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通讯作者:

陈允梓,E-mail:chenyunzi@njmu.edu.

中图分类号:R392.11

文献标识码:A

文章编号:1007-4368(2022)08-1073-07

DOI:10.7655/NYDXBNS20220804

参考文献 1
CHRISTAKOS S,DHAWAN P,VERSTUYF A,et al.Vi⁃ tamin D:metabolism,molecular mechanism of action,and pleiotropic effects[J].Physiol Rev,2016,96(1):365-408
参考文献 2
弓玉祥,倪海锋,张思宇,等.活性维生素D3对脓毒血症小鼠急性肝损伤的保护作用及机制[J].南京医科大学学报(自然科学版),2021,41(9):1289-1321
参考文献 3
DUAN A,MA Z,LIU W,et al.1,25⁃Dihydroxyvitamin D inhibits osteoarthritis by modulating interaction between vitamin D receptor and NLRP3 in macrophages[J].J In⁃ flamm Res,2021,14:6523-6542
参考文献 4
VOUTSADAKIS I A.Vitamin D receptor(VDR)and me⁃ tabolizing enzymes CYP27B1 and CYP24A1 in breast cancer[J].Mol Biol Rep,2020,47(12):9821-9830
参考文献 5
LAVERNY G,PENNA G,USKOKOVIC M,et al.Synthe⁃ sis and anti ⁃inflammatory properties of 1alpha,25⁃ dihy⁃ droxy⁃16⁃ene⁃20⁃cyclopropyl⁃24⁃oxo⁃vitamin D3,a hypo⁃ calcemic,stable metabolite of 1alpha,25⁃ dihydroxy ⁃16⁃ ene⁃20⁃cyclopropyl⁃vitamin D3[J].J Med Chem,2009,52(8):2204-2213
参考文献 6
LEE S M,MEYER M B,BENKUSKY N A,et al.The im⁃ pact of VDR expression and regulation in vivo[J].J Ste⁃ roid Biochem Mol Biol,2018,177:36-45
参考文献 7
DE ALBUQUERQUE BORBOREMA M E,OLIVEIRA D C,DE AZEVEDO SILVA J.Down regulation of VDR gene expression in metabolic syndrome and atherosclero⁃ sis’patients:cause or consequence?[J].Gene,2021,771:145341
参考文献 8
SHANG M,SUN J.Vitamin D/VDR,probiotics,and gas⁃ trointestinal diseases[J].Curr Med Chem,2017,24(9):876-887
参考文献 9
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参考文献 10
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参考文献 11
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参考文献 12
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参考文献 13
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参考文献 14
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参考文献 15
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参考文献 16
HUSS L,BUTT S T,BORGQUIST S,et al.Vitamin D re⁃ ceptor expression in invasive breast tumors and breast cancer survival[J].Breast Cancer Res,2019,21(1):84
参考文献 17
FATHI N,AHMADIAN E,SHAHI S,et al.Role of vita⁃ min D and vitamin D receptor(VDR)in oral cancer[J].Biomed Pharmacother,2019,109:391-401
参考文献 18
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参考文献 19
BAKKE D,SUN J.Ancient nuclear receptor VDR with new functions:microbiome and inflammation[J].Inflamm Bowel Dis,2018,24(6):1149-1154
参考文献 20
ZENATA O,VRZAL R.Fine tuning of vitamin D receptor(VDR)activity by post ⁃transcriptional and post ⁃transla⁃ tional modifications[J].Oncotarget,2017,8(21):35390-35402
参考文献 21
MICHAEL B.Targeted regulation of nuclear lamins by ubiquitin and ubiquitin⁃like modifiers[J].Cells,2020,9:1340
参考文献 22
ANTJE R,DANIELA H,CAROLINE B,et al.PRMT1 pro⁃ motes thetumor suppressor function of p14ARF and is indic⁃ ative for pancreatic cancer prognosis[J].EMBO J,2021,40(13):e106777
目录contents

