Construction of Validating Vectors for Investigation of MSI2-miRNA Direct Interactions

Background and Aim: MSI2 is one of the members of Musashi family,considered as key regulators in the maintenance and self-renewal of stemcells. MSI2 higher expression level is associated with poor prognosis ofleukemia. It has been shown that myeloid leukemias are very diversediseases that are associated with expression aberrations of somemicroRNAs. According to previous studies, knocking down of MSI2sensitizes cancer cells to treatments. Therefore, MSI2 can be consideredas a promising target for cancer treatment. In this study, several targetingmiRNAs for MSI2 were predicted by bioinformatic studies.Methods: Prediction of MSI2-targeting miRNAs was performed by severalonline bioinformatic software such as TargetScan, miRWalk, PicTar, andmiRDB. 3’UTR of MSI2 (5.1 kb) and precursors of predicted miRNAswere amplified by specific primers. Amplified fragments were subclonedinto pTZ57R/T vector. After sequencing analysis, 3’UTR of MSI2 andmiRNA precursors were subcloned into psiCHECK-2 and pBud-EGFP,respectively. Also, the 3’UTR fragments containing mutations in miRNAbindingsites for each miRNA were amplified using SOEing PCR andsubcloned into psiCHECK-2 vector separately. Last constructs are beingused as the negative controls. The accuracy of constructed vectors wasconfirmed with restriction digestion.Results: According to bioinformatic studies, the highly conserved miRNAswere confirmed. The miRNAs were selected according to the results ofdifferent software as the candidates for further studies. Precursor structureof each selected miRNA was subcloned successfully in the pBud-EGFPvector. Also, the 3’UTR of MSI2 gene was subcloned into psiCHECK-2downstream of Renilla luciferase in order to apply luciferase reporterassay. These constructions will be used to explore the direct interactionsbetween selected miRNAs and 3’UTR of MSI2 in HEK293T cells.Conclusion: Some of the predicted miRNAs were previously reportedas the tumor suppressors in hematopoietic malignancies. Based onbioinformatic data, we predict that the selected miRNAs have thepotential to directly target MSI2 gene. For verifying this capability, theaforementioned vectors should be employed to transfect both HEK293Tand hematopoietic cells.

کلیدواژه ها:

MSI2 ، miRNA ، Myeloid leukemia