The Role Of M⁶A RNA Methylation Of TGF-β1 In NaAsO₂-mediated Rat Hepatic Stellate Cell Activation

Objective:Exposure to inorganic arsenic（i As）can cause liver fibrosis（LF）in rats.The activation of hepatic stellate cells（HSCs）is a crucial event in the occurrence of liver fibrosis.Our group previously confirmed that i As can induce the activation of HSCs,but the specific molecular mechanism is still unclear.Study showed that i As can upregulate the expression of transforming growth factorβ1（TGF-β1）,and TGF-β1 played an important role in HSCs activation.N6-methyladenosine（m⁶A）may also participate in the activation of HSCs,however the relationship between them till worth to explore.Therefore,this study mainly explored the effect of m⁶A methylation on HSCs activation and its specific mechanism,as well as the relationship and the function between them under sodium arsenite（chemical formula:NaAsO₂）exposure.Methods:10-week and 220 g～250 g weight male Sprague-Dawley（SD）rats were used as the study object,and randomly divided into control group and sodium arsenite treatment group（5 mg/kg BW NaAsO₂）.The method of gavage was used for 9 months,twice a day,with an interval of 12 h.During the period,rats ate and drank freely,and liver tissues of rats were collected after 9 months.The serum levels of glutamic-pyruvic transaminase（ALT）and Glutamic oxaloacetic transaminase（AST）were detected by microplate.Collagen deposition levels were detected by Masson staining.Western blot was used to detect the expression level of a marker of HSCs activation,α-smooth muscle actin（α-SMA）and m⁶A methylation-related proteins methyltransferase 3（METTL3）,methyltransferase14（METTL14）,insulin-like growth factor 2 m RNA binding protein 1（IGF2BP1）,insulin-like growth factor 2 m RNA binding protein 2（IGF2BP2）,alkylation repair homolog 5（ALKBH5）and fat mass and obesity-associated protein（FTO）protein,and the TGF-β/Smad pathway related protein mothers against decapentaplegic homolog2（Smad2）,mothers against decapentaplegic homolog 3（Smad3）and mothers against decapentaplegic homolog 4（Smad4）.The m RNA expression level of TGF-β1 was detected by quantitative reverse transcription polymerase chain reaction（q RT-PCR）.And the level of m⁶A methylation modification of total RNA was detected by colorimetry.Hepatic stellate cell line（HSC-t6）of rat was used to establish an vitro exposure model.After HSC-t6 cells were treated with 0,1,2,4μM NaAsO₂ for 24 h,we used western blot to detect the expression level of TGF-β1.After treating HSC-t6 cells with 4μM NaAsO₂ for 24 h,Western blot was used to detect the expression of HSCs activation proteinα-SMA and Collagen type I（Collagen-I）,the related protein Smad2/3/4 of TGF-β/Smad pathway,and m⁶A methylation-related proteins METTL3,METTL14,IGF2BP1/2,ALKBH5 and FTO protein.The m RNA expression level of TGF-β1 was detected by quantitative reverse transcription PCR（q RT-PCR）.And the level of m⁶A methylation modification of total RNA was detected by colorimetry.RNA Binding Protein Immunoprecipitation（RIP）was used to detect the binding level of METTL14 and IGF2BP2 with m RNA of TGF-β1.Lately,after 12 h of pretreatment with si TGF-β1,si METTL14 or si IGF2BP2 respectively,then using 4μM NaAsO₂ to treat HSC-t6 cells for 24 h,the expression of Smad2/3 and hepatic stellate cell activation indexα-SMA and Collagen-I were detected by western blot.After 12 h of pretreatment with si METTL14 or si IGF2BP2,using 4μM NaAsO₂ to treat HSC-t6 cells for 24 h,5μg/m L Actinomycin D（ACTD）was treated for 0,3 and 6 h,the m RNA expression level of TGF-β1 was detected by q RT-PCR.Results:Western blot demonstrated that after treating rat liver tissue and HSC-t6cells with NaAsO₂,the protein expression level ofα-SMA,p-Smad2/3,METTL14 and IGF2BP2 in NaAsO₂ treatment group were significantly higher,but Smad4,METTL3,ALKBH5 and FTO remained unchanged.The results of q RT-PCR showed that the m RNA expression level of TGF-β1 was significantly increased in NaAsO₂ treatment group.After NaAsO₂ treatment,the serum ALT and AST levels of rats were significantly increased,and Masson staining showed that collagen deposition increased significantly in the liver tissue of rats treated with NaAsO₂.The protein expression level of Collagen-I in HSC-t6cells treated with NaAsO₂ was significantly increased.After we used si TGF-β1,si METTL14 or si IGF2BP2 to transfect HSC-t6 cells for 12 h and NaAsO₂ for 24 h,Western blot results showed that p-Smad2/3,α-SMA and Collagen-I were decreased in si RNA treatment group.When transfected HSC-t6 cells with si METTL14 or si IGF2BP2for 12 h and NaAsO₂ for 24 h,adding 5μg/m L actinomycin D（ACTD）for 0,3 and 6 h,q RT-PCR results revealed that the half-life time of the TGF-β1 m RNA was shortened in si RNA treatment group.The results of RIP experiment indicated that METTL14 and IGF2BP2 were interacted with TGF-β1 m RNA.Conclusion:NaAsO₂ enhanced TGF-β1 m RNA stability through METTL14 and reading protein IGF2BP2 in an m⁶A-dependent manner,also promoted its expression,thus activating TGF-β/Smad pathway and inducing activation of HSCs.