Galectin-9调控SHH信号通路影响结直肠癌HT29细胞凋亡
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摘要
目的:探讨半乳糖凝集素9(galectin-9)对结直肠癌细胞凋亡的影响及机制。方法:在结直肠癌细胞系HT29中分别转染galectin-9过表达载体pcDNA3.1-Galectin-9和对照载体pcDNA3.1,用real-time PCR和Western blot检测过表达效果。Annexin V-FITC/PI双染法检测细胞凋亡变化,Western blot测定细胞中活化的caspase-3水平,以及SHH信号通路蛋白Smo、Gli1和SHH的表达变化。用SHH信号通路特异性抑制剂cyclopamine处理上调galectin-9表达的结直肠癌细胞,用上述方法检测细胞凋亡、细胞中活化的caspase-3水平,以及SHH信号通路蛋白Smo、Gli1和SHH的表达变化。结果:pcDNA3.1-Galectin-9可显著上调结直肠癌HT29细胞中galectin-9的mRNA和蛋白水平。上调galectin-9表达后的结直肠癌HT29细胞凋亡率升高,细胞中活化的caspase-3蛋白水平升高,同时细胞中SHH信号通路蛋白Smo、Gli1和SHH的表达水平降低(P<0.05)。Cyclopamine处理可以进一步诱导上调galectin-9表达的结直肠癌HT29细胞凋亡,促进细胞中活化的caspase-3蛋白水平上调,降低细胞中SHH信号通路的激活水平(P<0.05)。结论:Galectin-9通过抑制SHH信号通路诱导结直肠癌HT29细胞凋亡。
AIM: To investigate the effect of galectin-9 on the apoptosis of colorectal cancer HT29 cells. METHODS: Galectin-9 over-expression vector(pcDNA3.1-Galectin-9) or control vector(pcDNA3.1) was transfected into the HT29 cells. The galectin-9 over-expression was detected by real-time PCR and Western blot. Annexin V-FITC/PI double staining was used to detect the apoptosis. The protein level of activated caspase-3 and the expression of SHH signaling pathway-related proteins Smo, Gli1 and SHH in the HT29 cells were determined by Western blot. SHH signaling pathway specific inhibitor cyclopamine was used to treat the HT29 cells with up-regulated galectin-9 expression, and the apoptosis, the protein level of cleaved caspase-3 and the expression of SHH signaling pathway-related proteins Smo, Gli1 and SHH in the HT29 cells were detected by the above methods. RESULTS: Transfection with pcDNA3.1-Galectin-9 up-regulated galectin-9 expression at mRNA and protein levels in the colorectal cancer HT29 cells(P<0.05). The apoptotic rate of the HT29 cells was increased after galectin-9 up-regulation. The protein level of cleaved caspase-3 in the cells was increased, while the expression levels of SHH signaling pathway-related proteins Smo, Gli1 and SHH were decreased. Cyclopamine treatment further induced the apoptosis of the HT29 cells with up-regulation of galectin-9, increased the protein le-vels of cleaved caspase-3, and decreased the activation level of SHH signaling pathway in the HT29 cells(P<0.05). CONCLUSION: Galectin-9 induces the apoptosis of colorectal cancer HT29 cells by inhibiting SHH signaling pathway.
AIM: To investigate the effect of galectin-9 on the apoptosis of colorectal cancer HT29 cells. METHODS: Galectin-9 over-expression vector(pcDNA3.1-Galectin-9) or control vector(pcDNA3.1) was transfected into the HT29 cells. The galectin-9 over-expression was detected by real-time PCR and Western blot. Annexin V-FITC/PI double staining was used to detect the apoptosis. The protein level of activated caspase-3 and the expression of SHH signaling pathway-related proteins Smo, Gli1 and SHH in the HT29 cells were determined by Western blot. SHH signaling pathway specific inhibitor cyclopamine was used to treat the HT29 cells with up-regulated galectin-9 expression, and the apoptosis, the protein level of cleaved caspase-3 and the expression of SHH signaling pathway-related proteins Smo, Gli1 and SHH in the HT29 cells were detected by the above methods. RESULTS: Transfection with pcDNA3.1-Galectin-9 up-regulated galectin-9 expression at mRNA and protein levels in the colorectal cancer HT29 cells(P<0.05). The apoptotic rate of the HT29 cells was increased after galectin-9 up-regulation. The protein level of cleaved caspase-3 in the cells was increased, while the expression levels of SHH signaling pathway-related proteins Smo, Gli1 and SHH were decreased. Cyclopamine treatment further induced the apoptosis of the HT29 cells with up-regulation of galectin-9, increased the protein le-vels of cleaved caspase-3, and decreased the activation level of SHH signaling pathway in the HT29 cells(P<0.05). CONCLUSION: Galectin-9 induces the apoptosis of colorectal cancer HT29 cells by inhibiting SHH signaling pathway.