    摘要

    目的:确定维生素D受体(vitamin D receptor,VDR)的核定位信号并验证其功能。方法:通过软件分析及文献调研预测VDR核定位信号,利用点突变试剂盒构建了VDR一系列点突变体,通过蛋白质免疫印迹检测VDR在胞质和胞核里的分布,并结合免疫荧光观察确定其预测突变点对VDR入核的影响,最后采用实时荧光定量链式聚合酶反应(quantitative real⁃time PCR,qPCR)检测 VDR 下游基因的表达,以判断 VDR 核定位突变体的活性。结果:确定 VDR 核定位信号序列为 49RRSMKRKALFLT61,构建的VDR一系列核定位序列(nuclear localization sequence,NLS)点突变体在细胞内的表达主要分布在胞质里,维生素D(vitamin D,VD)既不能诱导这些点突变体入核,也不能促进VD下游基因表达。结论:VDR核定位确定信号能有效控制VDR进入细胞核发挥转录因子作用,进而影响其下游基因的表达,为探索VD及VDR相关疾病的治疗提供新平台和思路。

    Abstract

    Objective:To determine the nuclear localization signal of vitamin D receptor(VDR)and verify its function. Methods: The nuclear localization signal of VDR was predicted by software analysis and literature investigation. A series of point mutants of VDR were constructed by a point mutation kit. The distribution of VDR in the cytoplasm and nucleus was obsened by Western blot and immunofluoresceme to determine the effect of mutation points in the entry of VDR into the nucleus. Finally,quantitatve real⁃time PCR (qPCR)was used to detect the expression of genes downstream of VDR to determine the activity of VDR nuclear localization mutants. Results:The nuclear localization signal sequence of VDR was determined to be49RRSMKRKALFLT61. The expression of a series of NLS (nuclear localization sequence)point mutants of VDR was mainly distributed in the cytoplasm. Vitamin D could neither induce these point mutants into the nucleus nor promote the expression of downstream genes of vitamin D. Conclusion:VDR nuclear localization signal can effectively control the entry of VDR into the nucleus,play the role of transcription factors,and then affect the expression of downstream genes,which provides a new platform and idea for exploring the treatment of vitamin D and VDR related diseases。

  • 维生素D(vitamin D,VD)是一种脂溶性维生素[1],不仅调节钙磷代谢和骨健康,在免疫系统[2]、神经系统和代谢系统中也发挥重要的调节作用,它通过与维生素D受体(vitamin D receptor,VDR)结合而发挥作用[3-4]。VD与VDR的结合能激活VDR发生核转位,在细胞核内发挥转录因子功能,启动下游基因的表达,从而参与细胞的生长分化与免疫应答的调控[5-7]

  • 核定位信号(nuclear localization sequence,NLS) 是蛋白质实现核转位的重要结构域,通常由4~8个氨基酸组成的短序列,含有Pro、Lys和Arg。有研究表明,VD在细胞内主要通过激活VDR发生核转位发挥转录因子功能[8-9],VDR核定位信号可能位于两个锌指结构之间,其氨基酸序列与核定位相关[10-11],但缺乏精确定位和实验鉴定。研究表明,VDR在胞质中也发挥功能,它能与IKKβ相互作用并抑制NF⁃ κB的激活[12]。因此,VDR的生物学功能与其细胞定位密切相关,确定VDR的NLS对深入了解VDR信号具有重要意义。本研究采用软件分析预测NLS并进行点突变验证,确定了VDR的NLS序列及其对VDR活性的影响,为VD及VDR的入核信号通路的调控提供理论依据。

  • 1 材料和方法

  • 1.1 材料

  • VD由美国芝加哥大学Yanchun Li教授赠送。胰酶、胎牛血清(Gibco公司,美国),PVDF膜(Bio⁃ Rad公司,美国),Triton X⁃100、Tris、NP40、甘油和吐温20(合肥Biosharp公司),NaCl、KCl、EDTA(上海凌峰化学试剂有限公司),胶回收试剂盒、质粒小提试剂盒(上海Omega Bio⁃tek公司),HA抗体(Abmart公司,美国),Tublin抗体、VDR抗体(Santa Cruz公司,美国),HRP标记抗兔IgG抗体、HRP标记抗鼠IgG抗体(Jackson Immune Research公司,美国),LaminB抗体、Tris⁃HCl pH 8.8、Tris⁃HCl pH 6.8、抗荧光猝灭剂、细胞核蛋白与细胞浆蛋白抽提试剂盒(上海碧云天技术有限公司),组织固定液(武汉赛维尔生物科技有限公司),Poly⁃JET(济南Signa⁃Gen),GAPDH抗体(武汉三鹰生物技术有限公司),所有引物由南京金斯瑞公司合成。SYBR Green QPCR试剂盒、逆转录酶试剂盒、点突变试剂盒(Mut Express Ⅱ Fast Mutagenesis Kit V2)(南京诺唯赞生物科技有限公司)。

