非对称RNA干扰抑制CDCA5基因表达对结直肠癌细胞增殖的影响
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摘要
目的探讨非对称抑制CDCA5基因的表达对人结直肠癌细胞增殖的影响,并探讨其可能的作用机制。方法收集2016年～2017年南充市中心医院行根治性手术的20例结直肠癌患者的癌组织标本及匹配的癌旁正常组织, Western blot检测结直肠癌组织和癌旁正常组织中CDCA5蛋白的表达。同时,Western blot检测结直肠癌细胞系HT29、HCT116及SW480细胞中CDCA5蛋白的表达,选择表达水平最高的细胞系进行后续研究。设计针对CDCA5基因的siRNA,阴性对照siRNA-NC及三条长短不同的非对称小干扰RNA,即aiRNA(15/21)、aiRNA(16/21)和aiRNA(17/21)。PCR检测各组对CDCA5基因的沉默效果。MTT检测siRNA、siRNA-NC和aiRNA(15/21)组的细胞增殖能力,流式细胞仪检测各组细胞周期的变化。结果 CDCA5在结直肠癌组织中的表达明显高于癌旁正常组织(P<0.05)。人结直肠癌细胞系HCT116中CDCA5蛋白的表达明显高于HT29和SW480细胞系(P<0.05)。aiRNA(15/21)在各组中有最强的基因沉默效果(P<0.05)。与siRNA和siRNA-NC组相比,aiRNA(15/21)组细胞增殖能力低于其他两组,处于细胞周期G_2/M期的细胞数量显著多于其他两组(均P<0.05)。结论 CDCA5可能是治疗结直肠癌的一个重要靶点,基于CDCA5基因的特异性非对称aiRNA(15/21)干扰在沉默效率上优于传统siRNA。
Objective To investigate the effect of the expression of the target silencing CDCA5 gene on the proliferation of human colorectal cancer cells and its possible mechanism. Methods Western blot was used to detect the expression of CDCA5 protein in colorectal cancer tissues, adjacent normal tissues and human colorectal cancer cell lines HT29, HCT116 and SW480 cells. siRNA for CDCA5 gene, negative control siRNA-NC and three different asymmetric small interference RNA, namely aiRNA(15/21), aiRNA(16/21) and aiRNA(17/21)were designed. The effect of CDCA5 gene silencing was detected by PCR. MTT assay was used to detect the proliferation of siRNA, siRNA-NC and aiRNA(15/21) cells. Flow cytometry was used to detect the changes of cell cycle in three groups. Results The expression of CDCA5 in colorectal cancer tissues was significantly higher than that of normal tissues adjacent to the cancer(P<0.05). The expression of CDCA5 protein in human colorectal cancer cell line HCT116 was significantly higher than that of HT29 and SW480 cell lines(P<0.05). AiRNA(15/21) had the strongest gene silencing effect in each group(P<0.05). Compared with siRNA and siRNA-NC group, the proliferation ability of aiRNA(15/21) group was significantly lower than that of the other two groups, and the number of cells in the G2/M phase of cell cycle was significantly more than that of the other two groups(all P<0.05). Conclusion CDCA5 may be an important target for the treatment of colorectal cancer. The specific asymmetric aiRNA(15/21) interference based on CDCA5 gene is superior to traditional siRNA in the efficiency of silence.
Objective To investigate the effect of the expression of the target silencing CDCA5 gene on the proliferation of human colorectal cancer cells and its possible mechanism. Methods Western blot was used to detect the expression of CDCA5 protein in colorectal cancer tissues, adjacent normal tissues and human colorectal cancer cell lines HT29, HCT116 and SW480 cells. siRNA for CDCA5 gene, negative control siRNA-NC and three different asymmetric small interference RNA, namely aiRNA(15/21), aiRNA(16/21) and aiRNA(17/21)were designed. The effect of CDCA5 gene silencing was detected by PCR. MTT assay was used to detect the proliferation of siRNA, siRNA-NC and aiRNA(15/21) cells. Flow cytometry was used to detect the changes of cell cycle in three groups. Results The expression of CDCA5 in colorectal cancer tissues was significantly higher than that of normal tissues adjacent to the cancer(P<0.05). The expression of CDCA5 protein in human colorectal cancer cell line HCT116 was significantly higher than that of HT29 and SW480 cell lines(P<0.05). AiRNA(15/21) had the strongest gene silencing effect in each group(P<0.05). Compared with siRNA and siRNA-NC group, the proliferation ability of aiRNA(15/21) group was significantly lower than that of the other two groups, and the number of cells in the G2/M phase of cell cycle was significantly more than that of the other two groups(all P<0.05). Conclusion CDCA5 may be an important target for the treatment of colorectal cancer. The specific asymmetric aiRNA(15/21) interference based on CDCA5 gene is superior to traditional siRNA in the efficiency of silence.
