Biological Effects Of UCA1 On Osteosarcoma And The Mechanism Of Induction Of Chemoresistance

Purpose:Long non-coding RNAs have been found to be involved in tumor development and chemoresistance.lnc RNA UCA1 was found to be involved in the progression of osteosarcoma in the previous study,and this study is to further investigate whether lnc UCA1 is involved in chemoresistance of osteosarcoma cells and the related mechanisms based on the previous study,in order to provide a new strategy for the treatment of osteosarcoma patients with drug resistance.Methods:（1）Clinical specimens of osteosarcoma tissues were collected,and the expression of lnc UCA1 in adjacent normal and osteosarcoma tissues as well as in osteosarcoma-resistant tissues was detected by real-time polymerase chain reaction（RT-q PCR）.（2）Cisplatin-resistant osteosarcoma cell lines（MG63/DDP,U2OS/DDP）were established using an intermittent stepwise induction protocol.The half inhibition rate of established cisplatin-resistant osteosarcoma cells to cisplatin was detected by CCK-8 assay,and the expression of resistance key genes MRP1 and P-gp in the resistant cell lines was detected by RT-q PCR and Western Blot to ensure the establishment of cisplatin-resistant cell lines.（3）The correlation between lnc UCA1and osteosarcoma progression and drug resistance was demonstrated by detecting the difference in lnc UCA1 expression in human osteoblasts,parental osteosarcoma cells and induced cisplatin-resistant cell lines by RT-q PCR,and by altering cell viability and half inhibition rate after knockdown of lnc UCA1 in drug-resistant cell lines.（4）Plasmid transfection technique was applied to overexpress lnc UCA1 in MG63 and U2OS cell lines,and lnc UCA1 in MG63/DDP and U2OS/DDP drug-resistant cell lines was knocked down by lentiviral transfection.（5）Cell proliferation ability was determined using CCK8.Cell migration,invasion and healing ability were detected by Transwell and Wound-healing assays.The effect of lnc UCA1 alteration on apoptosis of osteosarcoma cells was detected by flow cytometry.In this study,RT-q PCR and Western Blot were performed to analyze the expression of key genes MRP1,P-gp,PCNA,MMP9,and Caspase-3.Results:lnc UCA1 was highly expressed in OS-resistant tissues and in cisplatin-induced resistant cell lines（P<0.05）.Knockdown of lnc UCA1 in resistant cell lines significantly inhibited cell viability（P<0.05）,and IC₅₀ values were significantly decreased after knockdown of lnc UCA1 compared with normal cisplatin-resistant osteosarcoma cells MG63/DDP and U2OS/DDP,28.07%and 29.57%,respectively（P<0.05）,indicating that lnc UCA1 was associated with osteosarcoma progression and cisplatin drug resistance was associated.Phenotypic assays showed that overexpression of lnc UCA1 in MG63 and U2OS cell lines promoted cell proliferation,migration,invasion and inhibition of apoptosis.PCR and WB results showed that lnc UCA1 overexpression altered the expression of MRP1,P-gp,PCNA,MMP9 and Caspase-3 and increased the cisplatin resistance of OS cells.The combination of knockdown of lnc UCA1 and cisplatin in cisplatin-resistant cells inhibited cell viability,significantly attenuated migration,invasion and promoted apoptosis,altered the expression of MRP1,P-gp,PCNA,MMP9 and Caspase-3,and restored the sensitivity of resistant cells to cisplatin.Conclusions:This study demonstrates that lnc UCA1 positively regulates osteosarcoma cell progression and leads to chemoresistance to cisplatin,and elucidates the mechanism by which lnc UCA1 affects osteosarcoma cell biological behavior and cisplatin resistance by altering the expression of key genes MRP1,P-gp,PCNA,MMP9,and Caspase-3.The clinical application value of the combined effect of lnc UCA1 downregulation and cisplatin in resistant cells on the inhibition of osteosarcoma cell progression and the restoration of sensitivity of resistant cells to cisplatin was revealed,providing a theoretical basis for the treatment of chemoresistance.