Preliminary Study Of LncRNA-miRNA-mRNA Interaction

Approximately 85% of the DNA in the human genome can be transcribed into RNA,but only less than 2% of the RNAs could be translated into proteins,and the remainder does not encode protein,which is called “non-coding RNA”.With the intensive researches,it has been found that non-coding RNA can regulate the expression of genes through different channels at different levels,and thus participate in the life processes of cells,tissues and even the whole body.These discoveries changed the concept “all life activities are based on protein”.Non-coding RNAs can be classified into small non-coding RNA,middle size non-coding RNA and long non-coding RNA(lncRNA)according to their length.The length of small non-coding RNA length is less than 50 nt,middle size non-coding RNA length is between 50-200 nt,and the long non-coding RNA length is over 200 nt.Recent studies have shown that there are interactions among RNA molecules,such as LncRNA and mi RNA,miRNA and mRNA,lncRNA and mRNA,and these interactions form a regulatory network of lncRNA-mi RNA-mRNA.Clarifying this complex and sophisticated regulatory network is essential to reveal the mechanism of interaction between RNA molecules and explain its function.Part 1: MiRNA accelerates the “molecular sponge” lncRNA degradationLong noncoding RNA(LncRNA)refers to a class of RNA with length longer more than 200 nucleotides without coding protein.It has been found that lncRNA is widely expressed in mammalian cells,and thousands of lncRNA have been identified.However,only about 100 lncRNA molecules function has been revealed so far and most of their functions and molecular mechanisms remained to be addressed.Compared with m RNA,the expression level of lncRNA is generally lower and the conservation is relatively poor.It is generally believed that lncRNA is mainly transcribed from intergenic regions,the upstream region of the gene,and anti-sense strand of the gene.LncRNAs can be classified into four categories base on its function: guidance molecules,decoy molecules,signal molecules and scaffolds molecules.LncRNA can regulate a number of biological activities of cells,including epigenetic regulation,regulation of gene transcription level,post-transcriptional regulation of genes,formation of sub-structure of cells,chromosome remodeling,cell differentiation even body development and so on.In addition,it is found that the abnormal expression of long noncoding RNA is associated with a number of serious illnesses,including cancer,neurological disorders,diabetes and autoimmune diseases.With the deepening of research,lncRNA was found to be a “molecular sponge” of miRNA,which aroused interests of researcher to study interaction between lncRNA and miRNA.LncRNA “molecular sponge” means that it binds to miRNAs and attenuates the “silencing effect” of miRNAs on the target gene,thereby regulating the expression of mi RNA target genes.In order to study the exact interaction between lncRNA and mi RNA,so we propose three questions: It has been reported that most of miRNAs are relatively stable and have long half-lives,so how about the stability of lncRNA which is miRNA sponge;what is the relative abundance between lncRNA and mRNA and whether it affects lncRNA “molecular sponge” funiction;and whether the lncRNA “molecular sponge” effect is universal.We selected five groups of lncRNAs which had been proved to be miRNA sponge.These lncRNAs can bind with miRNAs competitively to regulate the level of mRNAs which were miRNA target.We selected HEK293 T and HeLa cells as cell models to detect the half-life of several lncRNAs and mRNAs in the cells.We used actinomycin D to inhibit intracellular transcription,and then collected the total RNA of the cells at five time points(0,4,8,12,24h).qRT-PCR was used to detect the expression of lncRNAs and target mRNAs at each time point.Results showed that not all half-lives of lncRNAs were long.Except for lncRNA-UCA1,the rest of lncRNAs half-lives were shorter than 12 h.Then we examined the relative abundance of lncRNAs and mRNAs and found that the relative abundance of some lncRNAs was higher than mRNAs,while that of the other was lower.The half-lives of lncRNAs were much shorter than the control group when overexpression of its sponged miRNA,which suggested that miRNA accelerated lncRNA degradation.Then we constructed the overexpression vector of lncRNA-UCA1,and also found that miR-216 b can accelerate degradation of both of endogenous and exogenous lncRNA-UCA1,Furtherstudy showed that mi R-216 b inhibitors could restrain the process of mi R-216 b accelerated the degradation of lncRNA-UCA1.These results suggested that the role of lncRNA as a “molecular sponge” of miRNAs may be played by competing with mRNA to bind with their complementary miRNA,and then reduce the content of free miRNAs to regulate target mRNA.After lncRNA binding with miRNA,lncRNA also served as a target of miRNAs,that is,miRNAs reduce the stability of lncRNA and promote its degradation.Part 2: LncRNA inhibits decay of miRNA imperfect complementary target mRNAMiRNA(microRNA)refers to a class of single-stranded non-coding RNA molecules about 18-22 nt in length.MiRNAs are highly conserved,and has tissue-specificity,and temporal and spatial specificity.MiRNAs can regulate gene expression at post-transcriptional levels by binding to the 3' untranslated region(3'UTR)of the target mRNA,resulting in target mRNA cleavage or translation inhibition.It is believed that miRNA mainly silences target mRNA through two ways: one is that the miRNA perfect complementarily binds with mRNA,causing Argonaute 2(Ago2)protein-mediated target mRNA cleavage,resulting in rapid degradation of mRNA molecules;another is imperfect complementation between mi RNA and target mRNA,causing target mRNA translation inhibition.MiRNA has been found to participate in a number of biological activities;it has played an important role for cell proliferation,cell differentiation,apoptosis and development of organisms.And miRNA aberrant expression has a closed relationship with and many diseases.LncRNA-GAS5,a natural “molecular sponge” for miR-21,could absorb miR-21 to inhibite effect of mi R-21 to target mRNA.PTEN and TPM1 are imperfect complementary target m RNA for miR-21.We confirmed that mi R-21 was able to regulate PTEN and TPM1 protein expression by incomplete complementation in HEK293 T and HeLa cells,but miR-21 did not affect PTEN and TPM1 mRNA levels.After overexpression of mi R-21,qRT-PCR was used to detect PTEN and TPM1 mRNA half-lives.Results showed that half-lives of these mRNAs were significantly shortened.And miR-21 accelerated the degradation of PTEN and TPM1 mRNA.When transfection of lncRNA-GAS5 overexpressing plasmid,we found that lncRNA-GAS5 could competitively bind with miR-21,which was able to prolong the half-life of PTEN and TPM1 mRNA.These results suggested that lncRNA-GAS5 as a “molecular sponge” of miR-21 can inhibit the degradation of PTEN and TPM1 mRNA,which are imperfect complementary targets of miR-21.The interaction between LncRNA and mRNA can help us understand and illuminate the lncRNA-miRNA-mRNA regulatory network.Part 3: Construction and verification for related expression vectorIn order to investigate the mechanism of whether mRNA can also regulate the stability and abundance of lncRNA,we need to construct the 3' UTR overexpression vector for target mRNA.We constructed eukaryotic overexpression vector for miR-21 target gene TPM1 3' UTR and miR-216 b target gene FGFR1 3' UTR,and then we verified the expression of the these vectors.We also constructed target m RNA PTEN and TPM1 3' UTR luciferase vectors.Our results showed that these overexpression vectors and luciferase vectors were successfully constructed,providing powerful tools for further study of lncRNA-miRNA-mRNA interactions.