靶向热休克蛋白-27基因沉默对5-氟尿嘧啶诱导SW480细胞凋亡的影响及机制

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摘要目的：通过RNA干扰抑制热休克蛋白-27（HSP27）基因的表达，探讨沉默该基因对5-氟尿嘧啶（5-FU）诱导大肠癌SW480细胞凋亡的影响及机制。方法：将细胞分为正常对照组（Normal组）、阴性siRNA转染组（NC组）、HSP27-siRNA转染组（siRNA组）、单纯5-FU组（5-FU组）、5-FU+阴性siRNA转染组（NC+5-FU组）、5-FU+HSP27-siRNA转染组（siRNA+5-FU组），通过脂质体LipofectamineTM 2000将HSP27-siRNA导入SW480细胞，CCK-8法检测靶向HSP27的siRNA和5-FU对SW480细胞增殖的抑制作用，流式细胞技术观察转染后细胞在5-FU作用下的早期凋亡情况，Western blot方法检测凋亡相关蛋白Active-casepase-3、Active-caspase-9及细胞色素C（Cytc）的表达。结果：CCK-8法检测结果显示siRNA+5-FU组细胞增殖抑制率明显高于同期siRNA组和5-FU组，表明联合应用抑制率明显升高（P＜0.05）。流式细胞仪检测结果表明siRNA组和5-FU组均可诱导SW480细胞凋亡，两者联合应用细胞早期凋亡率明显升高，与Normal组及NC组比较，差异均有统计学意义（均P＜0.05）；Western blot检测结果显示siRNA+5-FU组Active-casepase-3、Active-caspase-9、Cytc蛋白相对表达水平均高于siRNA组和5-FU组，差异均有统计学意义（均P＜0.05）。结论：靶向HSP27-siRNA能有效抑制大肠癌SW480细胞HSP27蛋白的表达，上调Active-casepase-3、Active-caspase-9、Cytc蛋白的表达，增强5-FU诱导SW480细胞凋亡，逆转肿瘤细胞对5-FU的耐药。

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	黄崇杰
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关键词 ：结直肠肿瘤, 热休克蛋白-27, RNA干扰, 5-氟尿嘧啶, 细胞凋亡

Abstract：Objective: To explore the effect and mechanism of 5-FU-induced apoptosis of colon cancer SW480 cells with HSP27 silenced by siRNA interference. Methods: Six groups of cells were used in this part of research, including Normal control group (Normal group), negative siRNA group (NC group), HSP27 siRNA group (siRNA group), 5-FU group, 5-FU with negative siRNA group (NC+5-FU group), 5-FU with HSP27 siRNA group (siRNA+5-FU group). HSP27-siRNA was transfected into SW480 cells by lipofectamine 2000. CCK-8 method was used to detect the siRNA targeted on HSP27 and 5-FU’s inhibition effect on SW480 cell proliferation. Early apoptotic rate was measured with flow cytometry technique, and Western blot analysis was applied to detect the expression of apoptosis-related protein of Active-casepase-3, Active-caspase-9 and Cytc. Results: Result detected by CCK-8 showed that the cell proliferation inhibition rate of siRNA+5-FU group was obviously higher than that of 5-FU group, which indicated that combined use of 5-FU and siRNA could significantly improve the inhibition rate (P<0.05). Result detected with flow cytometry (FCM) showed that both siRNA group and 5-FU group could induce SW480 cell apoptosis, and the combined use of 5-FU and siRNA could greatly improve the early apoptotic rate. Comparing the siRNA and 5-FU group with Normal and NC group, the difference had statistically significant (P<0.05). Western blot analysis showed that the expression level of Active-casepase-3, Active-
caspase-9 and Cytc in combination group were higher than that of siRNA group and 5-FU group, the difference had statistically significant (P<0.05). Conclusion: HSP27-siRNA can significantly inhibit HSP27 expression in SW480 cells, and improve the expression of protein Active-casepase-3, Active-caspase-9 and Cytc, then enhance the apoptosis-inducing effect of 5-FU on SW480 cells, and reverse the drug resistance of SW480 cells to 5-FU.

