Bioinformatics Analysis Of MiRNA Expression Profile In Rat's Damaged Kidney Caused By Cadmium Exposure

Objective:?1?To explore the relationship between renal function changes and miRNA differential expression after cadmium exposure;to analyze the GO enrichment and KEGG pathways that these miRNA target genes mainly involved in.?2?To build up the foundation for the mechanisms of miRNA on damaged renal caused by cadmium exposure and for searching the early biomarkers of kidney damage caused by cadmium exposure.Materials and methods:?1?20 SPF SD male rats were randomly divided into 4 groups:5 male control group,5male 0.5 mg/kg·bw CdCl₂ group,5 male 1.0 mg/kg·bw CdCl₂ group and 5 male 2.0mg/kg·bw CdCl₂ group.The exposure group received CdCl₂ and the control group received saline through subcutaneous injection 5 days per week for 2 weeks,respectively.The dosage of Cd were adjusted for the body weights.After the end of 2 weeks,urine was collected before the time of death.Kidneys were extracted and weighted.The viscera coefficient of kidney were calculated?kidney weight/weight×1000??2?The kidney issue homogenate was made,the MDA was measured by TBA,the SOD was measured by WST-1,the ROS was measured by chemical fluorescence,the GSH-Px was measured by enzymatic reaction,protein was determined with BCA.ELISA assay kits were used to detect kidney function indexes?Kim-1,NAG,ALB?,and urinary creatinine was measured by the ammonia iminohydrolase.?3?The total RNA of kidney issue was extracted,The gene chip technology were employed to screen the differential expression of miRNA,P<0.05 means significant different,FC>2 means up-regulated,FC<0.05 means down-regulated;RT-PCR was used to verify the differential miRNA,2^-??Ct>1 means up-regulation,2^-??Ct<1 means down-regulation;Ten databases including miRwalk,Targetscan were used to predict the target genes.The different target genes of miRNA were subjected to gene ontology?GO?enrichment and KEGG pathways analysis to predict the target pathway.?4?SPSS17.0 was used to analysis the data.All results were presented as means and standard deviations,The data were analyzed by ANOVA and nonparametric tests.LSD-t was performed to determine significant differences among the groups.Pearson was used to analyze the correlation.P<0.05 was considered statistically significant.Results:?1?There was significant difference in the viscera coefficient of kidney among the four groups?P=0.001?,the coefficient of 2.0 mg/kg·bw group were significantly higher than other groups.uNAG,uALB,Kim-1 did not vary among these groups?P>0.05?,the uNAG,uALB and Kim-1 of the 2.0 mg/kg·bw group are significantly higher than the control group.It suggested that the 2.0 mg/kg·bw group kidney was damaged.?2?MDA,a known lipid-peroxidation related marker of oxidative stress,was significantly decrease in 2.0 mg/kg·bw group,compared to the control group?P=0.022?.In addition,no difference was found in ROS,GSH-Px and SOD.the correlation between SOD and renal dysfunction was found,It suggested that there is a little relationship between oxidative stress and kidney.Whether the oxidative stress indicators can be used as early biomarkers of kidney injury or not required further research.?3?The miRNA chip screening results showed that compared with control group,three differential expressed miRNAs were found in the 2.0 mg/kg·bw group?miR-21?FC=2.411,P<0.001?,miR-29b?FC=3.415,P<0.001?,miR-29c?FC=2.228,P<0.001??,two differential expressed miRNAs?miR-29b?FC=2.904,P<0.001?and miR-451?FC=2.551,P<0.001??in the 1.0 mg/kg·bw group as well.But no differential expression of miRNAs was found in the 0.5 mg/kg·bw group compared to control group.Only one differential expressed miRNA was revealed in the 2.0 mg/kg·bw group compared to the 1.0 mg/kg·bw group,that is miR-206?FC=0.184,P<0.001?.The RT-PCR results showed that the expression of miR-21,miR-29 and miR-29c was elevated,the miR-451 and miR-206 have no obvious difference.?4?The miRNA bioinformatics analysis results suggested miRNA regulating target genes mainly occurred in biological process,such as DNA transcription,amino acid protein and phosphorylation;mainly occurred in the molecule functions,such as oxidative stress and combination of transcription factor binding molecules;existed in cell components,such as the nucleus,cell membrane,cytoplasm,plasma membrane,and other cellular components;mainly involved in MAPK signaling pathways,TGF-beta signaling pathways,the relevant miRNA control other pathways needs further validation.Conclusions:The kidney of 2.0 mg/kg·bw group has a significantly damage after two-week cadmium exposure.And the balance between oxidation and anti-oxidation was destroyed in the 2.0 mg/kg·bw group.The expressions of miR-21,miR-29b and miR-29c were significantly up-regulated in the damaged renal induced by cadmium exposure.And the bioinformatics results implied that these miRNA will participate in the MAPK and TGF-beta signal pathway.