosis rate, PUMA positivity and E cadherin positivity were obviously increased, and the differences were all statistically significant （P〈0.05）.ConclusionsInhibiting miR-29 expression can decrease cell proliferation, migration and metastasis of PANC1 cells, and the potential mechanism may be associated with the upregulation of PUMA and E-cadherin.