引文
[1] Malapelle U, Pisapia P, Sgariglia R, et al. Less frequently mutated genes in colorectal cancer: evidences from next-generation sequencing of 653 routine cases[J]. J Clin Pathol, 2016, 69(9):767-771.
[2] Douard R, Moutereau S, Pernet P, et al. Sonic Hedgehog-dependent proliferation in a series of patients with colorectal cancer[J]. Surgery, 2006, 139(5):665-670.
[3] 张丰, 计骏, 蔡劬, 等. 结直肠癌中Galectin-9的表达及其意义[J]. 肿瘤, 2008, 28(5):419-422.
[4] Irie A, Yamauchi A, Kontani K, et al. Galectin-9 as a prognostic factor with antimetastatic potential in breast cancer[J]. Clin Cancer Res, 2005, 11(8):2962-2968.
[5] 程亚玉, 谭雪莹, 黄飞, 等. Galectin-9蛋白过表达对卵巢癌细胞增殖、迁移及凋亡的影响[J]. 中国医药导报, 2018,15(3):13-16.
[6] Manning JC, García Caballero G, Knospe C, et al. Network analysis of adhesion/growth: regulatory galectins and their binding sites in adult chicken retina and choroid[J]. J Anat, 2017, 231(1):23-37.
[7] Conrad ML, Freitag N, Diessler ME, et al. Differential spatiotemporal patterns of galectin expression are a hallmark of endotheliochorial placentation[J]. Am J Reprod Immunol, 2016, 75(3):317-325.
[8] Takaku S, Niimi N, Kadoya T, et al. Galectin-1 and galectin-3 as key molecules for peripheral nerve degeneration and regeneration[J]. AIMS Mol Sci, 2016, 3(3):325-337.
[9] D’Haene N, Maris C, Sandras F, et al. The differential expression of galectin-1 and galectin-3 in normal lymphoid tissue and non-Hodgkin's and Hodgkin's lymphomas[J]. Int J Immunopathol Pharmacol, 2005, 18(3):431-443.
[10] Okura R, Fujihara S, Iwama H, et al. MicroRNA profiles during galectin-9-induced apoptosis of pancreatic cancer cells[J]. Oncol Lett, 2018, 15(1):407-414.
[11] Liang M, Ueno MS, Arikawa T, et al. Galectin-9 expression links to malignant potential of cervical squamous cell carcinoma[J]. J Cancer Res Clin Oncol, 2008, 134(8):899-907.
[12] Fu H, Liu Y, Xu L, et al. Galectin-9 predicts postoperative recurrence and survival of patients with clear-cell renal cell carcinoma[J]. Tumor Biol, 2015, 36(8):5791-5799.
[13] 董超, 徐正丰, 邱涵, 等. Galectin-9在乳腺癌组织中的表达及其对癌细胞侵袭转移的影响[J]. 山东医药, 2016, 56(41):81-83.
[14] 张丰. Galectin-9在结直肠癌中的表达及其在肿瘤转移中作用机制的研究[D]. 上海:上海交通大学, 2008.
[15] Yang J, Zhu L, Cai Y, et al. Role of downregulation of galectin-9 in the tumorigenesis of gastric cancer.[J]. Int J Oncol, 2014, 45(3):1313-1320.
[16] Fukumori T, Oka N, Takenaka Y, et al. Galectin-3 regulates mitochondrial stability and antiapoptotic function in response to anticancer drug in prostate cancer[J]. Cancer Res, 2006, 66(6):3114-3119.
