Molecular Mechanism Of DNMT3A Mediated LncRNA ANRIL Methylation Promoted Liver Fibrosis

BackgroundLiver fibrosis is a pathological process in which various damage factors cause continuous damage and repair reactions,which lead to abnormal deposition of extracellular matrix（ECM）in liver tissues,and further cause abnormal changes of liver structure and function.At present,the clinical treatment of liver fibrosis is not effective,which seriously endangers human life and health.Therefore,clarifying the pathogenesis of liver fibrosis has important clinical significance.Studies have confirmed that activated hepatic stellate cells（HSCs）are the main source of ECM,and HSCs activation and proliferation is the central link in the development of liver fibrosis.Under various injury stimuli,HSCs transdifferentiate from stationary,vitamin A-storing cells to proliferative and fibroblasts,and secreteα-smooth muscle actin（α-SMA）and type I Collagen（Collagen I,Col1a1）,etc.,which produce cytokines that promote fibrosis such as transforming growth factor-β1（TGF-β1）,platelet derived growth factor（Platelet derived growth factor-BB,PDGF-BB）,etc.,are now recognized as mainly liver damage driving factor.However,up to now,the molecular mechanism regulating HSCs proliferation and liver fibrosis is still unclear.Therefore,exploring the molecular mechanism of HSCs proliferation in the pathogenesis of liver fibrosis is of great significance to the prevention and treatment of liver fibrosis.Various factors are involved in regulating the HSCs proliferation.Epigenetic modification of DNA methylation is an important epigenetic marker,which plays a key role in the formation of liver fibrosis.Our research team found that DNA methylation is involved in regulating the HSCs proliferation,but it is still unclear how DNMT3A,one of the DNA methyltransferases,plays a regulatory role in the pathogenesis of liver fibrosis.In addition,studies have shown that epigenetic modification of long non-coding RNA（Lnc RNA）has the effect of regulating the HSCs proliferation,and the antisense non-coding RNA（Antisense Non-Coding RNA in the INK4 Locus,ANRIL）in the INK4 locus is known as one of the key molecules with the function of regulating cell activation and proliferation.It is reported that Lnc RNA ANRIL can regulate cell proliferation activity by mediating the expression of related cell proliferation pathway proteins.However,the specific molecular mechanism of how Lnc RNA ANRIL regulates the HSCs proliferation is unclear and needs to be elucidated.Based on how the two epigenetic regulation modes of DNMT3A and Lnc RNA ANRIL regulate the HSCs proliferation,the interaction,target and regulation mode between them,are still unclear and need to be clarified.This article will explore the molecular mechanism of DNMT3A-mediated Lnc RNA ANRIL methylation in liver fibrosis,in order to provide scientific basis,new molecular diagnostic indicators and intervention targets for the treatment of clinical liver fibrosis.In this study,serum and tissue specimens of clinical liver fibrosis patients were collected,and the classic carbon tetrachloride（CCl₄）subcutaneous injection was used to establish a mice liver fibrosis model as the in vivo research object,HSCs as the in vitro research object,and fluorescence in situ Hybridization,methylation-specific PCR（MSP）,5-bromodeoxyuridine（Brd U）fluorescent staining and other techniques were used to study the molecular mechanism of DNMT3A-mediated Lnc RNA ANRIL methylation in liver fibrosis.The full text consists of three parts:Part one The differential expression of DNMT3A,ANRIL,α-SMA and Collagen I in clinical liver fibrosis patientsObjectiveTo explore the differential expression of DNMT3A,ANRIL,α-SMA and Collagen I in clinical liver fibrosis samples.Methods:A total of 20 patients with chronic liver disease were included in this study.Grouping:according to the pathological diagnosis of liver biopsy with or without symptoms of fibrosis,they were divided into control group（non fibrosis group）and fibrosis group.The pathological diagnosis of liver biopsy refer to“consensus on diagnosis and treatment of liver fibrosis（2019）”.The relevant information of patients,such as age and gender,was recorded,and the serum and liver tissue samples were collected for testing in strict accordance with the requirements of the biomedical ethics committee of Anhui Medical University.