KISS-1基因对结肠癌细胞株HT29体外表达的影响

Anti-proliferation and inducing apoptosis effects of KISS-1 on HT29 colon cancer cells

目的 观察KISS-1基因对结肠癌细胞株HT29体外表达的作用和对其生物学功能的影响.方法 通过基因转染法使KISS-1在结肠癌细胞株HT29中稳定表达,应用噻唑蓝（MTT）法和Transwell侵袭实验检测KISS-1表达对结肠癌细胞株HT29的增殖和侵袭能力的作用和影响;通过免疫组织化学法和实时定量聚合酶链反应（Real-time PCR）技术检测KISS-1基因mRNA在体外结肠癌细胞株HT29中的表达水平,并用Real-time PCR测定转染后结肠癌细胞株HT29中c-fos、c-jun mRNA水平表达变化,并通过显微镜、透射电镜等观察凋亡细胞的形态学改变.结果 （1）Real-time PCR和免疫细胞组织化学证实转染后结肠癌细胞中有KISS-1稳定表达.（2）MTt法表明：培养0、24 h时各组HT29细胞数差异无统计学意义（P＞0.05）,48 h后,随着培养时间延长,转染组HT29细胞数明显低于空白对照组与阴性对照组（P＜0.05）,而空白对照组与阴性对照组HT29细胞数差异无统计学意义（P＞0.05）.（3） Transwell侵袭实验结果：转染KISS-1基因的结肠癌细胞HT29穿透Matrigel基底膜的细胞数显著低于空质粒转染组及空白对照组细胞（P＜0.01）,侵袭指数显著降低（P＜0.01）,侵袭力抑制率达到56.5％,显著低于空质粒转染组及空白对照组细胞（P＜0.01）.空质粒转染组及空白对照组细胞比较,侵袭穿膜细胞数差异无统计学意义（P＞0.05）.（4）KISS-1作用于结肠癌细胞株HT29 24 h后,形态学观察结肠癌细胞株HT29发生凋亡或坏死.（5）Real-time PCR：转染组c-jun和c-fos吸光度值为0.87 ±0.16和0.44±0.12,阴性对照组吸光度值为1.82±0.39和1.76±0.34,空白对照组吸光度值为1.65±0.36和1.58±0.31.与对照组比较,转染组c-jun和c-fos扩增产物条带吸光度值均显著降低（P＜0.01）.结论 KISS-1基因对体外结肠癌细胞株HT29具有抑制增殖和促进凋亡作用,其可能是通过负性调控信号通路降低结肠癌细胞中c-fos、c-jun的表达.

Objective To study the anti-proliferation and inducing apoptosis effects of KISS-1 on HT29 colon cancer cells.Methods Effects of the expression of the gene transfection method for detection of KISS-1 on colon cancer cell line HT29 anti proliferation and biological function;the stable expression in colon cancer cell line KISS-1 by HT29 gene transfection method, by detecting the expression level of KISS-1 gene mRNA immunohistochemical method and real-time quantitative polymerase chain reaction （Real-time PCR） technique in colon cancer cells in vitro in HT29, and the changes of c-fos, c-jun were determined by Real-time PCR mRNA HT29 expression level in colon cancer cell line transfected cells, apoptotic morphological changes under microscope, HE staining, transmission electron microscope.Results Real-time PCR and immune cell histochemistry confirmed after transfection of colon cancer primary KISS-1 stable expression have lesions and metastases in Real-time PCR transfected cells;confirmed colon cancer primary c-fos, c-jun mRNA levels of the lesion and metastatic cells decreased significantly （P 〈 0.01）.There was no significant difference in the number of HT29 cells in culture 0 and 24 h, and the number of HT29 cells in the transfected group was significantly lower than that in control group （P 〈 0.05）, while the number of HT29 cells in control group was significantly lower than that in control group （P 〉 0.05）.The effect of KISS-1 on HT29 24 h colon cancer cell line, morphological observation of colon cancer cell line HT29 apoptosis or necrosis.Real-time PCR confirmed that c-jun and mRNA c-fos levels （0.87 ± 0.16 and 0.44 ± 0.12） were significantly decreased in colon cancer primary lesion and metastasis cells （1.82 ± 0.39 and 1.76 ± 0.34, P 〈 0.01）.Conclusion KISS-1 gene can inhibit proliferation and promote apoptosis effect on colon cancer cells in vitro HT29, which may be decreased expression of colon cancer cells c-fos, c-jun through negative regulation of signal transduction pathway.