Cloning And Expression Analysis Of Porcine MiRNAs And Construction Of A Common MiRNA Vector

MicroRNAs(miRNA) are a kind of small RNAs which are somewhat like siRNAs. They have a length of 20-24nt with a mutation of 1-2nt on the 3’side. MiRNAs play an important role in creatures as depicted, however, there is little report about miRNAs in pig. Our study cloned four miRNAs in pig and identified their expression in ten pig tissues. In order to study the relationship between miRNAs and their targets, we constructed a common miRNA vector and testified its function. Our study got some conclusions below:1. By stem-loop primers, four miRNAs in pig were isolated. They are ssc-miR-181b, ssc-miR-155, ssc-miR-26a-1, ssc-let-7e.2. Using real-time polymerase chain reaction, we identified the expression patterns of the four miRNAs. The results showed that ssc-miR-181b was all expressed in ten tissues with a high expression in lung and muscle. Despite ssc-miR-155 was suggested to widely express in all the tissues, it had a high expression level in spleen. Meanwhile, ssc-miR-26a-1 and ssc-let-7e can not be found in intestine.3. A common miRNA vector was constructed.4. To test if miR-29 negatively regulates PTEN we used PTEN-psi-CHECK-2 vector. The results showed that miR-29 had a significant effect on PTEN with the p value was less than 0.01. We can use them as a positive control for our vector.5. In order to testify the newly recombined vector, we inserted PTEN 3’UTR of mouse into our vector, than transfected miR-29 and vector into BHK cells.24 hours later, numbers of the GFP is deeply decent,130 times less than before in details with ELISA. By observing the green fluorescence and ELISA test, indicating the newly constructed vector can be good reflecting the miRNA and its target gene interactions both from qualitative and quantitative aspect.