（1）To detect the changes of AST,ALT and HA in serum of patients with liver fibrosis;（2）HE staining was used to observe the pathological changes of liver tissue;（3）Masson staining and Sirius red staining were used to observe the changes of collagen deposition in liver tissue of patients with liver fibrosis;（4）Western blotting and Immunohistochemistry were used to detect the differential expression of DNMT3A,α-SMA and Collagen I in liver tissues of patients with liver fibrosis;（5）RT-qPCR was used to detect the differential expression of DNMT3A,ANRIL,α-SMA and Collagen I m RNA in liver tissues of patients with liver fibrosis.Results:1.The expression of DNMT3A was significantly increased and the expression of ANRIL was significantly decreased in patients with clinical liver fibrosis（1）Compared with the control group,AST/ALT ratio and HA content in serum of liver fibrosis group were significantly increased;（2）Compared with the control group,collagen deposition,inflammatory infiltration and fibrosis were significantly increased in clinical liver fibrosis patients;（3）Western blotting and Immunohistochemistry results showed that the protein expression of DNMT3A,α-SMA and Collagen I in clinical liver fibrosis patients was significantly higher than that in the control group;（4）RT-qPCR results showed that the expression of DNMT3A,α-SMA,Collagen I m RNA in clinical liver fibrosis patients was significantly higher than that in the control group,while the expression of ANRIL m RNA was significantly lower.（5）Preason analysis showed that the expression of DNMT3A andα-SMA showed a significantly positive correlation,and the expression between ANRIL andα-SMA showed a significantly negative correlation in tissues of liver fibrosis patients.Summary of Part One:High expression of DNMT3A,low expression of ANRIL and impaired liver function may be related to the formation of fibrosis in patients with liver fibrosis.Part Two Expression of DNMT3A,ANRIL,α-SMA,Collagen I and methylation level of ANRIL promoter region in mice liver fibrosis tissue and HSCsObjectiveTo further explore the expression of DNMT3A,ANRIL,α-SMA,Collagen I and the methylation level of ANRIL promoter region in mice liver fibrosis tissue and HSCs.Methods:1.30 male mice（18～22g）were randomly divided into two groups:normal group（n=15）and CCl₄treatment group（n=15）.From the day of treatment,male mice in each group were subcutaneously injected with 50%CCl₄olive oil solution（1ml/kg）except the normal group（1ml/kg）At the end of the 12th week,the weight changes of mice were observed and recorded.At the end of the 12th week,Liver tissue and blood samples were collected to detect the related indicators after anesthetized the mice.（1）The levels of AST,ALT,HA and TGF-β1 in serum of mice liver fibrosis were detected;（2）HE staining was used to observe the histopathological changes of liver fibrosis in mice;（3）Masson staining and Sirius red staining were used to observe the changes of collagen deposition in mice liver fibrosis tissues;（4）The expressions of DNMT3A,α-SMA and Collagen I in mice liver fibrosis tissues were detected by Western blotting and Immunohistochemistry;（5）RT-qPCR was used to detect the m RNA expression of DNMT3A,ANRIL,α-SMA and Collagen I in mice liver fibrosis tissues;（6）The expression of ANRIL in mice liver fibrosis tissues was detected by FISH;（7）MSP was used to detect the methylation level of ANRIL promoter in mice liver fibrosis tissues.2.HSCs were used as the research object in vitro,and the proliferation of HSCs was induced by TGF-β1（5ng/ml）,to establish the HSCs proliferation mode.the following experiments were carried out（1）The levels of HA and PIIIP in the supernatant of HSCs treated with TGF-β1 were detected;（2）The differential expression of DNMT3A,α-SMA and Collagen I in TGF-β1 treated HSCs was detected by Western blotting and Immunofluorescence assay;（3）RT-qPCR was used to detect the differential expression of DNMT3A,ANRIL,α-SMA and Collagen I m RNA in TGF-β1 treated HSCs;（4）MTT,CCK8 and Brd U fluorescence staining were used to observe the effect of TGF-β1 treatment on the proliferation of HSCs;（5）BSP was used to detect the methylation sites of ANRIL promoter in HSCs treated with TGF-β1.Results:1.The expression of DNMT3A in mice liver fibrosis tissue is significantly increased,and the expression of ANRIL is significantly decreased.The decreased expression of ANRIL may be related to the increase in the level of methylation in the promoter region of the ANRIL gene.（1）Serum levels of AST/ALT ratio,HA and TGF-β1 in mice with liver fibrosis were significantly higher than those in the control group;（2）Compared with the control group,the collagen deposition in the liver fibrosis tissue of mice increased,the inflammatory infiltration increased,and the fibrosis changes were obvious;（3）Compared with the control group,the expression of DNMT3A,α-SMA,Collagen I protein and m RNA was significantly increased in mice liver fibrosis tissue,while the expression of ANRIL m RNA in mice liver fibrosis tissue was significantly reduced;（4）Compared with the control group,the methylation level of ANRIL promoter region in mice liver fibrosis tissue was significantly increased.2.In the in vitro HSCs proliferation model,DNMT3A is highly expressed and ANRIL is low.The