Study On M~6A/Ythdc2 Regulates The PPER Pathway In Cadmium-induced Pancreatic β-cell Death

ObjectiveClarifying the effects of m⁶A modification on Protein Processing in Endoplasmic Reticulum（PPER）pathway signaling molecules in cadmium-induced isletβ-cells death,and exploring the relationship between YTH domain-containing protein 2（Ythdc2）and signaling molecule proteins of PPER pathway.Methods1.CCK8 and Hoechst staining assay were used to detect cell viability and apoptosis rates in isletβ-cells（NIT-1）treated by cadmium sulfate（Cd SO₄）,real-time quantitative PCR（RT-PCR）technology was utilized to determine the m RNA levels of PPER signaling molecules such as B-cell lymphoma/leukemia-2 gene（Bcl-2）,transcription factor C/EBP homologous protein（Chop）,heat shock protein 40kd family member B1（Dnajb1）,Bcl-2-associated X protein（Bax）,heat shock protein90kdαfamily member A1 protein（Hsp90aa1）,type III collagen structural protein（Col3a1）and X-box-binding protein 1（Xbp1）,and the protein expressions of PPER pathway signaling molecules was detected by Western Blot（WB）.2.CCK8 assay was conducted to screen the optimal treatment concentration of the m⁶A agonist entacapone（Ent）.In order to clarify the effects of Ent on Cd SO₄-induced m RNA levels and protein expressions of the PPER pathway signaling molecules,the levels of m RNA and protein of the PPER pathway signaling molecules were detected respectively by RT-PCR and WB in isletβ-cells treated with 20μmol/L Ent,4μmol/L Cd SO₄,20μmol/L Ent+4μmol/L Cd SO₄ for 24 h.Calculating the protein translation efficiency of PPER pathway signaling molecules under the treatment of Cd SO₄and Ent.3.Screening the optimal experimental concentration of m⁶A inhibitor 3-deazaadenosine（DAA）by CCK8 assay.In order to clarify the effects of DAA on Cd SO₄-induced m RNA levels and protein expressions of the PPER pathway signaling molecules,the levels of m RNA and protein of the PPER pathway signaling molecules were detected respectively by RT-PCR and WB in isletβ-cells treated with 25μmol/L DAA,4μmol/L Cd SO₄ and 25μmol/L DAA+4μmol/L Cd SO₄ for 24 h.The protein translation efficiency of PPER pathway signaling molecules,which were mediated by DAA in Cd SO₄-induced changes,was calculated and measured.4.The expression levels of Ythdc2 during Cd SO₄-induced isletβ-cell death were detected by WB.And Pearson correlation was used to analyze the relationship between the levels of Ythdc2 and PPER signaling proteins（Bcl-2,Bax,Chop,Dnajb1,Hsp90aa1,Xbp1 and Col3a1）.Co-immunoprecipitation（Co IP）assay was used to detect the interactions of Ythdc2 protein and PPER signaling proteins,which were influenced by m⁶A modification level was also further evaluated.Results1.Changes in PPER pathway protein expression induced by Cd SO₄:Cd SO₄treatment decreased the cell viability of isletβ-cells and significantly increased apoptosis rates in a dose-dependent manner.Results from WB showed that the protein levels of Bcl-2,Xbp1 and Col3a1 were 0.57,0.60 and 0.67-fold of that in the control group after being treated with 4μmol/L Cd SO₄;the differences were statistically significant（P<0.05）.The protein levels of Bax and Chop,Dnajb1 and Hsp90aa1 were1.78,1.74,1.67 and 1.93-fold when compared with that in the control group;the differences were statistically significant（P<0.05）.2.Effect of m⁶A agonist Ent on changes in the PPER pathway molecules induced by Cd SO₄:Co-treatment of 20μmol/L Ent was able to attenuate the cell viability,which was inhibited by Cd SO₄.And Ent also reversed Cd SO₄-induced apoptosis and significantly increased the level of m⁶A modification in the Cd SO₄ treatment group.Results from WB showed that