siRNA干扰人宫颈癌基因(HCCR)的表达对胰腺癌PANC1细胞增殖?凋亡和侵袭的影响

目的:建立稳定转染人宫颈癌癌基因(human cervical cancer oncogene,HCCR)siRNA真核表达质粒的人胰腺癌PANC1细胞株,探讨siRNA干扰HCCR的表达后对人胰腺癌PANC1细胞增殖、凋亡和侵袭的影响.方法:通过脂质体转染法将含HCCRsiRNA的真核表达质粒pGCsi-HCCR稳定转染至人胰腺癌细胞株PANC1,抗生素G418筛选获得稳转细胞株;Western blot检测PANC1细胞中HCCR的表达,同时检测肿瘤相关基因p53蛋白表达的变化;流式细胞仪检测PANC1细胞的细胞周期和凋亡率变化;MTT比色法检测siRNA干扰后对PANC1细胞增殖能力的影响:Transwell侵袭实验观察siRNA干扰后对PANC1细胞侵袭能力的影响.结果:Western blot证实siRNA稳转组的PANC1细胞株和空载体稳转组比较HCCR蛋白表达水平下调,稳转细胞株建立成功.siRNA稳转组p53蛋白表达下降.siRNA稳转组的S期细胞数目减少而G0/G1期细胞数目增加,细胞凋亡增加.MTT结果显示siRNA稳转组1和稳转组2细胞吸光度分别为空载体组细胞的0.65倍和0.68倍,细胞增殖能力下降.Transwell侵袭实验显示siRNA稳转组细胞和空载体组细胞穿膜数分别为24.4±9.9和49.1±15.4(P＜0.01),稳转组细胞侵袭能力下降.结论:siRNA干扰HCCR的表达后能抑制胰腺癌PANC1细胞增殖和侵袭,促进其凋亡.

The effect of down-regulating the expression of HCCR gene by siRNA on cell proliferation, invasion and apoptosis in pancreatic cancer PANC1 cells

Objective:To obtain the PANC1 pancreatic cell line which was stably transfected with HCCR siRNA plasmid and investigate the effect of proliferation and invasion on PANC1 cell by knockdown of HCCR expression through RNA interference. Methods:The siRNA targeting HCCR was designed and the plasmid of pGCsi-HCCR was synthesized. Plasmid of pGCsi-HCCR was transfected into PANC1 cells. The vector pGCsi was used as the negtive control. G418-resistant clone was selected and obtained. Proteins of the stable-transfected cell lines was detected by western blot using HCCR antibody. p53 protein in the stably transfected cell lines was also detected. Flow cytometry(FCM), MTT and Transwell assay were examined to observe the apoptotic rate, proliferation and invasion of HCCR siRNA transfectant. Results:Plasmid of pGCsi-HCCR can inhibit the expression of HCCR. The protein level of P53 was decreased in HCCR siRNA cells compared with the vector cells. Plasmid of pGCsi-HCCR can induce a more significant G0/G1 stage arrest and higher apoptotic rate compared with control cells. The proliferation of HCCRsiRNA cells were decreased by 0.65 times and 0.68 times in HCCRsiRNA cells compared with control cells. The number of invasion cells were 24.4 ± 9.9 and 49.1 ± 15.4 respectively, which were significant difference. Conclusion:Down-regulating the expression of HCCR gene by siRNA can inhibit proliferation and invasion and induce apoptosis in PANC1 cells.