沉默DEPTOR表达促进胰岛细胞释放胰岛素
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摘要
背景:mTOR复合物是调控胰岛β细胞质量与功能的关键蛋白,Deptor是复合物中的共有组分,Deptor的丢失是否会影响胰岛β细胞的胰岛素分泌功能,目前尚缺乏相关研究。目的:利用si RNA干扰技术沉默小鼠胰岛素瘤NIT-1细胞DEPTOR基因的表达,探讨其对胰岛素瘤细胞分泌功能的影响及背后的机制。方法:设计合成3对针对DEPTOR基因的si RNA序列,利用脂质体法将si RNA转染至NIT-1细胞。实验组设为6组:(1)空白转染组(NIT-1细胞,转染复合液仅加Lipofectamin);(2)阴性对照转染组(NC-FAM);(3)阳性对照转染组(GAPDH);(4)si RNA deptor 1组(脂质体介导的转染复合+si RNA deptor385);(5)si RNA deptor 2组(脂质体介导的转染复合+si RNA deptor766);(6)si RNA deptor 3组(脂质体介导的转染复合+si RNA deptor1275)。荧光显微镜下观察转染效率;QPCR检测DEPTOR m RNA的相对表达量;ELISA胰岛素试剂盒检测NIT-1细胞胰岛素分泌情况;Western blot检测DEPTOR下游关键蛋白的表达。结果与结论:(1)在荧光显微镜下观察,si RNA能有效转入到NIT-1细胞内,呈绿色点状荧光;(2)PCR检测干扰后DEPTOR m RNA的相对表达量,发现设计合成3对si RNA序列中有两对si RNA序列(si DEPTOR385and si DEPTOR766)对DEPTOR基因有干扰效果,干扰效果与阴性对照差异有显著性意义(P<0.05);(3)ELISA胰岛素测定结果显示,在NIT-1细胞中沉默DEPTOR后,有效转染组细胞的胰岛素分泌显著升高(P<0.05);(4)Western-blot检测结果显示,Deptor下游关键蛋白S6、4EBP-1磷酸化明显增加,AKT磷酸化略有减少;(5)结果说明,利用RNAi技术在NIT-1细胞上可以有效地沉默DEPTOR基因的表达,并促进细胞胰岛素的分泌,该现象可能与mTORC1通路的激活相关。
BACKGROUND: Mammalian target of rapamycin(mTOR) complexes are a key regulator of pancreatic beta cells mass and function. DEP-domain containing mTOR-interacting protein(DEPTOR) is a common part of mTOR complexes and whether DEPTOR loss in islet cells affects insulin-secreting function hasnever been identified. OBJECTIVE: To assess the alternation of insulin secretion by silencing DEPTOR gene in pancreatic β cells NIT-1 and to explore the underlying mechanism. METHODS: Three si RNA sequences for silencing DEPTOR gene were designed and constructed, which were transfected with lipofectamine into NIT-1 cells. There were six groups: blank transfection group(NIT-1 cells plus Lipofectamin), negative control group(NC-FAM), positive control group(GAPDH), si RNA deptor 1 group(si RNA deptor385), si RNA deptor 2 group(si RNA deptor766), and si RNA deptor 3 group(si RNA deptor1275). The transfection efficiency was determined by fluorescence microscope. The relative expression level of DEPTOR m RNA was detected by quantitative-PCR. Insulin secretion in the cell conditioned medium was determined by insulin ELISA kit. The expression level of DEPTOR downstream key protein was detected by western blot assay. RESULTS AND CONCLUSION: Specific green fluorescence accumulated in a punctated pattern under fluorescence microscope, indicating that the effectiveness of transfection was eligible. Quantitative-PCR results showed two(si DEPTOR385 and si DEPTOR766) of the three si RNA sequences could significantly disrupt the expression of DEPTOR m RNA, which had significant difference with negative control group(P < 0.05). The ELISA results showed that the total amount of insulin secretion in the effective transfected groups was significantly increased(P < 0.05). Western blot assay results showed the grey levels of p-s6 and p-4 EBP-1 proteins were significantly elevated, while p-AKT of those former was slightly decreased. These findings suggest that si RNA technology can effectively silence the DEPTOR gene in NIT-1 cells, which improves β-cell insulin secretion in a manner of mTORC1 activation.