[17] Takano J, Morishita A, Fujihara S, et al. Galectin-9 suppresses the proliferation of gastric cancer cells in vitro[J]. Oncol Reports, 2016, 35(2):851-860.
[18] Sarkar A, Balakrishnan K, Chen J, et al. Molecular evidence of Zn chelation of the procaspase activating compound B-PAC-1 in B cell lymphoma[J]. Oncotarget, 2016, 7(3):3461-3476.
[19] Zhang R, Li L, Yuan L, et al. Hypoxic preconditioning protects cardiomyocytes against hypoxia/reoxygenation-induced cell apoptosis via sphingosine kinase 2 and FAK/AKT pathway[J]. Exp Mol Pathol, 2016, 100(1):51-58.
[20] 陈喜德, 魏波, 杨柳菁, 等. 不同病期骨关节炎大鼠关节软骨中caspase-3与细胞凋亡的相关性[J]. 广东医学, 2018, 39(2):181-186.
[21] Solanki A, Lau CI, Saldaňa JI, et al. The transcription factor Gli3 promotes B cell development in fetal liver through repression of Shh[J]. J Exp Med, 2017, 214(7):2041-2058.
[22] Suyama K, Onishi H, Imaizumi A, et al. CD24 suppresses malignant phenotype by downregulation of SHH transcription through STAT1 inhibition in breast cancer cells[J]. Cancer Lett, 2016, 374(1):44-53.
[23] 张苑, 张伟伟, 陈磊磊. Sonic Hedgehog信号通路在心血管系统疾病中的研究进展[J]. 医学综述, 2017, 23(20):3989-3994.
[24] Cui FQ, Wei ZQ. Expression of Shh,Gli1 and VEGF proteins in colorectal cancer and their clinical significance[J]. Life Sci Res, 2010, 14(4):345-349.
[25] Syed IS, Pedram A, Farhat WA. Role of sonic hedgehog (Shh) signaling in bladder cancer stemness and tumorigenesis[J]. Curr Urol Rep, 2016, 17(2):1-7.
[26] 冀凯伦, 刘琪, 牟作峰, 等. BRD4通过SHH信号通路诱导甲状腺癌细胞增殖、侵袭与迁移[J]. 中国癌症杂志, 2017, 27(11):860-866.
[27] Cheng XL, Li ZD, Jiang BQ, et al. Interactions between the Sonic Hedgehog signaling pathway and the cAMP/PKA signaling pathway in LKB1 transfected breast cancer cells[J]. Fudan University J Med Sci, 2011, 38(6):485-491.
[2] Douard R, Moutereau S, Pernet P, et al. Sonic Hedgehog-dependent proliferation in a series of patients with colorectal cancer[J]. Surgery, 2006, 139(5):665-670.
[3] 张丰, 计骏, 蔡劬, 等. 结直肠癌中Galectin-9的表达及其意义[J]. 肿瘤, 2008, 28(5):419-422.
[4] Irie A, Yamauchi A, Kontani K, et al. Galectin-9 as a prognostic factor with antimetastatic potential in breast cancer[J]. Clin Cancer Res, 2005, 11(8):2962-2968.
[5] 程亚玉, 谭雪莹, 黄飞, 等. Galectin-9蛋白过表达对卵巢癌细胞增殖、迁移及凋亡的影响[J]. 中国医药导报, 2018,15(3):13-16.
[6] Manning JC, García Caballero G, Knospe C, et al. Network analysis of adhesion/growth: regulatory galectins and their binding sites in adult chicken retina and choroid[J]. J Anat, 2017, 231(1):23-37.
[7] Conrad ML, Freitag N, Diessler ME, et al. Differential spatiotemporal patterns of galectin expression are a hallmark of endotheliochorial placentation[J]. Am J Reprod Immunol, 2016, 75(3):317-325.
[8] Takaku S, Niimi N, Kadoya T, et al. Galectin-1 and galectin-3 as key molecules for peripheral nerve degeneration and regeneration[J]. AIMS Mol Sci, 2016, 3(3):325-337.
