| Chemotherapy routinely plays an important role in the treatment of advanced osteosarcoma.Now,although the percentage of patients cured has increased between 60% and 70% by neoadjuvant chemotherapy coupled with limb-sparing surgery,the prognosis remains poor for most patients with metastatic or recurrent osteosarcoma.This poor prognosis is mostly due to the development of drug resistance by osteosarcoma cells after chemotherapy.Currently,neoadjuvant chemotherapy drugs for osteosarcoma include cisplatin(CDDP),adriamycin(ADM),methotrexate(MTX)and ifosfamide(IFO).C isplatin or cis-diamminedichloroplatinum(II)is one of the most active anticancer agents,being widely used against variou tumors including osteosarcoma.Because osteosarcoma cell often acquire resistance to drugs and even develop multiple drug resistance,which results in treatment failure.In this regard,CDDP-resistance has become one of majo clinical problem to overcome.Though extensive study has been done on drug resistance,there is no mechanism completely to explain the clinical response to drug resistance.Chemotherapeutic insensitivity remains a major obstacle to osteosarcoma treatment.Recently,increasing evidence has suggested that long non-coding RNAs(lncRNAs)play an essential role in tumourigenesis.However,the potential biological roles and regulatory mechanisms of novel lncRNAs in response to cisplatin treatment are poorly understood.Here,we found that lnc RNA LINC00161 was induced by cisplatin in osteosarcoma cells.Elevated LINC00161 increased cisplatin-induced apoptosis and reversed the cisplatinresistant phenotype of osteosarcoma cells by upregulating IFIT2.In the process of drug resistance produced,due to the expression of LINC00161 suppressed osteosarcoma cells produce drug resistance and apoptosis.Further mechanistic studies revealed that LINC00161 could sponge endogenous miR-645 and inhibit its activity leading to IFIT2 increase.In addition,we identified that LINC00161 enhanced cisplatin-induced apoptosis through regulation of the miR-645-IFIT2 pathway.Thus,these findings demonstrate that LINC00161 is an essential regulator in cisplatininduced apoptosis,and the LINC00161-miR-645-IFIT2 signalling axis plays an important role in reducing osteosarcoma chemoresistance.Through this study,LINC00161 can be used as a biomarker resistance of osteosarcoma,but also can serve as targets for treatment of osteosarcoma,these help to cisplatin treatment of osteosarcoma and reducing drug resistance to provide new ideas.Experiment 1 cisplatin-induced U2 OS apoptosis regulated by LINC00161Purpose: LINC00161 in cisplatin-induced OS apoptosisMethod: Target Lnc RNAs in cisplatin-induced OS apoptosis are selected via RNA chip technology.Thus,cisplatin-induced expression of mRNA at posttranscriptional level in different periods(0,6h,12 h and 24h)is detected by q-RT-PCR in U2 OS cells,while expression of apoptosis biomarkers is analyzed by Western Blot during each period.Also,expression level of mRNA is detected by q-RT-PCR after treating with different doses(0??,5??,10??)of cisplatin in U2 OS cells and expression of apoptosis biomarkers is analyzed by Western Blot in doses 0??,5??and 10??.MRNA expression of LINC00161 in MG-63 cells is detected via the same way as mentioned above.Role of LINC00161 in cisplatin-induced U2 OS apoptosis is to be identified by a gene silencer of LINC00161 expression through transfection and by overexpression of LINC00161 on the other hand,with expression and apoptosis biomarkers detected through methods of q-RT-PCR and Western Blot.Results: Apoptosis biomarkers PARP expression and Cleaved-PARP expression are increased as doses(0??,5??,10??)of cisplatin added and moments(0,6h,12 h and 24h)going by,which is showed in Western Blot detection.Besides,mRNA expression of LINC00161 is also upregulated in q-RT-PCR detection.Conclusion: 1.In the process of drug resistance produced,due to the expression of LINC00161 suppressed osteosarcoma cells produce drug resistance and apoptosis.2.Cisplatin-induced U2 OS apoptosis is promoted by LINC00161.Experiment 2 drug resistance of U2 OS enhanced by downregulated LINC00161 and expression of protein IFIT2 regulated by downregulated LINC00161Purpose: Role of LINC00161 in drug-resistant cells of U20S(U2SR),as well