BACKGROUND: Mammalian target of rapamycin(mTOR) complexes are a key regulator of pancreatic beta cells mass and function. DEP-domain containing mTOR-interacting protein(DEPTOR) is a common part of mTOR complexes and whether DEPTOR loss in islet cells affects insulin-secreting function hasnever been identified. OBJECTIVE: To assess the alternation of insulin secretion by silencing DEPTOR gene in pancreatic β cells NIT-1 and to explore the underlying mechanism. METHODS: Three si RNA sequences for silencing DEPTOR gene were designed and constructed, which were transfected with lipofectamine into NIT-1 cells. There were six groups: blank transfection group(NIT-1 cells plus Lipofectamin), negative control group(NC-FAM), positive control group(GAPDH), si RNA deptor 1 group(si RNA deptor385), si RNA deptor 2 group(si RNA deptor766), and si RNA deptor 3 group(si RNA deptor1275). The transfection efficiency was determined by fluorescence microscope. The relative expression level of DEPTOR m RNA was detected by quantitative-PCR. Insulin secretion in the cell conditioned medium was determined by insulin ELISA kit. The expression level of DEPTOR downstream key protein was detected by western blot assay. RESULTS AND CONCLUSION: Specific green fluorescence accumulated in a punctated pattern under fluorescence microscope, indicating that the effectiveness of transfection was eligible. Quantitative-PCR results showed two(si DEPTOR385 and si DEPTOR766) of the three si RNA sequences could significantly disrupt the expression of DEPTOR m RNA, which had significant difference with negative control group(P < 0.05). The ELISA results showed that the total amount of insulin secretion in the effective transfected groups was significantly increased(P < 0.05). Western blot assay results showed the grey levels of p-s6 and p-4 EBP-1 proteins were significantly elevated, while p-AKT of those former was slightly decreased. These findings suggest that si RNA technology can effectively silence the DEPTOR gene in NIT-1 cells, which improves β-cell insulin secretion in a manner of mTORC1 activation.
引文
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[13]Gu Y,Lindner J,Kumar A,et al.Rictor/m TORC2 is essential for maintaining a balance between beta-cell proliferation and cell size.Diabetes.2011;60(3):827-837.
[14]Rachdi L,Balcazar N,Osorio-Duque F,et al.Disruption of Tsc2 in pancreatic beta cells induces beta cell mass expansion and improved glucose tolerance in a TORC1-dependent manner.Proc Natl Acad Sci U S A.2008;105(27):9250-9255.
[15]Pirollo KF,Chang EH.Targeted delivery of small interfering RNA:approaching effective cancer therapies.Cancer Res.2008;68(5):1247-1250.
[16]袁松,孙会敏,丁丽霞.脂质体物理化学稳定性研究进展[J].中国药事,2011,25(4):384-388.
[17]张旭,丁会芹,王冰,等.脂质体介导RNAi的研究[J].生物医学工程学杂志,2012,29(4):722-726.
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[19]Cota D.Mammalian target of rapamycin complex 1(m TORC1)signaling in energy balance and obesity.Physiol Behav.2009;97(5):520-524.
[20]Le Bacquer O,Petroulakis E,Paglialunga S,et al.Elevated sensitivity to diet-induced obesity and insulin resistance in mice lacking 4E-BP1 and 4E-BP2.J Clin Invest.2007;117(2):387-396.
[21]Demidenko ZN,Shtutman M,Blagosklonny MV.Blagosklonny,Pharmacologic inhibition of MEK and PI-3K converges on the m TOR/S6 pathway to decelerate cellular senescence.Cell Cycle.2009;8(12):1896-900.
