The Study On The Regulation Of MiRNA And Its Interactive Proteins Of GSTP1 In The Liver Of The White Spotted Bamboo Shark

The liver is well known for its powerful regenerative capability. The mechanism of liver regeneration has been the focus in related research areas for many years. A number of experimental models and techniques have been used to study the mechanism of liver regeneration at the level of organic, cellular and molecular basis in mice, rats, baboons, dogs and humans in the past two decades. However, little is known so far about shark liver regeneration. The white spotted bamboo shark（Chiloscyllium plagiosum） is a kind of small shark that is widely distributed in the South China Sea and can be collected throughout the year. Meanwhile, it possesses an adaptive immune system. These advantages made white spotted bamboo shark a perfect material for us to reveal the mechanism of liver regeneration.In our study, 359 differentially expressed unigenes（DEGs） was obtained by screening between normal liver transcriptome and the damaged hepatic transcriptome: 217 up-regulated and 142 down-regulated. Then,the putative functions related to liver regeneration of unigenes in all liver libraries were analyzed and screened by GO Enrichment and clustering analysis. Finally, 13 DEGs having different expression patterns were selected for qRT-PCR analysis: 10 up-regulated（slc22a16, Slc39a4,Slc1a2, NR5A2, hoga1, ESR2, DIO1, Atp2a2, comp51558_c0, comp43528_c0） and 3 down-regulated（GSTP1,comp44386_c0, comp60069_c3）, and GSTP1 unigene was the most down-regulated one.In order to study the miRNA regulation of GSTP1,small RNA library was screened by miRanda and Target Scan, and 3 target miRNAs was obtained: chp-let-7a、chp-miR-143-3p R+1₁ss21CA and chp-miR-143. Finally, we demostrated GSTP1 unigene interact with chp-miR-143 by dual luciferase report gene assay system.In our study, we first constructed the prokaryotic expression plasmid of pET-28a（+）-Shark GSTP1 and then the recombinant protein was abundantly expressed in the bacteria induced by IPTG. Moreover, the purified recombinant protein was utilized to immunize rabbit to produce polyclonal antibodies. The antibody titer of the purified recombinant protein measured by directed ELISA was 1:51200. In order to reduce the influence of impurities in serum, we purify serum by protein A agrose. Afterwards, we first identify the existence of GSTP1 protein in the liver of Chiloscyllium plagiosum by Western Blotting and MALDI-TOF MS. At last, we successfully detected GSTP1 and its interacting protein through Co-IP with purified serum in the shark liver,which laid a foundation for identification of the proteins interacted with GSTP1.