| Objective:A variety of factors such as viral infection,drugs,alcohol and metabolic abnormality can lead to liver damage which develop into inflammation and fibrosis.Without effectively controlled,it may develop into cirrhosis or even hepatocellular carcinoma.Hepatic stellate cells(HSCs)play an important role in hepatic fibrosis,especially activated HSCs with the ability of proliferation and migration as well as secret the excessive secretion of extracellular matrix which is composed of Collagen I.The activation of HSCs involves multiple molecular and pathway that is extremely complicated.Therefore,elucidating the specific molecular mechanism of hepatic stellate cells activation as well as inhibiting the activation of HSCs are the focus of hepatic fibrosis.Recently,multiple long non-coding RNAs(LncRNAs)were found upregulated in activated HSCs.Although LncRNAs cannot encode protein,they can play an important role in activation of hepatic stellate cells by regulating the encoded gene.Our previous study found that LncRNAs expression was significantly different in liver tissues with different degrees of fibrosis.To explore the most important LncRNA in the activation of hepatic stellate cells and investigate their mechanisms can provide valuable suggestion for the future therapeutic targets of hepatic fibrosis.Liver tissue with different fibrosis degree were analyzed by second generation sequencing aim to investigate LncRNA which closely related to hepatic stellate cell activation.Then established activated hepatic stellate cell model and explored the function such as activation,proliferation,migration and apoptosis in order to provide directions for further exploration of the pathogenesis of hepatic fibrosis.Contents and Methods:Part Ⅰ: Screened and identified LncRNA that closely related to the activation of hepatic stellate cells1.To detect the LncRNA which are closely related to liver fibrosis and hepatic stellate cells activation in liver tissue: 1)Liver tissue of the Department of Infection,Shengjing Hospital Affiliated to China Medical University from 2019 to 2020 were divided into 3 cases in the S stage ≥2 group(liver fibrosis group)and 3 cases in the S stage=0 group(control group)according to their fibrosis degree.LncRNAs closely related to liver fibrosis were selected by fold change and relative expression in liver fibrosis group.2)The expression of LncRNA closely related to liver fibrosis and hepatic stellate cells activation was verified by RT-qPCR in liver fibrosis group.2.Detection of LncRNA which is closely related to the activation of hepatic stellate cells.1)Construction the activated hepatic stellate cell model: different concentrations of TGF-β were used to stimulate human hepatic stellate cells,and the expression of SMA and Col1a1,the activation markers of hepatic stellate cells was detected by RTq PCR and Western Blot,which confirmed the activated hepatic stellate cells are successfully established.2)LncRNA expression was detected and determined in activated hepatic stellate cells by RT-qPCR.According to the relative expression of LncRNA,LncRNA NONHSAT152502.1 was selected.Part Ⅱ: To clarify the role of LncRNA NONHSAT152502.1 in the activation of hepatic stellate cells1.Lentivirus was used as vector to construct a cell line with stable knockdown of LncRNA NONHSAT152502.1.2.The role of LncRNA NONHSAT152502.1 plays in the activation of hepatic stellate cells:RT-qPCR and Western Blot were performed to evaluate the markers(SMA,Col1a1,MMP2 and Vimentin)of hepatic stellate cell activation in m RNA and protein expression in sh-NC and sh-LncRNA152502.1.3.The role of LncRNA NONHSAT152502.1 plays in the proliferation of hepatic stellate cells:CCK8 was used to measure value at 450 nm and flow cytometry was applied to measure cell cycle of sh-NC and sh-LncRNA152502.1 to evaluate the proliferation of hepatic stellate cells.4.The role of LncRNA NONHSAT152502.1 plays in the migration of hepatic stellate cells:wound healing assays were used to measure the wound healing area in sh-NC and sh-LncRNA152502.1 to evaluate the migration of hepatic stellate cells.5.The role of LncRNA NONHSAT152502.1 plays in the apoptosis of hepatic stellate cells: Annexin-V-APC+7-AAD and flow cytometry was used to