| Rainbow trout(Oncorhynchus mykiss)is considered as a typical cold-water fish,its optiomal growth condition is 12-18℃,When the water temperature exceeds 25℃,rainbow trout will gradually die.Therefore,high temperature has become one of the biggest threat to rainbow trout production and death in summer in most areas of China.In recent years,the regulation of target gene m RNA mediated by mi RNA may be the main strategy to respond to heat stimulation and adaptability,so mi RNA plays an important regulatory role in the process of growth,development and stress of the body,which has become a hot spot.Based on the previous transcriptome data,this study found that mi R-8159-x differentially expressed between heat and non-heat stress,speculated that mi R-8159-x may play a regulatory role in the heat stress of rainbow trout,but the mechanism remains unclear.To explore the regulatory mechanism of mi RNA involved in heat stress of rainbow trout,primary hepatocytes of rainbow trout was successfully constructed.Besides,we constructed recombinant vectors of wild type(hsp90a1 WT)and mutants type(hsp90a1 mut),and mi R-8159-x mimics and inhibitors were also succeeded in synthesizing.Dual-luciferase reporter gene system was used to verify targeting relationship,RT-q PCR,Ed U labeling and flow cytometry had verified that mi R-8159-x played a regulatory role in rainbow trout primary hepatocyte,and the molecular mechanism of mi R-8159-x involved in heat stress(24℃)was verified by RTq PCR and Western blot.The specific research content is as follows:1.Rainbow trout liver as material,hepatocytes were separated by trypsin digestion and cultured for primary hepatocyte.The cells grew in good condition and had passed to more than 20 generations.The optimal medium of rainbow trout hepatocyte was DMEM medium;the optimal growth temperature was 18℃;the optimal FBS was 15%.2.The target relationship was predicted by mi Randa and our laboratory’s transcriptome data,the potential target gene hsp90a1 was screened.The wild-type and mutant dual-luciferase vectors were constructed to detect the relationship between mi RNA and targets.These results of dual luciferase report showed that: in the experiment of transfection wild-type recombinant vector plasmid,after transfection into HEK 293 T cell for 48 h,compared with the mimics NC group,the mi R-8159-x mimics group down-regulated luciferase activity and the difference was extremely significant(**P<0.01);But In the experiment of transfection mutant recombinant vector,compared with the mimics NC group,the mi R-8159-x mimics group had no significant difference.These results indicated that mi R-8159-x had a targeting relationship with hsp90a1.3.Transfected mi R-8159-x mimics,mi R-8159-x mimics NC,mi R-8159-x inhibitor,and mi R-8159-x inhibitor NC into rainbow trout hepatocyte.RT-q PCR detection of hsp90a1 gene expression showed that: compared with mimics NC,mi R-8159-x mimics group was significantly reduced by 48.05%(P<0.05),but the mi R-8159-x inhibitor group expression level of hsp90a1 increased by 1.17 times(P>0.05).Ed U detected cell proliferation.The results showed that the number of positive cells proliferating was weaker after mi R-8159-x overexpression,and the positive cell signals proliferating was also weaker after mi R-8159-x was inhibited.The above results indicated that mi R-8159-x had almost no effect on cell proliferation.Flow cytometry detected cell apoptosis.The results showed that the apoptotic rate of rainbow trout hepatocytes increased from 9.75%to 12.24%(P<0.05)when mi R-8159-x was overexpressed,but decreased from 9.75% to7.42%(P<0.05)when mi R-8159-x was inhibited,these results indicated that mi R-8159-x promoted the apoptosis of rainbow trout hepatocyte.The above studies showed that mi R-8159-x had no effect on the proliferation of rainbow trout liver cells,but promoted cell apoptosis.4.In order to further understand the regulation mechanism of mi R-8159-x respond to heat stress,the hepatocytes were heat-treated(24℃).After 4 h,8 h,12 h,24 h and 48 h acute heat treatment,detected the relative hsp90a1 m RNA in the hepatocyte.After 4 h of heat stress,compared with the control group,the m RNA level of hsp90a1 in the mi R-8159-x mimics group was significantly decreased by 20.2%(P<0.05),and the m RNA level of hsp90a1 in the mi R-8159-x inhibitior group was increased by 1.31 times(P>0.05).These results suggested that mi R-8159-x had a negative regulation effect on hsp90a1 gene under heat stress.Western Blot results showed that:After 12 h of heat stress,compared with the control group,mi R-8159-x mimics gropup Hsp90a1 protein level was significantly decreased by 36.5%(P<0.05),and inhibitor group level was increased by1.08 times(P>0.05).These results indicated that mi R-8159-x also had negative regulation on Hsp90a1 protein expression,but lagged behind gene expression.This study showed that mi R-8159-x is involved in the regulation mechanism of rainbow trout heat stress through negative regulation of hsp90a1. |
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