  • pCMV ⁃ HA ⁃ VDR质粒由本实验室提供[6], HEK293T和HeLa细胞来自美国ATCC。

  • 1.2 方法

  • 1.2.1 引物的设计

  • 对质粒引入单碱基或多碱基的定点突变,引物设计如表1,采用点突变试剂盒在质粒pCMV⁃HA⁃ VDR上构建了突变体HA⁃VDR(R49W/R50G)、HA⁃ VDR(K53Q/R54G/K55EP)、HA ⁃VDR(R49W/R50G/K53Q/R54G/K55E)。

  • 表1 VDR基因点突变引物

  • Table1 VDR gene point mutation primers

  • 1.2.2 实时荧光定量PCR分析

  • 在质粒转染及VD刺激后,收集HeLa细胞,采用TRIzol试剂提取总RNA,并根据逆转录酶试剂盒说明书将其逆转录为cDNA,然后采用SYBR Green QPCR试剂盒,在StepOnePlus(Applied Biosystems) 上分析cDNA,进行靶基因的扩增(表2)。

  • 1.2.3 VDR的亚细胞定位分析

  • HEK293T细胞进行质粒转染,经VD处理后收集细胞。采用细胞核蛋白与细胞浆蛋白抽提试剂盒分别获得胞质和胞核组分,点样10%聚丙烯酰胺凝胶(SDS⁃PAGE)进行电泳分离,再经湿转法将蛋白转移到PVDF膜上,进行5%BSA室温封闭1h。一抗分别采用VDR、Tubulin和Lamin B抗体,4℃孵育过夜,二抗为辣根过氧化物酶标记的羊抗免多聚抗体,室温孵育1h。最后采用ECL化学发光法检测,在蛋白分析仪拍摄下进行结果分析。

  • 表2 实时荧光定量PCR引物

  • Table2 Real⁃time fluorescent quantitative PCR primers

  • 1.2.4 免疫荧光分析

  • 细胞样品采用4%多聚甲醛固定,室温15min, PBS清洗2遍,再用0.02%NP⁃40处理15min。然后用QuickBlockTM免疫染色封闭液封闭15min,进行一抗HA抗体孵育,4℃过夜。次日,PBS(含0.5%Tri⁃ tion X⁃100)清洗后,加入荧光标记的二抗室温孵育1h。最后复染DAPI,细胞样品进行荧光显微镜的图像采集并分析。

  • 1.3 统计学方法

  • 实验数据用GraphPad Prism 7.0软件进行统计学分析,各组数据用均数±标准差(x-±s)表示,多组定量资料比较采用单因素方差分析(one⁃way ANOVA)检验,多组间数据两两比较采用LSD法。两组定量数据比较用t检验。P< 0.05为差异有统计学意义。

  • 2 结果

  • 2.1 VDR核定位信号的预测

  • 为了初步确定VDR的核定位信号位置,借助在线预测网站对VDR氨基酸全序列进行了分析 (http://nls ⁃ mapper.iab.keio.ac.jp/cgi ⁃ bin/NLS_Map⁃ per_form.cgi),预测结果显示核定位信号序列为 49RRSMKRKALFLT61(图1A)。对不同物种来源的VDR进行序列比对,发现预测的核定位信号序列具有很好的保守性(图1B)。

  • 图1 预测VDR核定位信号

  • Fig.1 Predicting the nuclear localization signal of VDR

  • 2.2 VDR突变体的构建

  • 根据预测的结果和文献报道[10],针对VDR核定位序列中的保守氨基酸精氨酸(R)和赖氨酸(K)进行点突变设计,采用点突变试剂盒(Mut Express Ⅱ Fast Mutagenesis Kit V2)以质粒pCMV⁃HA⁃VDR为模板,构建了一系列突变体(R49W/R50G、K53Q/R54G/K55E和R49W/R50G/K53Q/R54G/K55E),通过Vector NTI软件对测序结果进行分析,结果显示VDR突变体成功引入突变点(图2)。

