MiR-103 Contributes To Cadmium-induced Apoptosis And Autophagy By Targeting FoxO In Common Carp Spleens

Cadmium?Cd?is a toxic heavy metal for organisms.Cd pollution is increasing which is caused by human activities.Therefore,Cd pollution has become the focus of attentions and researches.Various forms of Cd pollution converged into the lakes with precipitation and surface runoff.Cd accumulated in aquatic animals and damaged animals,and it eventually affected human health through the food chains.The common carp?Cyprinus carpio L.?is one of the most widely distributed fish in the world and it can be used as a biomarker of environmental pollution.Micro RNA?miRNA?,as a non-encoding RNA,participates post-transcriptional regulation of animal genes.In recent years,researchers found that miRNAs were involved in the regulation of environment pollution poisoning and diseases,which were considered as a potential biomarker of environment pollution.However,there were rarely reports on the mechanisms of miRNA regulating apoptosis and autophagy induced by Cd in common carp spleens Therefore,in this study,vivo and vitro experiments were conducted to investigate the mechanism of apoptosis and autophagy regulated by Cd exposure in carp spleen cells.162 healthy common carp were divided into two groups?the control group and the Cd group?.The Cd content in the water of the Cd group was 0.26 mg/L Cd?1/25 of 96 h LC50?.We collected test samples of the common carp spleens at the 10^th day,20^th day,and 30^th day respectively.We used the methods of microstructural and ultrastructural observation,transcriptome sequencing,and bioinformatics technology and predicted the target relationship between miR-103 and FOXO.Furthermore,we used the methods of TUNEL assay,detecting oxidative stress and inflammation indexes,real time quantitative PCR?RT-q PCR?and western blot?WB?to study the mechanism of apoptosis and autophagy related pathway genes and protein expressions induced by Cd exposure in carp spleen cells.In vitro,to study the mechanism,determining the Cd concentration in experiments of carp epithelial?EPC?cells by CCK-8 test;verifying the targeting relationship between miR-103and FOXO by dual luciferase gene reporter assay;detecting the indexes of oxidative stress and inflammation by Cd exposing EPC cells for 1 h,6 h and 12 h;knockdown and overexpression test of miR-103;AO/EB staining;flow cytometry;MDC/EB staining;RT-q PCR;and western blot were used to study apoptosis and autophagy.The main results were as follows:?1?By observing the Cd poisoning carp in the model,the surface color of carp in the Cd treatment group became lighter than that in the control group,the swimming routes in the water were disordered and irregular,the spirit was depressed,the intake of food decreased,and the growth of carp was significantly inhibited by the Cd exposure.The areas of red pulps were larger than those of white pulps,the boundary between red pulps and white pulps were not obvious,and the color of spleen was even.After 10 days of Cd treatment,the areas of red pulps decreased and white pulp increased in spleens,and lymphocyte aggregation and cellulosic swelling of vascular endothelial cells appeared.In addition to the above pathological changes,bleeding and heme appeared after 20and 30 days of Cd treatment.Moreover,there were vacuoles in the spleens of carp after Cd treatment for 30 days.The results of electron microscopy showed that the nuclei of some cells were irregular and shrunk,the cytoplasm were dense,and the chromatin were surrounded by membrane to form apoptotic bodies.At the same time,some spleen cells were irregular in morphology,and a large number of autophagy and lysosomes appeared in the cells.These results indicated that Cd exposure caused inflammatory injury,apoptosis and autophagy in spleen cells.?2?The spleen tissues of carp were sequenced after Cd exposure for 30 days.miRNA sequencing showed that 23 miRNAs were differentially expressed,of which 17 were significantly up-regulated and 6 were significantly down-regulated.3794 differentially expressed m RNA were found by m RNA sequencing,of which 1848 were significantly