LncRNA ANRIL Regulates The Cisplatin Chemoresistance Of Osteosarcoma Cells By MiR-125a-5p/STAT3 Molecular Axis

Objective: Osteosarcoma is the most common osteosarcoma among all primary malignant bone tumors,with an incidence of approximately 35%.With the establishment of multidisciplinary treatment,the 5-year survival rate of non-metastatic patients can reach over 60%.Neoadjuvant chemotherapy combined with surgical resection of primary tumor has become the standard treatment for newly diagnosed osteosarcoma.Cisplatin is a commonly used drug for osteosarcoma.It interacts with the nucleophilic N7 site of purine bases in DNA and inducing DNA damage and subsequently cell death.However,the drug resistance mechanism of cisplatin in osteosarcoma limited the efficacy of chemotherapy drugs,so the long-term survival rate of patients with metastatic or recurrent osteosarcoma is still low.Long non-coding RNAs(lnc RNAs)are non-coding RNA subsets with no protein-coding ability and m RNA transcripts with a length of more than 200 nucleotides.It has been reported that lnc RNA is involved in the mechanism of tumor resistance.Lnc RNA ANRIL is the antisense non-coding RNA of the INK4 site(ANRIL),and is transcribed into 3834 nt lnc RNA,which is considered as a risk factor for tumorigenesis.Recent studies have reported that lnc RNA ANRIL can promote the malignant biological behavior of tumor cells in cancers such as prostate cancer,colorectal cancer and gastric cancer.In this study,it was found that lnc RNA ANRIL was abnormally expressed in osteosarcoma cells.And it can regulate the resistance of osteosarcoma cells to cisplatin drugs.But the specific mechanism is still unclear.mi R-125a-5p,located at 19q13.41,is generally expressed in tissues and is involved in endothelium-mediated angiogenesis in aging mice and vascular homeostasis in apoplexy prone hypertensive rats.Abnormal expression of mi R-125a-5p has been found in glioblastoma,hepatocellular carcinoma,gastric cancer,non-small cell lung cancer and other cancers and it plays a role in tumor inhibition.Multiple studies have shown that mir-125a-5p is an important tumor suppressor gene,which is low expressed in laryngeal cancer,juvenile angiofibroma,colorectal cancer,breast cancer,lung cancer,cervical cancer,prostate cancer,pancreatic cancer and other human cancers.In addition,mi R-125a-5p inhibits the occurrence of cancer by targeting oncogenes such as vascular endothelial growth factor A and E2 F transcription factor 3.mi R-125a-5p has also been confirmed as a prognostic factor for gastric cancer,and overexpression of mir-125a-5p can inhibit the proliferation and migration of gastric cancer cells.However,the mechanism of mir-125a-5p in drug resistance of osteosarcoma cells has not been reported.Signal transducers and transcriptional activators 3(STAT3)are key components of various oncogenic signaling pathways,and STAT3 is most closely related to tumor development among the members of the STAT protein family.It was found that in various hematologic diseases,solid tumors,cancers and other diseases,STAT3 was abnormally activated,and was closely related to the clinical pathological phenotype and poor prognosis of patients.Since the disorder of STAT3 drives the occurrence and development of tumors,we believe that STAT3 targeted therapy can control tumors with high expression and high activity.However,the molecular mechanism of STAT3 resistance to cisplatin in osteosarcoma has not been fully elucidated.This study mainly discussed the expression of lnc RNA ANRIL in osteosarcoma cells and its role in the development of osteosarcoma.At the same time,the regulatory effect of lnc RNA ANRIL on cisplatin drug resistance in osteosarcoma cells through the molecular axis of mi R-125a-5p/STAT3 was discussed,and the possible molecular mechanism was analyzed.Methods:1.The expression of lncRNA ANRIL in osteosarcoma cells MG-63,U2-OS and Saos-2 were detected by q RT-PCR.2.lnc RNA ANRIL knockdown plasmid was transfected into U2-OS and Saos-2 cells,and the transfection efficiency was determined by q RT-PCR.3.CCK-8 assay and clonal formation assay were used to detect the effect of knockdown lnc RNA ANRIL on the proliferation of U2-OS and Saos-2 cells.4.CCK-8assay,cell apoptosis assay and Western blotting assay were used to detect the effect of knockdown lnc RNA ANRIL on cisplatin drug resistance.5.q RT-PCR was used to detect the effect of knockdown lnc RNA ANRIL on the expression of mi R-125a-5p.6.The targeted relationship between lnc RNA ANRIL and mi R-125a-5p was confirmed by dual luciferase reporter gene assay.7.CCK-8 assay,cell apoptosis assay and Western blotting assay were used to detect the effect of knockdown of mi R-125a-5p on cisplatin resistance of lnc RNA ANRIL knockdown's U2-OS and Saos-2 cells.8.The effect of knockdown lnc RNA ANRIL on STAT3 expression was detected by q RT-PCR and Western blotting assay.9.The targeted relationship between STAT3 and mi R-125a-5p was confirmed by dual luciferase reporter gene assay.10.CCK-8 assay,cell apoptosis assay and Western blotting assay were used to detect the effect of overexpression of STAT3 on cisplatin resistance of lnc RNAANRIL knockdown's U2-OS and Saos-2 cells.Results: 1.lncRNA ANRIL was increased in osteosarcoma cells MG-63,U2-OS and Saos-2;the stable knockdown lnc RNA ANRIL U2-OS and Saos-2 cell lines were successfully constructed.2.Knockdown of lnc RNA ANRIL inhibits the proliferation of U2-OS and Saos-2 cells.3.Knockdown of lnc RNA ANRIL inhibits cell proliferation induced by cisplatin and promotes apoptosis induced by cisplatin.4.Knockdown of lnc RNA ANRIL enhanced the inhibitory effect of cisplatin on cell proliferation and promoted the induction effect of cisplatin on cell apoptosis.5.The results of dual luciferase reporter gene assay confirmed that the 3'UTR of lnc RNA ANRIL can target to mi R-125a-5p.6.In U2-OS and Saos-2 cells knockdown with lnc RNA ANRIL,knockdown of mi R-125a-5p can reduce the inhibitory effect of cisplatin on cell proliferation and apoptosis induced by cisplatin.7.Knockdown of lnc RNA ANRIL can inhibit STAT3 expression in U2-OS and Saos-2 cells.8.The results of dual luciferase reporter gene assay confirmed that the 3'UTR of STAS3 can target to mi R-125a-5p.9.Overexpression of STAT3 in lnc RNA ANRIL knockdown U2-OS and Saos-2 cells reduce the inhibitory effect of cisplatin on cell proliferation and inhibit cisplatin-induced apoptosis.Conclusion: Long non-coding RNA ANRIL enhances cisplatin resistance in osteosarcoma cells by mi R-125a-5p/STAT3 molecular axis.