Characteristic Analysis Of Long Non-coding RNA Serpina3h And Study Of Its Relationship With SerpinA3 Sub-family Genes

Serine protease inhibitor(Serpin)is the largest protease inhibitor super family so far,and subtribe SerpinA3,which serves as the important branch of Serpin,participates in multiple physiological and pathological processes.Now,there is still less information about Serpina3h,the only long non-coding RNA which belongs to subtribe SerpinA3,has a good potential,although it has received attention.To improve the characterization of Serpina3h and preliminarily explore the function,this study was performed on mouse endometrial stromal cells(ESCs),and adopted the method comprised bioinformatics and technologies of rapid amplification of cDNA ends(RACE),RNA fluorescence in situ hybridization(FISH),quantitative real-time PCR(qPCR),flow cytometry and so on.The main results were as follows:1.We obtained 5'-and 3'-cDNA ends of Serpina3h by RACE assay,and the whole length was 2072 bp by splicing sequence.Bioinformatics analysis showed that Serpina3h was a pseudogene,and an intergenic lncRNA as well,which had 31 ORFs included,high coding potential and high conservative property,and KEGG predicted that Serpina3h might be involved in the K04525 pathway.2.Serpina3h was enriched in cytoplasmic fraction through the experiment of RNA-FISH.Through transfection of constructed pcDNA3.1-Serpina3h in mouse ESCs,we discovered that Serpina3h could increase relative expression of Serpina3g(P<0.05),Serpin3c(P<0.001),Serpin3m(P<0.001)and Serpin3n(P<0.01).Compared to control group,the detailed ration of relative expression(experimental group value:control group value)was 7.917±0.442(Serpina3h),2.077±0.880(Serpina3g),64.756±0.075(Serpina3c),91.205±0.823(Serpina3m)and 72.028±2.386(Serpina3n)respectively.3.Apoptosis rate of experimental group and control group was detected by flow cytometry.Meanwhile,transcriptional level of apoptosis-related genes Caspase-3,Caspase-9,P53,Bcl-2,Bax and c-myc was detected by qPCR between experimental group and control group.Results showed that Serpina3h could not significantly affect the apoptosis rate of mouse ESCs(P>0.05).Serpina3h had no significant influence on relative expression of Caspase-3,Caspase-9,Bax and c-myc(P>0.05).The comparison of control group and experimental group showed P53 was significantly increased(P<0.05)and Bcl-2 was significantly decreased(P<0.01)in transcription level in experimental group,and the ration of Bax/Bcl-2(P<0.0001)was increased as well.The conclusions can be acquired according to the above experimental results:(1)Serpina3h is concentrated in the cytoplasmic with polyA,and the full-length is 2072 bp.(2)Serpina3h can increase expression level of Serpina3g,Serpina3c,Serpina3m and Serpina3n,moreover,it can efficient increase the transcription frequency of Serpina3c,Serpina3m and Serpina3n like an enhancer.(3)Serpina3h is associated with some apoptosis-related genes,it can significantly increase expression level of P53 and ration of Bax/Bcl-2,decreases expression level of Bcl-2.This research completes and improves the sequence of Serpina3h,and primarily analyzes its function to provide some help for further study.Next we will conduct the experiment of Serpina3h pull down to hunt for some protein or other substance which combined to Serpina3h for further studying the biological functions.