Study on the Function of Circular RNA – Over Expression Vector

Circular RNA is a kind of RNA molecule with closed circular structure. As early as twentieth Century, there were related reports. But because of its low abundance, it was difficult to many more reports in the literature, and it had been considered a rare RNA transcription error to be neglected.

Until the beginning of 2012, researchers began to identify circRNAs from bacterial, nematode, mouse and human cells, revealing that circRNAs exist in eukaryotic transcriptome largely, which was a common phenomenon of gene overexpression, and might a play important role in biological science.

Further studies show that circRNAs are highly conserved in different species. Most of the linear RNA circRNAs overexpression cell level is equivalent to that of the corresponding RNA. For others, the expression level of circRNAs is more than 10 times that of the linear RNA expression, and the expression of the protein is specific. CircRNAs has a closed ring structure, which is not easy to be degraded by exonuclease, and is more stable than linear RNA.

Studies have shown that circRNAs play an important role in brain development, Parkinson, Alzheimer’s disease and carcinogenesis.

In recent years, it has been proved that the main function of circRNAs is:

1. As ceRNA or miRNA sponge;

2. Direct regulation of other RNA expression by base pairing;

3. Protein binding;

4. Protein synthesis as a template for translation;

5. Regulation of host transcriptional activity by interaction with polymerase II.

For the functional overexpression study of circRNAs, researchers constructed the expression vector of the CMV promoter based on the flanking sequence features of circRNAs, PCR amplified target circRNAs Alu sequence or 200-1000bp sequence and circRNAs sequence. The amplification step is complex; the construction cycle is long; the expression times are limited.

The circRNAs expression vector was used to construct the over expression vector of more than 100 target circRNAs. After transfected cell was finished, circRNAs molecules could be formed by RNA, and was verified in HEK293, SH-SY5Y, Hela, Ej, T24, HepG2 and MHCC-97H cell lines.

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