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
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摘要
背景:骨髓间充质干细胞在体外可以被诱导分化为肝样细胞,但是细胞功能不成熟。研究表明,长链非编码RNA可以影响间充质干细胞的分化,但是关于长链非编码RNA肝癌高表达转录本是否能够参与骨髓间充质干细胞向肝细胞分化还未有报道。目的:探讨长链非编码RNA肝癌高表达转录本在人骨髓间充质干细胞向肝样细胞分化中的作用。方法:体外添加细胞因子诱导人骨髓间充质干细胞(购自上海中科院细胞库)向肝样细胞分化,诱导4周后倒置显微镜观察细胞的形态学变化,免疫荧光技术检测肝细胞特异性蛋白的表达,qRT-PCR检测分化过程中肝癌高表达转录本的表达;然后通过慢病毒转染构建稳定表达肝癌高表达转录本的人骨髓间充质干细胞株,以转染阴性对照病毒作为对照组,两组同时添加细胞因子进行肝向细胞诱导分化,q RT-PCR、Western blot检测过表达组与对照组肝细胞特异性基因、蛋白表达的差异。结果与结论:①人骨髓间充质干细胞在体外添加细胞因子下可以成功被诱导分化为肝样细胞,在分化过程中肝癌高表达转录本的表达逐渐增高;②过表达肝癌高表达转录本后促进了人骨髓间充质干细胞诱导分化后肝细胞特异性基因及蛋白的表达;③结果表明,肝癌高表达转录本在人骨髓间充质干细胞向肝样细胞分化中发挥着至关重要的作用,将为细胞治疗肝脏疾病奠定坚实的理论基础。
BACKGROUND: Human bone marrow mesenchymal stem cells can be induced to differentiate into hapatocytes without mature function.Long-chain non-coding RNA(lncRNA) plays an important part in the differentiation of mesenchymal stem cells. To date, there is still no report on whether the lncRNA highly up-regulated in liver cancer(HULC) is involved in the differentiation of bone marrow mesenchymal stem cells into hepatocytes.OBJECTIVE: To investigate the mechanism of lncRNA HULC in the differentiation of human bone marrow mesenchymal stem cells into hepatocyte-like cells.METHODS: Human bone marrow mesenchymal stem cells were purchased from the Cell Bank of Chinese Academy of Sciences, Shanghai,China and were cultured in the hepatic differentiation medium containing cytokines in vitro. After 4 weeks of induction, cell morphology was observed under inverted microscope. Hepatocyte specific proteins were analyzed by immunofluorescence. The expression of IncRNA HULC was examined by qRT-PCR. Bone marrow mesenchymal cell lines stably expressing IncRNA HULC were then produced through lentiviral transfection. Bone marrow mesenchymal stem cells transfected with negative lentivirus were used as controls. Two groups of cells were concurrently induced with the hepatic differentiation medium containing cytokines. The expressions of hepatocyte specific genes and proteins were detected and compared between the two groups by qRT-PCR and western blot, respectively.RESULTS AND CONCLUSION: Human bone marrow mesenchymal stem cells could be successfully induced into hepatocytes in vitro through the addition of cytokines. The expression of IncRNA HULC was gradually increased during the differentiation. Overexpression of IncRNA HULC caused a marked increase in the expression of hepatocyte specific genes and proteins. To conclude, IncRNA HULC plays an important role in the differentiation of human bone marrow mesenchymal stem cells into hepatocytes. It will provide a theoretical basis for the application of cell therapy in liver diseases.