measure the apoptotic rate of sh-NC and sh-LncRNA152502.1 to evaluate the apoptosis of hepatic stellate cells.Part Ⅲ: Molecular mechanism of LncRNA NONHSAT152502.1 in the activation of hepatic stellate cell1.Screened and identified the binding proteins of LncRNA NONHSAT152502.1:1)Predicted the binding proteins of LncRNA NONHSAT152502.1 by cat RAPID and RBPsuite.2)Identified the proteins which bind to LncRNA NONHSAT152502.1 by RNA pull-down and mass spectrometry and preliminarily discussed the function of the protein by bioinformatics analysis.3)Combined the result of bioinformatics and RNA pull-down,Western Blot were applied to detect UPF1 expression in RNA pull-down protein,which is aim to confirm LncRNA NONHSAT152502.1 bind to UPF1.2.The role of UPF1 in the activation of hepatic stellate cells: silenced and overexpressed UPF1 by transfection of si RNA and UPF1 plasmid respectively.Divided the hepatic stellate cells into four groups: si-NC,si-UPF1,NC,UPF1.1)The role of UPF1 in the activation of hepatic stellate cells: The m RNA and protein expression of SMA,Col1a1,MMP2 and Vimentin in each group were detected by RTq PCR and Western Blot.3)The role of UPF1 in the proliferation of hepatic stellate cells:OD value at 450 nm was measured by CCK8 and cell cycle was measured by flow cytometry in each group.4)The effect of UPF1 in the migration of hepatic stellate cells:wound healing assays were performed to measure the wound healing area in each group.5)The effect of UPF1 in the apoptosis of hepatic stellate cell: Annexin-V-APC+7-AAD flow cytometry was used to evaluate the apoptotic rate of hepatic stellate cells in each group.3.The role of LncRNA NONHSAT152502.1 and UPF1 in the activation of hepatic stellate cells: 1)The stable knockdown of LncRNA NONHSAT152502.1 hepatic stellate cell line was transfected with overexpressed UPF1 plasmid,and the expression of SMA and Col1a1 were measured by Western Blot to evaluate the role of LncRNA NONHSAT152502.1 and UPF1 in the activation of hepatic stellate cells.2)The stable knockdown of LncRNA NONHSAT152502.1 hepatic stellate cell line was transfected with overexpressed UPF1 plasmid,OD value at 450 nm was measured by CCK8,and cell cycle was measured by flow cytometry to evaluate the role of LncRNA NONHSAT152502.1 and UPF1 in proliferation of hepatic stellate cells.3)The stable knockdown of LncRNA NONHSAT152502.1 hepatic stellate cell line was transfected with overexpressed UPF1 plasmid,the wound healing area was measured by wound healing assays to evaluate the role of LncRNA NONHSAT152502.1 and UPF1 in migration of hepatic stellate cells.4.The role of LncRNA NONHSAT152502.1 and UPF1 on TGF-β/SMAD: Western Blot was performed to measure SMAD7,p-SMAD2 and p-SMAD3,SMAD2,SMAD3 expression in sh-NC,sh-LncRNA152502.1 and sh-LncRNA 152502.1+UPF1.Results:Part Ⅰ: Screened and identified LncRNA that closely related to the activation of hepatic stellate cell1.1)179 LncRNAs were closely related to liver fibrosis,131 LncRNAs were positively correlated with the fibrosis,and 48 LncRNAs were negatively correlated with the fibrosis.According to their fold change and relative expression in liver fibrosis group,LncRNA NONHSAT152502.1,NONHSAT221105.1,NONHSAT121643.2,NONHSAT151398.1,NONHSAT216122.1,NONHSAT158981.1,NONHSAT185104.1,NONHSAT242288.1,NONHSAT242287.1,NONHSAT152444.1,NONHSAT213706.1,NONHST169386.1 may be related to the formation of liver fibrosis.2)RT-qPCR showed LncRNA NONHSAT152502.1,NONHSAT151398.1,NONHSAT185104.1,NONHSAT242288.1,NONHSAT242287.1,NONHSAT152444.1,NONHSAT213706.1 and NONHSAT169386.1 significantly increased in liver fibrosis group(P<0.05).2.1)Selected 10ng/ml TGF-β for 48 h as the optimal condition,and successfully constructed the activated hepatic stellate cell model.2)The expression of LncRNA NONHSAT152502.1 was significantly increased in activated hepatic stellate cells(P<0.001).Part Ⅱ: To clarify the role of LncRNA NONHSAT152502.1 in the activation of hepatic stellate cells1.Successfully constructed the stable cell line with the knockdown of LncRNA NONHSAT152502.1 in hepatic stellate cells.2.RT-qPCR and Western Blot showed that comparing with sh-NC,the m RNA and protein expression of hepatic stellate cell activation markers SMA,Col1a1,MMP2 and Vimentin