引文
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[2] Siegel R,Desantis C,Jemal A.Colorectal cancer statistics[J].CA Cancer J Clin,2014,64(2):104-117.
[3] Siegel RL,Miller KD,Jemal A.Cancer statistics[J].CA Cancer J Clin,2015,65(1):5-29.
[4] Armaghany T,Wilson JD,Chu Q,et al.Genetic alterations in colorectal cancer[J].Gastrointest Cancer Res,2012,5(1):19-27.
[5] Zhang N,Pati D.Sororin is a master regulator of sister chromatid cohesion and separation[J].Cell cycle,2012,11(11):2073-2083.
[6] Zhang N,Pati D.Handcuff for sisters:A new model for sister chromatid cohesion[J].Cell cycle,2009,8(3):399-402.
[7] Cheng IW,Lin VC,He HL,et al.CDCA5 overexpression is an indicator of poor prognosis in patients with urothelial carcinomas of the upper urinary tract and urinary bladder[J].Am J Transl Res,2015,7(4):710-722.
[8] Nguyen MH,Koinuma J,Ueda K,et al.Phosphorylation and activation of cell division cycle associated 5 by mitogen-activated protein kinase play a crucial role in human lung carcinogenesis[J].Cancer Res,2010,70(13):5337-5347.
[9] Kuo HS,Hsu FN,Chiang MC,et al.The role of CdkS in retinoic acid-induced apoptosis of cervical cancer cell line[J].Chin J Physiol,2009,52(1):23-30.
[10] Hsu FN,Chen MC,Chiang MC,et al.Regulation of androgen receptor and prostate cancer growth by cyclin-dependent kinase 5[J].J Biol Chem,2011,286(38):33141-33149.
[11] Hwang J,Chang C,Kim JH,et al.Development of Cell-Penetrating Asymmetric Interfering RNA Targeting Connective Tissue Growth Factor[J].J Invest Dermatol,2016,136(11):2305-2313.
[12] Jin Y,Chen W,Yang H,et al.Scutellaria barbata D.Don inhibits migration and invasion of colorectal cancer cells via suppression of PI3K/AKT and TGF-β/Smad signaling pathways[J].Exp Ther Med,2017,14(6):5527-5534.
[13] Sano M,Sierant M,Miyagishi M,et al.Effect of as etric terminal structures of short RNA duplexes on the RNA interference activity and strand selection[J].Nucleic Acids Res,2008,36(18):5812-5821.
[14] Sun X,Rogoff HA,Li CJ.Asymmetric RNA duplexes mediate RNA interference in mammalian cells[J].Nat Biotechnol,2008,26(12):1379-1382.
[15] Norihiko T,Koh-ichi N,Hirshi T,et al.Therapeutic potential of targeting cell division cycle associated 5 for oral squamous cell carcinoma[J].Oncotarget,2016,7(3):2343-2353.
[16] Wang L,Tang H,Thayanithy V,et al.Gene networks and microRNAs implicated in aggressive prostate cancer[J].CancerRes,2009,69(24):9490-9497.
[17] Cavard C,Colnot S,Audard V,et al.Wnt /beta-catenin pathway in hepatocellular carcinoma pathogenesis and liver physiology[J].Future Oncol,2008,4(5):647-660.
[18] Rankin S,Ayad NG,Kirschner MW.Sororin,a substrate of the anaphase-promoting complex,is required for sister chromatid cohesion in vertebrates[J].Molecular Cell,2005,18(2) :185-200.
[19] Schmitz J,Watrin E,Lenart P,et al.Sororin is required for stable binding of cohesin to chromatin and for sister chromatid cohesion in interphase[J].Current Biology,2007,17(7):630-636.
[20] Nishiyama T,Ladurner R,Schmitz J,et al.Sororin mediates sister chromatid cohesion by antagonizing Wapl[J].Cell,2010,143(5):737-749.