low expression of ANRIL may be related to the increase in the level of methylation in the promoter region of ANRIL（1）The contents of HA and PIIIP in the supernatant of HSCs cells after TGF-β1treatment were significantly higher than those in the control group;（2）Compared with the control group,the proliferation activity of HSCs（24h,48h）induced by TGF-β1 was significantly enhanced;（3）Compared with the control group,the expression of DNMT3A,α-SMA,Collagen I protein and m RNA was significantly increased in HSCs induced by TGF-β1,while the expression of ANRIL m RNA was significantly decreased;（4）At the same time,TGF-β1 induced and stimulated the methylation level of ANRIL promoter region in HSCs was significantly higher than that of the control group.Summary of Part Two:The high expression of DNMT3A and the down-regulation of ANRIL expression caused by the hypermethylation modification of ANRIL may be related to the formation of liver fibrosis in mice and the HSCs proliferation.However,how DNMT3A and ANRIL regulate the formation of liver fibrosis and the HSCs proliferation,and whether they have a regulatory effect is still unclear,and it is worthy of further study.Part Three Molecular mechanism of DNMT3A-mediated ANRIL methylation in liver fibrosis and HSCs proliferationObjective:Explore how DNMT3A mediates ANRIL methylation to regulate the molecular mechanism of HSCs proliferation and liver fibrosis.Method:1.60 male mice（18～22g）were randomly divided into 4 groups:normal group,CCl₄treatment group,CCl₄+LV3 lentivirus empty vector group,CCl₄+LV3-DNMT3A lentivirus group,15 mice in each group.From the day of treatment,except for the normal group（1 ml/kg）,the mice in the other groups were given subcutaneous injection of 50%CCl₄olive oil solution（1 ml/kg）twice a week for a total of 12 Weeks;11 weeks after modeling,mice in the LV3 empty vector group and LV3-DNMT3A lentivirus group were divided into 30ul lentiviral empty vector LV3 and 30ul lentiviral LV3-DNMT3A tail vein injection treatment（Slow the titer of the virus particles was1×10⁹TU/ml）.The mice were sacrificed one week later,and liver tissue and blood samples were collected to detect related indicators.（1）HE staining was used to observe histopathological changes of liver fibrosis in mice with recombinant lentivirus silencing DNMT3A;（2）Masson staining and Sirius red staining were used to observe the changes of collagen deposition in liver fibrosis tissue in mice with recombinant lentivirus silencing DNMT3A;（3）Western blotting and Immunohistochemistry experiments were used to detect the expression change of DNMT3A,α-SMA,Collagen I protein in mice liver fibrosis tissue with recombinant lentivirus silencing DNMT3A;（4）RT-qPCR to detect the expression change of DNMT3A,ANRIL,α-SMA,Collagen I m RNA in mice liver fibrosis tissue with recombinant lentivirus silencing DNMT3A;（5）FISH detects the expression change of ANRIL in mice liver fibrosis with recombinant lentivirus silencing DNMT3A.2.Cell experiment,application of DNMT3A overexpression plasmid,DNMT3A small interfering RNA and DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine（5-Azad C）1μmol/L was transfected with HSCs after TGF-β1 stimulation.Cell experiment grouping:controlgroup,TGF-β1（5ng/ml）treatmentgroup,TGF-β1（5ng/ml）+DNMT3A overexpression plasmid treatment group,TGF-β1（5ng/ml）+DNMT3A small interfering RNA treatment Group,TGF-β1（5ng/ml）+5-Azad C（1μmol/L）treatment group,and the following experiment was performed:（1）Western blotting experiment to detect the expression changes of DNMT3A,α-SMA,Collagen I protein in DNMT3A overexpression plasmid,DNMT3A small interfering RNA and 5-Azad C intervention treatment in HSCs;（2）RT-qPCR experiment to detect the expression changes of DNMT3A,ANRIL,α-SMA,Collagen I m RNA in DNMT3A overexpression plasmid,DNMT3A small interfering RNA and 5-Azad C（1μmol/L）intervention in HSCs;（3）Use MTT,CCK8 and Brd U fluorescent staining to observe the effects of DNMT3A overexpression plasmid,DNMT3A small interfering RNA and 5-Azad C treatment on HSCs proliferation.3.Use ANRIL overexpression plasmid and ANRIL small interfering RNA transfection to treat HSCs stimulated by TGF-β1 for 24 hours.Cell experiment grouping:control group,TGF-β1（5ng/ml）treatment group,TGF-β1（5ng/ml）+ANRIL overexpression plasmid treatment group,TGF-β1（5ng/ml）+ANRIL small interfering RNA treatment group,and perform the following experiment:（1）Western blotting was used to detect the expression changes ofα-SMA,Collagen I,AMPK,and Phospho-AMPK（p-AMPK）proteins after ANRIL overexpression plasmid and ANRIL small interfering RNA treated activated HSCs respectively;（2）RT-qPCR to detect the expression changes of ANRIL,α-SMA,Collagen I m RNA after ANRIL overexpression plasmid and ANRIL small interfering RNA respectively treat activated HSCs;（3）Use MTT,CCK8 and Brd U fluorescence