Role of long non-coding RNA nuclear-enriched abundant transcript 1 in the differentiation of human umbilical cord mesenchymal stem cells into hepatocytes

doi:10.12307/2022.765

Abstract

Abstract: BACKGROUND: Human umbilical cord mesenchymal stem cells can be induced to differentiate into hepatocytes with the help of cytokines in vitro, but the resulting hepatoid cells are not yet functionally mature. It has been gradually found that long non-coding RNA (lncRNA) is closely related to cell differentiation and the purpose of promoting cell differentiation is achieved by interfering with the expression of some lncRNAs.
OBJECTIVE: To explore the role of long non-coding RNA nuclear-enriched abundant transcript 1 (lncRNA NEAT1) in the differentiation of human umbilical cord mesenchymal stem cells into hepatocytes.
METHODS: A differentiational cell line system of human umbilical cord mesenchymal stem cells into hepatocytes was established. Periodic Acid-Schiff staining and indocyanine green intake test were used to assess hepatocellular functional characteristics of the cells. Quantitative real-time reverse transcription polymerase chain reaction was used to detect the expression of hepatocyte specific genes and lncRNA NEAT1. The lentivirus was constructed to interfere with NEAT1 expression, and stably transfected cell lines were obtained 7 days after transfection, and hepatotropic differentiation was induced for 4 weeks. A negative control group for transfection with empty plasmids and a control group without virus transfection were set up. The expression levels of hepatocyte specific genes and proteins were detected by quantitative real-time reverse transcription polymerase chain reaction and western blot assay.
RESULTS AND CONCLUSION: (1) After Periodic Acid-Schiff staining, there were red and purple glycogen particles in the cytoplasm of cells, and a large number of indocyanine green positive cells were observed after indocyanine green ingestion. (2) With the extension of induction time, the mRNA expression levels of albumin and cytochrome enzyme P450 3A4 mRNA increased gradually, while the mRNA expression levels of NEAT1 decreased gradually. (3) Compared with the negative control group and the control group, the mRNA and protein expression levels of albumin and cytochrome enzyme P450 3A4 were significantly increased after the interference of NEAT1. (4) The results confirm that lncRNA NEAT1 may be involved in the differentiation of human umbilical cord mesenchymal stem cells into hepatocytes.

Key words: long non-coding RNA NEAT1, mesenchymal stem cell, hepatocyte, differentiation, hepatocyte specific genes, transfection

CLC Number:

Wang Dandan, Yu Yabin, Liu Shiqi, Yan Yulou, Zhang Jianhuai. Role of long non-coding RNA nuclear-enriched abundant transcript 1 in the differentiation of human umbilical cord mesenchymal stem cells into hepatocytes[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(30): 4847-4851.

