The Effect Of MiR-122 On Goat Placental Trophoblast Cells During Apoptosis Induced By Swainsonine

microRNAs(miRNA) are a class of small non-coding RNAs of 19-23 nucleotides in length that regulate numerous biological processes through binding with 3′UTR of target mRNA at the post-translational level.Swainsonine(SW) is an indolizidine triol that causes loco-disease. SW treatment induced apoptosis in GTC via mitochondria pathway. However, the link between miRNAs and SW induced apoptosis in GTC is still unknown. We find out the suitable time and concentration of SW treatment first,performed microRNA sequencing and analyze the information. We filter the miRNA and choose a remarkable one after get the different expression of miRNA. We analyzed the role of miRNAs in the process of apoptosis induced by SW treatment in GTC. The results were as follows:1. Treated GTC with 0.8 μg/mL、1.6 μg/mL、2.4 μg/mL、3.2 μg/mL SW for 24 h,using MTT method to test cell viability and found decreased GTC cell viability with86.48%、71.69%、45.71% and 29.89%. Using1.6 μg/mLand 2.4 μg/mL SW treated GTC for 24 h then used flow cytometry to test apoptotic rate,found that SW treatment can increase GTC apoptotic rate with 35.46% and 77.9%;Using 2.4 μg/mL SW treated GTC for 0 h、12 h、24 h、36 h and 48 h,found apoptosis begins at 24 h. So we decided treat GTC with 2.4 μg/mL SW for 24 h then ready for microRNA sequencing.2. Used clean reads to analyze the information after microRNA sequencing. The length of miRNA majorly contributed to 22 nt which means the acceptability of sample. According to the different expression(DE)of miRNA after SW treated,we found 25 miRNAs were changed remarkably(fold change>2, p<0.05). Among them 16 miRNAs were up-regulated and 9 miRNAs were down-regulated. To validate the results of differentially expressed miRNAs, we tested the expression levels of 3 up-regulated miRNAs and 4 down-regulated miRNAs using real-time PCR,The results were correlated with sequencing. We choose miR-122 which was related to apoptosis for further research. Used mireap,miranda,targetscan to predict the target genes of DE miRNA and pick up the intersection of three software.The predictions were then applied to analyze the KEGG Orthology using the KEGG database,51 significantly enriched pathways(P-value < 0.05) were picked out.3. according to the database,we choose miR-122 for further investigation to figure out whether miR-122 is involved in the apoptosis induced by SW. Cells were transfected with miR-122 mimics or miR-122 inhibitors and treated with SW,then test apoptotic rate、caspase-3 viability and cell viability. The results showed that over-expression of miR-122 could decrease cell viability,increase apoptotic rate and enhanced the viability of caspase-3. Down-expression of miR-122 could increase cell viability,decreased the apoptotic rate and reduced the viability of caspase-3. We filter the apoptosis related candidate target,then the 3′UTR of candidate target were cloned downstream of the Renilla luciferase gene in the psiCheck-2 plasmid. miR-122 mimics and recombinant plasmid were co-transfected into GTC and measure the luciferase activity. We identified that the miR-122 directly bind to the 3′UTR of Bcl-w mRNA, western blotting detected the protein level of Bcl-w after transfected with miR-122 mimics or mimics control showed Bcl-w decreased remarkably which indicates Bcl-w is a direct target gene of miR-122.This study illustrates SW treatment caused the change of miRNAs profile and miR-122 was changed remarkably. We proved miR-122 could promote SW induced GTC apoptosis and verificated it target gene Bcl-w. This study illustrates the effect of miR-122 on GTC apoptosis induced by SW.The results provide theoretical basis for the pathogenesis of SW.