lnc-NONHSAT074080调控靶基因ARL6IP4参与RA-UIP发病的机制研究

doi:10.11958/20220644

天津医药 ›› 2023, Vol. 51 ›› Issue (1): 1-7.doi: 10.11958/20220644

• 细胞与分子生物学 • 下一篇

lnc-NONHSAT074080调控靶基因ARL6IP4参与RA-UIP发病的机制研究

刘媛¹^,²(), 鲁芙爱¹, 王乐², 杨有国², 李小芬², 肖运平³^,^∆()

1 包头医学院第一附属医院风湿免疫科（邮编014010）
2 广西医科大学附属柳州市人民医院风湿免疫科
3 广西医科大学附属柳州市人民医院放射科

收稿日期:2022-04-26 修回日期:2022-07-21 出版日期:2023-01-15 发布日期:2023-01-17
通讯作者: 肖运平 E-mail:liuyuanem@163.com;xyp22133@sina.com
作者简介:刘媛（1982），女，副主任医师，主要从事风湿病发病机制方面研究。E-mail：liuyuanem@163.com
基金资助:
国家自然科学基金资助项目(81960304);柳州市科技计划项目(2020NBAA0804)

Study on the mechanism of lnc-NONHSAT074080 regulating target gene ARL6IP4 involved in the pathogenesis of RA-UIP

LIU Yuan¹^,²(), LU Fuai¹, WANG Le², YANG Youguo², LI Xiaofen², XIAO Yunping³^,^∆()

1 Department of Rheumatology, First Affiliated Hospital of Baotou Medical College, Baotou 014010, China
2 Department of Rheumatology, Liuzhou People's Hospital, Guangxi Medical University
3 Department of Radiology, Liuzhou People's Hospital, Guangxi Medical University

Received:2022-04-26 Revised:2022-07-21 Published:2023-01-15 Online:2023-01-17
Contact: XIAO Yunping E-mail:liuyuanem@163.com;xyp22133@sina.com

刘媛, 鲁芙爱, 王乐, 杨有国, 李小芬, 肖运平. lnc-NONHSAT074080调控靶基因ARL6IP4参与RA-UIP发病的机制研究[J]. 天津医药, 2023, 51(1): 1-7.

LIU Yuan, LU Fuai, WANG Le, YANG Youguo, LI Xiaofen, XIAO Yunping. Study on the mechanism of lnc-NONHSAT074080 regulating target gene ARL6IP4 involved in the pathogenesis of RA-UIP[J]. Tianjin Medical Journal, 2023, 51(1): 1-7.

摘要/Abstract

摘要：

目的探讨lnc-NONHSAT074080在类风湿关节炎（RA）相关普通型间质性肺炎（UIP）发病中的作用及调控机制。方法按是否合并UIP分为RA肺纤维化组4例及RA对照组4例。留全血，Affymetrix基因芯片筛选RA肺纤维化发病相关的长链非编码RNA（lncRNA）进行生物信息学分析，并对表达上调明显的10个lncRNA进行实时荧光定量聚合酶链反应（qPCR）验证。选择经qPCR验证，在RA肺纤维化组表达明显升高，且生物信息学分析与肺纤维化进程相关的lncRNA作为目标lncRNA。构建目标lncRNA的shRNA，荧光素酶基因报告验证目标lncRNA与靶基因的关系。以目标lncRNA shRNA转染转化生长因子（TGF）-β1诱导的人胚肺成纤维细胞系，CCK-8法检测细胞的增殖能力，细胞免疫荧光检测α平滑肌肌动蛋白（α-SMA）的表达，qPCR检测细胞中目标lncRNA及靶基因的表达，酶联免疫吸附试验检测细胞中Ⅰ型胶原及纤连蛋白表达。结果与RA对照组相比，RA肺纤维化组患者全血中存在192个表达明显上调的lncRNA，qPCR结果显示lnc-NONHSAT074080在RA肺纤维化组患者全血中的表达升高（P<0.05）;生物信息学分析及荧光素酶基因报告表明lnc-NONHSAT074080与纤维化进程相关，作用的靶基因是ARL6IP4，可能通过分裂原激活蛋白激酶（MAPK）、信号转导子和激活因子（STAT）等纤维化相关信号通路发挥作用;与空白对照组相比，TGF-β1组细胞增殖水平、α-SMA、lnc-NONHSAT074080、ARL6IP4、Ⅰ型胶原和纤连蛋白表达水平明显升高（P<0.05）;与TGF-β1组比较，TGF-β1+NONHSAT074080 shRNA组上述变化趋势相反（P<0.05）。结论 lnc-NONHSAT074080与RA-UIP的发病及纤维化进程相关，可能通过调控靶基因ARL6IP4参与纤维化进程。

