RNAi沉默整合素连接激酶基因的表达对膀胱癌BIU-87细胞生长及凋亡的影响
摘要
目的构建针对ILK基因的特异性siRNA表达质粒,转染人膀胱癌BIU-87细胞,并检测其对人膀胱癌BIU-87细胞生长及凋亡的影响。
方法构建针对ILK基因的特异性siRNA表达质粒siRNA ILK及对照质粒siRNA-NC;将siRNA ILK及siRNA-NC分别转染膀胱癌BIU-87细胞,G418筛选稳定表达细胞株,并分别命名为BIU-87 si ILK及BIU-87 vector,并经RT-PCR,Western Blot及细胞免疫荧光检测ILK的表达变化;采用Am-Blue法比较BIU-87,BIU-87 si ILK及BIU-87 vector细胞的增殖能力并绘制生长曲线, Tunel细胞凋亡检测试剂盒检测各组细胞的凋亡,流式细胞术分析各组细胞的细胞周期及凋亡率,细胞免疫荧光检测p-Akt和p-GSK 3β的表达水平;Western Blot检测下游信号通路分子及凋亡相关蛋白Akt, p-Akt, GSK 3(α/β), p-GSK 3β,β-catenin, Caspase 3, Bax及Bcl-2的表达水平;激光共聚焦显微镜观察各组细胞中RI的表达水平及共定位。将BIU-87,BIU-87 si ILK及BIU-87 vector细胞分别注射至裸鼠背部皮下,30天后处死裸鼠,取出瘤组织并称重;HE染色观察瘤组织的形态学改变;CD31抗体检测肿瘤微血管密度变化;免疫组织化学检测各组瘤组织中ILK, p-Akt,p-GSK-3β,β-catenin及RI蛋白的表达差异;Tunel细胞凋亡检测试剂盒检测各组瘤组织中的细胞凋亡率。
结果经测序证实ILK基因的siRNA表达质粒siRNA ILK及对照质粒siRNA-NC构建成功,RT-PCR,Western Blot及免疫细胞荧光分析BIU-87细胞中ILK基因的RNAi效果显示,siRNA ILK能显著地抑制ILK基因的表达(P<0.05),而siRNA-NC中ILK的表达无明显变化(P>0.05),表明成功获得稳定表达细胞株,即BIU-87 si ILK及BIU-87 vector细胞; Am-Blue结果显示BIU-87 si ILK细胞较BIU-87 vector及BIU-87细胞增殖能力明显降低;Tunel细胞凋亡检测结果证实BIU-87 si ILK细胞较BIU-87 vector和BIU-87细胞凋亡明显增加;流式细胞分析结果表明BIU-87 si ILK细胞生长停滞于G1期,细胞凋亡率明显增高;细胞免疫荧光结果显示,BIU-87 si ILK细胞中p-Akt和p-GSK 3β的表达较BIU-87 vector和BIU-87细胞明显减少(P<0.05),Western Blot显示,与BIU-87细胞相比,BIU-87 si ILK细胞中p-Akt, p-GSK 3β,β-catenin及Bcl-2的表达明显减少(P<0.05),Caspase 3和Bax蛋白的表达明显增加(P<0.05),Akt, GSK 3(α/β)蛋白的表达无明显变化(P>0.05);激光共聚焦显微镜观察显示,ILK和RI均位于细胞质,BIU-87 si ILK细胞中RI的表达较BIU-87 vector和BIU-87细胞明显增加(P<0.05)。与BIU-87组相比,BIU-87 si ILK组裸鼠的瘤重显著降低(P<0.05),抑瘤率为64.73%,BIU-87 vector组无显著差异(P>0.05), HE染色显示,BIU-87 si ILK组癌细胞较其它两组体积减小,核质比例明显缩小,核分裂相显著减少;BIU-87 si ILK组中CD31阳性细胞(棕黄色染色)较其他两组明显减少甚至无血管出现;免疫组化结果显示,p-Akt,p-GSK3β及RI蛋白均位于细胞质,BIU-87 si ILK组中p-Akt和p-GSK3β较BIU-87和BIU-87 vector组明显减少,RI明显增加。Tunel细胞凋亡检测结果表明,BIU-87 siILK组中凋亡细胞较其他两组明显增加。
结论成功构建针对ILK基因的特异性siRNA表达质粒,并通过调控p-Akt, p-GSK-3β,β-catenin, Caspase 3, Bax及Bcl-2蛋白的表达抑制BIU-87细胞的增殖并促进其凋亡;裸鼠实验证实ILK siRNA能显著抑制BIU-87细胞在体内的生长能力,其机制可能与ILK siRNA降低p-Akt, p-GSK-3β,β-catenin蛋白的表达有关。
Objective To construct the specific siRNA vectors of ILK gene and transfect bladder cancer BIU-87 cells. And study the effects of ILK gene silencing by siRNA on growth and apoptosis.
