lncRNA MALAT1对肝星状细胞活化的调节作用及其机制

doi:10.13481/j.1671-587X.20230319

摘要/Abstract

摘要：

目的探讨长链非编码RNA（lncRNA）肺腺癌转移相关转录本1（MALAT1）在肝纤维化进展过程中对肝星状细胞（HSC）活化的调节作用，并阐明其作用机制。方法收集25例健康志愿者（健康组，n=25）和25例肝纤维化患者［轻度肝纤维化组（n=12）和重度肝纤维化组（n=13）］血清样本。小鼠HSC分为对照组、转化生长因子β1（TGF-β1）组、TGF-β1+si-NC组、TGF-β1+si-MALAT1组、TGF-β1+si-MALAT1+anti-miR-150-5p组和TGF-β1+si-MALAT1+CXCL14组。采用实时荧光定量PCR（RT-qPCR）法检测各组研究对象血清和各组HSC中MALAT1 mRNA、miR-150-5p和CXC趋化因子配体14（CXCL14）mRNA表达水平，Western blotting法检测各组研究对象血清中CXCL14蛋白表达水平和各组HSC中CXCL14、α-平滑肌肌动蛋白（α-SMA）和Ⅰ型胶原蛋白α1（COL1A1）蛋白表达水平，CCK-8法检测各组HSC增殖活性，免疫荧光法检测各组HSC中α-SMA和COL1A1蛋白表达量，双荧光素酶报告系统检测miR-150-5p与MALAT1和CXCL14 3′-UTR基因的靶向关系。结果 RT-qPCR法检测，与健康组比较，轻度和重度肝纤维化组患者血清中MALAT1 mRNA和CXCL14 mRNA表达水平升高（P<0.05），miR-150-5p表达水平降低（P<0.05）；与轻度肝纤维化组比较，重度肝纤维化组患者血清中MALAT1 mRNA和CXCL14 mRNA表达水平升高（P<0.05），miR-150-5p表达水平降低（P<0.05）；与对照组比较，TGF-β1组HSC中MALAT1 mRNA和CXCL14 mRNA表达水平均升高（P<0.05），miR-150-5p表达水平降低（P<0.05）；与TGF-β1+si-NC组比较，TGF-β1+si-MALAT1组HSC中MALAT1 mRNA和CXCL14 mRNA表达水平均降低（P<0.05），miR-150-5p表达水平升高（P<0.05）；与TGF-β1+si-MALAT1组比较，TGF-β1+si-MALAT1+anti-miR-150-5p组HSC中miR-150-5p表达水平降低（P<0.05），CXCL14 mRNA表达水平升高（P<0.05）；与TGF-β1+si-MALAT1组比较，TGF-β1+si-MALAT1+CXCL14组HSC中CXCL14 mRNA表达水平升高（P<0.05）。Western blotting法检测，与健康组比较，轻度和重度肝纤维化组患者血清中CXCL14蛋白表达水平升高（P<0.05）；与轻度肝纤维化组比较，重度肝纤维化组患者血清中CXCL14蛋白表达水平升高（P<0.05）；与对照组比较，TGF-β1组HSC中CXCL14、α-SMA和COL1A1蛋白表达水平升高（P<0.05）；与TGF-β1+si-NC组比较，TGF-β1+si-MALAT1组HSC中CXCL14、α-SMA和COL1A1蛋白表达水平降低（P<0.05）；与TGF-β1+si-MALAT1组比较，TGF-β1+si-MALAT1+anti-miR-150-5p组和TGF-β1+si-MALAT1+CXCL14组HSC中CXCL14、α-SMA和COL1A1蛋白表达水平升高（P<0.05）。CCK-8法检测，与对照组比较，TGF-β1组HSC增殖活性升高（P<0.05）；与TGF-β1+si-NC组比较，TGF-β1+si-MALAT1组HSC增殖活性降低（P<0.05）；与TGF-β1+si-MALAT1组比较，TGF-β1+si-MALAT1+anti-miR-150-5p组和TGF-β1+si-MALAT1+CXCL14组HSC增殖活性升高（P<0.05）。免疫荧光检测，各组HSC中α-SMA和COL1A1蛋白表达与Western blotting法检测结果一致。MALAT1和CXCL14 3'-UTR与miR-150-5p存在靶向关系。双荧光素酶报告基因测定，与miR-NC组比较，与MALAT1 WT或CXCL14 WT共转染的miR-150-5p组HSC中荧光素酶活性降低（P<0.05）。结论敲低MALAT1可抑制TGF-β1诱导HSC活化，其机制可能与miR-150-5p/CXCL14信号通路有关。