in sh-LncRNA152502.1 was decreased(P<0.01).3.CCK8 showed that the OD value at 450 nm of sh-LncRNA152502.1 was significantly decreased in 24 h,48h,72 h and 96 h than that of sh-NC.Flow cytometry showed that the number of hepatic stellate cells in G2/M phase was significantly decreased in sh-LncRNA152502.1 compared with sh-NC(P<0.05).4.Wound healing assays showed that the percentage of wound healing area in sh-LncRNA152502.1 was significantly decreased compared with sh-NC(P< 0.01).5.Flow cytometry showed that the apoptotic rate of hepatic stellate cells in sh-LncRNA152502.1 was increased compared with sh-NC(P< 0.05).Part Ⅲ: Molecular mechanism of LncRNA NONHSAT152502.1 in the activation of hepatic stellate cell1.1)cat RAPID and RBPsuite predicted that LncRNA NONHSAT152502.1 may bind to UPF1,RBM15,FAM120 A,HNRNPU and SUPV3L1.2)RNA pull-down combined with mass spectrometry confirmed the presence of UPF1 in the pull-down protein.Bioinformatics suggested that LncRNA NONHSAT152502.1 binding proteins were involved in m RNA splicing,protein stabilization and other biological processes as well as m RNA surveillance.Western Blot confirmed that LncRNA NONHSAT152502.1 bind to UPF1.2.1)RT-qPCR and Western Blot indicated that m RNA of SMA,Col1a1,MMP2 and Vimentin was significantly decreased when UPF1 was knockdown(P<0.05).Simultaneously,the protein expression of SMA and Col1a1 was significantly decreased(P<0.05).There was no significant difference in the expression of MMP2 and Vimentin(P>0.05).The m RNA of SMA,Col1a1,MMP2 and Vimentin was significantly increased when UPF1 was overexpressed(P<0.05),while the protein expression of SMA and Col1a1 was increased as well(P<0.05).However,there was no significant difference in the protein level of MMP2 and Vimentin(P>0.05).2)The OD value at 450 nm in si-UPF1 group was significantly decreased compared with si-NC group at 48 h,72h and 96h(P<0.05),there was no significant difference at 24h(P>0.05).At the same time,the number of hepatic stellate cell in G2/M phase in si-UPF1 significantly was decreased(P<0.05).When UPF1 was overexpressed,OD value at450 nm was increased compared with NC group at 48 h,72h and 96h(P<0.05),there was no significant difference at 24h(P>0.05).At the same time the number of hepatic stellate cell in G2/M phase was increased compare with NC(P<0.05).3)The rate of wound healing area was significantly decreased when UPF1 was knocked down(P<0.01);Compared with NC group,the percentage of wound healing area was increased when UPF1 was overexpressed(P<0.01).4)There was no significant difference in apoptotic rate of hepatic stellate cells when UPF1 was silenced or overexpressed(P>0.05).3.1)Knocked down LncRNA NONHSAT152502.1 combined with UPF1 overexpressed,the protein expression of SMA and Col1a1 were increased than knocked down LncRNA NONHSAT152502.1 alone(P<0.05).2)Knocked down LncRNA NONHSAT152502.1 combined with UPF1 overexpressed,the OD value at 450 nm was increased than knocked down LncRNA NONHSAT152502.1 alone at 48 h,72h and 96h(P<0.05),there was no significant difference in the OD value at 450 nm measured for24h(P>0.05),while flow cytometry indicated the number of hepatic stellate cell in G2/M phase was increased compared with knocked down LncRNA NONHSAT152502.1 alone(P<0.05).3.Knocked down LncRNA NONHSAT152502.1combined with UPF1 overexpressed,the percentage of wound healing area was increased than knocked down LncRNA NONHSAT152502.1 alone(P<0.01).4.Knocked down LncRNA NONHSAT152502.1,the protein expression of SMAD7 increased,while the protein expression of p-SMAD2 /SMAD2 and p-SMAD3/SMAD3 decreased.However,knocked down LncRNA NONHSAT152502.1 combined with UPF1 overexpressed,SMAD7 decreased,p-SMAD2/SMAD2 and p-SMAD3/SMAD3 increased compared with knocked down LncRNA NONHSAT152502.1 alone.(P<0.05).Conclusion:1.LncRNA NONHSAT152502.1 is closely related to the activated hepatic stellate cells.2.LncRNA NONHSAT152502.1 promotes the activation of hepatic stellate cells and enhances its proliferation and migration.3.LncRNA NONHSAT152502.1 inhibits the apoptosis of hepatic stellate cells.4.LncRNA NONHSAT152502.1 regulates TGF-β/SMAD and the activation of hepatic stellate cells through bind to UPF1. |
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