Methods One siRNA vector specific to ILK gene and one non-homologous negative control vector were designed and constructed;then stably transfected into bladder cancer BIU-87 cells via Lipofectamine 2000; Stably expression cell lines are screened by G418, and were named BIU-87 si ILK and BIU-87 vector;Detected the interferential efficiency by RT-PCR , Westeron Blot and cell Immunofluorescence;The inhibitory effect on cell proliferation was assessed by Am-Blue and Flow Cytometry, cell apoptosis was assessed by Tunel Kit and Flow Cytometry, The expression level of p-Akt and p-GSK-3βwas assessed by cell Immunofluorescence;The expression level of Akt, p-Akt, GSK 3(α/β), p-GSK 3β,β-catenin, Caspase 3, Bax and Bcl-2 were assessed by Western Blot;The expression level of RI and location were assessed by laser Scanning Confocal。The BIU-87 cells, BIU-87 vector cells, and BIU-87 si ILK cells were respectively injected subcutaneously into the backs of the BALB/C nude mice. The time of tumor formation was recorded,and 30 days after injection, the mice were sacrificed ,and tumors were excised and weighed. The change of morphology was detected by HE dying;The density of tumor microvessel was detected by CD31 antibody;The expression of ILK, p-Akt,p-GSK-3β,β-catenin and RI protein were assessed by Immunohistochemistry;Xenograft tumor cell apoptosis was assessed by Tunel Kit .
Results The siRNA ILK vectors were verified by enzyme digestion and DNA sequencing, the expression of mRNA and protein significantly decreased in bladder cancer BIU-87 cells by transfected siRNA ILK vectors compared with negative control, It is indicated that stably expression cell lines BIU-87 si ILK and BIU-87 vector were successfully obtained; The ability of cell proliferation stably decreased in BIU-87 si ILK cells compared with BIU-87 vector and BIU-87 cells;cell apoptosis significantly increased in BIU-87 si ILK cells compared with BIU-87 vector and BIU-87 cells; The proliferation of BIU-87 si ILK cells was inhibited with G1 phase arresting, S phase reducing and G2-M phase delaying; The expression of p-Akt, p-GSK 3β,β-catenin及Bcl-2 significantly reduced in BIU-87 si ILK cells compared with BIU-87 vector and BIU-87 cells(P<0.05), The expression of Caspase 3 and Bax protein stably increased (P<0.05), but, The expression of Akt, GSK 3(α/β)do not significantly changed(P>0.05);Laser Scanning Confocal assay showed that ILK and RI could colocalized in cytoplasm and the expression of RI in transfected BIU-87 si LIK cells group markedly increase compared with the control cells(P<0.05)。xenograft tumor showed that the BIU-87 si ILK cells group significantly inhibited the growth of tumors compared with the other two control groups(P<0.05),The inhibiting rate of tumor was 64.73% or 66.73%(,P>0.05), HE staining showed significantly decrease in volume of tumor cells, in the ratio of nucleus to cytoplasm in the tumor of BIU-87 si ILK group, compared to the BIU-87 vector and BIU-87 group; BIU-87 si ILK group resulted in apparent inhibition of angiogenesis and much higher RI expression as well as lower ILK, p-Akt, p-GSK3βandβ-catenin expression in tumor tissue, whereas numerous small vessels and much higher ILK, p-Akt, p-GSK3βandβ-catenin expression were seen in tumor tissue of the control groups.