  • 2.3 VDR点突变抑制其入核能力

  • 为了验证点突变是否对VDR核定位产生影响,将VDR突变体(HA ⁃VDR、HA ⁃VDR ⁃R49W/R50G、 HA⁃VDR⁃K53Q/R54G/K55E、HA⁃VDR⁃R49W/R50G/K53Q/R54G/K55E)转染HEK 293T细胞进行表达。在VD 100nmol/L处理前后,分别获取细胞样品进行核质分离,并采用Western blot分析,其中Tubulin为胞质内参,Lamin B为胞核内参。结果表明,VD处理能诱导野生型VDR转位细胞核内(图3A),而VDR突变体VDR(R49W/R50G),VDR(K53Q/R54G/K55E)和VDR(R49W/R50G/K53Q/R54G/K55E)的表达主要停留在胞质中,不能被VD诱导转位入细胞核(图3B~D)。由此可见,在预测的NLS里这些保守位点的突变抑制了VDR进入细胞核,初步确定它们是VDR入核的关键位点。

  • 接着,采用免疫荧光法直接观察VDR点突变对其空间分布的影响。在HeLa细胞中转染VDR及其突变体载体,观察这些蛋白的表达在VD刺激前后在细胞空间上的分布差异,以DAPI标记细胞核,红色荧光标记HA⁃tag指示VDR及变体的所在位置。如图4结果所示,在VD刺激下,野生型HA⁃VDR发生明显转位进入细胞核,而VDR突变体在VD刺激前后,细胞内的空间分布并未发生明显变化,与上述Western blot结果一致,表明这些点突变位点是VDR核定位的关键位点。

  • 图2 构建VDR点突变

  • Fig.2 Constructing VDR point mutations

  • 图3 VDR的点突变抑制其核定位

  • Fig.3 VDR point mutations inhibit the transfer of VDR to the nucleus

  • 2.4 VDR点突变减弱了对下游基因表达的调控

  • VD的生物学功能主要由VDR下游基因实现。 Cyp24a1、Atp2b1是VDR调控的下游基因[13-14],而Cyp27A不受VDR调控作为阴性对照。在HEK 293T细胞里瞬时表达HA⁃VDR及点突变体,采用100nmol/L VD处理,收集细胞样品进行qPCR检测。如图5所示,在VDR表达的细胞里,VD可以有效诱导Cyp24a1mRNA、Atp2b1mRNA水平上调;而在VDR点突变表达的细胞中,VD诱导Cyp24a1mRNA、Atp2b1mRNA表达的能力显著降低,说明VDR的点突变能削弱了VD的生物学功能,不能有效响应VD信号启动下游基因的转录。

  • 3 讨论

  • VDR的活性受VD调控,在一些疾病如癌症、神经系统疾病、免疫性疾病的研究中已成为预防、诊断和治疗的重要靶点分子[15-19]。VD与VDR结合能激活VDR入核发挥转录因子功能,确定其NLS有助于进一步了解VDR在细胞核和细胞质中的不同功能,为研究相关疾病提供参考依据。

  • 图4 VDR点突变影响VDR在细胞内的分布

  • Fig.4 VDR point mutations affect the spatial distribution of VDR in cells

  • 图5 VDR的点突变影响了VDR对下游基因的转录

  • Fig.5 VDR point mutations affect the transcription of downstream genes by VDR

  • 根据在线网站预测分析VDR的核定位信号位置,提示VDR的NLS为49RRSMKRKALFLT61,并且通过序列比对显示在不同种属VDR序列里该NLS具有很好的保守性。根据NLS保守性分析,针对49/50位、53/54/55位、49/50/53/54/55位的保守氨基酸进行突变,通过Western blot和免疫荧光等方法进行检测,结果显示:当VD刺激后,野生型VDR能快速入核;而VDR的NLS突变都能有效抑制VDR入核,证明了这些氨基酸位点参与调控VDR的入核。据文献报道,VD与VDR结合能启动生物反应,启动下游基因如CYP24A1、ATP2B1等的转录。通过qPCR检测这些下游基因,结果表明突变体VDR明显抑制了CYP24A1、ATP2B1的mRNA表达,确定了这5个位点是VDR入核并发挥转录调节作用的关键位点。基于这些VDR点突变体不能入核的性质,提示这些点突变体可作为研究胞质VDR功能的新工具,应用于VDR的相关研究[20]