[22]Fritsche L,Neukamm SS,Lehmann R,et al.Insulin-induced serine phosphorylation of IRS-2 via ERK1/2 and m TOR:studies on the function of Ser675 and Ser907.Am J Physiol Endocrinol Metab.2011;300(5):E824-836.
[23]Jacinto E,Loewith R,Schmidt A,et al.Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive.Nat Cell Biol.2004;6(11):1122-1128.
[24]Elghazi L,Balcazar N,Blandino-Rosano M,et al.Decreased IRS signaling impairs beta-cell cycle progression and survival in transgenic mice overexpressing S6K in beta-cells.Diabetes.2010;59(10):2390-2399.
[25]Briaud I,Dickson LM,Lingohr MK,et al.Insulin receptor substrate-2 proteasomal degradation mediated by a mammalian target of rapamycin(m TOR)-induced negative feedback down-regulates protein kinase B-mediated signaling pathway in beta-cells.J Biol Chem.2005;280(3):2282-2293.
[26]Efeyan A,Sabatini DM.m TOR and cancer:many loops in one pathway.Curr Opin Cell Biol.2010;22(2):169-176.
[27]Copps KD,White MF.egulation of insulin sensitivity by serine/threonine phosphorylation of insulin receptor substrate proteins IRS1 and IRS2.Diabetologia.2012;55(10):2565-2582.
[28]Chau GC,Im DU,Kang TM,et al.m TOR controls Ch REBP transcriptional activity and pancreatic beta cell survival under diabetic stress.J Cell Biol.2017;216(7):2091-2105.
[29]Koyanagi M,Asahara S,Matsuda T,et al.Ablation of TSC2enhances insulin secretion by increasing the number of mitochondria through activation of m TORC1.PLo S One.2011;6(8):e23238.
[30]Gurevitch D,Boura-Halfon S,Isaac R,et al.Elimination of negative feedback control mechanisms along the insulin signaling pathway improves beta-cell function under stress.Diabetes,2010;59(9):2188-2197.
[2]Yang W,Lu J,Weng J,et al.Prevalence of diabetes among men and women in China.N Engl J Med.2010.362(12):1090-1101.
[3]Dinesh Shah A,Langenberg C,Rapsomaniki E,et al.Type 2diabetes and incidence of cardiovascular diseases:a cohort study in 1.9 million people.Lancet Diabetes Endocrinol.2015;3(2):105-113.
[4]Yagihashi S,Inaba W,Mizukami H.Dynamic pathology of islet endocrine cells in type 2 diabetes:beta-Cell growth,death,regeneration and their clinical implications.J Diabetes Investig.2016;7(2):155-165.
[5]Peterson TR,Laplante M,Thoreen CC,et al.DEPTOR is an m TOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival.Cell.2009;137(5):873-886.
[6]Zhang HR,Chen JM,Zeng ZY,et al.Knockdown of DEPTOR inhibits cell proliferation and increases chemosensitivity to melphalan in human multiple myeloma RPMI-8226 cells via inhibiting PI3K/AKT activity.J Int Med Res.2013;41(3):584-595.
[7]Proud CG.Dynamic balancing:DEPTOR tips the scales.J Mol Cell Biol.2009;1(2):61-63.
[8]Lai EY,Chen ZG,Zhou X,et al.DEPTOR expression negatively correlates with m TORC1 activity and tumor progression in colorectal cancer.Asian Pac J Cancer Prev.2014;15(11):4589-4594.
[9]Meng ZX,Li S,Wang L,Ko HJ,et al.Baf60c drives glycolytic metabolism in the muscle and improves systemic glucose homeostasis through Deptor-mediated Akt activation.Nat Med.2013;19(5):640-645.
[10]Bernal-Mizrachi E,Kulkarni RN,Scott DK,et al.Human beta-cell proliferation and intracellular signaling part 2:still driving in the dark without a road map.Diabetes.2014;63(3):819-831.
[11]Vergès B,Cariou B.m TOR inhibitors and diabetes.Diabetes Res Clin Pract.2015;110(2):101-108.