the protein levels of Bcl-2,Xbp1 and Col3a1 in Cd SO₄combined with the Ent treatment group were 0.77,0.80 and 0.82-fold compared to that in the control group.Co-treatment with Ent also significantly alleviated the reduction in protein levels,which led by Cd SO₄ treatment（0.57,0.60 and 0.69-fold）;the differences were statistically significant（P<0.01）.The protein expressions of Bax,Chop,Dnajb1 and Hsp90aa1 in the co-treatment group were 1.38,1.42,1.43 and1.67-fold of that in the control group,the increase factors were significantly lower than the increase in protein levels caused by Cd SO₄ treatment（1.78,1.76,1.66,1.94-fold）（P<0.01）;the changes trends of protein expression were consistent with those of m RNA.3.Effect of the m⁶A inhibitor DAA on changes in the PPER pathway molecules induced by Cd SO₄:The combination of 25μmol/L DAA and Cd SO₄ significantly enhanced the death of Cd SO₄-induced isletβ-cells,being indicated by cell viability reduction and apoptosis elevation.DAA co-treatment also reduced the level of m⁶A modification.Results from WB also showed that combined administration of DAA and Cd SO₄significantly down-regulated the protein levels of Bcl-2,Xbp1 and Col3a1,of which changes were 0.44,0.55,and 0.51-fold of that in the control group,further aggravating the reduction in protein levels caused by Cd SO₄ treatment（0.58,0.68,0.67-fold）.On the contrary,combined administration of DAA and Cd SO₄ remarkably increased the protein expression levels of Bax,Chop,Dnajb1 and Hsp90aa1 to 2.08,2.11,1.91 and 2.16-fold of that in the control group,respectively,and indicating aggravating the elevation of protein levels induced by Cd SO₄ treatment（1.78,1.74,1.73,1.93-fold）（P<0.01）,the change trends of protein expression were consistent with those of m RNA.4.Interaction of Ythdc2 with the PPER pathway molecules:The results of WB showed that the protein expression levels of Ythdc2 were 0.93,0.80 and 0.59-fold of that in the control group after cells treated with 1,2 and 4μmol/L Cd SO₄ for 24 h.Pearson correlation showed that there were positive correlations between Ythdc2 and Bcl-2,Col3a1 and Xbp1,of which correlation coefficient“r”were 0.9447、0.9469and 0.9782,respectively.On the contrary,negative correlations were found between Ythdc2 and Bax,Chop,Hsp90aa1 and Dnajb1,and the correlation coefficient“r”were-0.9722、-0.98、-0.9576 and-0.9692,respectively.Results from the Co IP experiment revealed that the protein levels of Bcl-2,Xbp1 and Col3a1 in the Cd SO₄group,Cd SO₄+DAA group and Cd SO₄+Ent group were 0.58,0.42,0.77-fold,0.66,0.52,0.85-fold and 0.68,0.56,0.84-fold of the control group,DAA co-treatment aggravated the changes in protein level Cd SO₄-induced significantly,while Ent co-treatment alleviated the decrease in protein levels（P<0.05）.The proteins expression levels of Bax,Chop,Dnajb1 and Hsp90aa1 in the Cd SO₄ group,Cd SO₄+DAA group and Cd SO₄+Ent group were 1.68,2.10,1.40-fold,1.75,2.00,1.49-fold,respectively.1.61,1.95,1.41-fold,and 1.92,2.09,1.70-fold of the control group,DAA co-treatment promoted the increase in protein levels induced by Cd SO₄,while Ent co-treatment inhibited the increase in protein levels obviously（P<0.05）.Conclusionm⁶A modification is involved in the process of isletβ-cell death induced by cadmium.Alterations in the level of m⁶A modification significantly affect the expression of the PPER pathway signaling molecules.m⁶A binding protein Ythdc2 is also involved in the process of isletβ-cells death induced by cadmium and closely related to the changes of PPER pathway signaling molecules,and Ythdc2 may modulate the PPER signaling pathway in an m⁶A modification-dependent manner.