吉林大学学报(医学版)  2020, Vol. 46 Issue (05): 955-962     DOI: 10.13481/j.1671-587x.20200510

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乔良伟, 曲青山, 李明
QIAO Liangwei, QU Qingshan, LI Ming
LncRNA-ATB通过调节miR-200c表达对肾移植大鼠急性排斥反应的影响
Effect of LncRNA-ATB on acute rejection of kidney transplanted rats by regulating miR-200c expression
吉林大学学报(医学版), 2020, 46(05): 955-962
Journal of Jilin University (Medicine Edition), 2020, 46(05): 955-962
10.13481/j.1671-587x.20200510

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收稿日期: 2020-02-26
LncRNA-ATB通过调节miR-200c表达对肾移植大鼠急性排斥反应的影响
乔良伟 , 曲青山 , 李明     
郑州人民医院器官移植中心肾脏移植科, 河南 郑州 450000
[摘要]: 目的: 探讨被转化生长因子β活化的长链非编码RNA(LncRNA-ATB)调节微小RNA(miRNA)-200c对肾移植大鼠移植后急性排斥反应(AR)和受体组织炎症反应的影响,分析肾移植AR潜在的分子机制,为临床诊断和治疗AR提供理论参考。方法: 30只SD大鼠为供体,30只Wistar大鼠为受体,通过手术将供体肾脏移植到受体大鼠体内,建立肾移植AR模型,将建模成功大鼠(27只)随机分为模型组、LncRNA-ATB短发夹RNA(shRNA)组和LncRNA-ATB-NC组,每组9只。选取10只Wistar大鼠为假手术组。术后2 h,LncRNA-ATB-shRNA组和LncRNA-ATB-NC组大鼠按每只200 μL分别尾静脉注射含LncRNA-ATB-shRNA和LncRNA-ATB-NC的psiCHECK2基因重组质粒脂质体复合物,假手术组和模型组大鼠按每只200 μL尾静脉注射Opti-MEM培养基。干预7 d后,RT-PCR法检测各组大鼠外周血中LncRNA-ATB和miR-200c表达水平,全自动生化分析仪检测各组大鼠血肌酐(Scr)和血尿素氮(BUN)水平,酶联免疫吸附法(ELISA)检测各组大鼠血清中白细胞介素6(IL-6)、白细胞介素8(IL-8)和肿瘤坏死因子α(TNF-α)水平,苏木精-伊红(HE)染色法观察各组大鼠肾组织病理形态表现并对AR进行诊断分级和半定量评分,RT-PCR法和Western blotting法检测各组大鼠肾组织中Toll样受体4(TLR4)、髓样分化因子88(MyD88)和核转录因子κB(NF-κB)mRNA及蛋白表达水平。结果: HE染色,与假手术组比较,模型组大鼠肾脏出现AR,表现为炎性细胞浸润、动脉炎症、间质细胞水肿和皮质出血等,LncRNA-ATB-NC组与模型组大鼠肾脏组织病理形态表现相似,LncRNA-ATB-shRNA组大鼠肾组织病理学形态表现较模型组和LncRNA-ATB-NC组均减轻。与假手术组比较,模型组、LncRNA-ATB-NC组和LncRNA-ATB-shRNA组大鼠外周血中LncRNA-ATB表达水平,血清中Scr、BUN、IL-6、IL-8和TNF-α水平,AR半定量评分,肾组织中TLR4、MyD88和NF-κB mRNA及蛋白表达水平均升高(P < 0.05),外周血中miR-200c表达水平均降低(P < 0.05);与模型组和LncRNA-ATB-NC组比较,LncRNA-ATB-shRNA组大鼠外周血中LncRNA-ATB表达水平,血清中Scr、BUN、IL-6、IL-8和TNF-α水平,AR半定量评分,肾组织中TLR4、MyD88和NF-κB mRNA及蛋白表达水平均降低(P < 0.05),外周血中miR-200c表达水平升高(P < 0.05);模型组与LncRNA-ATB-NC组大鼠外周血中LncRNA-ATB和miR-200c表达水平,血清中Scr、BUN、IL-6、IL-8和TNF-α水平,AR半定量评分,肾组织中TLR4、MyD88和NF-κB mRNA及蛋白表达水平比较差异均无统计学意义(P>0.05)。结论: 沉默LncRNA-ATB可以减轻肾移植大鼠移植后AR,抑制受体组织发生炎症反应,其机制可能是通过上调miR-200c,抑制TLR4、MyD88和NF-κB mRNA及蛋白表达而发挥炎症抑制作用。
关键词: 被转化生长因子β活化的长链非编码RNA    微小RNA    肾移植    急性排斥反应    炎症反应    大鼠, Wistar    
Effect of LncRNA-ATB on acute rejection of kidney transplanted rats by regulating miR-200c expression
QIAO Liangwei , QU Qingshan , LI Ming     
Department of Kidney Transplantation, Organ Transplant Center, Zhengzhou People's Hospital, Zhengzhou 450000, Henan
[ABSTRACT]: Objective: To investigate the influence of long non-coding RNA activated by TGF-β (LncRNA-ATB) by regulating microRNA (miRNA)-200c in the acute rejection (AR) and the inflammatory response of recipient tissue after transplantation in the kidney transplantation rats, and to analyze the potential molecular mechanism of AR of kidney transplantation. Methods: Thirty SD rats were used as donors and 30 Wistar rats were used as recipients. The donor kidneys were transplanted into the recipient rats by surgery to establish the models of kidney transplantion AR. The successful modeling rats (n=27) were randomly divided into model group, LncRNA-ATB short hairpin RNA (shRNA) group, and LncRNA-ATB-NC group; there were 9 rats in each group. Ten Wistar rats were selected and used as sham operation group. Two hours after transplantation, the rats in LncRNA-ATB-shRNA group and LncRNA-ATB-NC group were injected with