MG 63 cells were co transfected with miR 33a or miR Vec control together with either TWIST 3 UTR luciferase reporter or TWIST mut33 luciferase reporter. The reduction of renilla lucifer ase activity caused by miRNA 33a was specifically abol ished by the mutation of the corresponding anti seed sequence, suggesting that miR 33a could suppress TWIST expression by acting on its predicted sequence in the 3 UTR. To confirm the findings, we determined miRNA 33a and TWIST protein levels in chemoresistant OS patients and control patients in the validation cohort. As shown in Figure 4A, the chemoresistant OS group presented a significantly higher range of miR 33a levels than the control group. On the other hand, the chemoresistant OS group presented a significantly lower range of TWIST protein levels than the control group.

Correlation analyses in the entire val idation cohort Promethazine HCl cost showed that the miR 33a level was negatively correlated with the TWIST protein level in the OS tissue. The miR 33a was nega tively correlated with the tumor necrosis rate, while the TWIST protein level was positively cor related with the tumor necrosis rate. Effect of overexpression and inhibition of miR 33a on TWIST expression in OS cells We next examined the effects of miRNA 33a on TWIST expression in human OS cells. As shown in Figure 5, miR 33a was highly expressed in Saos 2 cells, which had a low constitutive expression of TWIST at both the mRNA and the protein levels. In contrast, MG 63 cells had a constitu tive low expression of miR 33a, and a high expression of TWIST at both the mRNA and the protein levels.

Thus, overexpression and PTC-209 HBr availability knockdown of TWIST were respectively performed in the two cell lines to approach the study objectives. As shown in Figure 6A, inhibition of miR 33a by antagomir 33a increased TWIST expression by over 1. 5 fold in Saos 2 cells. On the other hand, overex pression of miR 33a decreased TWIST expression by about 30%. Overexpression of TWIST led to an approxi mately two fold increase of TWIST expression in Saos 2 cells, which was largely reversed by overexpression of miR 33a and doubled by antagomir 33a. As shown in Figure 6B, overexpression of miR 33a decreased TWIST expression by nearly 70% in MG 63 cells, while antagomir 33a in creased TWIST expression by 0. 4 fold. Knockdown of TWIST by shRNA resulted in an approximately 80% de crease of endogenous TWIST expression in MG 63 cells, which was partially reversed by antagomir 33a.

Functional role of miR 33a in TWIST inhibited OS cell survival against cisplatin TWIST reportedly decreases OS cell survival against cisplatin, an apoptosis inducing chemotherapeutic agent commonly used to treat OS. To explore the effect of interaction between miR 33a and TWIST on OS chemoresistance, we examined cell apoptosis rate in both cell lines treated with cisplatin using TUNEL assays.