as the relationship between targeting protein IFIT2 expression regulated by LINC00161 and OS apoptosisMethod: Expression of LINC00161 is upregulated through transfection of U2 SR which is as treatment while U2 SR non-transfection is as comparison sample.Diversities of LINC00161 expression and diversities of related apoptosis biomarkers PARP and casepase-3 are detected by q-RT-PCR and Western Blot.Diversities of the two group cells apoptosis are detected in Annexin V and PI cotransfection as well as cell colony formation experiment.Targeting protein IFIT2 which is the highest expression of related diversities of LINC00161 is detected by i TRAQ,verified by Western Blot in different periods and doses.Protein IFIT2 is found to be silent and overexpressed by the regulation of LINC00161 in U20 S cells in pros and cons through the two experiment groups detected via Western Blot.Results: LINC00161 expression is upregulated by transfection of exogenous LINC00161 in treatment group,and in contrast with the other group,its drug resistance to cisplatin is reduced when treated with cisplatin again.Therefore,downregulated expression of LINC00161 is very closely related to drug resistance showed in U2 OS drug-resistant generation process.U2 OS drug resistance is strengthened by downregulated LINC00161.Expression of protein IFIT2 is elevated as does of cisplatin and periods increased in Western Blot detection.Thus,protein IFIT2 expression is positively regulated by LINC00161 no matter in U20 S cells or in MG-63 cells.Drug-induced U2 OS apoptosis regulated by IFIT2 is verified through protein level of U2 OS and U2 OSR in pros and cons,in which cisplatin-induced U2 OS apoptosis increased by upregulated IFIT2 is also testified.Resultly,it is showed that drug-induced U2OS/U2 OSR apoptosis is under control of upregulated IFIT2 expression by LINC00161 while drug-induced U2OS/U2 OSR apoptosis is interfered directly by the inhibition of endogenous IFIT2 expression for in view of mRNA level of U2 OS and U2 OSR in the two experiment groups.Conclusion: 1.Drug resistance of U2 OS is enhanced by downregulated LINC00161.2.Targeting protein IFIT2 apoptosis is regulated by LINC00161,and expression of targeting protein IFIT2 is positively correlated with OS apoptosis.3.MRNA level of IFIT2 is not influenced by action mechanism of LINC00161 for an independent transcription level of IFIT2,which further proves that apoptosis is impacted when mRNA level of IFIT2 changes.Experiment 3 cells apoptosis and drug resistance impacted by LINC00161 via miR-645-IFIT2 axis regulatedPurpose: Role of LINC00161 in OS apoptosis and drug resistance with the regulation of protein IFIT2.Method: Immune protein deposition is formed in treatment group by Ago2 antibody with interference of RNAi in U2 OS,and level of LINC00161 expression is detected by q-RT-PCR in that group.The existence of competitive combination with miR-645 is detected through interaction between LINC00161 and miR-645 certified in luciferase reporter gene assay.Cisplatin-induced U2 OS apoptosis regulated by LINC00161 and mechanism of drug resistance is analyzed through the relationship among miR645 and IFIT2 as well as LINC00161 researched by Western Blot in U2 OS and in U2 OSR.Results: LINC00161 is analogous to ce RNA in function found in Ago2 complex while there is a competitive combination with micro RNA,which is showed in RNAi interfered experiment.The combination of miR-645 and LINC00161 is certified in luciferase reporter gene assay,and U2 OS apoptosis is strengthened by downregulated miR-645 while inhibited by upregulated miR-645 as a result of Western Blot detection.Overexpressed IFIT2 can be counteracted by overexpressed miR-645.Cells apoptosis is promoted by overexpressed LINC00161 with regulation of inhibited miR-645.Therefore,protein IFIT2 expression is finally facilitated when mRNA level of IFIT2 is out of miR-645 inhibition after competitive combination of LINC00161 and miR-645 via which cisplatin drug resistance is also decreased.Conclusion: 1.LINC00161 is ce RNA likeness in functional mechanism and it can be competitively combined with micro RNA.2.U2 OS apoptosis and drug resistance is impacted by LINC00161 via miR-645-IFIT2 axis regulated. |
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