LncRNA-ATB-shRNA and LncRNA-ATB-NC gene recombinant plasmid liposome complex in the tail vein (200 μL/rat), and the rats in sham operation group and model group were injected with Opti-MEM medium (200 μL/rat). After 7 d, the relative expression levels of LncRNA-ATB and miR-200c in peripheral blood of the rats in various groups were detected by RT-PCR method, and the levels of serum creatinine (Scr) and blood urea nitrogen (BUN) of the rats in various groups were detected by automatic biochemical analyzer; the levels of serum interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α(TNF-α) of the rats in various groups were measured by enzyme-linked immunosorbent assay(ELISA); HE staining was used to observe the pathomorphology of kidney tissue of the rats in various groups, and the diagnosis grade of AR and the AR semi-quantitative scores were performed; the expression levels of Toll-like receptor-4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear transcription factor κB (NF-κB) mRNA and proteins in kidney tissue of the rats in various groups were detected by RT-PCR and Western blotting methods. Results: The HE staining results showed that compared with sham operation group, there was AR in kidney tissue of the rats in model group, existing inflammatory cell infiltration, arterial inflammation, interstitial cell edema, and cortical hemorrhage, etc; the phathomorphology of kidney tissue of the rats of the rats in LncRNA-ATB-NC group was similar to model group; the pathomorphology of the kidney tissue in LncRNA-ATB-shRNA group was improved compared with model group and LncRNA-ATB-NC group. Compared with sham operation group, the expression levels of LncRNA-ATB in peripheral blood, the levels of serum Scr, BUN, IL-6, IL-8 and TNF-α, the AR semi-quantitative scores, the expression levels of TLR4, MyD88, NF-κB mRNA and proteins in kidney tissue of the rats in model group, LncRNA-ATB-NC group, and LncRNA-ATB-shRNA group were decreased(P < 0.05), the expression levels of miR-200c in peripheral blood were decreased (P < 0.05). Compared with model group and LncRNA-ATB-NC group, the expression level of LncRNA-ATB in peripheral blood, the levels of serum Scr, BUN, IL-6, IL-8 and TNF-α, the AR semi-quantitative score, the expression levels of TLR4, MyD88, NF-κB mRNA and proteins of the rats in LncRNA-ATB-shRNA group were reduced (P < 0.05), and the expression level of miR-200c in peripheral blood was increased (P < 0.05). There were no significant differences in the expression levels of LncRNA-ATB and miR-200c in peripheral blood, the levels of serum Scr, BUN, IL-6, IL-8 and TNF-α, the AR semi-quantitative scores, and the expression levels of TLR4, MyD88, NF-κB mRNA and proteins in kidney tissue of the rats between model group and LncRNA-ATB-NC group (P>0.05). Conclusion: Silencing LncRNA-ATB can reduce AR after kidney transplantation in the rats and inhibit the inflammatory reactions in recipient tissue. Its mechanism may be related to up-regulating the expression of miR-200c kidney transplanted and inhibiting the expressions of TLR4, MyD88, NF-κB mRNA and proteins to exert the inflammatory suppression.
KEYWORDS: long non-coding RNA activated by transforming growth factor β    microRNA    kidney transplantion    acute rejection    inflammation response    rats, Wistar    