BACKGROUND: Human bone marrow mesenchymal stem cells can be induced to differentiate into hapatocytes without mature function.Long-chain non-coding RNA(lncRNA) plays an important part in the differentiation of mesenchymal stem cells. To date, there is still no report on whether the lncRNA highly up-regulated in liver cancer(HULC) is involved in the differentiation of bone marrow mesenchymal stem cells into hepatocytes.OBJECTIVE: To investigate the mechanism of lncRNA HULC in the differentiation of human bone marrow mesenchymal stem cells into hepatocyte-like cells.METHODS: Human bone marrow mesenchymal stem cells were purchased from the Cell Bank of Chinese Academy of Sciences, Shanghai,China and were cultured in the hepatic differentiation medium containing cytokines in vitro. After 4 weeks of induction, cell morphology was observed under inverted microscope. Hepatocyte specific proteins were analyzed by immunofluorescence. The expression of IncRNA HULC was examined by qRT-PCR. Bone marrow mesenchymal cell lines stably expressing IncRNA HULC were then produced through lentiviral transfection. Bone marrow mesenchymal stem cells transfected with negative lentivirus were used as controls. Two groups of cells were concurrently induced with the hepatic differentiation medium containing cytokines. The expressions of hepatocyte specific genes and proteins were detected and compared between the two groups by qRT-PCR and western blot, respectively.RESULTS AND CONCLUSION: Human bone marrow mesenchymal stem cells could be successfully induced into hepatocytes in vitro through the addition of cytokines. The expression of IncRNA HULC was gradually increased during the differentiation. Overexpression of IncRNA HULC caused a marked increase in the expression of hepatocyte specific genes and proteins. To conclude, IncRNA HULC plays an important role in the differentiation of human bone marrow mesenchymal stem cells into hepatocytes. It will provide a theoretical basis for the application of cell therapy in liver diseases.
引文
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[6]Lin H,Xu R,Zhang Z,et al.Implications of the immunoregulatory functions of mesenchymal stem cells in the treatment of human liver diseases.Cell Mol Immunol.2011;8(1):19-22.
[7]Chew JRJ,Chuah SJ,Teo KYW,et al.Mesenchymal stem cell exosomes enhance periodontal ligament cell functions and promote periodontal regeneration.Acta Biomater.2019 Mar 13.doi:10.1016/j.actbio.2019.03.021.[Epub ahead of print]
[8]Zhou X,Yang J,Liu Y,et al.Observation of the effect of bone marrow mesenchymal stem cell transplantation by different interventions on cirrhotic rats.Braz J Med Biol Res.2019;52(3):e7879.
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[11]He H,Liu X,Peng L,et al.Promotion of hepatic differentiation of bone marrow mesenchymal stem cells on decellularized cell-deposited extracellular matrix.Biomed Res Int.2013;2013:406871.
[12]Chen S,Jia L,Zhang S,et al.DEPTOR regulates osteogenic differentiation via inhibiting MEG3-mediated activation of BMP4 signaling and is involved in osteoporosis.Stem Cell Res Ther.2018;9(1):185.
[13]Huang Y,Zheng Y,Jin C,et al.Long Non-coding RNA H19Inhibits Adipocyte Differentiation of Bone Marrow Mesenchymal Stem Cells through Epigenetic Modulation of Histone Deacetylases.Sci Rep.2016;6:28897.
[14]Zhang W,Dong R,Diao S,et al.Differential long noncoding RNA/mRNA expression profiling and functional network analysis during osteogenic differentiation of human bone marrow mesenchymal stem cells.Stem Cell Res Ther.2017;8(1):30.
[15]Cao Z,Huang S,Li J,et al.Long noncoding RNA expression profiles in chondrogenic and hypertrophic differentiation of mouse mesenchymal stem cells.Funct Integr Genomics.2017;17(6):739-749.
[16]Yang Q,Jia L,Li X,et al.Long Noncoding RNAs:New Players in the Osteogenic Differentiation of Bone Marrow-and Adipose-Derived Mesenchymal Stem Cells.Stem Cell Rev.2018;14(3):297-308.
[17]Xie Z,Li J,Wang P,et al.Differential Expression Profiles of Long Noncoding RNA and mRNA of Osteogenically Differentiated Mesenchymal Stem Cells in Ankylosing Spondylitis.J Rheumatol.2016;43(8):1523-1531.
[18]Huo S,Zhou Y,He X,et al.Insight into the Role of Long Non-coding RNAs During Osteogenesis in Mesenchymal Stem Cells.Curr Stem Cell Res Ther.2018;13(1):52-59.
[19]Panzitt K,Tschernatsch MM,Guelly C,et al.Characterization of HULC,a novel gene with striking up-regulation in hepatocellular carcinoma,as noncoding RNA.Gastroenterology.2007;132(1):330-342.
[20]Wang J,Liu X,Wu H,et al.CREB up-regulates long non-coding RNA,HULC expression through interaction with microRNA-372 in liver cancer.Nucleic Acids Res.2010;38(16):5366-5383.
[21]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.
[22]Xu C,Zhang Y,Wang Q,et al.Long non-coding RNA GAS5controls human embryonic stem cell self-renewal by maintaining NODAL signalling.Nat Commun.2016;7:13287.