  • 通常,精氨酸(R)可以被甲基化修饰,赖氨酸 (K)可以被泛素化修饰,而这些蛋白质的后加工修饰形式也可以调控蛋白质的核定位行为,例如: Laims蛋白能发生泛素化调节其核定位[21],PRMT1甲基化酶能使p14ARF蛋白发生甲基化修饰调节该蛋白的核定位[22]。因此,对于VDR点突变体不能入核的现象,推测还存在另一个可能:当精氨酸和赖氨酸位点进行点突变后,可能抑制了VDR的泛素化或甲基化的后加工形式,从而影响VDR的入核行为。是否VDR的这些位点会发生后加工修饰参与VDR核定位的调节呢?这也是一个值得探究的问题。

  • 总之,本研究通过预测分析,点突变体的构建,结合亚细胞定位分析和qPCR等方法,确定了VDR的核定位信号为 49RRSMKRKALFLT61,并且证明了这5个位点(49/50/53/54/55)的突变能够影VD/VDR信号的生物学活性,这一发现为探索VDR在细胞质和细胞核中的作用提供研究平台,为探讨VD/VDR相关疾病的预防、诊断和治疗提供了一定的理论基础。

  • 参考文献

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    • [8] SHANG M,SUN J.Vitamin D/VDR,probiotics,and gas⁃ trointestinal diseases[J].Curr Med Chem,2017,24(9):876-887

    • [9] CUI C,CUI J,JIN F,et al.Induction of the vitamin D re⁃ ceptor attenuates autophagy dysfunction ⁃ mediated cell death following traumatic brain injury[J].Cell Physiol Biochem,2017,42(5):1888-1896

    • [10] PELEG S,NGUYEN C V.The importance of nuclear im⁃ port in protection of the vitamin D receptor from polyubiq⁃ uitination and proteasome ⁃ mediated degradation[J].J Cell Biochem,2010,110(4):926-934

    • [11] KLOPOT A,HANCE K W,PELEG S,et al.Nucleo⁃cyto⁃ plasmic cycling of the vitamin D receptor in the enterocyte⁃ like cell line,Caco⁃2[J].J Cell Biochem,2007,100(3):617-628

    • [12] KELLERMANN L,JENSEN K B,BERGENHEIM F,et al.Mucosal vitamin D signaling in inflammatory bowel disease[J].Autoimmun Rev,2020,19(11):102672

    • [13] SCHLINGMANN K P,KAUFMANN M,WEBER S,et al.Mutations in CYP24A1 and idiopathic infantile hypercal⁃ cemia[J].N Engl J Med,2011,365(5):410-421

    • [14] CARLBERG C,CAMPBELL M J.Vitamin D receptor sig⁃ naling mechanisms:integrated actions of a well ⁃ defined transcription factor[J].Steroids,2013,78(2):127-136

    • [15] RAO Z,CHEN X,WU J et al.Vitamin D receptor inhibits NLRP3 activation by impeding its BRCC3⁃mediated deu⁃biquitination[J].Front Immunol,2019,10:2783

    • [16] HUSS L,BUTT S T,BORGQUIST S,et al.Vitamin D re⁃ ceptor expression in invasive breast tumors and breast cancer survival[J].Breast Cancer Res,2019,21(1):84

    • [17] FATHI N,AHMADIAN E,SHAHI S,et al.Role of vita⁃ min D and vitamin D receptor(VDR)in oral cancer[J].Biomed Pharmacother,2019,109:391-401

    • [18] JEON S M,SHIN E A.Exploring vitamin D metabolism and function in cancer[J].Exp Mol Med,2018,50(4):1-14

    • [19] BAKKE D,SUN J.Ancient nuclear receptor VDR with new functions:microbiome and inflammation[J].Inflamm Bowel Dis,2018,24(6):1149-1154

    • [20] ZENATA O,VRZAL R.Fine tuning of vitamin D receptor(VDR)activity by post ⁃transcriptional and post ⁃transla⁃ tional modifications[J].Oncotarget,2017,8(21):35390-35402