staining to observe the effects of ANRIL overexpression plasmid and ANRIL small interfering RNA on the proliferation of HSCs.Results:1.After the intervention of recombinant lentivirus DNMT3A,the expression of ANRIL was significantly increased,and the degree of liver fibrosis in mice was significantly improved（1）Compared with the control group,the collagen deposition in the liver fibrosis tissue of mice increased,the inflammatory infiltration increased,and the fibrosis changes were obvious.Compared with the lentiviral empty vector group,the liver cells could be seen after the recombinant lentivirus silenced DNMT3A intervention,the structure is improved,collagen fibers are reduced,and the pathological improvement of liver fibrosis is more obvious;（2）Compared with the control group,the expression of DNMT3A,α-SMA,Collagen I protein and m RNA in the liver fibrosis tissue of mice was significantly increased.Compared with the lentiviral empty vector group,the recombinant lentivirus silenced DNMT3A after intervention,The expression of DNMT3A,α-SMA,Collagen I protein and m RNA in mice liver fibrosis tissue was significantly reduced;（3）However,compared with the control group,the expression of ANRIL m RNA in the mice liver fibrosis tissue was significantly reduced.Compared with the lentiviral empty vector group,the recombinant lentivirus silenced DNMT3A in the mice liver fibrosis tissue after intervention,the expression of ANRIL m RNA was significantly increased.2.Overexpression of DNMT3A significantly inhibit the expression of ANRIL and promote the proliferation of HSCs.While silencing of DNMT3A significantly promote the expression of ANRIL and inhibit the proliferation of HSCs.Suggested that DNMT3A affect the proliferation activity of HSCs through negatively regulating the expression of ANRIL.（1）Compared with the negative control group,the expression of DNMT3A m RNA in HSCs was significantly reduced after transfection with DNMT3A small interfering RNA,while the expression of ANRIL m RNA was significantly increased.（2）However,compared with the empty plasmid group,DNMT3A m RNA expression in HSCs was significantly increased after DNMT3A overexpression plasmid was transfected,while ANRIL m RNA expression was significantly decreased.（3）Compared with the TGF-β1 stimulation group,the expression of ANRIL was significantly increased after HSCs were treated with DNA methylation inhibitor5-Azad C.（4）Compared with the empty plasmid group,the proliferation of HSCs can be significantly enhanced after transfection with DNMT3A overexpression plasmid;（5）Compared with the negative control group,DNMT3A small interfering RNA transfection can significantly inhibit the proliferation of HSCs;in addition,compared with the TGF-β1 stimulation group,5-Azad C treatment can significantly inhibit the proliferation of HSCs.3.Overexpression of ANRIL can significantly inhibit AMPK phosphorylation,at the same time,significantly reduce the proliferation activity of HSCs.Silencing of ANRIL can significantly increase the phosphorylation of AMPK,at the same time,significantly promote the proliferation activity of HSCs.（1）Compared with the empty plasmid group,ANRIL overexpression plasmid transfected HSCs with ANRIL expression significantly increased,while the expression ofα-SMA,Collagen I and p-AMPK decreased significantly;（2）In addition,compared with the empty plasmid group,the ANRIL overexpression plasmid significantly inhibited the proliferation of HSCs after transfection.（3）However,compared with the negative control group,the expression of ANRIL was significantly reduced after HSCs were transfected with ANRIL small interfering RNA,while the expression ofα-SMA,Collagen I and p-AMPK were significantly increased;（4）Compared with the negative control group,ANRIL small interfering RNA transfection significantly promoted the proliferation of HSCs.Summary of Part Three:Lnc RNA ANRIL may be down-regulated due to DNMT3A-mediated high methylation modification,thereby weakened the inhibitory effect on the downstream AMPK signaling pathway core protein AMPK,activating the AMPK signaling pathway,enhanced the proliferation of HSCs,and promoting the formation of liver fibrosis.Conclusion:1.High expression of DNMT3A,low expression of ANRIL and impaired liver function may be related to the formation of liver fibrosis.2.The high expression of DNMT3A and the down-regulation of ANRIL expression caused by the hypermethylation modification of ANRIL may be related to the formation of liver fibrosis and the HSCs proliferation.3.Lnc RNA ANRIL may be down-regulated due to DNMT3A-mediated high methylation modification,thereby weakened the inhibitory effect on the downstream AMPK signaling pathway core protein AMPK,activating the AMPK signaling pathway,enhanced the proliferation of HSCs,and promoting the formation of liver fibrosis.