Role And Molecular Mechanism Of MiR-22 In Response To Pingyangmycin In Tongue Cancer

Background and Aim:Chemotherapy plays very important role in Tongue cancer's comprehensive treatment; however, the effectiveness of chemotherapy can be attenuated because of intrinsic and/or acquired drug resistance, but the mechanism remains unclear.miRNA, which can negatively regulates gene expression in the post-transcriptional level, has been widely proved to play crucial roles not only in tumor development and progression, but also in drug-resistance. However, pingyangmycin drug resistance mediated by miRNA in tongue cancer has not been reported right now.Based on the differentially expression profile of miRNA in this study, we will explore miRNA's regulatory role in tongue cancer, and identify a new class of drug resistance-related biomarker, with the aim of providing theoretical evidence for cancer therapy, which is useful to reverse chemoresistance and improve the therapeutic effect.METHODS:microRNA expression profiles between drug-resistant cell line Tca8113/PYM and its parental cell line Tca8113 were detected by miRNA microarray for screening drug-resistant-related miRNAs. The results obtained by microarray profiling were validated using real-time quantitative PCR analysis. (2) The eukaryotic expression vector of miR-22 were transfected into Tca8113 and Tca8113/PYM cells using liposome to establish stable cell lines, and then anti-has-miR-22 was used to down-regulate its expression; the cytotoxicity effect of pingyangmycin on these transfected cells were determined by MTS assay; clone formation experiment was used to test the clonality of the overexpr -essiong cells. (3)Bioinformatic approaches have been used to predict mRNA targets of miR-22, then determine the candidates by combining their biological function and the results of gene chips. RT-PCR and Western blot were used to detect the changes of MYST4 after tansfecting miR-22 and anti-mir-22. The sequence containing putative binding sites for miR-22,some flanking sequences and Hindâ…¢and Speâ… enzyme site were inserted into the pMIR-REPORT miRNAExpression Reporter vector. Report construction containing 3'UTR binding sites of target gene MYST4 or not, Beta-gal Reporter Control Vector and miR-22 were Co-transfected into 293 T cells, and the change of luciferase activity was assayed after 24 h. MYST4 inhibitor was transfected into Tca8113 cells, Cell proliferation was assayed by MTS method. (4) Western blot was used to detect the changes of PI3K/AKT pathway related proteins beween Tca8113 and Tca8113/PYM cells; Tca8113/PYM cells were interfered with LY294004, and the expression of miR-22 were determined by real-time quantitative PCR analysis. MTS was used to detect the cytotoxicity effect of pingyangmycin on those cells. Western blot was used to detect the change of PI3K/Akt pathway related protein with miR-22 and MYST4 changed.(5) Real-time quantitative PCR was used to detect the expression of miR-22 when Tca8113 cells exposured to PYM for Oh,24h,36h. Western blot was used to detect the expression of PI3K/Akt.Results:There are 65 miRNAs differentially expressed (>2 fold) in multidrug-resistant tongue cancer cell line Tca8113/PYM and its parental cell line Tca8113. Among them,41 miRNAs were up-regulated and 24 were down-regulated. We confirmed the expression of two down -regulated miRNAs (miR-181b and miR-491-3p) and four up-regulated miRNAs(miR-22, miR-203, miR-221 and miR-21) by real-time quantitative PCR. The results were consistent with results from miRNA microarray analysis.2. Overexpression of miR-22 could increase the sensitivity of Tca8113 cells to PYM and inhibited its clonogenicity in vitro, without significant affects on Tca8113/PYM cells. Conversely, inhibiting miR-22 enabled both Tca8113 and Tca8113/PYM cells more resistant to PYM.(3) MYST4 might be the candidate target gene of miR-22 by bioinformatic prediction. RT-PCR and Western blot were used to detect the changes of MYST4 mRNA and protein when miR-22 was transfected into Tca8113 and Tca8113/PYM or when miR-22 inhibitor was transfected into Tca8113/PYM. MYST4 mRNA and protein were increased in stable transfected Tca8113 cells, while decreased in transient interference cells. (3) We found miR-22 directly modulated MYST4 expression in both mRNA and protein levels, and MYST4 silencing with MYST4 antisense oligonucleotides contributed to chemosensitivity to PYM in Tca8113 cells. (4) Moreover, PI3K/Akt pathway was more active in Tca8113/PYM cells and PI3K inhibitor LY294004 markedly downregulated miR-22 dose-dependently, and PI3K/Akt pathway inhibition and miR-22 overexpression cooperated to enhance the sensitivity to PYM.Conclusion:(1) drug resistance-related miRNA expression profile was first constructed. (2)miR-22 can lead to cleavage of MYST4 mRNA, and increase sensitivity of tongue cancer to PYM. (3) Our study suggested a negative feedback between PI3K/Akt pathway and miR-22 that maybe mediated by MYST4. PI3K/Akt pathway inhibition and miR-22 overexpression cooperated to increase the anticancer effect of PYM. (4) miR22 and PI3K/Akt both participate in the response of primary tongue cancer to PYM. PYM can affect the expression of miR-22 independent of PI3K/Akt pathway in primary tongue cancer, and PI3K/Akt's activity does not depend on miR-22/MYST4 pathway in drug-resistant cells.