Mechanism Of Long Non-coding RNA ODRUL In Regulating The Doxorubicin-resistance In Osteosarcoma

Background: Osteosarcoma is the most common primary malignant bone tumor in adolescent.Chemotherapy is its most important adjuvant therapy,however,the emergence of doxorubicin-resistance has greatly impeded the treatment of osteosarcoma.It has been reported that long non-coding RNAs(lncRNAs)are emerging in molecular biology as crucial regulators of cancer.The efficacy of doxorubicin--based chemotherapy in osteosarcoma(OS)is limited by acquired drug resistance.Identification of the lncRNAs related to doxorubicin resistance in OS may help to improve clinical response rates.Objective: To explore the underlying mechanism of doxorubicin-resistance of osteosarcoma in terms of lncRNA and to further discuss the reasons for chemoresistance and potential strategies for overcoming this resistance in OS.Methods: Using the third-generation human lncRNA-mRNA combined microarray,differentially expressed lncRNAs and mRNAs were detected in human primary osteosarcoma doxorubicin-resistant MG63/DXR cells and paired parental MG63 cells.Fifteen lncRNAs were randomly selected to validate the consistency with the microarray data by q RT-PCR.The expression of two most up-regulated lncRNAs and the most down-regulated lncRNA was evaluated in two pairs of doxorubicin-resistant and doxorubicin-sensitive osteosarcoma cell lines(MG63/DXR vs MG63 and KH-OS/DXR vs KH-OS)and other doxorubicin-sensitive osteosarcoma cell lines(Sao S2、U-2OS)by q RT-PCR.Then the expression of the most up-regulated lncRNAs--lncRNA ENST00000563280,which was named after lncRNA ODRUL(Osteosarcoma doxorubicin-resistance related up-regulated lncRNA),was examined in 60 primary osteosarcoma tissue samples by q RT-PCR to explore its potential clinical significance.CCK-8 assay was used to examine the inhibition of cell proliferation after the si-lncODRUL transfection in the MG63/DXR and KH-OS/DXR cell lines.Flow cytometry was used to evaluate the cell apoptosis and cell cycle.Wound Healing and Transwell assay were used to evaluate the cell migration and cell invasion respectively.To further explore the molecular mechanism of lncRNA ODRUL regulating the doxorubicin-resistance in osteosarcoma,bioinformatics analysis was used to analyze the correlation of lncRNA and some classical drug-resistance related genes,such as ABCB1,EGFR,ABCC3,IL-6;then PCR and WB were used to detect the expression of these genes in terms of mRNA and protein level after knocking down the lncRNA ODRUL by si RNA in the MG63/DXR and KH-OS/DXR cell lines.Results: We identified 3,465 lncRNAs(1,761 up and 1,704 down)and 3,278 mRNAs(1,607 up and 1,671 down)aberrantly expressed in all three doxorubicin-resistant MG63/DXR cell lines(fold-change>2.0,P<0.05 and FDR<0.05)through the genome-wide lncRNA and mRNA expression profile analysis.And we found that only the most up-regulated lncRNA-lncRNA ENST00000563280(lncRNA ODRUL)presented the consistent trend in different osteosarcoma cell lines,namely higher expression in the doxorubicin-resistant osteosarcoma cell lines and lower expression in the doxorubicin-sensitive osteosarcoma cell lines.Besides,we demonstrated that lncRNA ODRUL was distinctly increased in specimens of OS patients with a poor chemoresponse and lung-metastasis at primary diagnosis compared to those with a good chemoresponse and lung non-metastasis.After knockdown lncRNA ODRUL by si RNA,the IC50 value of MG63/DXR and KH-OS/DXR greatly decreased than the controlled group and the ability of cell migration and invasion both decreased.The rate of late apoptosis of si-ODRUL transfected cells increased than the controlled group and the ratio of G1 phase also increased than before.Furthermore,bioinformatics analysis(co-expression analysis)found the expression correlation coefficients between lncRNA ODRUL and ABCB1,EGFR,ABCC3,IL-6 or other drug-related genes were more than 0.995 or less than-0.995 and the expression of classical drug-resistance related gene ABCB1 other than other related genes,such as EGFR,IL-6 and ABCC3,greatly decreased after lncRNA ODRUL knockdown through PCR and WB assay,which may suggest the potential mechanism of lncRNA ODRUL regulating the chemoresistance of osteosarcoma through affecting the expression of ABCB1 gene.Conclusion: In our study,we revealed the differentially expressed lncRNAs and mRNAs in human primary osteosarcoma doxorubicin-resistant MG63/DXR cells and their paired parental MG63 cells.We identified lncRNA ODRUL may play a vital role in the occurrence and development of doxorubicin –resistance of osteosarcoma,which may serve as a potential biomarker to classify the chemosensitive and chemoresistant osteosarcoma patients and a possible target for reversing the doxorubicin –resistance in osteosarcoma.These results provide important insights about the lncRNAs involved in osteosarcoma chemoresistance and lay a solid foundation for uncovering their regulatory function and mechanism.