Effects Of LncRNA-GTL2 On C2C12 Proliferation And Research On Its Mechanism

The growth of skeletal muscle is a trait of primary importance for domestic animal agriculture. Identification of the causative genes and their biological function in traits altering normal growth has important guiding significance on livestock production. Previous studies have found some important major dominant gene that regulates muscle growth and development, such as myogenesis regulating factor, the pair-box gene family and MSTN. In addition to these important encoding genes, skeletal muscle growth and development was regulated by noncoding RNAs. Though noncoding RNA cannot as main effect genes directly involved in skeletal muscle growth, such RNA through its special regulatory mechanism well illustrates on the premise of the same genotype muscle growth will appear bigger difference phenomenon. Existing research reported on muscle growth regulation put emphasis on micro RNA. But in recent years, with the progress of technology, people gradually revealed another kind of non-coding RNA, once considered transcriptional noise RNA-long non-coding RNA(lnc RNA). Micro RNA constitute only a small part of the non-coding RNAs, while the lnc RNA is the largest subfamily. In-deep study found that lnc RNA plays an important role in regulation lots of life activities, it has become the one of the most popular research fields of molecular biology. Therefore, its particularly important to find and reveal the lnc RNA role in understanding the mechanism of skeletal muscle growth and development.Anterior articles reported linc-MD1, lnc RNA-H19 isometric long noncoding RNA can regulate muscle growth and development, but there are still a lot of muscle specific lnc RNA has not been found. Our team had screen a muscle specific long noncoding RNA- lnc RNA- GTL2 by gene chip. And through the muscle damage model experiment preliminary confirmed that lnc RNA-GTL2 was involved in muscle growth and development process. But about the specific biological function of the gene and its function mechanism has yet to do in-depth research. In This study we test it’s effects on C2C12 cells proliferation through GTL2 expression and si RNA transfection experiment. The results showed that the lnc RNA-GTL2 can significantly inhibit the proliferation of C2C12 cells. This was in compliance with prior research in other cells. Transcription regulation is the most important and complex link in eukaryotic Gene expression progress. As an important cis element, Promoter is critical in gene expression. Promoter regions is recognized by RNA polymerase in transcriptional activation process, it control of gene expression(transcription) start time and expression level. In order to further clarify lnc RNA-GTL2 C2C12 cell proliferation inhibition mechanism, a 2074 bp upstream sequence of GTL2 was cloned, and connected to the PGL3 carrier, through transfection PK15, PIEC, C2C12 and so on three different cell lines to detect the promoter activity. Experimental results show that the sequence and positive control group have similar strong promoter activity. With recombinant plasmid as the template again cloned 6 different sizes of the promoter sequences, and connect to PGL3 carrier, then transfected C2C12 cells, through dual luciferase activity changes, concluded the core promoter regions. The experimental results show that the core promoter of GTL2 gene located in 200-300 bp upstream of the transcriptional start site.Given the complexity and diversity of lnc RNA function mechanism, we have explored the mi RNA and GTL2 interactions. Firstly, we carried out joint analysis using lnc RNA and mi RNA chip datas, the result showed a series of mi RNA that may interact with GTL2. The cell transfection and fluorescence quantitative experiment showed that mi R- 1, mi R- 22, mi R- 133- a and mi R- 29 may interact with GTL2, in the form of "sponge". This study reveals the GTL2 biological function that inhibit proliferation of C2C12 cells, and explore the mechanism. All in all these results provide theory basis for further research on the important role of lnc RNA-GTL2 in the muscle growth.