Conclusion Specific siRNA of ILK expression vectors were constructed successfully, and significately inhibited proliferation and induced apoptosis of bladder cancer BIU-87 cells by regulated the expression of p-Akt, p-GSK-3β,β-catenin, Caspase 3, Bax and Bcl-2, Nude mouse experiment showed that ILK siRNA may significately inhibit BIU-87 cell growth in vivo,the mechanism of its may be ILK siRNA can decrease the expression of p-Akt, p-GSK-3βandβ-catenin.
方法构建针对ILK基因的特异性siRNA表达质粒siRNA ILK及对照质粒siRNA-NC;将siRNA ILK及siRNA-NC分别转染膀胱癌BIU-87细胞,G418筛选稳定表达细胞株,并分别命名为BIU-87 si ILK及BIU-87 vector,并经RT-PCR,Western Blot及细胞免疫荧光检测ILK的表达变化;采用Am-Blue法比较BIU-87,BIU-87 si ILK及BIU-87 vector细胞的增殖能力并绘制生长曲线, Tunel细胞凋亡检测试剂盒检测各组细胞的凋亡,流式细胞术分析各组细胞的细胞周期及凋亡率,细胞免疫荧光检测p-Akt和p-GSK 3β的表达水平;Western Blot检测下游信号通路分子及凋亡相关蛋白Akt, p-Akt, GSK 3(α/β), p-GSK 3β,β-catenin, Caspase 3, Bax及Bcl-2的表达水平;激光共聚焦显微镜观察各组细胞中RI的表达水平及共定位。将BIU-87,BIU-87 si ILK及BIU-87 vector细胞分别注射至裸鼠背部皮下,30天后处死裸鼠,取出瘤组织并称重;HE染色观察瘤组织的形态学改变;CD31抗体检测肿瘤微血管密度变化;免疫组织化学检测各组瘤组织中ILK, p-Akt,p-GSK-3β,β-catenin及RI蛋白的表达差异;Tunel细胞凋亡检测试剂盒检测各组瘤组织中的细胞凋亡率。
结果经测序证实ILK基因的siRNA表达质粒siRNA ILK及对照质粒siRNA-NC构建成功,RT-PCR,Western Blot及免疫细胞荧光分析BIU-87细胞中ILK基因的RNAi效果显示,siRNA ILK能显著地抑制ILK基因的表达(P<0.05),而siRNA-NC中ILK的表达无明显变化(P>0.05),表明成功获得稳定表达细胞株,即BIU-87 si ILK及BIU-87 vector细胞; Am-Blue结果显示BIU-87 si ILK细胞较BIU-87 vector及BIU-87细胞增殖能力明显降低;Tunel细胞凋亡检测结果证实BIU-87 si ILK细胞较BIU-87 vector和BIU-87细胞凋亡明显增加;流式细胞分析结果表明BIU-87 si ILK细胞生长停滞于G1期,细胞凋亡率明显增高;细胞免疫荧光结果显示,BIU-87 si ILK细胞中p-Akt和p-GSK 3β的表达较BIU-87 vector和BIU-87细胞明显减少(P<0.05),Western Blot显示,与BIU-87细胞相比,BIU-87 si ILK细胞中p-Akt, p-GSK 3β,β-catenin及Bcl-2的表达明显减少(P<0.05),Caspase 3和Bax蛋白的表达明显增加(P<0.05),Akt, GSK 3(α/β)蛋白的表达无明显变化(P>0.05);激光共聚焦显微镜观察显示,ILK和RI均位于细胞质,BIU-87 si ILK细胞中RI的表达较BIU-87 vector和BIU-87细胞明显增加(P<0.05)。与BIU-87组相比,BIU-87 si ILK组裸鼠的瘤重显著降低(P<0.05),抑瘤率为64.73%,BIU-87 vector组无显著差异(P>0.05), HE染色显示,BIU-87 si ILK组癌细胞较其它两组体积减小,核质比例明显缩小,核分裂相显著减少;BIU-87 si ILK组中CD31阳性细胞(棕黄色染色)较其他两组明显减少甚至无血管出现;免疫组化结果显示,p-Akt,p-GSK3β及RI蛋白均位于细胞质,BIU-87 si ILK组中p-Akt和p-GSK3β较BIU-87和BIU-87 vector组明显减少,RI明显增加。Tunel细胞凋亡检测结果表明,BIU-87 siILK组中凋亡细胞较其他两组明显增加。
结论成功构建针对ILK基因的特异性siRNA表达质粒,并通过调控p-Akt, p-GSK-3β,β-catenin, Caspase 3, Bax及Bcl-2蛋白的表达抑制BIU-87细胞的增殖并促进其凋亡;裸鼠实验证实ILK siRNA能显著抑制BIU-87细胞在体内的生长能力,其机制可能与ILK siRNA降低p-Akt, p-GSK-3β,β-catenin蛋白的表达有关。
Objective To construct the specific siRNA vectors of ILK gene and transfect bladder cancer BIU-87 cells. And study the effects of ILK gene silencing by siRNA on growth and apoptosis.
