P-25: Developing a Simple Method for Amplification of miR-302s in Bladder Tumor Samples Using Real-Time PCR


Khayatzadeh H *, Monfared H , Rafiee MR , Ziaee SAH , Hashemitabar M , Mowla SJ ,

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Objective: MiR302-367 is a cluster of microRNAs that are exclusively expressed at high levels in embryonic stem (ES) cells. Indeed, miR302-367 cluster may play an essential role in maintaining hESC pluripotency and self-renewal. In addition, promoter of miR302-367 is transcriptionally regulated by the ES cell master regulators, e.g. Oct-3/4, Sox2, and Nanog (OSN). Previous studies indicated that expression of OSN could be detected in tumor samples. Therefore, expression of miR302s as a downstream component of OSN is also conceivable in the cancer cells and has the merit of being studied as a potent tumor marker. Materials and Methods: Generally, amplification and detection of microRNAs by PCR is not straightforward, because of having short length (20-24nt) in one hand, and high similarity of miR302-family members on the other hand, make it difficult to detect individual members of miR302s by using PCR. In the current study, a stretch of A-nucleotides were added to the 3'-end of the extracted RNAs by using poly-A polymerase. cDNA was then synthesized using an oligo-dT primer that was anchored to a tag sequence on its 5'-end. The tag could be used as a reverse primer in the subsequent stages. Additionally, the forward primer was selected such that it could specifically amplify miR302b.Tumor and marginal tissues of bladder samples were collected from Labafinejad Hospital and mir302b was detected by Real-Time PCR in those samples. Results: Specificity of the PCR was examined using a vector containing miR-302a, miR-302c, and miR-302d but not miR-302b. Based on our data, the miR302b-PCR system was specific, at least in the presence of 6×106 copies of the vector in 45 cycles. Confirming the specificity of the primers to miR302b, we used them to compare the expression of miR-302b in tumor vs. non-tumor samples of bladder and various stem and tumor cell lines. Interestingly, the expression of miR302b was detected in some brain and bladder tumor cell lines. Our data revealed high expression of miR-302b in stem cell lines, but low or undetectable level of expression in bladder tumor samples. Conclusion: Our data revealed that the expression of miR-302b is restricted to stem cells and cancer stem cells with very low or undetectable expression in tumor cell lines and tissues. These finding suggest that miR-302b is better correlated with the state of pleuripotency rather that the state of tumorigenesis