FTO对小鼠肾小球系膜细胞m6A修饰及增殖能力的影响
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摘要
目的:构建表达脂肪与肥胖相关(fat mass and obesity associated,FTO)基因的重组质粒,并检测其对小鼠肾小球系膜细胞(mice mesangial cell,MMC)N6-甲基腺嘌呤(m6A)修饰及增殖能力的影响。方法:PCR法扩增FTO基因片段,并将其插入表达载体pCMV-MCS-EGFP质粒中以构建重组质粒pCMV-FTO。将目的质粒pCMV-FTO及对照质粒pCMV分别转染MMC,采用qRT-PCR法检测FTO mRNA表达水平,蛋白质印迹法检测细胞FTO蛋白和相关增殖标志物的表达,CCK8法检测细胞增殖,m6A RNA甲基化定量试剂盒检测m6A含量。结果:菌落PCR鉴定以及测序结果证实重组质粒pCMV-FTO构建成功。RT-qPCR及蛋白印迹结果显示转染目的质粒pCMV-FTO后FTO表达明显增加。过表达FTO后,m6A含量、细胞增殖水平及细胞周期蛋白D1(Cyclin D1)、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)蛋白水平均显著下降。结论:FTO可以降低MMC中m6A修饰水平及抑制细胞增殖。
Objective:This study aims to construct a recombinant plasmid expressing fat mass and obesity associated(FTO)gene and to detect its effects on m6 A modification and proliferation of mouse mesangial cells(MMCs). Methods:The FTO gene fragment was amplified by PCR and inserted into the expression vector pCMV-MCS-EGFP plasmid to construct the recombinant plasmid pCMVFTO. The target plasmid pCMV-FTO and the control plasmid pCMV were transfected into MMCs,respectively,and the mRNA expression of FTO was measured with real-time quantitative PCR(RT-qPCR). The total protein was extracted,and FTO,EGFP,proliferation markers of Cyclin D1 and PCNA were detected by Western blotting. The proliferation of MMCs were studied with CCK8 method. The m6 A content was measured using the m6 A RNA methylation quantification kit. Results:Colony PCR identification and sequencing confirmed that the recombinant plasmid pCMV-FTO was successfully constructed. The results showed that the mRNA and protein level of FTO was significantly increased after transfection of the target plasmid pCMV-FTO. After overexpression of FTO,m6 A content,the proliferation of MMCs and Cyclin D1,PCNA protein levels decreased significantly. Conclusion:FTO can reduce the level of m6 A modification and inhibit cell proliferation in MMCs.
Objective:This study aims to construct a recombinant plasmid expressing fat mass and obesity associated(FTO)gene and to detect its effects on m6 A modification and proliferation of mouse mesangial cells(MMCs). Methods:The FTO gene fragment was amplified by PCR and inserted into the expression vector pCMV-MCS-EGFP plasmid to construct the recombinant plasmid pCMVFTO. The target plasmid pCMV-FTO and the control plasmid pCMV were transfected into MMCs,respectively,and the mRNA expression of FTO was measured with real-time quantitative PCR(RT-qPCR). The total protein was extracted,and FTO,EGFP,proliferation markers of Cyclin D1 and PCNA were detected by Western blotting. The proliferation of MMCs were studied with CCK8 method. The m6 A content was measured using the m6 A RNA methylation quantification kit. Results:Colony PCR identification and sequencing confirmed that the recombinant plasmid pCMV-FTO was successfully constructed. The results showed that the mRNA and protein level of FTO was significantly increased after transfection of the target plasmid pCMV-FTO. After overexpression of FTO,m6 A content,the proliferation of MMCs and Cyclin D1,PCNA protein levels decreased significantly. Conclusion:FTO can reduce the level of m6 A modification and inhibit cell proliferation in MMCs.
