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Objective To screen the expression profile of circular RNA associated with chemo-resistance in osteosarcoma,and to analyze and identify its possible molecular functions.Methods Three pairs of matched drug-resistant and sensitive human osteosarcoma cell lines (MG63/DXR vs MG63,U2OS/DXR vs U2OS,KHOS/DXR vs KHOS) were first tested by CCK-8 assay for three commonly used osteosarcoma chemotherapy drugs (doxorubicin,cisplatin,and methotrexate);then,using next-generation high-throughput RNA sequencing technology,comparative analysis of circRNA expression was performed in three pairs of matched multidrug-resistant and sensitive human osteosarcoma cell lines;followed by real-time quantitative PCR (qRT-PCR) to confirm the reliability and accuracy of sequencing data in chemotherapy-resistant osteosarcoma cell lines and tissues.In addition,a variety of bioinformatics analyses,including GO,KEGG pathway analysis and the construction of circRNA-miRNA networks,were performed to predict potential molecular functions of differentially expressed circRNAs and to construct relevant regulatory pathways or networks.Results Three osteosarcoma-resistant cells (MG63/DXR,U2OS/DXR,KHOS/DXR) were significantly resistant to the three common osteosarcoma chemotherapy drugs compared with control cells (MG63,U2OS,KHOS),which laid a solid foundation for the further experiments.RNA sequencing revealed a total of 80 circRNAs differentially expressed between the two groups.Compared with drug-sensitive osteosarcoma cells,57 circRNAs were upregulated and 23 circRNAs weredownregulated in the drug-resistant osteosarcoma cell lines (fold change≥ 2 or ≤0.5,P ＜ 0.05).Tenrandomly selected circRNAs were verified by qRT-PCR and the results showed that 9 of the 10 circRNAswere consistent with the sequencing results.In addition,KEGG pathway analysis showed that 56 pathways were significantly enriched in differentially expressed circRNAs,including Glycolysis/gluconeogenesis,ABC transporter,VEGF signaling pathway,and so on.Moreover,thedifferently expressedcircRNA-hsa_circ_0004674 with the highestfold change was highly expressedin the chemotherapy-resistant osteosarcoma cells and tissues,associated with poor prognosis in osteosarcoma patients.Some potential endogenous competitive RNA (ceRNA) regulatory pathways associated with hsa_circ_0004674,such as hsa_circ_0004674-miR-490-3p-ABCC2 and hsa_circ_0004674-miR-1254-EGFR,were constructed by use of the authoritative databases (target scan and miRanda) and literature searching and the miRNA response element sequences (MREs) between miRNAs that have a potential ceRNA regulatory relationship with hsa_circ_0004674 were alsopredicted.Conclusion CircRNA is closely related to tumor progression and may play a role in ostoosarcoma chemo-resistance.Besides,hsa_circ_0004674 may be a potential candidate for reversing drug resistance of osteosarcoma.
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Objective@#To investigate the mechanism of long non-coding RNA (LncRNA) NR_036444 mediated sensitivity of multidrug-resistant osteosarcoma to doxorubicin.@*Methods@#LncRNA-mRNA combined microarray was used to screen the differential expressions of lncRNA and mRNA in doxorubicin-resistant MG63/DXR osteosarcoma cells and their paired doxorubicin-sensitive MG63 cells; qRT-PCR was used to check the consistency of microarray. LncRNA NR_036444 was over-expressed in MG63/DXR cells by lentrvirus. CCK-8 array was used to evaluate cell proliferation and the sensitivity of cells to doxorubicin; Flow cytometry was used to evaluate cell cycle and apoptosis. The expressions of lncRNA NR_036444 in 60 cases of tumor tissues resected from osteosarcoma patients were detected by qRT-PCR and correlation analyses administrator were conducted.@*Results@#Compared with those in MG63 cells, 1, 761 lncRNAs were significantly up-regulated and 1, 704 lncRNAs were dramatically down-regulated in MG63/DXR cells (P<0.05). 15 lncRNAs were selected randomly and their expressions were confirmed by qRT-PCR. The data showed that the differences in expression of 15 lncRNAs were consistent with microarray result. lncRNA NR_036444 with 22 folds down-regulation was identified as the most significant differential lncRNA in MG63/DXR cells. Half maximal inhibitory concentrations (IC50) in the blank group, negative control group and lncRNA NR_036444 overexpressed group were (12.73±0.50) μg/ml、(12.12±0.31) μg/ml and (5.77±0.25) μg/ml, respectively. Compared with that in negative control group, IC50 in overexpressed group decreased by 49.6% (P<0.001). The percentages of G1 phase cells in the overexpressed group, negative control group and blank group were(90.12±0.08)%, (33.36±0.85)% and (39.38±1.02)%. Compared with the negative control group and blank control group, the overexpression group had significantly more cells arrested in G1 phase (both P<0.001). In the overexpressed group, negative control group and blank group, the percentages of late-apoptotic cells were (11.40±1.08)%, (4.23±1.12)% and (0.28±0.12)%, respectively. The percentages of early-apoptotic cells were (86.40±1.80)%, (4.35±2.03)% and (0.25±0.02) %, respectively. The percentages of early- and late-apoptotic cells were significantly increased in the overexpressed group when compared with those in control groups (P<0.01). Meanwhile, the expression of lncRNA NR_036444 in the tissues of chemoresistance group and chemosensitivity group was 19.67±1.53 and 77.67±2.52, respectively, with a statistically significant difference (P<0.01). The postoperative overall survival time of osteosarcoma patients with low expression of lncRNA NR_036444 was (24.6±2.4) months, significantly shorter than (48.2±1.8) months of patients with high expression of lncRNA NR_036444 (P<0.001).@*Conclusions@#LncRNA NR_036444 may play a vital role in regulating osteosarcoma doxorubicin resistance and it may be a useful biomarker to assess the chemosensitivity and predict the prognosis of osteosarcoma patients in the future.
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Long non-coding RNA (lncRNA) is a group of length more than 200 nucleotides without coding protein RNA.It plays an important role in cell proliferation,differentiation,senescence,death,tumor occurrence and development.This article reviews the main characteristics of lncRNA and its role in tumor drug resistance.
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OBJECTIVE:To observe therapeutic efficacy of adjuvant treatment of Xuebijing injection for severe acute pancreati-tis. METHODS:80 cases of severe acute pancreatitis were randomly divided into control group and observation group with 40 cas-es in each group. Control group was given symptomatic and supportive treatment,and observation group was additionally given in-travenous injection of Xuebijing injection 100 ml,twice a day,on the basis of control group. The levels of TNF-α,IL-6 and hs-CRP,organ failure were compared between 2 groups before and after treatment. RESULTS:There was no statistical significance in the levels of TNF-α,IL-6 and hs-CRP in 2 groups before treatment(P>0.05);after treatment,the levels of TNF-α,IL-6 and hs-CRP in observation group was significantly lower than control group and before treatment,with statistical significance (P<0.05). The proportion of renal function,respiration function and circulating failure in observation group were significantly lower than in control group,with statistical significance (P<0.05). CONCLUSIONS:Adjuvant treatment of Xuebijing injection for se-vere acute pancreatitis can significantly reduce the level of inflammation in the body and reduce organ damage.