Methods One siRNA vector specific to ILK gene and one non-homologous negative control vector were designed and constructed;then stably transfected into bladder cancer BIU-87 cells via Lipofectamine 2000; Stably expression cell lines are screened by G418, and were named BIU-87 si ILK and BIU-87 vector;Detected the interferential efficiency by RT-PCR , Westeron Blot and cell Immunofluorescence;The inhibitory effect on cell proliferation was assessed by Am-Blue and Flow Cytometry, cell apoptosis was assessed by Tunel Kit and Flow Cytometry, The expression level of p-Akt and p-GSK-3βwas assessed by cell Immunofluorescence;The expression level of Akt, p-Akt, GSK 3(α/β), p-GSK 3β,β-catenin, Caspase 3, Bax and Bcl-2 were assessed by Western Blot;The expression level of RI and location were assessed by laser Scanning Confocal。The BIU-87 cells, BIU-87 vector cells, and BIU-87 si ILK cells were respectively injected subcutaneously into the backs of the BALB/C nude mice. The time of tumor formation was recorded,and 30 days after injection, the mice were sacrificed ,and tumors were excised and weighed. The change of morphology was detected by HE dying;The density of tumor microvessel was detected by CD31 antibody;The expression of ILK, p-Akt,p-GSK-3β,β-catenin and RI protein were assessed by Immunohistochemistry;Xenograft tumor cell apoptosis was assessed by Tunel Kit .
Results The siRNA ILK vectors were verified by enzyme digestion and DNA sequencing, the expression of mRNA and protein significantly decreased in bladder cancer BIU-87 cells by transfected siRNA ILK vectors compared with negative control, It is indicated that stably expression cell lines BIU-87 si ILK and BIU-87 vector were successfully obtained; The ability of cell proliferation stably decreased in BIU-87 si ILK cells compared with BIU-87 vector and BIU-87 cells;cell apoptosis significantly increased in BIU-87 si ILK cells compared with BIU-87 vector and BIU-87 cells; The proliferation of BIU-87 si ILK cells was inhibited with G1 phase arresting, S phase reducing and G2-M phase delaying; The expression of p-Akt, p-GSK 3β,β-catenin及Bcl-2 significantly reduced in BIU-87 si ILK cells compared with BIU-87 vector and BIU-87 cells(P<0.05), The expression of Caspase 3 and Bax protein stably increased (P<0.05), but, The expression of Akt, GSK 3(α/β)do not significantly changed(P>0.05);Laser Scanning Confocal assay showed that ILK and RI could colocalized in cytoplasm and the expression of RI in transfected BIU-87 si LIK cells group markedly increase compared with the control cells(P<0.05)。xenograft tumor showed that the BIU-87 si ILK cells group significantly inhibited the growth of tumors compared with the other two control groups(P<0.05),The inhibiting rate of tumor was 64.73% or 66.73%(,P>0.05), HE staining showed significantly decrease in volume of tumor cells, in the ratio of nucleus to cytoplasm in the tumor of BIU-87 si ILK group, compared to the BIU-87 vector and BIU-87 group; BIU-87 si ILK group resulted in apparent inhibition of angiogenesis and much higher RI expression as well as lower ILK, p-Akt, p-GSK3βandβ-catenin expression in tumor tissue, whereas numerous small vessels and much higher ILK, p-Akt, p-GSK3βandβ-catenin expression were seen in tumor tissue of the control groups.