关键词: 关节炎，类风湿, 肺疾病，间质性, RNA，长链非编码, 胶原Ⅰ型, 纤连蛋白类, lnc-NONHSAT074080

Abstract:

Objective To investigate the role and regulatory mechanism of lnc-NONHSAT074080 on the pathogenesis of rheumatoid arthritis (RA)-related common interstitial pneumonia (UIP). Methods According to whether combined with UIP, patients were divided into the RA pulmonary fibrosis group (n = 4) and the RA control group (n = 4). The long non coding RNA (lncRNA) related RA pulmonary fibrosis was analyzed by Affymetrix gene chip. The bioinformatics analysis was carried out, and lncRNAs with significantly up-regulated expression were verified by real-time auantitative polymerase chain reaction (qPCR). The lncRNA, which was verified by qPCR and showed significantly increased expression in RA pulmonary fibrosis, bioinformatics analysis showed the relationship with the process of pulmonary fibrosis, was selected as the target lncRNA. Then target lncRNA shRNA was constructed，as well as the relationship between the target lncRNA and the target gene was verified by luciferase gene report. Moreover, TGF-β1-induced human embryonic lung fibroblasts were transfected with target lncRNA shRNA. CCK8 was used to detect cell proliferation, and immunofluorescence was used to detect the expression of α smooth muscle actin (α-SMA). qPCR was used to detect the expression of target lncRNA and target genes, and enzyme-linked immunosorbent assay was used to detect the expression of ? collagen and fibronectin in cells. Results There were 192 significant upregulated lncRNA in the whole blood of RA pulmonary fibrosis group compared to the RA control group. qPCR showed that the expression of lnc-NONHSAT074080 in RA pulmonary fibrosis group was significantly increased (P<0.05). Bioinformatics analysis and luciferase gene report showed that lnc-NONHSAT074080 was related to fibrosis progression. The possible target gene including ARL6IP4 may play a role through fibrosis related pathway such as mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription (STAT) signal pathways. Compared with the blank control group, the proliferation level, α-SMA, lnc-NONHSAT074080, ARL6IP4, type I collagen and fibronectin expression levels were significantly increased in the TGF-β1 group (P<0.05). Compared with the TGF-β1 group, the TGF-β1+NONHSAT074080 shRNA group showed the opposite trend (P<0.05). Conclusion lnc-NONHSAT074080 is associated with the pathogenesis and fibrosis process of RA-UIP, and may participate in fibrosis in the fibrosis process by regulating the target gene ARL6IP4.

