Screening Of Osteosarcoma Drug Resistant Cell Lines Using Regulatory Small Rna Library And The Resistant Mechanism

Background:Osteosarcoma(OS)is the most common primary malignant bone tumor in adolescents.It mainly occurs in the metaphysis of long bones,especially in the distal femur and proximal tibia.With the appearance and application of chemotherapeutic drugs,neoadjuvant chemotherapy+surgery+adjuvant chemotherapy has become the standard treatment for OS patients.The5-year survival rate has increased from less than 20%in the 1970s to 80%,however,the prognosis of patients with metastasis or relapsed diseases are still very poor,and with the times of chemotherapy increasing,patients will evitably harbor multi-drug resistance(MDR),chemotherapy resistance is the main reason for the failure of treatment of patients with OS.The mechanism of resistance fuor commonly used antitumor drug has not been understood clearly,the current study shows that multi-factors,multi-levels and multi-genes involve in the regulation drug resistance.To find out the key virulence factors of tumor resistance and the interaction network between these virulence factors efficiently from genome scale is urgent needed.Therefore,it’s important to elucidate the molecular mechanisms and regulatory networks of drug resistance by using functional-based high-throughput phenotyping-based screening methods to obtain the key drug-resistant molecules and to explore the network regulation between molecules.Previous scientists have succeeded in performing high-throughput gene function analyzes using genome-wide RNAi libraries.More recently,there have been reports of gain-of-function screening through the CRISPR library.Therefore,we speculated that using random regulatory small RNA(rsRNA)library also can find out the key drug resistant molecules after specific rsRNA regulation.Cisplatin is one of the most commonly used drugs for standardizing treatment of OS.Screening and studying cisplatin resistance-related molecules has important clinical value.The construction and application of the rsRNA library will provide a new method for the study of OS resistance and bring new opportunities for the comprehensive understanding of the mechanism of tumor resistance.The discovery and extensive study of new multi-drug resistant genes will provide potential target for the treatment of OS.Objective:1.To construct a random rsRNA library.2.Screening of drug resistant phenotypes in osteosarcoma cells 143B.3.To verify the role of rsRNA in drug resistance of osteosarcoma cells.4.To investigate the role of candidate target molecules in cisplatin resistance and their possible molecular mechanisms.Methods:1.Construct retroviral vector plasmid of rs RNA library by molecular cloning technique,identified by clone counting,colony PCR screening,plasmid DNA sequencing.2.The rsRNA library was packaged to retrovirus,using the RV-containing medium to infect OS cell line 143B,then cisplatin was added for drug resistance screening.WST-1,crystal violet staining,colony formation assay,migration and invasion assay,Hoechst stanning for apoptosis,cell cycle assay,qPCR were performed for drug resistance phenotype verification.3.The genomic DNA of drug-resistant cells was extracted for high-throughput sequencing,the role of enriched rsRNA fragments were verified.4.Obtaining lnc RNAs related to drug resistance of OS by bioinformatics analysis,detecting the expression of candidate lncRNAs by q PCR and exploring the potential mechanism of OS resistance.Results:1.Random rs RNA retrovirus library was successfully constructed.The fragment containing 29 randomly bases was chemically synthesized and double stranded DNA was amplified by PCR as the original fragment for Gibson assembly.The retroviral backbone pSOKd plasmid was used as a vector to assemble a RNA fragment containing a 29-base random sequence into the plasmid by Gibson assembly between the plasmid U6-H1 promoter.The number of plasmids assembled in the rsRNA library was about 1.07×10~7,and the positive rate of plasmids was 95%by electroporation,colony counting,colony PCR validation and plasmid sequencing.Twenty randomly selected positive colonies were sequenced and none of them overlapped.2.Screening of Osteosarcoma Resistant Cells Using Randomized rsRNA Retrovirus Library.(1)50ug random rs RNA library plasmid was transfected into 293PA cells and packaged.The osteosarcoma143B cells were infected with retrovirus containing rs RNA library and screened by BSD and dose-increaseing cisplatin.After 4 months,drug-resistant cell lines were obtained.(2)The proliferation rate of drug-resistant cells was significantly lower than parental cells,the median lethal dose was significantly higher than the parental cells.(3)Crystal violet staining showed that drug-resistant cells were more resistant to cisplatin,also showed cross-resistance to ifosfamide,doxorubicin and methotrexate.(4)The colony formation ability,migration and invasion ability of drug-resistant cells were more stronger than the parental cells.(5)Under drug treatment,the percentage of apoptosis cell in drug-resistant cells was significantly lower than that in parental cells,and cell cycle analysis showed that cisplatin blocked the parental cells in G1 phase.(6)qPCR results showed that the expression of drug resistance-related genes(such as ABCB1,ABCC1,ABCC2,LRP,GST-π,ATP7B,CTR2,etc.)were significantly increased in drug-resistant cells.3.Role of rsRNA fragments in drug resistance of osteosarcoma.(1)Three highly enriched rsRNA fragments obtained after high-throughput sequencing of the genomic DNA extracted from drug-resistant cells.(2)Increased cisplatin resistance can be observed after single or multi-overexpression of the fragment in the parental cells.4.Bioinformatics analysis of lncRNAs related to cisplatin resistance in osteosarcoma and the potential mechanism.(1)Ten candidate lncRNAs were found by Ensemble blast.(2)qPCR detection after regular RT and gene-specific RT all confirmed the highly expression level of the ten lncRNAs in drug resistant cells.Conclusion:We constructed a completely randomized rsRNA retrovirus library and performed high-throughput screening using this rsRNA libraries to perform phenotypic-based functional screening of cisplatin-resistant molecules associated with osteosarcoma for the first time.Three rsRNA fragments were obtained.After reverse verification,the fragments could play a role in cisplatin resistance.Bioinformatics analysis revealed 10 lncRNAs related to cisplatin resistance in osteosarcoma.These lncRNAs had some roles in cisplatin resistance.This rsRNA library provides a new tool for studying osteosarcoma drug resistance and provides a new opportunity for understanding the mechanism of drug resistance in osteosarcoma.It will provide new targets and prognostic indicators for the treatment of osteosarcoma.