Conclusion Specific siRNA of ILK expression vectors were constructed successfully, and significately inhibited proliferation and induced apoptosis of bladder cancer BIU-87 cells by regulated the expression of p-Akt, p-GSK-3β,β-catenin, Caspase 3, Bax and Bcl-2, Nude mouse experiment showed that ILK siRNA may significately inhibit BIU-87 cell growth in vivo,the mechanism of its may be ILK siRNA can decrease the expression of p-Akt, p-GSK-3βandβ-catenin.
引文
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[17] Jenny Chan, Frankie Chi Fat Ko, Yin-Shan Yeung, et al. Integrin-Linked Kinase Overexpression and Its Oncogenic Role in Promoting Tumorigenicity of Hepatocellular Carcinoma [J]. PLoS One. 2011, 6(2): 6984.
[18] Schmitz KJ, Grabellus F, Callies R, et al.Relation and prognostic significance of phosphor-(serine 166)-murine double minute 2 and Akt activation in node-negative breast cancer with regard to p53 expression[J]. Arch. 2006,448(1):16-23.
[19]廖永德,龙庆红.蛋白激酶B在肺鳞癌和腺癌中的表达及临床意义[J].中华肿瘤杂志. 2005, 27(3):156-9.
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[1] Younes MN, Yigitbasi OG, Yazici YD, Jasser SA, Bucana CD, El-Naggar AK, et al. Effects of the integrin-linked kinase inhibitor QLT0267 on squamous cell carcinoma of the head and neck[J]. Arch Otolaryngol Head Neck Surg. 2007,133 (1): 15-23.
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[7]陈瑜,刘超.整合素连接激酶与糖尿病肾病[J].医学研究生学报. 2009,22:100-3.
[8] Dedhar S. Cell2substrate interactions and signaling through ILK [ J ]. CurrOp in Cell Biol. 2000, 12 (2) : 2502256.
[9] Wong RP, Ng P, Dedhar S, Li G. The role of integrin-linked kinase in melanoma cell migration, invasion, and tumor growth [J]. Mol Cancer Ther. 2007, 6 (6): 1692-700.
[10] S. Attwell, C. Roskelley and S. Dedhar, The integrin-linked kinase (ILK)suppresses anoikis [J]. Oncogene. 2000, (19): 3811-3815.
[11] F. Acconcia, C.J. Barnes, R.R. Singh, A.H. Talukder and R. Kumar, Phosphorylation-dependent regulation of nuclear localization and functions of integrin-linked kinase[J]. Proc Natl Acad Sci USA. 2007,(104):6782–6787.
[12] F. Hess, D. Estrugo, A. Fischer, C. Belka and N. Cordes, Integrin-linked kinase interacts with caspase-9 and -8 in an adhesion-dependent manner for promoting radiation-induced apoptosis in human leukemia cells [J]. Oncogene 2007,(26):1372–1384.
[13] Franke TF, Yang SI, Chan TO, Datta K, Kazlauskas A, et al. The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase [J]. Cell. 1995;81:727.
[14] Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, et al. Mechanism of activation of protein kinase B by insulin and IGF-1 [J]. Embo J. 1996, 15:6541.
[15] Fayard E, Tintignac LA, Baudry A, Hemmings BA. Protein kinase B/Akt at a glance [J]. J Cell Sci. 2005, 118:5675.
[16] Yoeli-Lerner M, Toker A. Akt/PKB signaling in cancer: a function in cell motility and invasion [J]. Cell Cycle. 2006, 5:603.
[17] Jenny Chan, Frankie Chi Fat Ko, Yin-Shan Yeung, et al. Integrin-Linked Kinase Overexpression and Its Oncogenic Role in Promoting Tumorigenicity of Hepatocellular Carcinoma [J]. PLoS One. 2011, 6(2): 6984.
[18] Schmitz KJ, Grabellus F, Callies R, et al.Relation and prognostic significance of phosphor-(serine 166)-murine double minute 2 and Akt activation in node-negative breast cancer with regard to p53 expression[J]. Arch. 2006,448(1):16-23.