关键词: 肝纤维化, 肝星状细胞, 长链非编码RNA肺腺癌转移相关转录本1, 微小RNA-150-5p, CXC趋化因子配体14

Abstract:

Objective To discuss the regulatory effect of long chain non-coding RNA （lncRNA） metastasis associated transcript 1 （MALAT1） on the activation of the hepatic stellate cells （HSC） during the progression of liver fibrosis， and to clarify its mechanism. Methods The serum samples were collected from 25 healthy volunteers （n=25，healthy group） and 25 liver fibrosis patients［ mild liver fibrosis group （n=12）and severe liver fibrosis group （n=13）］. The mouse HSC were divided into control group，transforming growth factor-β1（TGF-β1） group， TGF-β1+si-NC group， TGF-β1+si-MALAT1 group，TGF- β 1+si-MALAT1+anti-miR-150-5p group，and TGF-β1 group+si-MALAT1+CXCL14 group. Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of MALAT1 mRNA， microRNA-150-5p （miR-150-5p）， and CXC chemokine ligand 14 （CXCL14） mRNA in serum and HSC of the subjects in various groups；Western blotting method was used to detect the expression levels of CXCL14， α-smooth muscle actin（α-SMA），and type Ⅰ collagen α 1 （COL1A1） proteins in serum of the subjects in various groups；CCK-8 method was used to detect the proliferation activities of the HSC in various groups； the expression levels of α- SMA，COL1A1 proteins in the HSC in various groups were detected by immunofluorescence；the targeting relationship between miR-150-5p and MALAT1 and CXCL14 3′-UTR genes was detected by dual luciferase reporting system. Results .The RT-qPCR results showed that compared with healthy group， the serum expression levels of MALAT1 mRNA and CXCL14 mRNA of the subjects in mild and severe liver fibrosis groups were increased（P<0.05）， while the expression levels of miR-150-5p were decreased （P<0.05）； compared with mild liver fibrosis group， the serum expression levels of MALAT1 mRNA and CXCL14 mRNA of the subjects in severe liver fibrosis group were increased （P<0.05），while the expression level of miR-150-5p was decreased （P<0.05）； compared with control group， the expression levels of MALAT1 mRNA and CXCL14 mRNA in the HSC in TGF- β1 group were increased （P<0.05）， while the expression level of miR-150-5p was decreased （P<0.05）； compared with TGF-β1+si-NC group， the expression levels of MALAT1 mRNA and CXCL14 mRNA in the HSC in TGF-β1+si MALAT1 group were decreased （P<0.05）， while the expression level of miR-150-5p was increased （P<0.05）； compared with TGF-β1+si MALAT1 group， the expression levels of miR-150-5p in the HSC in TGF- β1+si-MALAT1 and anti-miR-150-5p groups were decreased （P<0.05）， while the expression levels of CXCL14 mRNA were increased （P<0.05）； compared with TGF-β1+si-MALAT1 group， the expression level of CXCL14 mRNA in the HSC in TGF-β1+si-MALAT1+CXCL14 group was increased （P<0.05）.The Western blotting results showed that compared with healthy group， the serum expression levels of CXCL14 protein of the subjects in mild and severe liver fibrosis groups were increased （P<0.05）； compared with mild liver fibrosis group， the serum expression level of CXCL14 protein of the subjects in severe liver fibrosis group was increased （P<0.05）； compared with control group， the expression levels of α-SMA and COL1A1 proteins in the HSC in TGF-β1 group were increased （P<0.05）； compared with TGF-β1+si-NC group， the expression levels of α- SMA and COL1A1 proteins in the HSC in TGF-β1+si-MALAT1 group were decreased （P<0.05）； compared with TGF-β1+si-MALAT1 group， the expression levels of CXCL14，α-SMA and COL1A1 proteins of the HSC in TGF-β1+si-MALAT1+anti-miR-150-5p group and TGF-β1+siMALAT1+CXCL14 group were increased （P<0.05）.The CCK-8 method results showed that compared with control group， the proliferation activity of the HSC in TGF-β1 group was increased （P<0.05）； compared with TGF-β1+si-NC group， the proliferation activity of the HSC in TGF-β1+si-MALAT1 group was decreased （P<0.05）； compared with TGF-β1+si-MALAT1 group，the proliferation activities of the HSC in TGF-β1+si-MALAT1+anti-miR-150-5p group and TGF-β1+si-MALAT1+CXCL14 group were increased （P<0.05）. The immunofluorescence results showed that the expressions of α-SMA and COL1A1 proteins in the HSC in various groups was consistent with the results detected by Western blotting method. There was a targeting relationship between MALAT1 and CXCL14 3 '-UTR and miR-150-5p. The double luciferase reporter gene assay results showed that compared with miR-NC group， the luciferase activity of the HSC in miR-150-5p group co-transfected with MALAT1 WT or CXCL14 WT was decreased （P<0.05）. Conclusion Knocking down of MALAT1 can inhibit the activation of the HSC induced by TGF- β 1，and the mechanism may be related to the miR-150-5p/CXCL14 signaling pathway.

