RT-PCR analysis of long non-coding RNAs expression in adriamycin-resistant osteosarcoma cells

ABSTRACT: One of the major challenges for chemotherapy is appearance of resistance to compounds. Despite several singling pathways have been implicated in development of Adriamycin (ADM) resistance, mechanisms involved in ADM-resistant osteosarcoma progression remain largely unknown. The present study attempts to illustrate the role of long noncoding RNA ARSR in development of ADM-resistance. We found lncRNA ARSR overexpressed in the Adriamycin resistant cell lines U2OS/ADM and MG63/ADM, accompanied with acquired multidrug resistance against to paclitaxel and cisplatin. Overexpression of lncRNA ARSR triggered rhodamine 123 efflux and survival, as well as migration of ADM resistant cells. Conversely, depletion of ARSR promoted rhodamine 123 retention and apoptosis while reduced motility of ADM resistant cells. Further investigation revealed that upregulation of ARSR enhanced Akt phosphorylation on Ser473 and increment of multidrug resistance-associated protein-1, apoptosis inhibitor Survivin and matrix metalloproteinase-2. Reduction of ARSR overcame the resistance to ADM and magnified the suppression of Akt-mediated signaling pathways by MK-2206. The current study gained novel evidence for understanding the mechanisms underlying adaptive ADM resistance, and provided rational to improve Akt-targeted therapy towards refractory osteosarcoma. Methods: The expression of lncRNA was analyzed by RT2 lncRNA PCR Arrays. The differential expressed lncRNAs were further verified by qRT-PCR. The relationship between lncRNA expression and clinical features was evaluated with Spearman correlation test. Cell proliferation was measured by MTT assay. Cell apoptosis percentage was detected by ANNEXIN V-PI analysis. The promoter activity was illustrated by Luciferase assay, and the change of microRNA and protein was detected by qRT-PCR and western blot, separately. Results: The expression of HAND2-AS1 declined in bladder cancer and correlated with depth of invasion and grades negatively. Restoration of HAND2-AS1 hampered cell growth by provoking cell apoptosis. Furthermore, one of the HAND2-AS1 sponge, miR-146, was found overexpression in bladder cancer tissue and cell lines. Expression of miR-146 related to HAND2-AS1 expression negatively. One of the targeted genes of miR-146, retinoic acid receptor beta (RARB) was downregulated in bladder cancer. In addition, the expression of RARB related to miR-146 negatively. Lost-of-function and gain-of-function experiments were used to identify the mechanisms underlying association of lncRNA HAND2-AS1: miR-146: RARB. miR-146 targeted RARB directly and hindered RARB-mediate apoptosis. However, the hindrance was impaired by HAND2-AS1 notably. Conclusion: HAND2-AS1 diminished miR-146 expression, thereby releasing the suppression of miR-146 on RARB-mediated apoptosis, promoting bladder cancer regression. Long noncoding RNA profiling by array

ORGANISM(S): Homo sapiens

PROVIDER: GSE142230 | GEO | 2020/05/19

REPOSITORIES: GEO