肾移植是治疗终末期肾病(end-stage kidney disease,ESRD)的有效手段,排斥反应是肾移植术后的主要难题和并发症,也是影响移植肾长期存活的主要危险因素,目前临床上最常见的排斥反应类型是急性排斥反应(acute rejection,AR)[1]。近年来,随着免疫抑制剂的出现,肾移植后排斥反应发生率不断下降,但仍时有发生。长链非编码RNA(long non-coding RNA,LncRNA)是一类不具有蛋白编码功能的RNA,广泛参与调节机体的各项基础生物进程,其表达水平异常与多种疾病的发生发展存在密切关联[2]。被转化生长因子β活化的长链非编码RNA(long non-coding RNA activated by TGF-β,LncRNA-ATB)是LncRNAs的重要成员,参与细胞增殖、凋亡、转移和侵袭等多种生理过程[3]。既往研究[4]表明:LncRNA-ATB在器官移植免疫排斥反应中起调控作用,但在肾移植方面报道较少。本研究通过沉默肾移植模型大鼠LncRNA-ATB的表达,探讨LncRNA-ATB对肾移植大鼠移植后AR和受体组织炎症反应的影响,分析肾移植AR潜在的分子机制,为临床诊断和治疗AR提供参考。

1 材料与方法 1.1 实验动物、主要试剂和仪器

供体:雄性SD大鼠30只,SPF级,7~8周龄,体质量250~300 g;受体:雄性Wistar大鼠40只,SPF级,7~8周龄,体质量230~300 g;均由北京维通利华实验动物技术有限公司提供,动物生产许可证号:SCXK(京)2016-0001。所有大鼠术前12 h禁食不禁水。含有LncRNA-ATB-短发夹(shRNA)和LncRNA-ATB-NC的psiCHECK2基因重组质粒(上海吉玛公司),Opti-MEM培养基和脂质体2000(Lipofectamine 2000)(美国Invitrogen公司),白细胞介素6(interleukin6,IL-6)、白细胞介素8(interleukin-8,IL-8)和肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)ELISA试剂盒(美国Abcam公司),兔抗大鼠Toll样受体4(Toll-like receptor-4,TLR4)、髓样细胞分化因子88(myeloid differentiation factor 88,MyD88)和核转录因子κB(nuclear transcription factor κB,NF-κB)多抗及辣根过氧化物酶标记的山羊抗兔MyD88和NF-κB单抗(英国Abcam公司)。7600全自动生化分析仪(日本Hitachi公司),Cary3500紫外分光光度计(美国安捷伦公司),550型全自动酶标仪和PowerPac系列电泳仪(美国Bio-Rad公司)。

1.2 大鼠肾移植AR模型建立和分组

供体和受体大鼠腹腔注射水合氯醛溶液(10%W/V)进行麻醉,固定于手术台上。供体30只SD大鼠经中线切口暴露腹主动脉、左肾及其所属动静脉,结扎肾上腺动静脉,游离左肾及肾动静脉,分离输尿管,用4℃含肝素生理盐水灌注左肾阻断肾脏血液供应,切断左肾静脉,待左肾颜色变为黄白色且左肾静脉流出清亮的灌注液后,切断左肾动脉,取出肾脏保存于4℃含肝素生理盐水中,剪去供肾多余脂肪及腔静脉,以利血管吻合。受体30只Wistar大鼠暴露左肾后移除,将供肾放置左肾窝,左肾静脉采用cuff套管吻合法,动脉采用端端吻合法。吻合完成后打开血管夹,数秒内可见左肾颜色变红,少量尿液从输尿管排出。术后肌肉注射青霉素10万单位,连续3 d。其余10只Wistar大鼠为假手术组,开腹后随即连续全层关闭,其他操作同上。术后所有大鼠均给予正常饮食和饮水。术后将移植成功的27只大鼠(死亡3只)随机分为模型组、LncRNA-ATB-shRNA组和LncRNA-ATB-NC组,每组9只。

1.3 干预方法

制备质粒脂质体复合物:按照Lipofectamine 2000转染试剂盒说明书,将5 μg含有LncRNA-ATB-shRNA(或LncRNA-ATB-NC)的psiCHECK2基因重组质粒、10 μL Lipofectamine 2000分别用Opti-MEM培养基稀释至总体积各为30 μL,室温静置5 min,然后将2种液体混合均匀,室温放置30 min,获得质粒脂质体复合物备用。肾移植术后2 h分别将2种质粒脂质体复合物用Opti-MEM培养基稀释至总体积200 μL,经尾静脉分别快速注入LncRNA-ATB-shRNA组和LncRNA-ATB-NC组大鼠体内。假手术组和模型组大鼠按每只200 μL尾静脉注射Opti-MEM培养基。

1.4 RT-PCR法检测大鼠外周血中LncRNA-ATB、微小RNA(microRNA,miRNA)-200c表达水平

各组大鼠干预7 d后,腹主动脉采血2 mL放入抗凝管中,按照TRIzol说明书提取总RNA,应用紫外分光光度计测定RNA纯度和浓度,将提取的总RNA按试剂盒说明书进行逆转录得到相对应的cDNA,进行产物扩增。以cDNA为模板,配制反应体系:SYBR GREEN MasterMix 10 μL,上游引物0.5 μL,下游引物0.5 μL,模板cDNA 1 μL,加双蒸水至总体积20 μL。反应条件:预变性94℃、10 min;变性94℃、10 s,退火60℃、30 s,延伸70℃、20 s,35个循环,每个样本检测3次,取平均数。引物均由上海吉玛制药技术有限公司设计合成,LncRNA-ATB以β-actin为内参,miR-200c以U6为内参,采用2-△△Ct法计算LncRNA-ATB和miR-200c表达水平。引物序列见表 1

