微小RNA-132对胰腺癌SWl990细胞增殖及凋亡的影响

Effects of microRNA-132 on the proliferation and apoptosis in human pancreatic cancer cells SW1990

目的观察微小RNA-132（miR-132）转染人胰腺癌细胞株SWl990后对细胞增殖及凋亡的影响，并探讨其作用机制。方法采用实时荧光定量PCR（RT-qPCR）法检测28例胰腺癌及匹配的癌旁正常胰腺组织miR-132的表达。采用脂质体将miR-132转染SWl990细胞，以未转染及转染错义miR．132的细胞分别作为空白对照和阴性对照。应用CCK-8法、DAPI染色法检测细胞的增殖及凋亡；将转染细胞种植于裸鼠皮下成瘤，应用TUNEL法检测种植瘤细胞凋亡；免疫组化法检测转染细胞的mucin-4、HER-2、P-FAK蛋白表达及种植瘤组织mucin-4、Ki-67蛋白表达。结果28例胰腺癌及癌旁正常胰腺组织miR．132的相对表达量分别为0．46±0．11和1．24±0．36，差异有统计学意义（P〈0．05）。转染细胞的miR-132表达量为2．95±0．46，显著高于阴性对照组的0．84±O．17（P〈0．05）；转染组细胞培养48、72、96h时的存活率分别为阴性对照组56．5％、44．7％、37．4％（P值均〈0．05）；细胞凋亡率为41．6％，显著高于阴性对照组的5．7％（P〈0．05）；转染细胞mucin-4、HER-2、p-FAK蛋白的表达较阴性对照组显著下调（0．76±0．1gLE2．94±0．42，0．34±0．04比1．75±O．33，0．27±0．03比2．74±O．24，P值均〈0．01）。与阴性对照组比较，转染组裸鼠皮下移植瘤生长明显被抑制[（0．23±O．05）g比（0．59±0．13）g，P〈0．05]，瘤内肿瘤细胞凋亡明显增加[（21．7±4．7）％LE（5．2-t-O．7）％，P〈0．05]，mucin-4和Ki-67蛋白表达显著下调（64．35±7．16比281．34±36．62，30．75±4．61LLl48．05±21．54，P值均〈0．01）。而阴性对照组与空白对照组间的差异均无统计学意义。结论转染miR-132对胰腺癌SWl990细胞有显著的增殖抑制和凋亡诱导作用，其机制可能与下调mucin-4、HER-2、p-FAK等蛋白的表达有关。

Objective To observe the effect of miR-132 transfection on proliferation and apoptosis of pancreatic cancer SW1990, and to explore the underlying mechanism. Methods The expression of miR-132 in the pancreatic cancer tissue and the adjacent tissues in 28 pancreatic cancer patients were detected by real- time quantitative polymerase chain reaction （RT-qPCR）. miR-132 was transfected into SW1990 cells by using liposome method, untransfected cells and cells with missense miR-132 transfection were used as black control and negative control. The proliferation and apoptosis was detected by CCK-8 assay and DAPI staining. The transfected cells were implanted in nude mice as xenograft tumor, and TUNEL was used to detect the apoptosis; immunohistochemistry was used to detect the expression of Ki-67 and mucin-4 protein in the xenograft tumors and mucin-4, HER-2, p-FAK protein in transfected cells. Results The expression levels of miR-132 in pancreatic cancer tissue and adjacent tissues were 0.46 ~ 0.11 and 1.24 ± 0.36, and the difference between the two groups was statistically significant （P 〈 0.05 ）. The expression level of miR-132 in transfeeted SW1990 ceils was 2.95 ± 0.46, which were significantly higher than those in negative control （ 0.84 ± 0.17 ） ; the survival rate of transfected cells at 48, 72, 96 h was 56.5% , 44.7% , 37.4% of negative control cells. The apoptosis rate in transfected ceils was 41.6% , and the corresponding value was 5.7% in negative control, and the difference was statistically significant （P 〈 0.05 ）. The expression levels of mucin-4, HER-2, p-FAK in nagative control were 2.94 ± 0.42, 1.75 ± 0.37 and 2.74 ± 0.24, and the corresponding values in transfected cells were 0.76 ± 0.14, 0.34 ± 0.04 and 0.27 ± 0.03, and the difference between the two groups was statistically significant （ P 〈 0.05 ）. In vivo, the growth of xenograft tumors in transfected nude mice was significantly inhibited [（0.23 ± 0.05 ） vs （0.59 ± 0.13 ） g, P 〈 O. 05 ], the apoptosis of xenograft tumor cells was significant increased [ （21.7 ± 4.7 ） % vs （ 5.2±0.7 ） % , P 〈 0.05 1. The expressions of mucin-4 and Ki-67 protein in nagative control was 281.34 ± 36.62 and 148.05 ± 21.34, and the corresponding values in transfection group were 64.35 ± 7.16 and 30.75 ± 4.61, and the difference was statistically significant （P〈O. 05）. Conclusions miR-132 transfection has an effect of inhibiting proliferation and promoting apoptosis on SW1990 cells, and the mechanism may be down-regulation of mucin-4, HER-2, p-FAK protein rxpression.