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

Abstract

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

CLC Number:

R575

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.

Figures/Tables 9

Tab.1

Tab.2

Tab.3

Fig. 1

Fig. 2

Tab.4

Fig. 3

Fig. 4

Tab.5

References 23

1	CASULLERAS M， ZHANG I W， LÓPEZ-VICARIO C，et al. Leukocytes， systemic inflammation and immunopathology in acute-on-chronic liver failure［J］. Cells， 2020， 9（12）： 2632.
2	BOLDEANU M V， SILOŞI I， BĂRBULESCU A L， et al.Host immune response in chronic hepatitis C infection： involvement of cytokines and inflammasomes［J］.Revue Roumaine De Morphol Embryol， 2020， 61（1）： 33-43.
3	JIANG Y， XIANG C， ZHONG F， et al. Histone H3K27 methyltransferase EZH2 and demethylase JMJD3 regulate hepatic stellate cells activation and liver fibrosis［J］. Theranostics， 2021， 11（1）： 361-378.
4	MORENO J A， HAMZA E， GUERRERO-HUE M， et al. Non-coding RNAs in kidney diseases： the long and short of them［J］. Int J Mol Sci， 2021， 22（11）： 6077.
5	HAN S， ZHANG T， KUSUMANCHI P， et al. Long non-coding RNAs in liver diseases： focusing on nonalcoholic fatty liver disease， alcohol-related liver disease， and cholestatic liver disease［J］. Clin Mol Hepatol， 2020， 26（4）： 705-714.
6	WU Y T， LIU X J， ZHOU Q， et al. Silent information regulator 1 （SIRT1） ameliorates liver fibrosis via promoting activated stellate cell apoptosis and reversion［J］. Toxicol Appl Pharmacol， 2015， 289（2）： 163-176.
7	JIANG R， ZHOU Y J， WANG S F， et al. Nicotinamide riboside protects against liver fibrosis induced by CCl₄ via regulating the acetylation of Smads signaling pathway［J］. Life Sci， 2019， 225： 20-28.
8	WANG Y H， MOU Q J， ZHU Z X， et al. MALAT1 promotes liver fibrosis by sponging miR‑181a and activating TLR4‑NF‑κB signaling［J］. Int J Mol Med， 2021， 48（6）： 215.
9	DAI X Y， CHEN C， XUE J C， et al. Exosomal MALAT1 derived from hepatic cells is involved in the activation of hepatic stellate cells via miRNA-26b in fibrosis induced by arsenite［J］. Toxicol Lett， 2019， 316： 73-84.
10	OU M H， ZHAO H D， JI G X， et al. Long noncoding RNA MALAT1 contributes to pregnancy-induced hypertension development by enhancing oxidative stress and inflammation through the regulation of the miR-150-5p/ET-1 axis［J］. FASEB J， 2020， 34（5）： 6070-6085.
11	LIAO J M， ZHANG Z， YUAN Q， et al. A lncRNA Gpr137b-ps/miR-200a-3p/CXCL14 axis modulates hepatic stellate cell （HSC） activation［J］. Toxicol Lett， 2021， 336： 21-31.
12	ZHAN W， LIAO X， CHEN Z S， et al. Leucine-rich repeats and immunoglobulin 1 （LRIG1） ameliorates liver fibrosis and hepatic stellate cell activation via inhibiting sphingosine kinase 1 （SphK1）/sphingosine-1-phosphate （S1P） pathway［J］. Iran J Allergy Asthma Immunol， 2020， 19（4）： 397-408.
13	VALLET-PICHARD A， MALLET V， POL S. FIB-4： a simple， inexpensive and accurate marker of fibrosis in HCV-infected patients［J］.Hepatology，2006，44（3）： 769.
14	JI P， DIEDERICHS S， WANG W B， et al. MALAT-1， a novel noncoding RNA， and thymosin beta4 predict metastasis and survival in early-stage non-small cell lung cancer［J］. Oncogene， 2003， 22（39）： 8031-8041.
15	WANG Z Q， WANG X J， ZHANG T Q， et al. LncRNA MALAT1 promotes gastric cancer progression via inhibiting autophagic flux and inducing fibroblast activation［J］. Cell Death Dis， 2021， 12（4）： 368.
16	WANG N， CAO S J， WANG X X， et al. lncRNA MALAT1/miR‑26a/26b/ST8SIA4 axis mediates cell invasion and migration in breast cancer cell lines［J］. Oncol Rep， 2021， 46（2）： 181.
17	TAN S， CHEN J L. Si-MALAT1 attenuates thymic cancer cell proliferation and promotes apoptosis via the miR-145-5p/HMGA2 pathway［J］. Oncol Lett， 2021， 22（2）： 585.
18	LIU P H， ZHANG B， CHEN Z， et al. m⁶A-induced lncRNA MALAT1 aggravates renal fibrogenesis in obstructive nephropathy through the miR-145/FAK pathway［J］. Aging， 2020， 12（6）： 5280-5299.
19	WANG H W， ZHENG X X， JIN J， et al. LncRNA MALAT1 silencing protects against cerebral ischemia-reperfusion injury through miR-145 to regulate AQP4［J］. J Biomed Sci， 2020， 27（1）： 40.
20	ZHOU X J， ZHAO S， LI W， et al. Tubular cell-derived exosomal miR-150-5p contributes to renal fibrosis following unilateral ischemia-reperfusion injury by activating fibroblast in vitro and in vivo ［J］. Int J Biol Sci， 2021， 17（14）： 4021-4033.
21	CHE H， WANG Y Q， LI Y， et al. Inhibition of microRNA-150-5p alleviates cardiac inflammation and fibrosis via targeting Smad7 in high glucose-treated cardiac fibroblasts［J］. J Cell Physiol， 2020， 235（11）： 7769-7779.
22	DU Z Y， WU T C， LIU L S， et al. Extracellular vesicles-derived miR-150-5p secreted by adipose-derived mesenchymal stem cells inhibits CXCL1 expression to attenuate hepatic fibrosis［J］. J Cell Mol Med， 2021， 25（2）： 701-715.
23	WANG S， SHUAI C， GAO S S， et al. Chemokine CXCL14 acts as a potential genetic target for liver fibrosis［J］. Int Immunopharmacol， 2020， 89（Pt A）： 107067.