1.5 全自动生化分析仪检测各组大鼠血肌酐(serum creatinine,Scr)和血尿素氮(blood urea nitrogen,BUN)水平

各组大鼠腹主动脉采血,离心半径12 cm,3 000 r·min-1离心20 min,取上清‒20℃冰箱保存。采用全自动生化分析仪检测大鼠血清中Scr和BUN水平。

表 1 LncRNA-ATB和miR-200c PCR扩增引物序列 Tab. 1 Sequences of LncRNA-ATB, miR-200c PCR amplification primer
Primer Sequence(5'-3')
LncRNAATB F:-ATTACCATCGATGCTAGCATGCA
R:ACAGATACCATTACATGGCGATC
β-actin F:ATTCGATCAGCTAATATCAGCGCTA
R:ACTGCATGCGATGCTAGCAGACTG
miR-200c F:CAGCTAGCATCGATCGCGATGAGCTA
R:CGCATAGCTAGCTACCATAGCAGCTA
U6 F:AACTCGATCAGATCGGCGACTACT
R:AGCTAGTACAGACTCGCTCATCCA
1.6 酶联免疫吸附测定(ELISA)法检测各组大鼠血清中IL-6、IL-8和TNF-α水平

取保存血清,严格按照ELISA试剂盒说明书操作步骤,检测大鼠血清中IL-6、IL-8和TNF-α水平,用酶标仪在490 nm波长处测量吸光度(A)值,以标准品浓度和A值绘制标准曲线,分别根据公式Y=0.970 1X+0.011 8、Y=1.025 3X-0.001 7和Y=0.974 6X+0.011 4计算待测样品IL-6、IL-8和TNF-α水平。

1.7 各组大鼠肾组织病理学观察及AR分级

采血完毕,处死各组大鼠,无菌摘取左肾并切分成两半,一半置于体积分数为10%中性甲醛保存备用。取中性甲醛中保存的肾组织,常规石蜡包埋、切片、苏木精-伊红(HE)染色,置于光镜下观察大鼠肾组织病理形态表现。AR的诊断参照Banff 2007移植肾病理学分类标准[5],按照严重程度分为7级:正常、临界性改变、Ⅰa级、Ⅰb级、Ⅱa级、Ⅱb级和Ⅲ级。AR分级半定量评分:正常为0分,临界性改变为1分,Ⅰa为2分,Ⅰb级为3分,Ⅱa级为4分,Ⅱb级为5分,Ⅲ级为6分。

1.8 RT-PCR法检测各组大鼠肾组织中TLR4、MyD88中NF-κB mRNA表达水平

采血完毕,处死大鼠,无菌摘取左肾并切分成两半,一半置于液氮中保存备用。取液氮中保存的肾组织100 mg,按照TRIzol试剂盒说明书提取总RNA,测定RNA纯度和浓度后进行cDNA逆转录,配制20 μL反应体系:模板cDNA 2 μL,上下游引物各2 μL,Taq DNA聚合酶10 μL,双蒸水6 μL。以转录产物cDNA为模板,以β-actin为内参,进行PCR扩增。扩增条件:预变性95℃、10 min,变性95℃、60 s,退火55℃、30 s,延伸65℃、30 s,共40个循环。各组大鼠肾组织中TLR4、MyD88和NF-κB mRNA表达水平按2-ΔΔCt法计算。引物序列见表 2

表 2 PCR扩增引物序列 Tab. 2 Sequences of PCR amplification primers
Gene Sequence(5'-3')
TLR4 F:5'-CTTAGATACCATCGATAATGCGCCA-3'
R:5'-AGCTAGCGCATAGCATCGATGACG-3'
MyD88 F:5'-ACGATTCATCGATCTGATGACTA-3'
R:5'-CGAACATAGGGCACTAGCTACAT-3'
NF-κB F:5'-ACGATCGAGCAGTAGCTAAATGCTATC-3'
R:5'-CTTACGATCGATGGATCGTAGCTAGAT-3'
β-actin F:5'-ACTTAACTAGTACTAGCGACAGG-3'
R:5'-CTATCAGCTATCGTCGAACTGAT-3'
1.9 Western blotting法检测各组大鼠肾组织中TLR4、MyD88和NF-κB蛋白表达水平

取液氮保存的肾组织100 mg,在液氮中研磨后离心取上清,BCA试剂盒检测蛋白浓度,制备蛋白样品,蛋白上样进行十二烷基硫酸钠聚丙烯酰氨(SDS-PAGE)凝胶电泳,电转至硝酸纤维素膜,5%脱脂牛奶孵育2 h,加入稀释的一抗TLR4(1︰1 000),MyD88(1︰1 000)、NF-κB(1︰1 000)和β-actin(1︰1 000),4℃摇床封闭过夜,洗膜后加入稀释的二抗(1︰4 000)于室温下摇床孵育1 h,洗膜后加入ELC试剂显影,暗室下曝光成像。采用Image J软件分析图像,计算TLR4、MyD88和NF-κB蛋白表达水平。目的蛋白表达水平=目的蛋白条带灰度值/β-actin蛋白条带灰度值。

