The Role Of Genome-wide SiRNA Library In Drug-resistant Osteosarcoma Cells And Its Biological Mechanism

Osteosarcoma,a histological form of primary bone cancer,has a high metastatic potential.The standard therapy is to use medications at first and then amputation.However,many patients gradually have chemotherapy resistance and even have multi-drug resistance during the treatment process,leading to the failure of treatment.Therefore,the priority is to explore the mechanism of drug resistance,and then to avoid or even reverse the therapeutic targets of drug-resistant.Our study aimed to construct effective co-delivery system for siRNAs and anti-cancer drugs,and to discover the essential genes which impact the efficacy of medicine.First,we used the siRNA library(cover 30,000 human genes)and drug to stimulate the osteosarcoma cells to Nutlin3a-resistant cell line.Afteracquired the enriched siRNA fragments via Illumina HiSeq,we transfected top 19 fragments into osteosarcoma cells and also observed drug resistance.Furthermore,we detected and compared the expression of important genes after the transfection of different siRNA sequences.At last,this research investigated the key genes and potential targets of Nutlin3a-resistant osteosarcoma cells.The main works and conclusions were included as follows:(1)We used genome-wide siRNA library plasmids to induce osteosarcoma cells SJSA1 to obtain drug-resistance(named n19-SJSA1-Nutlin3a).The lethal dose of Nutlin3 a to SJSA1 was 4?M,but the established stable drug-resistant cell line could tolerate 40?M Nutlin3 a.At the same time,the expression trends of drug-resistant genes in n19-SJSA1-Nutlin3 a were consistent to that in p53I232 S and p53E258Q(two drug-resistant cell linesas positive controls).Because p53I232 S and p53E258 Q expressed mutant P53 s,we tested the genotype of P53 inn19-SJSA1-Nutlin3 afor 30 samples to assure there was no mutation on the hot spot sequence of P53.It was indicated that the drug-resistance was stimulated by our siRNA library,and not resulted from the mutation of P53.(2)We used different concentrations of Nutlin3 a to treat the Nutlin3a-resistant SJSA1 cells,with SJSA1 parental cell line as the negative control and p53I232 S and p53E258 Q cell lines as positive controls.Through crystal violets staining,WST-1,Hoechst-stained apoptotic bodies,cleaved Caspase-3 protein immunofluorescence staining,cell cycle arrest assay and qPCR to check apoptotic pathway/ cell cycle relative genes,we found that n19-SJSA1-Nutlin3 a,p53I232S and p53E258 Q have similar drug sensitivity.In the meanwhile,the drug tolerance for these three kinds of cell lines were obviously enhanced compared to the parental cell line.(3)Epithelial–mesenchymal transition(EMT)is a process that relate to drug resistance.Through the expression of six marker genes of EMT and scratch healing experiments,the drug-resistant cell lines showed improved EMT phenomenon compared to parental SJSA1.(4)We selected cisplatin,doxorubicin,ifosfamide,methotrexate and estradiol to treat drug-resistant cell lines.Our data indicated that n19-SJSA1-Nutlin3 aalso resistant to cisplatin,doxorubicin,ifosfamide and methotrexate,while became more sensitive to estradiol.(5)The illumine HiSeq investigated enriched 19-base sequences of siRNA library plasmid in n19-SJSA1-Nutlin3 a.Sequencing results showed 7547 sequences,and we chose top 19 enriched fragments that over 1% based on different two primers TH169_PerTags(Index I)and TH170_PerTags(Index II)which used for amplification.(6)We analyzed the drug resistance after the retroviral expression vectors of 19 enriched fragments were constructedand transfected intoSJSA1 parental cell lines,separately.We found the best three fragments were NRT1,NRT2,NRT15.According to the efficiency of these fragments,we rearranged them into 3 mix sequences.Next,we over-expressed mix sequences in SJSA1,MHM and U2 OS cell lines,and used lethal dose drugs to treat these cell lines for several generations..Finally,almost all the MHM cell line were died,but the ability of drug resistance for SJSA1 and U2 OS cell lines were observed significantly.(7)The analysis of RNA-seq data and NCBI database provided drug-resistant related mRNAs and lnc RNAs which matched with NRTs enriched fragments.We used qPCR assay to compare the expression of these genes in parentalanddrug-resistant cell lines/enriched fragmentstransiently over-expressed cell lines.The results confirmed that the expression of MEG3,GAS5 and LRRC8 A decreased markedly.(8)After down-regulated the expression of MEG3,GAS5 and LRRC8 A in SJSA1 and U2 OS parental cell lines,we treated the cells with IC50 and lethal dose of Nutlin3 a.Crystal violets staining revealed that down-expression of GAS5 in SJSA1 cell line or down-expression of MEG3 and GAS5 in U2 OS cells could lose the sensitivity to Nutlin3 a.