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

Regulatory effect of lncRNA MALAT1 on activation of hepatic stellate cell and its mechanism

RichHTML

PDF (PC)

Like

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 9

References 23

Related Articles 14

Metrics

Comments

Recommended 10

[1]	Mingliu GU,Fengyuan LIN,Xuefeng WU,Jianing ZHOU,Xuechun LU,Peige DU,Liping AN. Improvement effect of Phellinus igniarius acidic polysaccharide on liver fibrosis induced by bile duct ligation in mice and its mechanism [J]. Journal of Jilin University(Medicine Edition), 2022, 48(5): 1167-1174.
[2]	Zhouguang WU,Bin WANG,Zhen CHENG,Wenjie ZHANG,Taoyan ZUO,Siqi CHEN,Jingru FU. Analysis on correlation of GPC3 and α-SMA expressions with liver function-related indexes in liver tissue of children with biliary atresia [J]. Journal of Jilin University(Medicine Edition), 2021, 47(5): 1237-1243.
[3]	Yang ZHENG,Jiahui WANG,Yue PENG,Xianling YUAN,Lei WANG,Tiejian ZHAO. Influence of curcumol in structure of liver sinusoidal endothelial cells of mice and its inhibitiory effect on intrahepatic angiogenesis [J]. Journal of Jilin University(Medicine Edition), 2021, 47(5): 1124-1130.
[4]	Rongjun JIA,Liman MA,Lihua LI. Protective effect of calcitriol on hepatic fibrosis induced by bile duct ligation in mice and its mechanism [J]. Journal of Jilin University(Medicine Edition), 2021, 47(2): 257-264.
[5]	YANG Fan, LI Lihua. Effect of vitamin D receptor activation on hepatic fibrosis induced by bile duct ligation in mice and its mechanism [J]. Journal of Jilin University(Medicine Edition), 2020, 46(04): 722-727.
[6]	XIE Jing, LI Lihua. Inhibitory effect of dehydroandrographolide on hepatocyte apoptosis induced by carbon tetrachloride in hepatic fibrosis model mice and its mechanism [J]. Journal of Jilin University(Medicine Edition), 2019, 45(05): 1009-1014.
[7]	BAI Jie, JI Wenjing, DING Yongnian, PENG Yuanyuan, CHEN Yuanwen. Changes of levelsof endogenous AcSDKP and its regulatory factors in liver tissue of model rats with liver fibrosis induced by bile duct ligation [J]. Journal of Jilin University(Medicine Edition), 2018, 44(05): 999-1004.
[8]	CHANG Caifang, HOU Yong, CONG Gang. Therapeutic effect of α-2b interferon on inflammation of liver tissue and liver fibrosis in hepatitis B model ducks [J]. Journal of Jilin University Medicine Edition, 2017, 43(01): 11-15.
[9]	YANG Kun, TIAN Zhenzhen, WANG Shuhua, XIU Ming, GUO Xiangling, QI Lina, LI Min, SUN Li, GAO Runping. Effect of LPS-TLR4 pathway in hepatic fibrogenesis of rats with chronic alcohol intake [J]. Journal of Jilin University Medicine Edition, 2015, 41(04): 751-755.
[10]	HAO Xiaofang, ZHANG Ronghua, WANG Lin, WANG Meimei, ZHEN Yongzhan, ZHANG Guangling, CHEN Jing. Therapeutic effect of rhein lysinate on liver fibrosis in cholestatic model rats and its molecular mechanisms [J]. Journal of Jilin University Medicine Edition, 2015, 41(03): 481-485.
[11]	ZHONG Zhi-hong,YAN Wen-zhen,DAI Dong,CHEN Tu-ming,HU Zu-chao. Therapeutic effect of rapamycin on liver fibrosis model rats induced by carbon tetrachloride oil  [J]. Journal of Jilin University Medicine Edition, 2013, 39(6): 1224-1227.
[12]	DAI Yong-Mei, CAI Li-Mian, LIN Ling. Inhibitory effect of octreotide on prolieration of rat hepatic stellate cells [J]. J4, 2009, 35(6): 1052-1056.
[13]	QI Xiao-yan,GAO Run-ping,ZHANG Rui-juan,LI Guo-feng,YANG Yong-guang. Effects of hammerhad ribozyme targeting connective tissue growth factor on collagen Ⅰ synthesis and cell cycle progression of human hepatic stellate cells [J]. J4, 2009, 35(1): 26-29.
[14]	ZHANG Yi-ning， NIU Jun-qi, DING Yan-hua, WANG Feng. Anti-oxidative stress effects of Taurine on rat hepatic stellate cells [J]. J4, 2005, 31(2): 179-181.