siRNA靶向沉默FLIP基因对前列腺癌PC3细胞的体外抑制作用

发布时间：2018-06-30 17:53

本文选题：FLIP + 前列腺癌　；参考：《南昌大学》2014年硕士论文

【摘要】：背景与目的： Fas相关死亡域样白介素1β转换酶抑制蛋白(cellular-FLICE inhibitoryprotein,c-FLIP)，简称FLIP，研究发现其过量表达于各类肿瘤细胞中，通过竞争性抑制Fas, TNFR-1, DR4及TRAILR等死亡受体介导的细胞凋亡途径中的caspase-8(半胱氨酸的天冬氨酸蛋白水解酶8)凋亡蛋白而阻止细胞凋亡程序，促使肿瘤细胞过量生长。FLIP过量表达于PCa细胞，抑制细胞凋亡，维持细胞过量生长，可能是PCa基因治疗的有效靶点。本研究旨在通过siRNA靶向沉默PC3细胞中FLIP基因的表达，探讨其作为PCa基因治疗靶点的可行性。方法：将体外培养的PC3细胞分为空白对照组(仅加培养基)，阴性对照组（NC-siRNA，脂质体）实验组(FLIPL-siRNA，脂质体)。QPCR检测PC3细胞FLIPLmRNA，caspase-8mRNA表达水平；western blot检测PC3细胞FLIPL蛋白表达水平；MTT法检测PC3细胞生长抑制率；流式细胞术检测PC3细胞凋亡率及Transwell模型检测PC3细胞侵袭性。结果： 1）FLIPL-siRNA转染PC3细胞后48h，QPCR检测FLIPLmRNA表达水平。相对于空白PC3组与阴性对照PC3组，实验组PC3细胞的FLIPLmRNA表达水平显著下降（P0.001）；siRNA-1，siRNA-2，siRNA-3和siRNA-4的抑制率分别为(69.9士2.2)%、(65.7士2.3)%、(75.4士1.6)%和(70.3士3.2)%，其中siRNA-3的抑制作用最强(P0.05）；而空白PC3组和阴性对照PC3组之间FLIPLmRNA的表达水平无显著差异(P0.05)。siRNA-3组的caspase-8mRNA表达水平较空白组和阴性对照组显著升高(P0.001)，且空白组与阴性对照组之间的mRNA表达水平差异无统计学意义(P0.05)。 2）FLIPL-siRNA转染PC3细胞后48h，Western blot法检测FLIPL蛋白表达水平。相对于空白PC3组，siRNA-3PC3组的FLIPL蛋白表达水平显著降低(P0.01)。 3）MTT，流式细胞术，Transwell模型检测发现siRNA-3对PC3细胞生长抑制率，凋亡率，侵袭性等生物学特征的影响有统计学意义（P0.01）。结论： 1）实验发现FLIP靶向特异性siRNA能在转录和翻译环节有效抑制PC3细胞中FLIP基因表达并诱导caspase8表达上调。 2）实验结果表明FLIPL-siRNA靶向沉默FLIPL基因可抑制PC3细胞生长，促进其凋亡并降低侵袭性。 3）实验结果提示FLIP基因可能是PCa基因治疗的有效靶点，，以FLIP基因为靶点的基因治疗可以成为现行PCa治疗方法的有效辅助治疗措施并提高PCa（尤其是AIPC）患者的生存率。
[Abstract]:Background and purpose:
Fas related death domain like interleukin 1 beta converting enzyme inhibitory protein (cellular-FLICE inhibitoryprotein, c-FLIP), referred to as FLIP, has been found to be overexpressed in various tumor cells by competitive inhibition of caspase-8 (cysteine aspartate protein water) in cell withering pathways mediated by Fas, TNFR-1, DR4 and TRAILR. Deapoptotic protein 8) apoptosis protein, which prevents cell apoptosis, excessively overgrowth of.FLIP in PCa cells, inhibits apoptosis and maintains cell growth, may be an effective target for PCa gene therapy. The aim of this study is to explore the expression of FLIP gene in the silent PC3 cells by targeting siRNA and explore the target as the target of PCa gene therapy. The feasibility of it.
Method:
The PC3 cells in vitro were divided into blank control group (only adding medium), and negative control group (NC-siRNA, liposome) test group (FLIPL-siRNA, liposome).QPCR was used to detect FLIPLmRNA, caspase-8mRNA expression level, Western blot detection of PC3 cell FLIPL protein expression level; MTT method was used to detect the inhibition rate of cell growth; flow cytometry was used. The apoptosis rate of PC3 cells was detected and Transwell cell model was used to detect the invasiveness of PC3 cells.
Result:
1) the expression level of FLIPLmRNA was detected by 48h and QPCR after transfection of FLIPL-siRNA to PC3 cells. The FLIPLmRNA expression level of PC3 cells in the experimental group was significantly lower than that in the blank PC3 group and the negative control group PC3 group (P0.001). SiRNA-1, siRNA-2, (65.7 and 2.3)%, (75.4 1.6)% and (70.3 3.2)%, respectively, (75.4) and (70.3 and 3.2)%, respectively. The inhibitory effect of 3 was the strongest (P0.05), while there was no significant difference in the expression level of FLIPLmRNA between the blank PC3 group and the negative control group (P0.05), the caspase-8mRNA expression level in the group.SiRNA-3 group was significantly higher than that in the blank group and the negative control group (P0.001), and there was no significant difference in the mRNA expression level between the blank group and the negative control group (P0.05).
2) after transfection of FLIPL-siRNA to PC3 cells, the expression level of FLIPL protein was detected by 48h and Western blot. The level of FLIPL protein expression in siRNA-3PC3 group was significantly lower than that in the blank PC3 group (P0.01).
3) MTT, flow cytometry, and Transwell model detection found that siRNA-3 had significant effects on the growth inhibition rate, apoptosis rate and invasiveness of PC3 cells (P0.01).
Conclusion:
1) it was found that FLIP targeted specific siRNA could effectively inhibit FLIP gene expression and induce the up regulation of caspase8 expression in PC3 cells during transcription and translation.
2) the experimental results showed that silencing of FLIPL gene by FLIPL-siRNA could inhibit the growth of PC3 cells, promote apoptosis and reduce invasiveness.
3) the experimental results suggest that FLIP gene may be an effective target for PCa gene therapy, and gene therapy targeting FLIP gene can be an effective adjuvant therapy for current PCa therapy and improve the survival rate of PCa (especially AIPC) patients.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25

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