Research On The Molecular Mechanism Of MiR-33a Modulates Osteosarcoma Chemoresistance Via TWIST/ET-1Signaling

Part Ⅰ construction of osteosarcoma cells with TWIST overexpressed or knocked down stablyObjectives:According to the different expression levels of TWIST in OS cells, we have constructed two stable cells in which TWIST were overexpressed or knocked down respectively for subsequent functional studies.Methods:Constructed eukaryotic expression vector pCDNA3.1(+)-TWIST using gene cloning, transfected OS cells Saos-2with this construted vector and screened cells by G418, then we successfully obtaine the stable cell line called Saos-2-TWIST in which TWIST was overexpressed by Western blot detection. Infected MG63cells with TWIST shRNA lentivirus and screened by puromycin, then we successfully obtained the stable cell line called MG63-TWIST-shRNA in which TWIST was knocked down by Western blot.Results:The results of restriction analysis and sequencing confirmed that we have successfully constructed the recombinant expression vector pCDNA3.1(+)-TWIST. After selected stable cells by G418or puromycin respectively and detected by Western bolt, we have constructed Saos-2cells in which TWIST gene was overexpressed stably and MG63-TWIST-shRNA in which the expression levels of TWIST was inhibited by82%。Conclusions:we have successfully constructed two stable cells in which TWIST were overexpressed or knocked down respectively using Western blot assay. Part Ⅱ TWIST interacts with endothelin-1/endothelin A receptor signaling in osteosarcoma cell survival against cisplatinObjectives:To investigate the interaction of TWIST and ET-1/ETAR signaling and molecular mechanisms for osteosarcoma chemoresistance.Methods:1. Detected ET-1expression in osteosarcoma cells with different expression levels of TWIST and the efforts of PI3K inhibitor LY294002on TWIST and ET-1expression by Realtime PCR. Western blot、ELISA respectively;2. The TUNEL assay was performed to detect cells apotosis rate against cisplatin in the OS stable cells in which TWIST was overexpressed and knocked down in the present of PI3K inhibitor LY294002and ETAR inhibitor BQ123;3. Detected the expression levels of p-AKT in the OS cells with PI3K inhibitor LY294002by western blot.Results:1. In Saos-2cells, overexpression of TWIST significantly decreased ET-1mRNA and protein expression levels, while in MG63cells, knockdown of TWIST significantly increased ET-1expression; In addition, ET-1expression of OS cells was significantly decreased by the PI3K inhibitor LY294002;2. The TUNEL results showed, in Saos-2cells treated with cisplatin, overexpressing TWIST led to a significantly increased cell apoptosis rate compared with the controls, which was reversed by exogenous ET-1(100pM). Additionally, the selective ETAR inhibitor (BQ123) was able to completely block the rescue effect of ET-1, while LY294002partially blocked the rescue effect; while in MG-63cells treated with cisplatin, knocking down TWIST significantly decreased the cell apoptosis rate. This effect was was abolished by BQ123or LY294002;3. The Western blot results suggested, in Saos-2cells, overexpression of TWIST significantly decreased phosphorylation at serine473(ser473) of Akt; LY294002treatment further decreased phosphorylation at this site; In MG-63cells, knockdown of TWIST increased the phosphorylation of Akt (P-Akt) at ser473≥2-fold compared with the controls, which was abolished by LY294003.Conclusions:In conclusion, we have demonstrated that TWIST decreases OS cell survival against cisplatin by downregulating ET-1/TAR signaling via inhibition of the PI3K/Akt pathway. PartⅢ miR-33a promotes osteosarcoma cell resistance to cisplatin by down-regulating TWISTObjectives:To profile miRNAs differentially expressed in chemoresistant OS, with a focus to identify miRNAs that regulate TWIST expression and OS chemoresistance.Methods:1. profile miRNAs differentially expressed in OS tissues from the chemoresistant OS patients compared with those from the control OS patients by miRNA microarray;2. Screen and identify miRNAs that regulate TWIST by dual luciferase assay;3. Detect miRNA-33a and TWIST protein levels in chemoresistant OS patients (n=35) and control patients (n=35) in the validation by Realtime PCR and Western blot respectively;4. TWIST expression and cell apoptosis against cisplantin in the OS cells in which TWIST was overexpressed and knocked down transfected with miR-33a or inhibition antagomir-33a were detected by Western blot and TUNEL assay respectively.Results:1. MiRNA microarray analyses showed that25miRNAs were differentially expressed in OS tissues from the chemoresistant OS patients compared with those from the control OS patients,16being up-regulated and9down-regulated;2. Screen12candidate miRNAs targeting TWIST gene by the TargetScan prediction software and miRNA microarray;3. Correlation analyses based the results of realtime PCR and Western blot in the entire validation cohort (n=70) showed that, the miR-33a level was negatively correlated with the TWIST protein level in the OS tissue (r=-0.627, p<0.001). The miR-33a was negatively correlated with the tumor necrosis rate (r=-0.352, p<0.001), while the TWIST protein level was positively correlated with the tumor necrosis rate (r=0.562, p<0.001).4. The TUNEL results showed, in Saos-2cells treated with cisplatin, inhibition of miR-33a by antagomir-33a markedly increased cell apoptosis, which was enhanced by overexpression of TWIST. The apoptosis-inducing effect of TWIST overexpression was reversed by overexpression of miR-33a. In MG-63cells, overexpression of miR-33a significantly decreased cisplatin-induced cell apoptosis, which was enhanced by knockdown of TWIST. Antagomir-33a significantly increased cisplatin-induced cell apoptosis, which was reversed by knockdown of TWIST.Conclusions:Overall, miR-33a is up-regulated in chemoresistant osteosarcoma and promotes osteosarcoma cell resistance to cisplatin by down-regulating TWIST. The findings suggest that inhibition of miR-33a/TWIST signaling could be a potential new strategy to enhance neoadjuvant chemotherapy for OS.