Key words: arthritis, rheumatoid, lung diseases, interstitial, RNA, long noncoding, collagen type I, fibronectins, lnc-NONHSAT074080

中图分类号:

R593.2

图/表 10

参考文献 16

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基因名称	引物序列（5＇→3＇）	产物大小（bp）
lnc-TMEM140-1：1	上游：GCCAGCCTGACTGACGCAATC	120
lnc-TMEM140-1：1	下游：CCGGTGTTCAGCCAGTGTTAATCC	120
lnc-NONHSAT071210	上游：ATGGTGCTGGGAAAACTGGC	173
lnc-NONHSAT071210	下游：TGCCCATGCCTATGTCCTGA	173
lnc-RP11-326I11.5	上游：CAGGCGGTGGATAGTCTTGATGC	94
lnc-RP11-326I11.5	下游：TGAATCTGCGTGCGTGGTGAAG	94
lnc-RP11-158I9.5.1-2：1	上游AAGAGGTGGACAGGTGGTATGAGG	146
lnc-RP11-158I9.5.1-2：1	下游：GCTTCTGGCAGTTCCGTGGTC	146
lnc-DPYD-IT1	上游：GGTCCTTACTGCCACTCCCA	657
lnc-DPYD-IT1	下游：ATCTCAGGGAACAGGGAGGC	657
lnc-NONHSAT074080	上游TCAACAAGGGAGAAGAAGGTTTGA	86
lnc-NONHSAT074080	下游：TGCTGTTGTTAGTTGGAGGCT	86
lnc-NONHSAT047350	上游：GATCGGTGACCTTACCCCCA	95
lnc-NONHSAT047350	下游：CAAAGCACATCTTGCACCGC	95
lnc-NONHSAT101272	上游：TAACGGTGGCTCTCTCAGCA	198
lnc-NONHSAT101272	下游：CTTGTAAGGCAGGCCTGGTG	198
lnc-NONHSAT115197	上游：TGGAAACTGAACAACCTGCTCC	2 270
lnc-NONHSAT115197	上游：TGGCTTCTGTTGCCATTGCT	2 270
lnc-NONHSAT082317	上游：TGGGCGGTGTCTGCTGTG	657
lnc-NONHSAT082317	下游：ACTCACTGCGCACACAGACT	657
ARL6IP4	上游：TCTAGTGTGTAGTCTGTGTGC	217
ARL6IP4	下游：ACCACACAGTACATACCACAT	217
GAPDH	上游：GTCTCCTCTGACTTCAACAGCG	131
GAPDH	下游：ACCACCCTGTTGCTGTAGCCAA	131

基因名称	引物序列（5＇→3＇）	产物大小（bp）
lnc-TMEM140-1：1	上游：GCCAGCCTGACTGACGCAATC	120
lnc-TMEM140-1：1	下游：CCGGTGTTCAGCCAGTGTTAATCC	120
lnc-NONHSAT071210	上游：ATGGTGCTGGGAAAACTGGC	173
lnc-NONHSAT071210	下游：TGCCCATGCCTATGTCCTGA	173
lnc-RP11-326I11.5	上游：CAGGCGGTGGATAGTCTTGATGC	94
lnc-RP11-326I11.5	下游：TGAATCTGCGTGCGTGGTGAAG	94
lnc-RP11-158I9.5.1-2：1	上游AAGAGGTGGACAGGTGGTATGAGG	146
lnc-RP11-158I9.5.1-2：1	下游：GCTTCTGGCAGTTCCGTGGTC	146
lnc-DPYD-IT1	上游：GGTCCTTACTGCCACTCCCA	657
lnc-DPYD-IT1	下游：ATCTCAGGGAACAGGGAGGC	657
lnc-NONHSAT074080	上游TCAACAAGGGAGAAGAAGGTTTGA	86
lnc-NONHSAT074080	下游：TGCTGTTGTTAGTTGGAGGCT	86
lnc-NONHSAT047350	上游：GATCGGTGACCTTACCCCCA	95
lnc-NONHSAT047350	下游：CAAAGCACATCTTGCACCGC	95
lnc-NONHSAT101272	上游：TAACGGTGGCTCTCTCAGCA	198
lnc-NONHSAT101272	下游：CTTGTAAGGCAGGCCTGGTG	198
lnc-NONHSAT115197	上游：TGGAAACTGAACAACCTGCTCC	2 270
lnc-NONHSAT115197	上游：TGGCTTCTGTTGCCATTGCT	2 270
lnc-NONHSAT082317	上游：TGGGCGGTGTCTGCTGTG	657
lnc-NONHSAT082317	下游：ACTCACTGCGCACACAGACT	657
ARL6IP4	上游：TCTAGTGTGTAGTCTGTGTGC	217
ARL6IP4	下游：ACCACACAGTACATACCACAT	217
GAPDH	上游：GTCTCCTCTGACTTCAACAGCG	131
GAPDH	下游：ACCACCCTGTTGCTGTAGCCAA	131

