应用siRNA敲低TPM3基因表达后对大鼠胰腺癌细胞侵袭转移能力的影响

Influence of small interfering RNA targeting TPM3 gene on capacities of migration and invasion of rat pancreatic cancer cells

摘要:

目的：探究原肌球蛋白3（tropomyosin alpha-3 chain，TPM3）在大鼠胰腺癌细胞中对其侵袭转移能力的影响并初步阐述其可能的分子机制。方法：（1）将70只大鼠随机分为手术组40只[将7，12-二甲基苯并蒽（7，12-dimethyl-1，2-benzanthracene，DMBA]植入大鼠胰腺被膜下后荷包缝合被膜），假手术组15只（只对胰腺进行荷包缝合而后关腹，不置入DMBA），阴性对照组15只（不做任何处理），建立大鼠胰腺癌模型。（2）通过组织免疫组化染色的方法对差异蛋白TPM3进行验证。（3）通过机械分离和酶阶段消化法分离胰腺癌组织的方法获取大鼠胰腺癌细胞，体外传代培养获得纯度较高的细胞后，应用siRNA敲低大鼠胰腺癌细胞中TPM3基因的表达；通过RT-PCR技术检测其沉默效果。（4）通过Transwell实验和平板克隆实验分别对细胞的侵袭、迁移能力及生长增殖能力进行观察。结果：（1）经病理验证模型组有37.83%（14/37）形成胰腺癌，证明大鼠胰腺癌模型建立成功。（2）免疫组化验证胰腺癌组织中TPM3阳性率（92.8%）明显高于正常胰腺组织（6.7%）。（3）RT-PCR结果显示，转染TPM3-siRNA的细胞中TPM3的表达量（0.31±0.02）明显低于其余各组，脂质体组（0.45±0.02），阴性对照组（0.45±0.02），空白对照组（0.43±0.02）（P＜0.05），其余各组之间TPM3表达量差异无统计学意义（P ＞0.05）。（4）转染TPM3-siRNA的细胞其侵袭[（161.63±4.94）个]、迁移能力[（206.87±4.21）个]及生长增殖能力[（51.5±2.327）个]较对照组明显降低[分别为（39.7±1.40）个，（67.27±1.76）个，（5.900±0.767）个]（P＜0.05）。结论：体外化学合成的TPM3-siRNA可有效的抑制大鼠胰腺细胞TPM3的表达，TPM3表达降低后其侵袭能力、迁移能力及生长增殖能力下降。

Abstract:

Objective：To investigate the biological effects of TPM3 in rat pancreatic cancer cells and related molecular mechanisms during tumorigenesis. Methods：（1）Totally 70 SD rat were randomly divided into three groups：operation group（n=40，DMBA was di－rectly implanted into the pancreatic parenchyma），sham operation group（n=15，the pancreatic parenchyma was dissected，but no DMBA was implanted），blank control group（n=15，received no treatment）. An animal model with pathological characteristics of human pan－creatic cancer was established. （2）TPM3 was verified by immunohistochemistry. （3）Combination of mechanical separation and en－zyme digestion and separation was used to get the rat pancreatic cancer cells，and then the expression of TPM3 in rat pancreatic can－cer cells was knocked down by transfecting TMP3-siRNA into cancer cells. Expression of TPM3 was confirmed by RT-PCR. （4）Tran－swell membrane assays were used to examine migration and invasion potentials. Colony formation assay was used to test cell growth capacity. Results：（1）37.83%（14/37） rats in model groups generated pancreatic carcinoma with pathologically verification. （2）The in－creased level of TPM3 protein in rat pancreatic cancer was further confirmed by immunohistochemical（92.8%）. （3）RT-PCR result showed that，compared with other groups（liposome group（0.45±0.02），negative control group（0.45±0.02），blank control group（0.43±0.02）），the ratios of relative TPM3 expression in inter－ference group were significantly reduced（0.31±0.02）（P<0.05）. （4）Silencing of TPM3 leaded to significantly inhibited capaci－ties of invasion（161.63±4.94 and 39.7±1.40 respectively） and migration （206.87±4.21 and 67.27±1.76 respectively） in rat pancreatic cells，and colony formation of PC cells was suppressed after TPM3 knockdown（51.5±2.327 and 5.9００±0.767 respectively）（P＜0.05）. Conclusion：Overexpression of TPM3 will increase poten－tials of invasion or migration of cancer cells during tumorigenesis.