1.10 统计学分析

采用SPSS 21.0统计软件进行统计学分析。采用KS检验数据正态分布,各组大鼠外周血中LncRNA-ATB和miR-200c表达水平,血清中Scr、BUN,IL-6、IL-8和TNF-α水平,肾组织中TLR4、MyD88和NF-κB mRNA及蛋白表达水平均符合正态分布,以x±s表示,多组间样本均数比较采用单因素方差分析,两两组间样本均数比较采用SNK-q检验。以P < 0.05为差异有统计学意义。

2 结果 2.1 各组大鼠外周血中LncRNA-ATB和miR-200c表达水平

各组大鼠外周血中LncRNA-ATB和miR-200c表达水平组间比较差异均有统计学意义(P < 0.01)。与假手术组比较,模型组、LncRNA-ATB-NC组和LncRNA-ATB-shRNA组大鼠外周血中LncRNA-ATB表达水平均升高(P < 0.05),miR-200c表达水平均降低(P < 0.05);与模型组和LncRNA-ATB-NC组比较,LncRNA-ATB-shRNA组LncRNA-ATB表达水平降低(P < 0.05),miR-200c表达水平升高(P < 0.05);模型组与LncRNA-ATB-NC组大鼠外周血中LncRNA-ATB和miR-200c表达水平比较差异无统计学意义(P > 0.05)。见表 3

表 3 各组大鼠外周血中LncRNA-ATB和miR-200c mRNA表达水平 Tab. 3 Expression levels of LncRNA-ATB and miR-200c mRNA in peripheral blood of rats in various groups
(x±s)
Group n LncRNA-ATB miR-200c
Sham operation 10 0.78±0.08 0.94±0.11
Model 9 2.31±0.25* 0.32±0.04*
LncRNA-ATB-NC 9 2.26±0.23* 0.29±0.03*
LncRNA-ATB-shRNA 9 1.32±0.15*△# 0.57±0.06*△#
F 149.884 181.786
P < 0.01 < 0.01
*P < 0.05 compared with sham operation group; P < 0.05 compared with model group; #P < 0.05 compared with LncRNA-ATB-NC group.
2.2 各组大鼠血清中Scr、BUN、IL-6、IL-8和TNF-α水平

各组大鼠血清中Scr、BUN、IL-6、IL-8和TNF-α水平组间比较差异有统计学意义(P < 0.01)。与假手术组比较,模型组、LncRNA-ATB-NC组和LncRNA-ATB-shRNA组大鼠血清中Scr、BUN、IL-6、IL-8和TNF-α水平均升高(P < 0.05);与模型组和LncRNA-ATB-NC组比较,LncRNA-ATB-shRNA组大鼠血清中Scr、BUN、IL-6、IL-8和TNF-α水平均降低(P < 0.05);模型组与LncRNA-ATB-NC组大鼠血清中Scr、BUN、IL-6、IL-8和TNF-α水平比较差异无统计学意义(P > 0.05)。见表 4

表 4 各组大鼠血清中Scr、BUN、IL-6、IL-8和TNF-α水平 Tab. 4 Levels of Scr, BUN, IL-6, IL-8, and TNF-α in serum of rats in various groups
(x±s)
Group n Scr
[cB/(μmol·L-1)]
BUN [cB/(mmol·L-1)] IL-6
[ρB/ (mg·L-1)]
IL-8
[ρB/ (mg·L-1)]
TNF-α [ρB/ (mg·L-1)]
Sham operation 10 73.64±9.51 8.56±1.39 65.26±7.02 215.61±22.75 45.31±4.75
Model 9 152.43±16.01* 49.52±5.51* 133.43±12.56* 268.52±27.51* 128.52±13.21*
LncRNA-ATB-NC 9 148.82±15.73* 53.21±6.02* 125.68±12.73* 273.49±28.46* 131.27±14.08*
LncRNA-ATB-shRNA 9 109.68±10.22*△# 20.15±2.87*△# 89.65±10.27*△# 238.47±24.87*△# 78.52±8.47*△#
F 76.291 242.250 82.377 10.416 143.621
P < 0.01 < 0.01 < 0.01 < 0.01 < 0.01
*P < 0.05 compared with sham operation group; P < 0.05 compared with model group; #P < 0.05 compared with LncRNA-ATB-NC group.
2.3 HE染色观察各组大鼠肾组织病理形态表现和AR分级半定量评分