引文
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[12]Ma JZ,Yang F,Zhou CC,et al. METTL14 suppresses the metastatic potential of hepatocellular carcinoma by modulating N6-methyladenosine-dependent primary MicroRNA processing[J]. Hepatology,2017,65(2):529-543
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[14] Batista PJ,Molinie B,Wang J,et al. m(6)A RNA modification controls cell fate transition in mammalian embryonic stem cells[J]. Cell Stem Cell,2014,15(6):707-719
[15] Dina C,Meyre D,Gallina S,et al. Variation in FTO contributes to childhood obesity and severe adult obesity[J].Nat Genet,2007,39(6):724-726
[16] Church C,Moir L,McMurray F,et al. Overexpression of Fto leads to increased food intake and results in obesity[J]. Nat Genet,2010,42(12):1086-1092
[17] McMurray F,Church CD,Larder R,et al. Adult onset global loss of the fto gene alters body composition and metabolisminthemouse[J].PLoSGenet,2013,9(1):e1003166
[18]Merkestein M,Laber S,McMurray F,et al. FTO influences adipogenesis by regulating mitotic clonal expansion[J]. Nat Commun,2015,6:6792
[19]Li Z,Weng H,Su R,et al. FTO plays an oncogenic role in acute myeloid leukemia as a N(6)-methyladenosine RNA demethylase[J]. Cancer Cell,2017,31(1):127-141
[20] Zhou S,Bai ZL,Xia D,et al. FTO regulates the chemo-radiotherapy resistance of cervical squamous cell carcinoma(CSCC)by targeting beta-catenin through mRNA demethylation[J]. Mol Carcinog,2018,57(5):590-597
[21] Shen F,Huang W,Huang JT,et al. Decreased N(6)-methyladenosine in peripheral blood RNA from diabetic patients is associated with FTO expression rather than ALKBH5[J]. J Clin Endocrinol Metab,2015,100(1):E148-E154
[22] Daoud H,Zhang D,McMurray F,et al. Identification of a pathogenic FTO mutation by next-generation sequencing in a newborn with growth retardation and developmental delay[J]. J Med Genet,2016,53(3):200-207
[23] Arora MK,Singh UK. Molecular mechanisms in the pathogenesis of diabetic nephropathy:an update[J]. Vascul Pharmacol,2013,58(4):259-271
[24]李萍,方莉,蔡秋萍,等.糖尿病肾病患者微炎症和氧化应激水平及其影响因素分析[J].南京医科大学学报(自然科学版),2016,36(2):214-217
[2] Jia G,Fu Y,Zhao X,et al. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO[J]. Nat Chem Biol,2011,7(12):885-887
[3] Boissel S,Reish O,Proulx K,et al. Loss-of-function mutation in the dioxygenase-encoding FTO gene causes severe growth retardation and multiple malformations[J]. Am J Hum Genet,2009,85(1):106-111
[4] Jia G,Fu Y,He C. Reversible RNA adenosine methylation in biological regulation[J]. Trends Genet,2013,29(2):108-115
[5] Liu J,Yue Y,Han D,et al. A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation[J]. Nat Chem Biol,2014,10(2):93-95
[6] Ping XL,Sun BF,Wang L,et al. Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase[J]. Cell Res,2014,24(2):177-189
[7] Zheng G,Dahl JA,Niu Y,et al. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility[J]. Mol Cell,2013,49(1):18-29
[8] Zhao X,Yang Y,Sun BF,et al. FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis[J]. Cell Res,2014,24(12):1403-1419
[9] Wang X,Lu Z,Gomez A,et al. N6-methyladenosine-dependent regulation of messenger RNA stability[J]. Nature,2014,505(7481):117-120
[10] Wang X,Zhao BS,Roundtree IA,et al. N(6)-methyladenosine modulates messenger RNA Translation Efficiency[J]. Cell,2015,161(6):1388-1399
[11] Weng H. METTL14 Inhibits Hematopoietic stem/progenitor differentiation and promotes leukemogenesis via mRNA m6A modification[J]. Cell Stem Cell,2018,22(2):191-205
[12]Ma JZ,Yang F,Zhou CC,et al. METTL14 suppresses the metastatic potential of hepatocellular carcinoma by modulating N6-methyladenosine-dependent primary MicroRNA processing[J]. Hepatology,2017,65(2):529-543
[13] Chen M,Wei L,Law CT,et al. RNA N6-methyladenosine methyltransferase METTL3 promotes liver cancer progression through YTHDF2 dependent post-transcriptional silencing of SOCS2[J]. Hepatology,2017,67(6):2254-2270
[14] Batista PJ,Molinie B,Wang J,et al. m(6)A RNA modification controls cell fate transition in mammalian embryonic stem cells[J]. Cell Stem Cell,2014,15(6):707-719
[15] Dina C,Meyre D,Gallina S,et al. Variation in FTO contributes to childhood obesity and severe adult obesity[J].Nat Genet,2007,39(6):724-726
[16] Church C,Moir L,McMurray F,et al. Overexpression of Fto leads to increased food intake and results in obesity[J]. Nat Genet,2010,42(12):1086-1092
[17] McMurray F,Church CD,Larder R,et al. Adult onset global loss of the fto gene alters body composition and metabolisminthemouse[J].PLoSGenet,2013,9(1):e1003166
[18]Merkestein M,Laber S,McMurray F,et al. FTO influences adipogenesis by regulating mitotic clonal expansion[J]. Nat Commun,2015,6:6792
[19]Li Z,Weng H,Su R,et al. FTO plays an oncogenic role in acute myeloid leukemia as a N(6)-methyladenosine RNA demethylase[J]. Cancer Cell,2017,31(1):127-141
[20] Zhou S,Bai ZL,Xia D,et al. FTO regulates the chemo-radiotherapy resistance of cervical squamous cell carcinoma(CSCC)by targeting beta-catenin through mRNA demethylation[J]. Mol Carcinog,2018,57(5):590-597
[21] Shen F,Huang W,Huang JT,et al. Decreased N(6)-methyladenosine in peripheral blood RNA from diabetic patients is associated with FTO expression rather than ALKBH5[J]. J Clin Endocrinol Metab,2015,100(1):E148-E154
[22] Daoud H,Zhang D,McMurray F,et al. Identification of a pathogenic FTO mutation by next-generation sequencing in a newborn with growth retardation and developmental delay[J]. J Med Genet,2016,53(3):200-207
[23] Arora MK,Singh UK. Molecular mechanisms in the pathogenesis of diabetic nephropathy:an update[J]. Vascul Pharmacol,2013,58(4):259-271
[24]李萍,方莉,蔡秋萍,等.糖尿病肾病患者微炎症和氧化应激水平及其影响因素分析[J].南京医科大学学报(自然科学版),2016,36(2):214-217