Cloning and functional research of microRNA-EC326 in human heart and human umbilical vein endothelial cells

Objectives Micro RNAs are a class of 19-25 nucleotides, non-protein-coding RNAs that base pair with specific mRNAs and inhibit translation or promote mRNA degradation. While hundreds miRNAs have been found, only a few have been studied in detail. We hypothesis that more microRNAs highly or even specifically expressed in cardiovascular system are undiscovered and essential in heart and vascular physiopathologic procedure. Methods To construct microRNA library, total RNAs of human heart tissue and human umbilical vein endothelial cells (HUVECs) were separated on a 15% denatured PAGE gel. 19-25 nt RNAs were recovered and ligated with 5’ and 3’ adaptors respectively, the products were amplified by RT-PCR then T-A cloned into vectors. Rat cardiomyocytes were digested by type II collagenase on a Langendorff apparatus. To detect the profile of cloned miRNAs by Northern Blotting and real time RT-PCR, total RNAs were separated on a 15% denatured PAGE gel and then transferred to a nylon membrane, after cross-link by UV, the membrane was hybrided withγ32P-ATP labeled specific oligo probes then developed under -80℃using X-ray films; total RNAs were reverse transcripted with specific stem-loop primers and then amplified with suitable primers and probes. Bioinformatics was used to locate and compare the sequence of mir-EC326 among several species. To verify the origin of mir-EC326, the intron contains the mir-EC326 along with the nearby exons was cloned in-framely into pEGFPcl. The plasmid was transfected into Hela and allowed to expression for 24 hours and then the transcription products were determined by PCR and Northern Blotting. To find mir-EC326 targets, gene microarray and bioinformatics assay were used. The putative targets were verified by luciferase reporter assay. Results We cloned hundreds of small RNAs sequences from human heart tissue and HUVECs. In heart, mir-1, mir-EC326, let-7b and mir-26a are the most abundant while in HUVECs, mir-26a, mir-EC326, mir-29c and mir-26b clone number are among the highest. Using Northern Blotting, we confirmed mir-1 was specifically expressed in rats cardiomyocytes and skeleton muscles. Mir-26a, let-7b, mir-29c, mir-26a and mir-EC326 are universally expressed in rat tissues. Interestingly, we found mir-EC326 which had not been verified in human before was both highly expressed in human heart and HUVECs. To our surprise, when detected in cell lines, mir-EC326 expression profile was restricted to HUVECs and rats cardiomyocytes but undetectable in human vascular smooth muscle cells and other cell lines by Northern Blotting. Next, we searched the location of mir-EC326 in human genome and found it seats in one of the introns of its host gene which plays important roles in angiogenesis and has the same expression pattern as mir-EC326. Otherwise, we found mir-EC326 precursor and its location are conserved among several mammalians like mouse and rat. Furthermore, we confirmed experimentally that mature mir-EC326 can be fonned after the pre-mRNA of host gene was spliced. Using bioinformatics and gene micorarray, we found VEGF, v-CRK and VCAM1 were the candidate targets gene of mir-EC326 which could repress the expression of a VEGF3’UTR containing reporter by 60% (P<0.05). We had also verified some genes such as TNFAIP3 and CXCLl 1 which may involve in angiogenesis and cell survival were dramatically upregulated in mir-EC326 overexpressed Hela cells. Conclusions We have constructed human heart and HUVECs microRNA library and verified for the first time of a novel miRNA-mir-EC326 which is highly expressed in HUVECs and cardiomyocytes. mir-EC326 precursor is conserved and located in one of the introns of a protein-coding gene and can be spliced out to process a mature miRNA. Finally, we have found that VEGF may be the target of mir-EC326 which on the other hand is able to induce TNFAIP3 and CXCL11. All these results indicate that mir-EC326 is highly expressed in cardiovascular system and may play pivotal roles in angiogenesis via a complicate regulation network.