[23]Wang L,Zhao Y,Bao X,et al.LncRNA Dum interacts with Dnmts to regulate Dppa2 expression during myogenic differentiation and muscle regeneration.Cell Res.2015;25(3):335-350.
[24]Kalwa M,H?nzelmann S,Otto S,et al.The lncRNA HOTAIRimpacts on mesenchymal stem cells via triple helix formation.Nucleic Acids Res.2016;44(22):10631-10643.
[25]Cui Y,Lu S,Tan H,et al.Silencing of Long Non-Coding RNANONHSAT009968 Ameliorates the Staphylococcal Protein A-Inhibited Osteogenic Differentiation in Human Bone Mesenchymal Stem Cells.Cell Physiol Biochem.2016;39(4):1347-1359.
[26]Cui M,Xiao Z,Wang Y,et al.Long noncoding RNA HULCmodulates abnormal lipid metabolism in hepatoma cells through an miR-9-mediated RXRA signaling pathway.Cancer Res.2015;75(5):846-857.
[27]Li J,Wang X,Tang J,et al.HULC and Linc00152 Act as Novel Biomarkers in Predicting Diagnosis of Hepatocellular Carcinoma.Cell Physiol Biochem.2015;37(2):687-696.
[28]Du Y,Kong G,You X,et al.Elevation of highly up-regulated in liver cancer(HULC)by hepatitis B virus X protein promotes hepatoma cell proliferation via down-regulating p18.J Biol Chem.2012;287(31):26302-26311.
[29]Li SP,Xu HX,Yu Y,et al.LncRNA HULC enhances epithelial-mesenchymal transition to promote tumorigenesis and metastasis of hepatocellular carcinoma via the miR-200a-3p/ZEB1 signaling pathway.Oncotarget.2016;7(27):42431-42446.
[30]Liu Y,Pan S,Liu L,et al.A genetic variant in long non-coding RNA HULC contributes to risk of HBV-related hepatocellular carcinoma in a Chinese population.PLoS One.2012;7(4):e35145.
[31]Lu Z,Xiao Z,Liu F,et al.Long non-coding RNA HULCpromotes tumor angiogenesis in liver cancer by up-regulating sphingosine kinase 1(SPHK1).Oncotarget.2016;7(1):241-254.
[32]Zhou C,Wu H,Liu Y,et al.Long non-coding RNA HULCaffects downstream-related targets to regulate migration and invasion of hepatoma cells.Zhonghua Gan Zang Bing Za Zhi.2018;26(7):513-518.
[33]Feng H,Wei B,Zhang Y.Long non-coding RNA HULCpromotes proliferation,migration and invasion of pancreatic cancer cells by down-regulating microRNA-15a.Int J Biol Macromol.2019;126:891-898.
[34]Yan H,Tian R,Zhang M,et al.High expression of long noncoding RNA HULC is a poor predictor of prognosis and regulates cell proliferation in glioma.Onco Targets Ther.2016;10:113-120.
[35]Yu X,Zheng H,Chan MT,et al.HULC:an oncogenic long non-coding RNA in human cancer.J Cell Mol Med.2017;21(2):410-417.
[36]Zhang Y,Song X,Wang X,et al.Silencing of LncRNA HULCEnhances Chemotherapy Induced Apoptosis in Human Gastric Cancer.J Med Biochem.2016;35(2):137-143.
[37]Zhao Y,Guo Q,Chen J,et al.Role of long non-coding RNAHULC in cell proliferation,apoptosis and tumor metastasis of gastric cancer:a clinical and in vitro investigation.Oncol Rep.2014;31(1):358-364.
[38]Uzan VR,Lengert Av,Boldrinié,et al.High Expression of HULC Is Associated with Poor Prognosis in Osteosarcoma Patients.PLoS One.2016;11(6):e0156774.
[39]Wang J,Ma W,Liu Y.Long non-coding RNA HULC promotes bladder cancer cells proliferation but inhibits apoptosis via regulation of ZIC2 and PI3K/AKT signaling pathway.Cancer Biomark.2017;20(4):425-434.
[40]Wang YF,Zhang S,Li XQ,et al.Expression of lncRNA HULCin cervical cancer and its correlation with tumor progression and patient survival.Eur Rev Med Pharmacol Sci.2016;20(19):3987-3991.
