A Mechanism Study On MiR-221/222-regulated Activation Of PI3K/AKT/mTOR Signaling Pathway And The Impacts On Multiple Myeloma

Part 1 mi R-221/222 play a regulatory role of PI3K/AKT/m TOR signaling pathways via targeting PTENObjective: To investigate the expression levels of mi R-221/222 in multiple myeloma(MM) cell lines. To evaluate the affections of mi R-221/222 on PI3K/AKT/m TOR signaling pathways via targeting PTEN.Methods: q RT-PCR analysis was used to analysis the levels of mi R-221/222 in MM NCI-H929?U266?OPM-2?MM.1S?MM.1R?RPMI8226 and ARH-77 cells.mi R-221/222 mimics was designed and synthesized for over-expression of mi R-221/222 and transfected into MM.1S cells by ribo FECT CP Reagent; while mi R221/222 inhibitor was designed and synthesized to silence endogenous mi R-221/222 and transfected into MM.1R and OPM-2 cells by ribo FECT CP Reagent.MM cells were allocated into 3 groups: blank group(non-tranfected group), negative control transfected group and mi R-221/222 mimics/inhibitor –transfected group. Themi R-221/222 level of the MM cells was assessed with q RT-PCR. The activity of PI3 K at cell level could be measured based on the amount of NADH by GENMED PI3 K Assay Kit. Western blot was performed to evaluate the expression of the total AKT and m TOR and the phosphorylation of AKT and m TOR.Results: High expression of mi R-221/222 level was found in MM OPM-2 cells, while the mi R-221/222 were detected in neither the RPMI-8226 cells nor the MM.1S cells.After transfected with mi R-221/222 mimics in MM.1S cells, we found a significant modulation of pathways such as the activity of PI3 K, apoptosis, cell cycle, Akt/m TOR signaling pathways, which may reveal the mechanism by which mi R-221/222 regulate MM cells growth. We next observed that the growth was significantly reduced in mi R-221/222-transfected MM.1R and OPM-2 cells compared with controls at both 48 and 96 hours, accompany with the low expression of p-Akt and p-m TOR without the change of the total Akt or m TOR.Conclusion: Abnormal expression of mi R-221/222 could target on the PTEN gene to increase the activity of PI3 K, thereby trigger the phosphorylation of the downstream AKT and m TOR. The antagonism of the abnormal expression mi R-221/222, on the other hand, would reduce the level of phosphorylation of PI3K/AKT/m TOR signaling pathways.Part 2 mi R-221/222 regulate the PI3K/AKT/m TOR signaling pathways via targeting PTEN, which affect the apoptosis and cell cycle of multiple myeloma cellsObjective: Use express/antagonism experiment in the different MM cell lines( MM.1R and MM.1S) to detect the influence of mi R-221/222 on the apoptosis and cell cycle of MM cells, and explore the underlying mechanism.Methods: mi R-221/222 mimics was designed and synthesized for over-expression of mi R-221/222 and transfected into MM.1S cells by ribo FECT CP Reagent; while mi R221/222 inhibitor was designed and synthesized to silence endogenous mi R-221/222 and transfected into MM.1R cells by ribo FECT CP Reagent. The MM cells were allocated into 3 groups: blank group(non-tranfected group), negative control transfected group and mi R-221/222 mimics/inhibitor –transfected group.Adding the Dexamethasone, Bortezomib and Rapamycin(the inhibitor of m TOR) to the 3 groups 24 hours after the transfection of mi R-221/222 mimics/inhibitor. The mi R-221/222 level of the MM cells was assessed with q RT-PCR. The activity of PI3 K at cell level could be measured based on the amount of NADH by GENMED PI3 K Assay Kit. Western blot was performed to evaluate the expression of the total AKT and m TOR and the phosphorylation of AKT and m TOR. CCK-8 assay was performed to evaluate the effect of mi R-221/222 up-regulating on the proliferation of MM cells.Flow cytometry was used to measure the effects of mi R-221/222 up-regulating on the cell cycle and apoptosis of MM cells.Results: After transfected with miR-221/222 mimics in MM.1S cells, we found a significant modulation of pathways such as the activity of PI3 K, apoptosis, cell cycle,AKT/m TOR signaling pathways, which may reveal the mechanism by which mi R-221/222 regulate MM cells growth. We next observed that the growth was significantly reduced in mi R-221/222 inhibitor-transfected MM.1R and OPM-2 cells compared with controls at both 48 and 96 hours, accompany with the low expression of p-AKT and p-m TOR without the change of the total AKT or m TOR. By Annexin V-FITC/PI flow cytometry analysis, we observed that mi R-221/222 inhibition could trigger apoptosis and improve the 48 and 96 hours inhibition rate and apoptosis rate by drug(Rapamycin). Moreover, mi R-221/222 inhibition induced S cell cycle arrest,which was associated with the activity of PI3 K, as well as the level of phosphorylated-Akt and phosphorylated-m TOR. Finally, we found that mi R-221/222 inhibitor and Rapamycin could effectively repress tumor growth synergistically.Conclusion: Silencing miR-221/222 in MM cells or the drug(Rapamycin) could induce deregulating of cell cycle regulators and proliferation rate. What's more, the mi R-221/222 inhibitor and Rapamycin could effectively repress tumor growth synergistically. Silencing mi R-221/222 in MM cells could reduce the IC50 of Rapamycin, thus effectively avoid the drug related side effects.