Figures/Tables 5

References

[1] TUSHER VG, TIBSHIRANI R, CHU G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A. 2001;98(9):5116-5121.
[2] DHAWAN A, PUPPI J, HUGHES RD, et al. Human hepatocyte transplantation: current experience and future challenges. Nat Rev Gastroenterol Hepatol. 2010;7(5):288-298.
[3] ZHOU WL, MEDINE CN, ZHU L, et al. Stem cell differentiation and human liver disease. World J Gastroenterol. 2012;18(17):2018-2025.
[4] HONG HS, KIM YH, SON Y. Perspectives on mesenchymal stem cells: tissue repair, immune modulation, and tumor homing. Arch Pharm Res. 2012;35(2):201-211.
[5] ZHONG YS, LIN N, DENG MH, et al. Deficient proliferation of bone marrow-derived mesenchymal stem cells in patients with chronic hepatitis B viral infections and cirrhosis of the liver. Dig Dis Sci. 2010; 55(2):438-445.
[6] MU N, LIU HB, MENG QH, et al. The differentiation of human multipotent adult progenitor cells into hepatocyte-like cells induced by coculture with human hepatocyte line L02. Ann Surg Treat Res. 2015; 88(1):1-7.
[7] ZHANG G, LAN Y, XIE A, et al. Comprehensive analysis of long noncoding RNA (lncRNA)-chromatin interactions reveals lncRNA functions dependent on binding diverse regulatory elements. J Biol Chem. 2019; 294(43):15613-15622.
[8] 张瑞欣, 董语迪, 肖建辉.lncRNA调控间充质干细胞向成骨细胞分化的研究进展[J].天津医药,2021,49(6):662-667.
[9] 李梦,周云,禹亚彬,等.长链非编码RNA肝癌高表达转录本促进人骨髓间充质干细胞向肝样细胞的分化[J].中国组织工程研究, 2019,23(29):4656-4661.
[10] 张晓宇,张赫.LncRNA在多能干细胞早期谱系分化中的作用与机制研究进展[J].同济大学学报(医学版),2021,42(2):147-151.
[11] LOEWEN G, JAYAWICKRAMARAJAH J, ZHUO Y, et al. Functions of lncRNA HOTAIR in lung cancer. J Hematol Oncol. 2014;7:90.
[12] YU X, LI Z, ZHENG H, et al. NEAT1: A novel cancer-related long non-coding RNA. Cell Prolif. 2017;50(2):e12329.
[13] COURCHAINE E, NEUGEBAUER KM. Paraspeckles: paragons of functional aggregation. J Cell Biol. 2015;210(4):527-528.
[14] BARRA J, GAIDOSH GS, Blumenthal E, et al. Integrator restrains paraspeckles assembly by promoting isoform switching of the lncRNA NEAT1. Sci Adv. 2020;6(27):eaaz9072.
[15] 张喜,段效军,李林瑞,等.lncRNA NEAT1通过靶向抑制miR-29a促进低氧低糖诱导的肺纤维化进程的机制研究[J].实用药物与临床, 2021,24(3):210-218.
[16] HUANG S, XU Y, GE X, et al. Long noncoding RNA NEAT1 accelerates the proliferation and fibrosis in diabetic nephropathy through activating Akt/mTOR signaling pathway. J Cell Physiol. 2019;234(7):11200-11207.
[17] CHI Y, WANG D, WANG J, et al. Long Non-Coding RNA in the Pathogenesis of Cancers. Cells. 2019;8(9):1015.
[18] DERRIEN T, JOHNSON R, BUSSOTTI G, et al. The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Res. 2012;22(9):1775-1789.
[19] CABILI MN, DUNAGIN MC, MCCLANAHAN PD, et al. Localization and abundance analysis of human lncRNAs at single-cell and single-molecule resolution. Genome Biol. 2015;16(1):20.
[20] XU C, ZHANG Y, WANG Q, et al. Long non-coding RNA GAS5 controls human embryonic stem cell self-renewal by maintaining NODAL signalling. Nat Commun. 2016;7:13287.
[21] GUTTMAN M, RINN JL. Modular regulatory principles of large non-coding RNAs. Nature. 2012;482(7385):339-346.
[22] WANG J, LIU S, SHI J, et al. The Role of lncRNAs in Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells. Curr Stem Cell Res Ther. 2020;15(3):243-249.
[23] LU T, SUN H, LV J, et al. EZH2 suppresses hepatocellular differentiation of mouse bone marrow mesenchymal stem cells. Genet Mol Res. 2014; 13(1):2231-2239.
[24] 刘莹,谢锦霞,李恒.长链非编码RNA NEAT1通过miR-34a靶向GAS1激活PI3K-AKT信号通路对宫颈癌的增殖、侵袭和上皮细胞-间充质转化的影响研究[J].中国性科学,2021,30(5):59-63.
[25] 陶正贵,杜静虎,田葵,等.干扰长链非编码RNA NEAT1上调miR-126抑制胃癌细胞的生长、间质转换及裸鼠肿瘤形成[J].实验动物科学,2021,38(2):8-16.
[26] 李宝辉,马辉,侯明明,等.长链非编码RNA NEAT1对非小细胞肺
癌增殖和凋亡的影响及其机制[J].中国免疫学杂志,2019,35(23): 2869-2872.
[27] 任晓阳.长链非编码RNA PVT1和NEAT1在食管鳞状细胞癌中的表达及意义[D].青岛:青岛大学,2017.
[28] 帅勇锋,王小军,张一中,等.长链非编码RNA NEAT1对胃癌细胞
增殖和凋亡的影响及其机制[J].中华实验外科杂志,2018,35(7): 1259-1261.
[29] CHEN H, XIA W, HOU M. Correction to: LncRNA-NEAT1 from the competing endogenous RNA network promotes cardioprotective efficacy of mesenchymal stem cell-derived exosomes induced by macrophage migration inhibitory factor via the miR-142-3p/FOXO1 signaling pathway. Stem Cell Res Ther. 2020;11(1):376.
[30] 喻大军,郭晨旭,李靖,等.lncRNA NEAT1/miR-103a/STAMBPL1轴对胃癌细胞增殖和侵袭的调控作用[J].山西医科大学学报,2020, 51(9):883-887.