[12]Ardestani A,Lupse B,Kido Y,et al.m TORC1 Signaling:A Double-Edged Sword in DiabeticβCells.Cell Metab.2018;27(2):314-331.
[13]Gu Y,Lindner J,Kumar A,et al.Rictor/m TORC2 is essential for maintaining a balance between beta-cell proliferation and cell size.Diabetes.2011;60(3):827-837.
[14]Rachdi L,Balcazar N,Osorio-Duque F,et al.Disruption of Tsc2 in pancreatic beta cells induces beta cell mass expansion and improved glucose tolerance in a TORC1-dependent manner.Proc Natl Acad Sci U S A.2008;105(27):9250-9255.
[15]Pirollo KF,Chang EH.Targeted delivery of small interfering RNA:approaching effective cancer therapies.Cancer Res.2008;68(5):1247-1250.
[16]袁松,孙会敏,丁丽霞.脂质体物理化学稳定性研究进展[J].中国药事,2011,25(4):384-388.
[17]张旭,丁会芹,王冰,等.脂质体介导RNAi的研究[J].生物医学工程学杂志,2012,29(4):722-726.
[18]许戈阳,刘芬婷,沈哲民,等.m TOR信号通路在糖代谢中作用[J].生理科学进展,2015,46(2):94-98.
[19]Cota D.Mammalian target of rapamycin complex 1(m TORC1)signaling in energy balance and obesity.Physiol Behav.2009;97(5):520-524.
[20]Le Bacquer O,Petroulakis E,Paglialunga S,et al.Elevated sensitivity to diet-induced obesity and insulin resistance in mice lacking 4E-BP1 and 4E-BP2.J Clin Invest.2007;117(2):387-396.
[21]Demidenko ZN,Shtutman M,Blagosklonny MV.Blagosklonny,Pharmacologic inhibition of MEK and PI-3K converges on the m TOR/S6 pathway to decelerate cellular senescence.Cell Cycle.2009;8(12):1896-900.
[22]Fritsche L,Neukamm SS,Lehmann R,et al.Insulin-induced serine phosphorylation of IRS-2 via ERK1/2 and m TOR:studies on the function of Ser675 and Ser907.Am J Physiol Endocrinol Metab.2011;300(5):E824-836.
[23]Jacinto E,Loewith R,Schmidt A,et al.Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive.Nat Cell Biol.2004;6(11):1122-1128.
[24]Elghazi L,Balcazar N,Blandino-Rosano M,et al.Decreased IRS signaling impairs beta-cell cycle progression and survival in transgenic mice overexpressing S6K in beta-cells.Diabetes.2010;59(10):2390-2399.
[25]Briaud I,Dickson LM,Lingohr MK,et al.Insulin receptor substrate-2 proteasomal degradation mediated by a mammalian target of rapamycin(m TOR)-induced negative feedback down-regulates protein kinase B-mediated signaling pathway in beta-cells.J Biol Chem.2005;280(3):2282-2293.
[26]Efeyan A,Sabatini DM.m TOR and cancer:many loops in one pathway.Curr Opin Cell Biol.2010;22(2):169-176.
[27]Copps KD,White MF.egulation of insulin sensitivity by serine/threonine phosphorylation of insulin receptor substrate proteins IRS1 and IRS2.Diabetologia.2012;55(10):2565-2582.
[28]Chau GC,Im DU,Kang TM,et al.m TOR controls Ch REBP transcriptional activity and pancreatic beta cell survival under diabetic stress.J Cell Biol.2017;216(7):2091-2105.
[29]Koyanagi M,Asahara S,Matsuda T,et al.Ablation of TSC2enhances insulin secretion by increasing the number of mitochondria through activation of m TORC1.PLo S One.2011;6(8):e23238.
[30]Gurevitch D,Boura-Halfon S,Isaac R,et al.Elimination of negative feedback control mechanisms along the insulin signaling pathway improves beta-cell function under stress.Diabetes,2010;59(9):2188-2197.