Study On The Role Of N~6-Methyladenosine And LncRNA Involved In Cadmium-induced Oxidative Damage Of Pancreatic β-cells

ObjectiveThrough in vitro experiments to identify the changes of m⁶A modification levels and Lnc RNAs during the oxidative damage of pancreaticβ-cells induced by cadmium.And to investigate the impact of changing the amount of m⁶A modification on oxidative damage as well as identify regulatory roles of m⁶A modification on Lnc RNAs.Methods（1）Cell viability was detected by CCK8 assay in pancreaticβ-cells（NIT1）which were treated with various concentrations of m⁶A agonist entacapone or m⁶A inhibitor 3-deazaadenosine.After NIT1 cells were treated with cadmium sulfate（Cd SO₄）in combination with entacapone or 3-deazaadenosine,CCK8 assay and Hoechst staining assay was respective used to measure the cell viability and apoptosis.Further,a fluorescence probe test was used for detecting intracellular reactive oxygen species,and the WST-8 test measure intracellular superoxide dismutase activity,and the thiobarbituric acid assay was utilized to determine cellular malondialdehyde content,and the level of m⁶A modification on total RNA was detected by Epi Quick TM RNA methylation quantification kit.（2）Protein immunoblotting was used to assess the effects of entacapone and 3-deaz aadenosine on the intracellular levels of superoxide dismutase 1（Sod1）,glutamate cyste ine ligase（Gclc）,nuclear factor E2-related factor（Nrf2）,and heme oxygenase 1（Ho-1）protein expression in NIT-1 cells which were treated with cadmium sulfate and/or entac apone and 3-deazaadenosine.The expression of NONMMUT065427,NONMMUT014565,NONMMUT036805,NONMMUT029382,ENSMUST00000162103 and ENSMUS T117235 were detected by fluorescence quantitative PCR after treatment with designed concentrations in NIT-1 cells.The correlation between Lnc RNAs and m⁶A modification levels was analyzed by Pearson’s linear correlation analysis.Results（1）Cell survival and apoptosis:The cell survival rate was 106.01%in the 20μmol/L entacapone treated group,78.43%in the 4μmol/L cadmium sulfate treated group,114.57%in the combined entacapone and cadmium sulfate treated group,indicating entacapone effectively antagonized cadmium sulfate-induced cell death（P<0.05）.Cell survival rate was 100.74%in the group treated with 25μmol/L of 3-deazaadenosine,51.86%in the group treated with the combination of 3-deazaadenosine and cadmium sulfate,demonstrating 3-deazaadenosine aggravated the cell death induced by cadmium sulfate（P<0.05）.The results from Hoechst staining showed that the apoptosis rates in the control,entacapone,cadmium sulfate,entacapone and cadmium sulfate co-treatment groups were 3.50%,4.00%,26.67%and 17.50%,respectively,and the apoptosis rate in the co-treatment group of entacapone and cadmium sulfate was obviously lower than that in the cadmium sulfate treatment group（P<0.05）.Apoptosis rates in the control,3-deazaadenosine,cadmium sulfate,3-deazaadenosine and cadmium sulfate co-treatment groups were 8.93%,11.84%,23.09%and 31.21%,respectively,the apoptosis rate in the 3-deazaadenosine and cadmium sulfate co-treatment group was apparently higher than that in the cadmium sulfate group（P<0.05）.