HE染色,假手术组大鼠肾组织无AR,结构清楚;模型组大鼠出现典型AR,表现为炎性细胞浸润、动脉炎症、间质细胞水肿和皮质出血等;LncRNA-ATB-NC组与模型组肾组织病理形态表现相似;LncRNA-ATB-shRNA组大鼠肾组织病理形态表现较模型组和LncRNA-ATB-NC组均减轻。假手术组、模型组、LncRNA-ATB-NC组和LncRNA-ATB-shRNA组的AR分级半定量评分分别为(0.00±0.00)分、(4.26±0.44)分、(4.54±0.45)分和(1.75±0.18)分。AR分级半定量评分组间比较差异均有统计学意义(F=425.923,P < 0.05)。与假手术组比较,模型组、LncRNA-ATB-NC组和LncRNA-ATB-shRNA组的AR分级半定量评分均升高(P < 0.05);与模型组和LncRNA-ATB-NC组比较,LncRNA-ATB-shRNA组的AR分级半定量评分降低(P < 0.05);模型组与LncRNA-ATB-NC组的AR分级半定量评分比较差异无统计学意义(P > 0.05)。见图 1插页二)。

A: Sham operation group; B: Model group; C: LncRNA-ATB NC group; D: LncRNA-ATB-shRNA group. 图 1 各组大鼠肾组织病理形态表现(HE, × 200) Fig. 1 Pathomorphology of kidney tissue of rats in various groups (HE, × 200)
2.4 各组大鼠肾组织中TLR4、MyD88和NF-κB mRNA表达水平

各组大鼠肾组织中TLR4、MyD88和NF-κB mRNA相对表达水平组间比较差异有统计学意义(P < 0.01)。与假手术组比较,模型组、LncRNA-ATB-NC组和LncRNA-ATB-shRNA组大鼠肾组织中TLR4、MyD88和NF-κB mRNA表达水平均升高(P < 0.05);与模型组和LncRNA-ATB-NC组比较,LncRNA-ATB-shRNA组大鼠肾组织中TLR4、MyD88和NF-κB mRNA表达水平均降低(P < 0.05);模型组与LncRNA-ATB-NC组大鼠肾组织中TLR4、MyD88和NF-κB mRNA表达水平比较差异无统计学意义(P > 0.05)。见表 5

表 5 各组大鼠肾组织中TLR4、MyD88和NF-κB mRNA表达水平 Tab. 5 Expression levels of TLR4, MyD88 and NF-κB mRNA in kidney tissue of rats in various groups
(x±s)
Group n TLR4 MyD88 NF-κB
Sham operation 10 0.68±0.07 0.64±0.08 0.73±0.09
Model 9 1.07±0.12* 1.13±0.11* 1.25±0.13*
LncRNA-ATB-NC 9 1.12±0.13* 1.08±0.12* 1.16±0.14*
LncRNA-ATB-shRNA 9 0.89±0.09*△# 0.87±0.09*△# 0.95±0.11*△#
F 34.913 47.159 36.608
P < 0.01 < 0.01 < 0.01
*P < 0.05 compared with sham operation group; P < 0.05 compared with model group; #P < 0.05 compared with LncRNA-ATB-NC group.
2.5 各组大鼠肾组织中TLR4、MyD88和NF-κB蛋白表达水平

各组大鼠肾组织中TLR4、MyD88和NF-κB蛋白表达水平组间比较差异均有统计学意义(P < 0.01)。与假手术组比较,模型组、LncRNA-ATB-NC组和LncRNA-ATB-shRNA组大鼠肾组织中TLR4、MyD88和NF-κB蛋白表达水平均升高(P < 0.05);与模型组和LncRNA-ATB-NC组比较,LncRNA-ATB-shRNA组大鼠肾组织中TLR4、MyD88和NF-κB蛋白表达水平均降低(P < 0.05);模型组与LncRNA-ATB-NC组大鼠肾组织中TLR4、MyD88和NF-κB蛋白表达水平比较差异无统计学意义(P > 0.05)。见表 6图 2

表 6 各组大鼠肾组织中TLR4、MyD88和NF-κB蛋白表达水平 Tab. 6 Expression levels of TLR4, MyD88, and NF-κB proteins in kidney tissue of rats in various groups
(x±s)
Group n TLR4 MyD88 NF-κB
Sham operation 10 0.35±0.04 0.32±0.03 0.24±0.03
Model 9 0.81±0.08* 0.78±0.09* 0.85±0.09*
LncRNA-ATB-NC 9 0.85±0.09* 0.76±0.08* 0.79±0.08*
LncRNA-ATB-shRNA 9 0.59±0.07*△# 0.57±0.06*△# 0.43±0.05*△#
F 98.609 94.539 183.702
P < 0.01 < 0.01 < 0.01
*P < 0.05 compared with sham operation group; P < 0.05 compared with model group; #P < 0.05 compared with LncRNA-ATB-NC group.
Lane 1 : Sham operation group; Lane 2 : Model group; Lane 3 : LncRNA-ATB-NC group; Lane 4: LncRNA-ATB-shRNA group. 图 2 各组大鼠肾组织中NF-κB、MyD88和TLR4蛋白表达电泳图 Fig. 2 Electrophoregram of expressions of NF-κB, MyD88, and TLR4 proteins in kidney tissue of rats in various groups
3 讨论