组别	NONHSAT074080	NONHSAT071210	RP11-158I9.5.1-2:1	NONHSAT047350	NONHSAT101272
RA对照组	1.000±0.002	1.000±0.317	1.000±0.054	1.000±0.253	1.000±0.002
RA肺纤维化组	6.108±2.180	2.447±0.212	0.637±0.162	3.868±0.728	2.885±0.517
t	2.343^＊	6.097^＊＊	2.239	3.911^＊＊	3.646^＊＊
组别	RP11-326I11.5	DPYD-IT1	TMEM140-1：1	NONHSAT115197	NONHSAT082317
RA对照组	1.000±0.003	1.000±0.006	1.000±0.065	1.000±0.012	1.000±0.004
RA肺纤维化组	3.396±1.487	2.418±0.447	3.453±1.300	1.341±0.207	2.099±0.237
t	1.611	3.172^＊	1.886	1.649	4.643^＊＊

组别	NONHSAT074080	NONHSAT071210	RP11-158I9.5.1-2:1	NONHSAT047350	NONHSAT101272
RA对照组	1.000±0.002	1.000±0.317	1.000±0.054	1.000±0.253	1.000±0.002
RA肺纤维化组	6.108±2.180	2.447±0.212	0.637±0.162	3.868±0.728	2.885±0.517
t	2.343^＊	6.097^＊＊	2.239	3.911^＊＊	3.646^＊＊
组别	RP11-326I11.5	DPYD-IT1	TMEM140-1：1	NONHSAT115197	NONHSAT082317
RA对照组	1.000±0.003	1.000±0.006	1.000±0.065	1.000±0.012	1.000±0.004
RA肺纤维化组	3.396±1.487	2.418±0.447	3.453±1.300	1.341±0.207	2.099±0.237
t	1.611	3.172^＊	1.886	1.649	4.643^＊＊

组别	0 h	24 h	48 h	72 h	96 h
空白对照组	0.063±0.009	0.496±0.034	0.844±0.040	1.088±0.019	1.183±0.091
TGF-β1组	0.061±0.006	0.689±0.010^a	0.975±0.012^a	1.336±0.016^a	1.528±0.055^a
TGF-β1+NONHSAT074080 shRNA组	0.059±0.005	0.804±0.018	0.739±0.113^b	1.065±0.025^b	1.098±0.025^b
F	0.524	51.234^＊＊	63.895^＊＊	39.174^＊＊	28.110^＊＊

lnc-NONHSAT074080调控靶基因ARL6IP4参与RA-UIP发病的机制研究

Study on the mechanism of lnc-NONHSAT074080 regulating target gene ARL6IP4 involved in the pathogenesis of RA-UIP

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图/表 10

参考文献 16

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组别	lnc-NONHSAT074080	ARL6IP4
空白对照组	1.000±0.072	1.000±0.039
TGF-β1组	3.617±0.150^a	1.862±0.080^a
TGF-β1+NONHSAT074080 shRNA组	1.676±0.177^ab	1.072±0.131^b
F	72.644^＊＊	32.106^＊

组别	I型胶原	纤连蛋白
空白对照组	2.490±0.098	22.906±1.688
TGF-β1组	7.008±0.344^a	40.517±1.978^a
TGF-β1+NONHSAT074080 shRNA组	3.160±0.081^ab	23.058±1.505^b
F	132.528^＊＊	34.056^＊＊

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