（2）m⁶A modification levels:After NIT1 cells treated by 1μmol/L,2μmol/L and 4μmol/L cadmium sulfate treatment,a decreasing trend was observed in m⁶A modification levels on total RNA.In detail,m⁶A modification levels in the control,1μmol/L,2μmol/L,and 4μmol/L cadmium sulfate treated groups were 0.85%,0.77%,0.49%,and 0.34%,respectively,the m⁶A modification levels in the 2μmol/L and 4μmol/L cadmium sulfate treated groups showing significantly lower m⁶A modification levels than that in the control group（P<0.05）.The m⁶A levels were 0.79%,0.84%,0.38%,and 0.56%in the control,entacapone,cadmium sulfate,and entacapone and cadmium sulfate co-treatment groups,respectively,with substantially greater levels in the co-treatment group of entacapone and cadmium sulfate than in the cadmium sulfate group（P<0.05）.The levels of m⁶A modification were 0.79%,0.74%,0.38%,and 0.18%in the control,3-deazaadenosine,cadmium sulfate,and 3-deazaadenosine and cadmium sulfate co-treatment groups,respectively,with significantly lower levels in the 3-deazaadenosine and cadmium sulfate co-treatment group than in the cadmium sulfate group（P<0.05）.（3）Oxidative damage indexes:ROS levels in the entacapone,cadmium sulfate and entacapone and cadmium sulfate co-treatment groups were 0.93-fold,4.50-fold,2.02-fold change of that in the control group,respectively,and the ROS levels in the entacapone and cadmium sulfate co-treatment group were remarkable lower than those in the cadmium sulfate group（P<0.05）.The ROS levels in the 3-deazaadenosine,cadmium sulfate and 3-deazaadenosine and cadmium sulfate co-treatment group were1.12-fold,3.93-fold,5.12-fold change of that in the control group,respectively,with obviously higher ROS levels in the 3-deazaadenosine and cadmium sulfate co-treatment group than in the cadmium sulfate group（P<0.05）.The SOD activities of the control,entacapone,cadmium sulfate,and co-treatment group with entacapone and cadmium sulfate were 19.09 U,20.37 U,12.78 U and 17.96 U,respectively,and the SOD activities of the co-treatment group were apparently higher than those of the cadmium sulfate group（P<0.05）.The MDA contents were 5.83μmol/mg protein,5.46μmol/mg protein,38.88μmol/mg protein,9.23μmol/mg protein,respectively,and the MDA content of the co-treatment group was considerably lower than those of the cadmium sulfate group（P<0.05）.The SOD activities of the control,3-deazaadenosine,cadmium sulfate,and co-treatment group with entacapone and cadmium sulfate were 18.63 U,18.06 U,10.56 U and 8.39 U,respectively,and the SOD activities of the co-treatment group were lower than those of the cadmium sulfate group（P<0.05）.The MDA contents were 9.03μmol/mg protein,11.56μmol/mg protein,16.42μmol/mg protein,20.04μmol/mg protein,respectively,and the MDA content of the co-treatment group was higher than that of the cadmium sulfate group（P<0.05）.（4）Oxidative damage-related proteins:Results from western blot were showed that the Sod1 protein expressions in the entacapone,cadmium sulfate,and entacapone and cadmium sulfate co-treatment groups were 0.88-fold,0.66-fold,and 1.19-fold in comparison to that in control group.The Gclc protein expressions were 1.19-fold,0.80-fold,1.39-fold change of that in the control group.The expression of Nrf2 protein were0.89-fold,2.01-fold,0.71-fold when compared with that in the control group.The expression of Ho-1 protein were 1.02-fold,8.19-fold,5.46-fold change of the control group.Sod1 and Gclc levels were at substantially greater levels,and Nrf2 and Ho-1levels were obviously lower in the group co-treated with entacapone and cadmium sulfate than in the cadmium sulfate group（P<0.05）.Sod1 expression in the 3-deazaadenosine,cadmium sulfate and entacapone and cadmium sulfate co-treatment groups were 0.86-fold,0.69-fold,0.48-fold change of the control group,respectively.Gclc expression were 0.88-fold,0.83-fold,0.69-fold in comparison to that in control group.Nrf2 expression were 1.04-fold,2.48-fold,4.79-fold when compared with that in the control group.Ho-1 expression were 0.93-fold,3.41-fold,8.02-fold change of that in the control group.Nrf2 and Ho-1 levels were substantially greater levels in the co-treatment group of entacapone and cadmium sulfate than in the cadmium sulfate group（P<0.05）,while the levels of Sod1 and Gclc levels were considerably lower than cadmium sulfate group（P<0.05）.