Study On The Target Gene Prediction Of MiR-30a-5p And Its Regulation Of Autophagy

Purpose: To predict the target genes of miR-30a-5p and further analyze the autophagy regulation function of miR-30a-5p and its target genes CAMKK2 in the endoplasmic reticulum stress(ER stress)Ca2+ channel.Methods: There were using Target Scan,miRDB,miRWalk,and other bioinformatics software to predict the target genes of miR-30a-5p.Using thapsigargin-induced Cellular ER stress and the expression level of the ER stress chaperone protein GRP78 was detected by Western blotting to verify the production of ER stress.Fluorescence quantitative PCR technology was used to detect the expression levels of miR-30a-5p and CAMKK2 genes under ER stress conditions.Fluorescence microscope and TEM transmission electron microscope were used to detect the process of autophagy after co-transfection with miR inhibitor + CAMKK2 activator.Results:(1)CAMKK2,the target gene of miR-30a-5p,was predicted and screened through bioinformatics analysis.And the protein function analysis and KEGG signal pathway analysis showed that CAMKK2 played a role in autophagy and interacted with the ER stress Ca2+channel.(2)Using 210 n M thapsigargin induces Cellular ER stress and using Western blotting to identify the expression level of GRP78,which is ER stress chaperone protein—confirming whether the production of ER stress.Under ER stress conditions,the expression levels of miR-30a-5p and CAMKK2 changed significantly(P <0.01).(3)Fluorescence quantitative PCR results showed that miR-30a-5p was significantly down-regulated after transfection with miR inhibitor,and CAMKK2 expression was significantly up-regulated after transfection with CAMKK2 activator(P <0.01).After cell transfection,the results of fluorescence microscopy showed that autophagosomes was the most significant in miR-30a-5p inhibitor group.TEM transmission electron microscope monitored that there formed cup-shaped membrane structure and autophagosomes in miR inhibitor +CAMKK2 activator group.miR and CAMKK2 were involved in autophagy regulation.Conclusion: CAMKK2,the target gene of miR-30a-5p,is screened.This gene participates in the regulation of the autophagy process through the ER stress Ca2+ channel.In the co-transfected with miR inhibitor + CAMKK2 activator group,the level of autophagy was very significant,and it also showed the production of autophagosomes.The results showed that miR-30a-5p and CAMKK2 were involved in autophagy regulation under ER stress conditions.