    • [21] MICHAEL B.Targeted regulation of nuclear lamins by ubiquitin and ubiquitin⁃like modifiers[J].Cells,2020,9:1340

    • [22] ANTJE R,DANIELA H,CAROLINE B,et al.PRMT1 pro⁃ motes thetumor suppressor function of p14ARF and is indic⁃ ative for pancreatic cancer prognosis[J].EMBO J,2021,40(13):e106777

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    • [6] LEE S M,MEYER M B,BENKUSKY N A,et al.The im⁃ pact of VDR expression and regulation in vivo[J].J Ste⁃ roid Biochem Mol Biol,2018,177:36-45

    • [7] DE ALBUQUERQUE BORBOREMA M E,OLIVEIRA D C,DE AZEVEDO SILVA J.Down regulation of VDR gene expression in metabolic syndrome and atherosclero⁃ sis’patients:cause or consequence?[J].Gene,2021,771:145341

    • [8] SHANG M,SUN J.Vitamin D/VDR,probiotics,and gas⁃ trointestinal diseases[J].Curr Med Chem,2017,24(9):876-887

    • [9] CUI C,CUI J,JIN F,et al.Induction of the vitamin D re⁃ ceptor attenuates autophagy dysfunction ⁃ mediated cell death following traumatic brain injury[J].Cell Physiol Biochem,2017,42(5):1888-1896

    • [10] PELEG S,NGUYEN C V.The importance of nuclear im⁃ port in protection of the vitamin D receptor from polyubiq⁃ uitination and proteasome ⁃ mediated degradation[J].J Cell Biochem,2010,110(4):926-934

    • [11] KLOPOT A,HANCE K W,PELEG S,et al.Nucleo⁃cyto⁃ plasmic cycling of the vitamin D receptor in the enterocyte⁃ like cell line,Caco⁃2[J].J Cell Biochem,2007,100(3):617-628

    • [12] KELLERMANN L,JENSEN K B,BERGENHEIM F,et al.Mucosal vitamin D signaling in inflammatory bowel disease[J].Autoimmun Rev,2020,19(11):102672

    • [13] SCHLINGMANN K P,KAUFMANN M,WEBER S,et al.Mutations in CYP24A1 and idiopathic infantile hypercal⁃ cemia[J].N Engl J Med,2011,365(5):410-421

    • [14] CARLBERG C,CAMPBELL M J.Vitamin D receptor sig⁃ naling mechanisms:integrated actions of a well ⁃ defined transcription factor[J].Steroids,2013,78(2):127-136

    • [15] RAO Z,CHEN X,WU J et al.Vitamin D receptor inhibits NLRP3 activation by impeding its BRCC3⁃mediated deu⁃biquitination[J].Front Immunol,2019,10:2783

    • [16] HUSS L,BUTT S T,BORGQUIST S,et al.Vitamin D re⁃ ceptor expression in invasive breast tumors and breast cancer survival[J].Breast Cancer Res,2019,21(1):84

    • [17] FATHI N,AHMADIAN E,SHAHI S,et al.Role of vita⁃ min D and vitamin D receptor(VDR)in oral cancer[J].Biomed Pharmacother,2019,109:391-401

    • [18] JEON S M,SHIN E A.Exploring vitamin D metabolism and function in cancer[J].Exp Mol Med,2018,50(4):1-14

    • [19] BAKKE D,SUN J.Ancient nuclear receptor VDR with new functions:microbiome and inflammation[J].Inflamm Bowel Dis,2018,24(6):1149-1154

    • [20] ZENATA O,VRZAL R.Fine tuning of vitamin D receptor(VDR)activity by post ⁃transcriptional and post ⁃transla⁃ tional modifications[J].Oncotarget,2017,8(21):35390-35402

    • [21] MICHAEL B.Targeted regulation of nuclear lamins by ubiquitin and ubiquitin⁃like modifiers[J].Cells,2020,9:1340

    • [22] ANTJE R,DANIELA H,CAROLINE B,et al.PRMT1 pro⁃ motes thetumor suppressor function of p14ARF and is indic⁃ ative for pancreatic cancer prognosis[J].EMBO J,2021,40(13):e106777

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