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Circular RNA hsa_circ_0003496 Contributes to Tumorigenesis and Chemoresistance in Osteosarcoma Through Targeting (microRNA) miR-370/Kruppel-Like Factor 12 Axis.

ABSTRACT: Background:Osteosarcoma (OS) is the most common primary malignancy of bone with a high incidence in children. Circular RNAs (circRNAs) play crucial roles in the carcinogenesis and chemoresistance of OS. In the current work, we focused on the function and mechanism of hsa_circ_0003496 (circ_0003496) in OS progression and chemoresistance. Materials and Methods:The expression levels of circ_0003496, miR-370 and Krüppel-like factor 12 (KLF12) mRNA were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The Cell Counting Kit-8 (CCK-8) assay was used to assess the 50% inhibitory concentration (IC50) value and cell proliferation. Cell migration, invasion and apoptosis were detected by transwell assay and flow cytometry, respectively. Western blot analysis was performed to assess the protein level. Targeted relationships among circ_0003496, miR-370 and KLF12 were validated by dual-luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull-down assays. Animal studies were carried out to observe the role of circ_0003496 in vivo. Results:Our results indicated that circ_0003496 up-regulation was associated with doxorubicin (DXR) resistance of OS. Circ_0003496 knockdown repressed DXR-resistant OS cell proliferation, migration and invasion, and enhanced apoptosis and DXR sensitivity. Circ_0003496 functioned as a sponge of miR-370, and miR-370 mediated the regulatory effect of circ_0003496 depletion on DXR-resistant OS cell progression and DXR sensitivity. KLF12 was a direct target of miR-370, and miR-370 overexpression suppressed cell progression and enhanced DXR sensitivity by KLF12. Moreover, circ_0003496 protected against KLF12 repression through sponging miR-370. Additionally, circ_0003496 knockdown hampered tumor growth and promoted DXR sensitivity in vivo. Conclusion:Our present work suggested that the knockdown of circ_0003496 suppressed OS progression and enhanced DXR sensitivity at least partially through modulating KLF12 expression via functioning as a miR-370 sponge, highlighting new opportunities for OS management.

SUBMITTER: Xie C

PROVIDER: S-EPMC7489950 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Circular RNA hsa_circ_0003496 Contributes to Tumorigenesis and Chemoresistance in Osteosarcoma Through Targeting (microRNA) miR-370/Krüppel-Like Factor 12 Axis.

Xie Chaofan C Liang Guanzhao G Xu Yinfeng Y Lin Erhu E

Cancer management and research 20200909

<h4>Background</h4>Osteosarcoma (OS) is the most common primary malignancy of bone with a high incidence in children. Circular RNAs (circRNAs) play crucial roles in the carcinogenesis and chemoresistance of OS. In the current work, we focused on the function and mechanism of hsa_circ_0003496 (circ_0003496) in OS progression and chemoresistance.<h4>Materials and methods</h4>The expression levels of circ_0003496, miR-370 and Krüppel-like factor 12 (KLF12) mRNA were measured by quantitative real-ti ...[more]

PMID: 32982419

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Project description:BACKGROUND:Circular RNAs (circRNAs) have emerged as a novel category of non-coding RNA, which exhibit a pivotal effect on regulating gene expression and biological functions, yet how circRNAs function in osteosarcoma (OSA) still demands further investigation. This study aimed at probing into the function of hsa_circ_0000282 in OSA. METHODS:The expressions of circ_0000282 and miR-192 in OSA tissues and cell lines were examined by quantitative real-time polymerase chain reaction (qRT-PCR), and the correlation between the expression level of circ_0000282 and clinicopathological features of OSA patients was analyzed. The expressions of X-linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in OSA cells were assayed by Western blot. The proliferation and apoptosis of OSA cells were examined by CCK-8, BrdU and flow cytometry, respectively. Bioinformatics analysis, dual-luciferase reporter gene assay and RIP experiments were employed to predict and validate the targeting relationships between circ_0000282 and miR-192, and between miR-192 and XIAP, respectively. RESULTS:Circ_0000282 was highly expressed in OSA tissues and cell lines, which represented positive correlation with Enneking stage of OSA patients and negative correlation with tumor differentiation degree. In vitro experiments confirmed that overexpression of circ_0000282 markedly facilitated OSA cell proliferation and repressed cancer cell apoptosis in comparison to control group. Besides, knockdown of circ_0000282 repressed OSA cell proliferation and promoted apoptosis. Additionally, the binding relationships between circ_0000282 and miR-192, and between miR-192 and XIAP were validated. Circ_0000282 indirectly up-regulated XIAP expression by adsorbing miR-192, thereby playing a role in promoting cancer in OSA. CONCLUSION:Circ_0000282 was a novel oncogenic circRNA in OSA. Circ_0000282/miR-192/XIAP axis regulated OSA cell proliferation apoptosis with competitive endogenous RNA mechanism.

Project description:Osteosarcoma is the most common type of pediatric bone tumor. Despite great advances in chemotherapy during the past decades, the survival rates of osteosarcoma patients remain unsatisfactory. Drug resistance is one of the main reasons, leading to treatment failure and poor prognosis. Previous reports correlated expression of cluster of differentiation 44 (CD44) with drug resistance and poor survival of osteosarcoma patients, however the underlying mechanisms are poorly defined. Here, we investigated the role of CD44 in the regulation of drug chemoresistance, using osteosarcoma cells isolated from mice carrying a mutation of the tumor suppressor neurofibromatosis type 2 (Nf2) gene. CD44 expression was knocked-down in the cells using CRISPR/Cas9 approach. Subsequently, CD44 isoforms and mutants were re-introduced to investigate CD44-dependent processes. Sensitivity to doxorubicin was analyzed in the osteosarcoma cells with modified CD44 expression by immunoblot, colony formation- and WST-1 assay. To dissect the molecular alterations induced by deletion of Cd44, RNA sequencing was performed on Cd44-positive and Cd44-negative primary osteosarcoma tissues isolated from Nf2-mutant mice. Subsequently, expression of candidate genes was evaluated by quantitative reverse transcription PCR (qRT-PCR). Our results indicate that CD44 increases the resistance of osteosarcoma cells to doxorubicin by up-regulating the levels of multidrug resistance (MDR) 1 protein expression, and suggest the role of proteolytically released CD44 intracellular domain, and hyaluronan interactions in this process. Moreover, high throughput sequencing analysis identified differential regulation of several apoptosis-related genes in Cd44-positive and -negative primary osteosarcomas, including p53 apoptosis effector related to PMP-22 (Perp). Deletion of Cd44 in osteosarcoma cells led to doxorubicin-dependent p53 activation and a profound increase in Perp mRNA expression. Overall, our results suggest that CD44 might be an important regulator of drug resistance and suggest that targeting CD44 can sensitize osteosarcoma to standard chemotherapy.

Dataset Information

Circular RNA hsa_circ_0003496 Contributes to Tumorigenesis and Chemoresistance in Osteosarcoma Through Targeting (microRNA) miR-370/Kruppel-Like Factor 12 Axis.

Publications

Circular RNA hsa_circ_0003496 Contributes to Tumorigenesis and Chemoresistance in Osteosarcoma Through Targeting (microRNA) miR-370/Krüppel-Like Factor 12 Axis.

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