（5）Levels of Lnc RNAs and their correlation with the m⁶A modification level:Lnc RNA expression were detected by fluorescence quantitative PCR,and the results showed that 2μmol/L and 4μmol/L cadmium sulfate treatment decreased the levels of NONMMUT065427（0.70 and 0.35-fold）,NONMMUT014565（0.46 and 0.53-fold）,4μmol/L cadmium sulfate treatment decreased NONMMUT014565（0.29-fold）levels,4μmol/L cadmium sulfate treatment elevated ENSMUST00000162103（2.16-fold）,NONMMUT029382（61.87-fold）,ENSMUST00000117235（5.40-fold）levels and the differences were statistically significant compared with the corresponding controls（P<0.05）.The levels of m⁶A modification were positive correlated to those of NONMMUT065427,NONMMUT014565 and NONMMUT036805,with correlation coefficients of 0.635,0.700,and 0.417,respectively.m⁶A modification levels were negatively correlated with NONMMUT029382,ENSMUST00000162103 and ENSMUST00000117235,with correlation coefficients of-0.630,-0.603 and-0.765.（6）Effects of alteration m⁶A modification levels on Lnc RNA changes induced by cadmium sulfate:The levels of NONMMUT065427 in the entacapone,cadmium sulfate and entacapone and cadmium sulfate co-treatment groups were 0.89-fold,0.38-fold,0.65-fold in comparison to that in control group.NONMMUT014565 levels were 0.61-fold,0.30-fold,0.54-fold change of the control group,respectively.NONMMUT036805levels were 1.00-fold,0.64-fold,0.86-fold when compared with that in the control group.ENSMUST00000162103 levels were 0.81-fold,4.47-fold,2.51-fold change of the control group.NONMMUT029382 levels were 2.03-fold,72.33-fold,51.18-fold in comparison to that in control group.ENSMUST00000117235 levels were 4.45-fold,16.72-fold,12.20-fold in comparison to that in control group.NONMMUT065427,NONMMUT014565 and NONMMUT036805 were substantially greater levels in the co-treatment group of entacapone and cadmium sulfate than in the cadmium sulfate group,and the levels of ENSMUST00000162103,NONMMUT029382 and ENSMUST00000117235 were considerably lower than those of the cadmium sulfate group（P<0.05）.NONMMUT065427 levels in the group treated with 3-deazaadenosine,cadmium sulfate and co-treatment group of 3-deazaadenosine and cadmium sulfate were 1.10-fold,0.54-fold,0.34-fold in comparison to that in control group.The levels of NONMMUT014565 were 1.28-fold,0.63-fold,0.36-fold change of the control group,respectively.NONMMUT036805 levels were 1.38-fold,0.74-fold,0.42-fold when compared with that in the control group.The levels of ENSMUST00000162103were 0.91-fold,3.76-fold,6.14-fold change of the control group.The levels of NONMMUT029382 were 0.69-fold,69.36-fold,118.76-fold in comparison to that in the control group.The levels of ENSMUST00000117235 were 1.55-fold,9.24-fold,16.01-fold change of the control group.NONMMUT065427,NONMMUT1014565,NONMMUT336805 levels were considerably lower in the co-treatment group of entacapone and cadmium sulfate than in the cadmium sulfate group,and ENSMUST00000162103,NONMMUT029382 and ENSMUST00000117235 levels were substantially higher.ConclusionThe m⁶A modification and multiple Lnc RNAs were changed and correlated during the oxidative damage of NIT-1 cells induced by cadmium sulfate.An increasing level of m⁶A modification could restore the oxidative damage of NIT-1 cells caused by cadmium,while a decrease in the level of m⁶A modification could aggravate the oxidative damage.