[19]廖永德,龙庆红.蛋白激酶B在肺鳞癌和腺癌中的表达及临床意义[J].中华肿瘤杂志. 2005, 27(3):156-9.
[20]郭瑞霞,魏丽惠,王建六,等. 17β-雌二醇对子宫膜癌细胞磷脂酰肌醇3激酶/蛋白激酶B信号传导通路的激活作用[J].中华妇产科杂志. 2004,39(7): 469-473.
[21] Karbowska J, Kochan Z, Smolenski RT. Peroxisome proliferator-activated receptoris downregulated in the failing human heart [J]. C ell Mol Biol Lett. 2003, 8:49 -53.
[22]周舟,张蕾,邓朝晖,等.辐射诱导IEC-6细胞GSK3β信号转导途径的激活与细胞凋亡的关系研究[J].辐射研究与辐射工艺学报. 2006, 24(3): 178-82.
[23] Bijur GN, and Jope RS. Proapoptotic stimuli induce nuclear accumulation of glycogen synthase kinase-3 beta [J]. J Biol Chem. 2001, 276: 37436-42.
[24] McCrea PD, Turck CW, Gumbiner B. A homolog of the armadillo protein in Drosophila (plakoglobin) associated with E-cadherin [J]. Science. 1991, 254(5036):1359-1361.
[25] Kraus C Liehr T, Hulsken J, Behrens J, Birchmeier W, Grzeschik KH, Ballhausen WG. Localization of the human beta-catenin gene (CTNNBI) to 3p21: a region implicated in tumor development [J]. Genomics. 1994, 23(1):272-274
[26] Nelson WJ, Nusse R, Convergence of ,Wnt,β-catenin, and cadherin pathways[J]. Science. 2004, 303: 1483-7.
[27] van Hengel J, Nollet F, Berx G, van Roy N, Speleman F , van Roy F .Assignment of the human beta-catenin gene (CTNNBI) to 3p22-->p21.3 by fluorescence in situ hybridization[J]. Cytogenet Cell Genet. 1995, 70 (1-2):68-70.
[28] ResnikE.β-catenin-one Player, two genes[J]. Nat Genet. 1997,16(l):9-11.
[29] Marotta A, Parhar K, Owen D, et al. Characterisation of integrin-linked kinase signalling in sporadic human colon cancer[J]. Br J Cancer.2003,88(11): 1755- 62
[30]杜菲,黄常志.整合素相关激酶(ILK)研究进展[J].国外医学?分子生物学分册. 2001,23(3): 142- 145
[31] White DE, Cardiff RD, Dedhar S, et al. Mammary epithelial-specific expression of the integrin- linked kinase (ILK) results in the induction of mammary gland hyperplasias and tumors in transgenic mice[J]. Oncogene. 2001,20(48):7064- 72
[32] Marotta A , Parhar K, Owen D , et al . Characterization of integrin-linked kinase signalling in sporadic human colon cancer[J]. Br J Cancer. 2003, 88 (11) :175521762.
[33] Berx G, Van RF. The E2cadherin / catenin complex: An important gatekeeper in
breast cancer tumorigenesis and malignant progression[J]. Breast Cancer Res. 2001, 3 (5) : 289 - 293.
[34] Oloumi A, Mcphee T, Dedhar S. Regulation of E2cadherin expression andβ2catenin /Tcf transcriptional. activity by the integrin-linked kinase [J]. Bioc Hem Biophys Acta. 2004, 1691(1) : 1 - 15.
[35] Guaita S, Puig I, Franci C, et al. Snail induction of epithelial to mesenchymal transition in tumor cells is accompanied by MUC1repression and ZEB1 expression [J]. J Biol Chem. 2002, 227(42) : 39209 - 39216.
[36]强占荣,杨国栋.整合素连接激酶在肿瘤发生及进展中的作用[J].临床肿瘤学杂志.2006,11(11):873-5.
[37] Ferrara N.VEGF and the quest for tumor angiogenesis factors[J]. Nat Rev Cancer. 2002, 2(10): 7952803.