Engineering An OSBPL2 Gene Knock-out HeLa Strain By CRISPR/Cas9 Technology And Primary Functional Study In The Cell Lines

Oxysterol binding protein related protein family(ORPs)are lipid binding proteins that are characterized by their oxysterol binding domain.They are implicated in many cellular processes including signaling,vesicular trafficking,lipid metabolism and nonvesicular sterol transport and so on.OSBPL2(Oxysterol binding protein like 2)is a member of the protein family.Previous study showed that OSBPL2 participate in cholesterol transport and lipid metabolism.However,it is still unclear how OSBPL2 participate in these functions.Our laboratory showed that OSBPL2 is a novel candidate causitive gene for progressive nonsyndromic hearing loss and we continued to explore the related functions with the successfully constructed cell lines.To father study the pathogenic molecular mechanism of OSBPL2,we used the CRISPR/Cas9 technology to effectively build the OSBPL2 knockout HeLa cell line.In our observation,in transcription and translation level,Bcl2/Bax ratio rose significantly within the OSBPL2 gene knock out HeLa cell,which showed antiapoptotic characteristic of the cell line and indicated OSBPL2's function in apoptosis regulation process.It is known that oxysterol binding protein(OSBP),another memer of ORPs family,participate in the regulation of ERK signaling pathway,and also ERK signaling pathway is significant for hearing loss in animal experiments,therefore we tried to explore the possibility of ERK signaling pathway's association with the features of OSBPL2 gene involved in regulation of apoptosis.In this case,we used ERK signaling pathway inhibitor U0126 to treat wild type HeLa cell and also OSBPL2 gene knock out HeLa cell.We found that the expression of Bax and Bc12 had no obvious change in translation level.Compared to the expression of p-ERK in wild type HeLa cell,the expression of p-ERK rose in OSBPL2 gene knock out HeLa cell.To investigate the function of ERK signaling pathway in this cell,there still need more research to support.Summarize the above experiments,we concluded that the CRISPR/Cas9 technology could effectively build OSBPL2 knock out cell lines,the cell lines have the feature of anti-apoptosis and OSBPL2 gene involved in cell apoptosis regulation,which provides some theoretical basis for further study on the molecular mechanism of the gene function.