[9] D’Haene N, Maris C, Sandras F, et al. The differential expression of galectin-1 and galectin-3 in normal lymphoid tissue and non-Hodgkin's and Hodgkin's lymphomas[J]. Int J Immunopathol Pharmacol, 2005, 18(3):431-443.
[10] Okura R, Fujihara S, Iwama H, et al. MicroRNA profiles during galectin-9-induced apoptosis of pancreatic cancer cells[J]. Oncol Lett, 2018, 15(1):407-414.
[11] Liang M, Ueno MS, Arikawa T, et al. Galectin-9 expression links to malignant potential of cervical squamous cell carcinoma[J]. J Cancer Res Clin Oncol, 2008, 134(8):899-907.
[12] Fu H, Liu Y, Xu L, et al. Galectin-9 predicts postoperative recurrence and survival of patients with clear-cell renal cell carcinoma[J]. Tumor Biol, 2015, 36(8):5791-5799.
[13] 董超, 徐正丰, 邱涵, 等. Galectin-9在乳腺癌组织中的表达及其对癌细胞侵袭转移的影响[J]. 山东医药, 2016, 56(41):81-83.
[14] 张丰. Galectin-9在结直肠癌中的表达及其在肿瘤转移中作用机制的研究[D]. 上海:上海交通大学, 2008.
[15] Yang J, Zhu L, Cai Y, et al. Role of downregulation of galectin-9 in the tumorigenesis of gastric cancer.[J]. Int J Oncol, 2014, 45(3):1313-1320.
[16] Fukumori T, Oka N, Takenaka Y, et al. Galectin-3 regulates mitochondrial stability and antiapoptotic function in response to anticancer drug in prostate cancer[J]. Cancer Res, 2006, 66(6):3114-3119.
[17] Takano J, Morishita A, Fujihara S, et al. Galectin-9 suppresses the proliferation of gastric cancer cells in vitro[J]. Oncol Reports, 2016, 35(2):851-860.
[18] Sarkar A, Balakrishnan K, Chen J, et al. Molecular evidence of Zn chelation of the procaspase activating compound B-PAC-1 in B cell lymphoma[J]. Oncotarget, 2016, 7(3):3461-3476.
[19] Zhang R, Li L, Yuan L, et al. Hypoxic preconditioning protects cardiomyocytes against hypoxia/reoxygenation-induced cell apoptosis via sphingosine kinase 2 and FAK/AKT pathway[J]. Exp Mol Pathol, 2016, 100(1):51-58.
[20] 陈喜德, 魏波, 杨柳菁, 等. 不同病期骨关节炎大鼠关节软骨中caspase-3与细胞凋亡的相关性[J]. 广东医学, 2018, 39(2):181-186.
[21] Solanki A, Lau CI, Saldaňa JI, et al. The transcription factor Gli3 promotes B cell development in fetal liver through repression of Shh[J]. J Exp Med, 2017, 214(7):2041-2058.
[22] Suyama K, Onishi H, Imaizumi A, et al. CD24 suppresses malignant phenotype by downregulation of SHH transcription through STAT1 inhibition in breast cancer cells[J]. Cancer Lett, 2016, 374(1):44-53.
[23] 张苑, 张伟伟, 陈磊磊. Sonic Hedgehog信号通路在心血管系统疾病中的研究进展[J]. 医学综述, 2017, 23(20):3989-3994.
[24] Cui FQ, Wei ZQ. Expression of Shh,Gli1 and VEGF proteins in colorectal cancer and their clinical significance[J]. Life Sci Res, 2010, 14(4):345-349.
[25] Syed IS, Pedram A, Farhat WA. Role of sonic hedgehog (Shh) signaling in bladder cancer stemness and tumorigenesis[J]. Curr Urol Rep, 2016, 17(2):1-7.
[26] 冀凯伦, 刘琪, 牟作峰, 等. BRD4通过SHH信号通路诱导甲状腺癌细胞增殖、侵袭与迁移[J]. 中国癌症杂志, 2017, 27(11):860-866.
[27] Cheng XL, Li ZD, Jiang BQ, et al. Interactions between the Sonic Hedgehog signaling pathway and the cAMP/PKA signaling pathway in LKB1 transfected breast cancer cells[J]. Fudan University J Med Sci, 2011, 38(6):485-491.