[2]Wang H,Liang X,Gravot G,et al.Visualizing liver anatomy,physiology and pharmacology using multiphoton microscopy.J Biophotonics.2017;10(1):46-60.
[3]?berg F,Isoniemi H,H?ckerstedt K.Long-term results of liver transplantation.Scand J Surg.2011;100(1):14-21.
[4]Locke JE,Shamblott MJ,Cameron AM.Stem cells and the liver:clinical applications in transplantation.Adv Surg.2009;43:35-51.
[5]Meirelles Júnior RF,Salvalaggio P,Rezende MB,et al.Liver transplantation:history,outcomes and perspectives.Einstein(Sao Paulo).2015;13(1):149-152.
[6]Lin H,Xu R,Zhang Z,et al.Implications of the immunoregulatory functions of mesenchymal stem cells in the treatment of human liver diseases.Cell Mol Immunol.2011;8(1):19-22.
[7]Chew JRJ,Chuah SJ,Teo KYW,et al.Mesenchymal stem cell exosomes enhance periodontal ligament cell functions and promote periodontal regeneration.Acta Biomater.2019 Mar 13.doi:10.1016/j.actbio.2019.03.021.[Epub ahead of print]
[8]Zhou X,Yang J,Liu Y,et al.Observation of the effect of bone marrow mesenchymal stem cell transplantation by different interventions on cirrhotic rats.Braz J Med Biol Res.2019;52(3):e7879.
[9]Lee KD,Kuo TK,Whang-Peng J,et al.In vitro hepatic differentiation of human mesenchymal stem cells.Hepatology.2004;40(6):1275-1284.
[10]Stock P,Brückner S,Winkler S,et al.Human bone marrow mesenchymal stem cell-derived hepatocytes improve the mouse liver after acute acetaminophen intoxication by preventing progress of injury.Int J Mol Sci.2014;15(4):7004-7028.
[11]He H,Liu X,Peng L,et al.Promotion of hepatic differentiation of bone marrow mesenchymal stem cells on decellularized cell-deposited extracellular matrix.Biomed Res Int.2013;2013:406871.
[12]Chen S,Jia L,Zhang S,et al.DEPTOR regulates osteogenic differentiation via inhibiting MEG3-mediated activation of BMP4 signaling and is involved in osteoporosis.Stem Cell Res Ther.2018;9(1):185.
[13]Huang Y,Zheng Y,Jin C,et al.Long Non-coding RNA H19Inhibits Adipocyte Differentiation of Bone Marrow Mesenchymal Stem Cells through Epigenetic Modulation of Histone Deacetylases.Sci Rep.2016;6:28897.
[14]Zhang W,Dong R,Diao S,et al.Differential long noncoding RNA/mRNA expression profiling and functional network analysis during osteogenic differentiation of human bone marrow mesenchymal stem cells.Stem Cell Res Ther.2017;8(1):30.
[15]Cao Z,Huang S,Li J,et al.Long noncoding RNA expression profiles in chondrogenic and hypertrophic differentiation of mouse mesenchymal stem cells.Funct Integr Genomics.2017;17(6):739-749.
[16]Yang Q,Jia L,Li X,et al.Long Noncoding RNAs:New Players in the Osteogenic Differentiation of Bone Marrow-and Adipose-Derived Mesenchymal Stem Cells.Stem Cell Rev.2018;14(3):297-308.
[17]Xie Z,Li J,Wang P,et al.Differential Expression Profiles of Long Noncoding RNA and mRNA of Osteogenically Differentiated Mesenchymal Stem Cells in Ankylosing Spondylitis.J Rheumatol.2016;43(8):1523-1531.
[18]Huo S,Zhou Y,He X,et al.Insight into the Role of Long Non-coding RNAs During Osteogenesis in Mesenchymal Stem Cells.Curr Stem Cell Res Ther.2018;13(1):52-59.
[19]Panzitt K,Tschernatsch MM,Guelly C,et al.Characterization of HULC,a novel gene with striking up-regulation in hepatocellular carcinoma,as noncoding RNA.Gastroenterology.2007;132(1):330-342.
[20]Wang J,Liu X,Wu H,et al.CREB up-regulates long non-coding RNA,HULC expression through interaction with microRNA-372 in liver cancer.Nucleic Acids Res.2010;38(16):5366-5383.
[21]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.
[22]Xu C,Zhang Y,Wang Q,et al.Long non-coding RNA GAS5controls human embryonic stem cell self-renewal by maintaining NODAL signalling.Nat Commun.2016;7:13287.