up-regulated and 1946 were significantly down-regulated.Through bioinformatics technology,23 miRNAs were predicted to target 2022 differential expressed m RNA,of which 1113 differentially expressed m RNA were down-regulated and 909 differentially expressed m RNA were up-regulated.Through the analysis of KEGG and GO,those were found that miRNA caused by Cd exposure were involved in the regulation of spleen function of carp by miRNA regulatory genes.?3?It was found that FoxO might be the target gene of miR-103 by bioinformatics prediction.Double luciferase reporter gene test confirmed that miR-103 targeted to regulate FOXO.Furthermore,FOXO was confirmed to be the target gene of miR-103 by the expression of FoxO m RNA and protein after knockdown and overexpression of miR-103.?4?After 10,20 and 30 days of Cd exposure in carp and 6 hours and 12 hours of Cd exposure in carp EPC cells,GSH content and CAT,GSH-Px,GST,SOD and T-AOC activity decreased;and H₂O₂,MDA and NO content and i NOS activity increased.The oxidative stress and inflammation were positively correlated with Cd exposure time.RT-q PCR results showed that the m RNA expression of CAT,GSH-Px and HO-1a was significantly down-regulated,and the m RNA expression of CYP26B1,COX-2 and PTGE were significantly up-regulated.It indicated that Cd induced oxidative stress and inflammation.?5?After 10,20 and 30 days of Cd exposure in carp and 6 and 12 hours of Cd exposure in carp EPC cells,RT-q PCR results showed that the m RNA expression of Foxp3,TGF-?,T-bet and IFN–?decreased,while the m RNA expression of GATA3 and IL4/13A increased.It indicated that Cd induced immunosuppression in carp spleens and carp EPC cells.?6?By knocking down the miR-103 of EPC cells and treating the cells with z-VAD-FMK,the results showed that the apoptotic rate of knockdown miR-103 cells increased significantly and could be inhibited by z-VAD-FMK.Overexpression of miR-103 could reduce the apoptosis rate of Cd treated cells.The above results showed that Cd induced the down-regulation of miR-103 expression and weakened the inhibition of FoxO.Furthermore,RT-q PCR and WB results showed that the m RNA and protein expression of apoptosis-related pathway genes?FoxO,Bim,Cyt C,caspase9 and caspase3?were up-regulated,which indicated that miR-103 targeted FoxO to induce mitochondrial oxidative stress and led to EPC cell apoptosis.RT-q PCR and WB results showed that the expression of apoptosis pathway related genes and proteins FoxO,Bim,Cyt C,caspase9 and caspase3 were up-regulated,indicating that Cd could cause apoptosis in carp tissues.?7?By knocking down the miR-103 of EPC cells and treating the cells with autophagy inhibitor3-MA,the results showed that the autophagy rate of knockdown miR-103 cells increased significantly and could be inhibited by autophagy inhibitor 3-MA.Overexpression of miR-103 could reduce the autophagy rate of Cd treated EPC cells.Furthermore,RT-q PCR and WB results showed that the expression of genes and proteins related to autophagy pathway was up-regulated,which indicated that miR-103 targeted FoxO to induce autophagy in carp EPC cells.RT-q PCR and WB results showed that the expression of genes and proteins related to autophagy pathway,FoxO,Beclin1,LC3-I and LC3-II,were up-regulated,indicating that Cd could cause autophagy in carp tissues cells.In conclusion,Cd exposure inhibited the growth of carp;Cd could induce apoptosis and autophagy of carp spleen cells by morphology;differential expression of miRNA and m RNA was found by transcriptome sequencing and bioinformatics analysis showed that miR-103 might target FoxO to regulate apoptosis and autophagy of spleen cells.The relationship between miR-103 and FoxO was confirmed by double luciferase reporter gene test in carp EPC cells.Further experiments showed that Cd exposure of spleen and EPC cells could induce oxidative stress,inflammation,immunity,apoptosis and autophagy.Apoptosis and autophagy pathway related genes and proteins FoxO,Bim,Cyt C,caspase9,caspase3,Beclin1,LC3-I and LC3-II were up-regulated.This study demonstrated that Cd exposure induced apoptosis and autophagy by miR-103/FoxO in carp spleen cells.