Key words: Liver fibrosis, Hepatic stellate cell, Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1, MicroRNA-150-5p, CXC chemokine ligand 14

中图分类号:

R575

徐菱遥,魏书堂,董勇,孙正路,赵俊波,韩大正. lncRNA MALAT1对肝星状细胞活化的调节作用及其机制[J]. 吉林大学学报(医学版), 2023, 49(3): 697-705.

Lingyao XU,Shutang WEI,Yong DONG,Zhenglu SUN,Junbo ZHAO,Dazheng HAN. Regulatory effect of lncRNA MALAT1 on activation of hepatic stellate cell and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2023, 49(3): 697-705.

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Group	n	Age(year)	Gender[n（η/%）]		Hepatitis C virus[n（η/%）]		Serum alanine aminotransferase[λ_B/（U·L^－1）]	Serum aspartate aminotransferase[λ_B/（U·L^－1）]
Group	n	Age(year)	Male	Female	Yes	No	Serum alanine aminotransferase[λ_B/（U·L^－1）]	Serum aspartate aminotransferase[λ_B/（U·L^－1）]
Healthy	25	54.39	12（48.0）	13（52.0）	－	－	35.47±17.10	37.58±18.23
Mild fibrosis	12	54.67	7（58.3）	5（41.7）	3（25.0）	9（72.0）	90.98±54.16	97.32±64.53
Severe fibrosis	13	55.58	6（46.2）	7（53.8）	4（30.8）	9（69.2）	134.25±86.29	148.65±74.82
“－”：No data.

Group	n	MALAT1 mRNA	miR-150-5p	CXCL14 mRNA
Healthy	25	1.02±0.04	1.05±0.04	1.04±0.04
Mild liver fibrosis	12	2.59±0.14^*	0.46±0.05^*	1.57±0.09^*
Severe liver fibrosis	13	4.18±0.22^*△	0.32±0.02^*△	2.03±0.14^*△

Group	MALAT1	miR-150-5p	CXCL14 mRNA
Control	1.05±0.03	1.02±0.04	1.01±0.05
TGF-β1	3.77±0.21^*	0.28±0.03^*	2.89±0.20^*
TGF-β1+si-NC	3.59±0.17	0.31±0.05	2.61±0.16
TGF-β1+si-MALAT1	1.64±0.12^△	0.75±0.06^△	1.42±0.11^△
TGF-β1+si-MALAT1+anti-miR-150-5p	1.56±0.13	0.46±0.04^#	1.89±0.12^#
TGF-β1+si-MALAT1+CXCL14	1.78±0.15	0.68±0.04	2.05±0.15^#

Group	CXCL14	α-SMA	COL1A1
Control	0.31±0.02	0.28±0.02	0.36±0.03
TGF-β1	0.89±0.06^*	0.95±0.07^*	1.03±0.08^*
TGF-β1+si-NC	0.80±0.07	0.88±0.06	0.96±0.07
TGF-β1+si-MALAT1	0.44±0.03^△	0.50±0.03^△	0.48±0.04^△
TGF-β1+si-MALAT1+anti-miR-150-5p	0.59±0.04^#	0.65±0.05^#	0.66±0.06^#
TGF-β1+si-MALAT1+CXCL14	0.65±0.05^#	0.71±0.06^#	0.76±0.05^#

Group	MALAT1 WT	MALAT1 MUT	CXCL14 WT	CXCL14 MUT
miR-NC	1.05±0.02	0.98±0.05	1.02±0.04	1.01±0.05
miR-150-5p	0.32±0.04^*	0.94±0.04	0.41±0.03^*	0.97±0.04