[23]Wang L,Zhao Y,Bao X,et al.LncRNA Dum interacts with Dnmts to regulate Dppa2 expression during myogenic differentiation and muscle regeneration.Cell Res.2015;25(3):335-350.
[24]Kalwa M,H?nzelmann S,Otto S,et al.The lncRNA HOTAIRimpacts on mesenchymal stem cells via triple helix formation.Nucleic Acids Res.2016;44(22):10631-10643.
[25]Cui Y,Lu S,Tan H,et al.Silencing of Long Non-Coding RNANONHSAT009968 Ameliorates the Staphylococcal Protein A-Inhibited Osteogenic Differentiation in Human Bone Mesenchymal Stem Cells.Cell Physiol Biochem.2016;39(4):1347-1359.
[26]Cui M,Xiao Z,Wang Y,et al.Long noncoding RNA HULCmodulates abnormal lipid metabolism in hepatoma cells through an miR-9-mediated RXRA signaling pathway.Cancer Res.2015;75(5):846-857.
[27]Li J,Wang X,Tang J,et al.HULC and Linc00152 Act as Novel Biomarkers in Predicting Diagnosis of Hepatocellular Carcinoma.Cell Physiol Biochem.2015;37(2):687-696.
[28]Du Y,Kong G,You X,et al.Elevation of highly up-regulated in liver cancer(HULC)by hepatitis B virus X protein promotes hepatoma cell proliferation via down-regulating p18.J Biol Chem.2012;287(31):26302-26311.
[29]Li SP,Xu HX,Yu Y,et al.LncRNA HULC enhances epithelial-mesenchymal transition to promote tumorigenesis and metastasis of hepatocellular carcinoma via the miR-200a-3p/ZEB1 signaling pathway.Oncotarget.2016;7(27):42431-42446.
[30]Liu Y,Pan S,Liu L,et al.A genetic variant in long non-coding RNA HULC contributes to risk of HBV-related hepatocellular carcinoma in a Chinese population.PLoS One.2012;7(4):e35145.
[31]Lu Z,Xiao Z,Liu F,et al.Long non-coding RNA HULCpromotes tumor angiogenesis in liver cancer by up-regulating sphingosine kinase 1(SPHK1).Oncotarget.2016;7(1):241-254.
[32]Zhou C,Wu H,Liu Y,et al.Long non-coding RNA HULCaffects downstream-related targets to regulate migration and invasion of hepatoma cells.Zhonghua Gan Zang Bing Za Zhi.2018;26(7):513-518.
[33]Feng H,Wei B,Zhang Y.Long non-coding RNA HULCpromotes proliferation,migration and invasion of pancreatic cancer cells by down-regulating microRNA-15a.Int J Biol Macromol.2019;126:891-898.
[34]Yan H,Tian R,Zhang M,et al.High expression of long noncoding RNA HULC is a poor predictor of prognosis and regulates cell proliferation in glioma.Onco Targets Ther.2016;10:113-120.
[35]Yu X,Zheng H,Chan MT,et al.HULC:an oncogenic long non-coding RNA in human cancer.J Cell Mol Med.2017;21(2):410-417.
[36]Zhang Y,Song X,Wang X,et al.Silencing of LncRNA HULCEnhances Chemotherapy Induced Apoptosis in Human Gastric Cancer.J Med Biochem.2016;35(2):137-143.
[37]Zhao Y,Guo Q,Chen J,et al.Role of long non-coding RNAHULC in cell proliferation,apoptosis and tumor metastasis of gastric cancer:a clinical and in vitro investigation.Oncol Rep.2014;31(1):358-364.
[38]Uzan VR,Lengert Av,Boldrinié,et al.High Expression of HULC Is Associated with Poor Prognosis in Osteosarcoma Patients.PLoS One.2016;11(6):e0156774.
[39]Wang J,Ma W,Liu Y.Long non-coding RNA HULC promotes bladder cancer cells proliferation but inhibits apoptosis via regulation of ZIC2 and PI3K/AKT signaling pathway.Cancer Biomark.2017;20(4):425-434.
[40]Wang YF,Zhang S,Li XQ,et al.Expression of lncRNA HULCin cervical cancer and its correlation with tumor progression and patient survival.Eur Rev Med Pharmacol Sci.2016;20(19):3987-3991.