ERSD是全球范围内重大公共卫生问题,随着肾移植的逐渐推广,部分患者得到了有效治疗,但术后AR仍然是一个不可忽视的问题,其不仅是造成移植肾失功的危险因素,同时也严重影响移植肾的长期存活。随着免疫抑制剂的不断出现和改进,器官移植排斥反应发生率明显下降,但大量使用免疫抑制剂会给患者带来不良反应[6]。因此,探讨移植免疫相关的分子机制对肾移植AR早期诊断和治疗具有重要意义。LncRNA和miRNA是最重要的2类非编码RNA,均在基因表达调控中起着关键作用。多项研究[7-9]表明:器官移植后LncRNA在免疫排斥受体体内异常表达,可以利用LncRNA作为早期诊断受体对同种异体移植物产生AR的标志物。既往研究[10-12]表明:LncRNA可作为内源竞争性RNA(competing endogenous RNA,ceRNA)与miRNA直接结合,从而抑制miRNA的活性,调控下游信号通路,进而影响疾病发展。研究[13]显示:LncRNA-ATB与miR-200s也存在上述相互作用,LncRNA-ATB可以与miR-200s结合,抑制miR-200s的表达,促进细胞增殖、迁移和侵袭。

本研究采用同种异体大鼠建立肾移植AR模型,与假手术组比较,模型组大鼠外周血LncRNA-ATB表达水平明显升高,表现出AR典型病理变化,经shRNA干扰载体沉默LncRNA-ATB后,LncRNA-ATB-shRNA组大鼠外周血中LncRNA-ATB表达水平、AR半定量评分及血清中Scr和BUN水平明显降低,肾组织AR病理形态改变减轻,提示肾移植AR大鼠LncRNA-ATB异常升高,可能参与AR发生发展过程,沉默LncRNA-ATB后可减弱肾移植后AR的发展。研究[14]表明:在肾移植术后AR受体肾组织切片中LncRNA-ATB表达水平明显高于对照组,促进供体器官炎症反应。QIU等[15]在对肾移植AR患者和对照肾移植患者的肾脏活检组织中检测到LncRNA-ATB表达,AR患者LncRNA-ATB表达水平较对照组明显上调,与本研究结果类似,进一步提示LncRNA-ATB可能参与大鼠肾移植术后AR进展,沉默LncRNA-ATB有利于减弱AR发展,减轻肾功能损害。

本研究结果显示:与模型组比较,LncRNA-ATB-shRNA组大鼠外周血中miR-200c表达水平明显升高,提示LncRNA-ATB可能参与调控miR-200c在肾移植大鼠中的表达。刘易[16]研究发现:LncRNA-ATB参与调控miR-200c的表达,LncRNA-ATB可以作为ceRNA竞争性结合miR-200c促进肺上皮细胞纤维化。赵伟等[17]研究显示:LncRNA-ATB与miR-200c竞争性地调控其下游通路发挥对miR-200c的调控作用。本研究与上述研究结果一致,表明LncRNA-ATB与miR-200c在疾病发生发展过程中有关联。miR-200c是一种能够抑制炎症反应的miRNA,与TLR4信号通路关系密切。TLR4信号通路与炎症反应密切相关,在依赖MyD88途径中,通过活化的结构域与MyD88结合,释放NF-κB,活化下游炎症因子(IL-6、IL-8和TNF-α等),促进炎症反应。过表达miR-200c可抑制TLR4信号通路,下调MyD88表达,阻断NF-κB活化,降低IL-6和TNF-α表达[18]。既往研究[19]显示:miR-200c可以通过改变NF-κB活性来调节IL-8表达,过表达miR-200c可以降低IL-8表达。在口腔鳞状细胞癌中,NF-κB激活的同时伴有促炎miRNA表达水平升高及miR-200c表达抑制[20]。本研究中经shRNA干扰载体沉默LncRNA-ATB后,大鼠外周血中miR-200c表达水平明显升高,IL-6、IL-8和TNF-α水平,TLR4、MyD88和NF-κB mRNA及蛋白表达水平均降低,提示沉默LncRNA-ATB可能通过上调miR-200c表达抑制TLR4/MyD88信号通路发挥抗炎作用。

综上所述,在大鼠肾移植后,沉默LncRNA-ATB可发挥抑制AR及炎症反应发生作用,其机制可能是通过上调miR-200c表达,阻断下游TLR4/MyD88信号通路,进而减弱AR发展,抑制受体组织炎症反应,本研究结果为临床早期诊断和治疗肾移植术后AR提供一定理论依据。