Key words： colorectal neoplasm heat shock protein-27 RNA interference 5-fluorouracil apoptosisC

收稿日期: 2016-05-04

基金资助:温州市科技局科研基金资助项目（Y20140107）。

通讯作者: 刘长宝，主任医师，硕士生导师，Email：lcb@wzhealth.com。

作者简介: 黄崇杰（1987-），男，浙江温州人，住院医师，硕士。

链接本文:

https://xb.wmu.edu.cn/CN/ 或 https://xb.wmu.edu.cn/CN/Y2016/V46/I12/873

[1] ZHANG N, YIN Y, XU S J, et al. 5-Fluorouracil: mechanisms of resistance and reversal strategies[J]. Molecules, 2008, 13(8):1551-1569.
[2] 黄崇杰, 刘长宝, 郑晨果, 等. 沉默热休克蛋白27基因增强大肠癌细胞对5-FU化疗敏感性[J]. 温州医科大学学报, 2015, 45(2): 106-111.
[3] LI S K, MARTIN A. Mismatch repair and colon cancer:mechanisms and therapies explored[J]. Trends Mol Med, 2016, 22(4): 274-289.
[4] HAYASHI R, ISHII Y, OCHIAI H, et al. Suppression of heat shock protein 27 expression promotes 5-fluorouracil sensitivity in colon cancer cells in a xenograft model[J]. Oncol Rep, 2012, 28(4): 1269-1274.
[5] 关蕾, 俞志高, 刘长宝. 辣椒素联合盐酸伊立替康诱导结肠癌细胞凋亡的机制研究[J]. 中华中医药学刊, 2011, 29(8): 1879-1882.
[6] 祝丽丽, 胡万乐, 张林君, 等. 辣椒素对结直肠癌裸鼠皮下移植瘤的抑制作用及机制[J]. 中华肿瘤杂志, 2013, 35(4):256-261.
[7] ZHUANG H, JIANG W, CHENG W, et al. Down-regulation of HSP27 sensitizes TRAIL-resistant tumor cell to TRAIL-induced apoptosis[J]. Lung Cancer, 2010, 68(1): 27-38.
[8] SONG T F, ZHANG Z F, LIU L, et al. Small interfering RNA-mediated silencing of heat shock protein 27 (HSP27) Increases chemosensitivity to paclitaxel by increasing production of reactive oxygen species in human ovarian cancer cells (HO8910)[J]. J Int Med Res, 2009, 37(5): 1375-1388.
[9] WANG H, LIU B, TIAN J H, et al. Monitoring early responses to irradiation with dual-tracer micro-PET in dual-tumor bearing mice[J]. World J Gastroenterol, 2010, 16(43):5416-5423.
[10] YANG C, CHOY E, HORNICEK F J, et al. Histone deacetylase inhibitor PCI-24781 enhances chemotherapy-induced apoptosis in multidrug-resistant sarcoma cell lines[J]. Anticancer Res, 2011, 31(4): 1115-1123.
[11] ZHANG Y, YUAN J, ZHANG H Y, et al. Natural resistance to apoptosis correlates with resistance to chemotherapy in colorectal cancer cells[J]. Clin Exp Med, 2012, 12(2): 97-103.
[12] OSHITA S E, CHEN F, KWAN T, et al. The small heat shock protein HspB2 is a novel anti-apoptotic protein that inhibits apical caspase activation in the extrinsic apoptotic pathway[J]. Breast Cancer Res Treat, 2010, 124(2): 307-315.
[13] CHEREAU D, ZOU H, SPADA A P, et al. A nucleotide binding site in caspase-9 regulates apoptosome activation [J]. Biochemistry, 2005, 44(13): 4971-4976.
[14] CONCANNON C G, ORRENIUS S, SAMALI A. HSP27 inhibits cytochrome c-mediated caspase activation by sequestering both pro-caspase-3 and cytochrome c[J]. Gene Expr, 2007, 9(4-5): 195-201.
[15] ROCCHI P, JUGPAL P, SO A, et al. Small interference RNA targeting heat-shock protein 27 inhibits the growth of p rostatic cell lines and induces apoptosis via caspase-3 activation in vitro[J]. BJU Int, 2006, 98(5): 1082-1089.