参考文献
[1]
中华医学会器官移植学分会, 中国医师协会器官移植医师分会. 中国肾移植排斥反应临床诊疗指南(2016版)[J]. 器官移植, 2016, 7(5): 332-338.
[2]
RAMNARINE V R, KOBELEV M, GIBB E A, et al. The evolution of long noncoding RNA acceptance in prostate cancer initiation, progression, and its clinical utility in disease management[J]. Eur Urol, 2019, 76(5): 546-559. DOI:10.1016/j.eururo.2019.07.040
[3]
JANG S Y, KIM G, PARK S Y, et al. Clinical significance of LncRNA-ATB expression in human hepatocellular carcinoma[J]. Oncotarget, 2017, 8(45): 78588-78597. DOI:10.18632/oncotarget.21094
[4]
CHEN W B, PENG W J, HUANG J R, et al. Microarray analysis of long non-coding RNA expression in human acute rejection biopsy samples following renal transplantation[J]. Mol Med Rep, 2014, 10(4): 2210-2216. DOI:10.3892/mmr.2014.2420
[5]
SOLEZ K, COLVIN R B, RACUSEN L C, et al. Banff 07 classification of renal allograft pathology:updates and future directions[J]. Am J Transplant, 2008, 8(4): 753-760. DOI:10.1111/j.1600-6143.2008.02159.x
[6]
王琴, 杨春兰, 冯丽娟, 等. 基因多态性与器官移植受者霉酚酸个体化治疗研究进展[J]. 安徽医科大学学报, 2018, 53(1): 161-166.
[7]
ZOU Y, ZHANG W, ZHOU H H, et al. Analysis of long noncoding RNAs for acute rejection and graft outcome in kidney transplant biopsies[J]. Biomark Med, 2019, 13(3): 185-195. DOI:10.2217/bmm-2018-0272
[8]
GU G X, HUANG Y J, WU C L, et al. Differential expression of long noncoding RNAs during cardiac allograft rejection[J]. Transplantation, 2017, 101(1): 83-91. DOI:10.1097/TP.0000000000001463
[9]
GE Y Z, XU T, Cao W J, et al. A molecular signature of two long non-coding RNAs in peripheral blood predicts acute renal allograft rejection[J]. Cell Physiol Biochem, 2017, 44(3): 1213-1223. DOI:10.1159/000485451
[10]
张扬.沉默LncRNA-ATB靶向miR-141-3p抑制乳腺癌的转移和侵袭[D].沈阳: 中国医科大学, 2018.
[11]
SONG Y X, YANG L X, GUO R W, et al. Long noncoding RNA MALAT1 promotes high glucose-induced human endothelial cells pyroptosis by affecting NLRP3 expression through competitively binding miR-22[J]. Biochem Biophys Res Commun, 2019, 509(2): 359-366. DOI:10.1016/j.bbrc.2018.12.139
[12]
TANG F C, LU Z C, WANG J M, et al. Competitive endogenous RNA(ceRNA) regulation network of LncRNAs, miRNAs, and mRNAs in Wilms tumour[J]. BMC Med Genomics, 2019, 12(1): 194. DOI:10.1186/s12920-019-0644-y
[13]
HAN F, WANG C H, WANG Y, et al. Long noncoding RNA ATB promotes osteosarcomacell proliferation, migration and invasion by suppressing miR-200s[J]. Am J Cancer Res, 2017, 7(4): 770-783.
[14]
LORENZEN J M, SCHAUERTE C, KÖLLING M, et al. Long Noncoding RNAs in urine are detectable and may enable early detection of acute T cell-mediated rejection of renal allografts[J]. Clin Chem, 2015, 61(12): 1505-1514. DOI:10.1373/clinchem.2015.243600
[15]
QIU J, CHEN Y, HUANG G, et al. Transforming growth factor-β activated long non-coding RNA ATB plays an important role in acute rejection of renal allografts and may impacts the postoperative pharmaceutical immunosuppression therapy[J]. Nephrology (Carlton), 2017, 22(10): 796-803. DOI:10.1111/nep.12851
[16]
刘易. LncRNA-ATB竞争性吸附miR-200c调控EMT促进矽尘诱导肺纤维化的机制研究[D].南京: 南京医科大学, 2018.
[17]
赵伟, 李晓明. 被转化生长因子β活化的长链非编码RNA对人淋巴瘤Raji细胞增殖、周期及凋亡的影响[J]. 中国现代医学杂志, 2017, 9(5): 91-96.
[18]
WENDLANDT E B, GRAFF J W, GIOANNINI T L, et al. The role of microRNAs miR-200b and miR-200c in TLR4 signaling and NF-κB activation[J]. Innate Immun, 2012, 18(6): 846-855. DOI:10.1177/1753425912443903
[19]
CHUANG T D, HO M, KHORRAM O. The regulatory function of miR-200c on inflammatory and cell-cycle associated genes in SK-LMS-1, a leiomyosarcoma cell line[J]. Reprod Sci, 2015, 22(5): 563-571. DOI:10.1177/1933719114553450
[20]
JOHNSON J J, MILLER D L, JIANG R, et al. Protease-activated receptor-2(PAR-2)-mediated Nf-κB activation suppresses inflammation-associated tumor suppressor MicroRNAs in oral squamous cell carcinoma[J]. J Biol Chem, 2016, 291(13): 6936-6945. DOI:10.1074/jbc.M115.692640