The Mechanistic Study Of Long Non-coding RNA SNHG15 Promoting Chemotherapeutic Drug Resistance In Osteosarcoma Cells

BackgroundOsteosarcoma(OS)is a kind of malignant bone tumor that mainly changes from osteoblast mesenchymal cells.The peak incidence of osteosarcoma occurs in children and adolescents.About three people per million are affected by OS,and the ratio of male patients to female patients is about 2.2:1.Whereas the recent adoption of multidisciplinary treatment methods for OS such as surgery and chemotherapeutic agents have enhanced the 5-year survival rate of patients,the overall survival remains less than 70%.Besides,drug resistance,especially to cisplatin(CDDP),has compounded the challenges in the treatment of OS.Therefore,to circumvent the resistance,it is important to understand the molecular mechanisms of the chemoresistance in OS.Tumor suppressor p53(TP53)is a transcription factor that can induce the transcription of genes related to cell cycle arrest,apoptosis,and metabolism,thereby acting as a tumor suppressor.In addition to its tumor suppressive function,p53 also plays an important role in the response of malignant and non-transformed cells to numerous anticancer therapies,particularly those that cause DNA damage.The researchers found that p53 mutations are present or absent in about 50% of human cancers,and p53 alterations including structural variation(SV)and somatic nucleotide variants(SNVs)are detected in 74% of human osteosarcoma.As a crucial sequence-specific transcription factor,p53 regulates the expression of many genes,such as micro RNAs,and long nc RNAs(lnc RNAs).Therefore,exploring the expression profile of lnc RNA regulated by p53 and its molecular mechanism in osteosarcoma cells is crucial to finding the relevant targets of osteosarcoma chemotherapy resistance.In recent years,the role of lnc RNA in chemotherapy resistance of tumor cells has gradually become a research hotspot.It is an important member of the non-coding RNA family,and its length is usually more than 200 nt,with no or limited protein coding ability.Studies have found that lnc RNA can modulate gene expression at the transcriptional,post-transcriptional and epigenetic levels.Among them,the most widely studied role is to act as a "sponge" of miRNA,adsorbing and inhibiting the expression of downstream miRNA.There are few reports about lnc RNA that regulate chemotherapy resistance in osteosarcoma cells.Therefore,the study of lnc RNA and related molecular mechanisms involved in chemotherapy resistance in osteosarcoma cells can shed a new light on theory instruction and therapeutic destinations for the clinical therapy of osteosarcoma.Methods and results Part 1 Tumor suppressor gene p53 downregulated the expression of SNHG15In this part of the experiment,to assess wild type p53-altered lnc RNA expression,we used H1299 cells,p53-deficient lung adenocarcinoma cells,to establish a stable expression of wild type p53 cells,in which the p53 expression of was controlled by doxycycline.After total RNA was extracted,high-throughput sequencing of the lnc RNAs based on the Pac Bio third-generation sequencing platform was performed.We found that the expression of 1161 lnc RNAs in the p53 expression group was down-regulated.Among them,the expression of lnc RNA SNHG15 decreased by 8.65 times compared with the control group.Furthermore,q RT-PCR and RT-PCR were used to verify the expression of SNHG15 in the two groups respectively,and it was confirmed that overexpression of p53 can inhibit the expression of SNHG15.Subsequently,we found that the ectopic expression of p53 down-regulated the RNA levels of SNHG15 in a dose-dependent manner.Additionally,unlike wild type p53,the R175 H and R273 H mutants did not decrease on the expression of SNHG15.To further confirm the downregulation of the SNHG15 gene by p53 in OS,we silenced the p53 gene using two different sh RNAs in U2 OS cells.The results indicated that p53 suppression dramatically elevated the SNHG15 expression.Taken together,this data demonstrated that p53 downregulates the expression of SNHG15.Part 2 p53 directly binds to the SNHG15 gene promoterTo determine the p53-binding regions on the SNHG15 gene,we first cloned the upstream sequence of the SNHG15 gene and then transfected them into p GL3-based luciferase reporter plasmids.Dual luciferase reporter gene assay imply that the region(-2000 to-1500 bp)was a critical for the SNHG15 expression by the p53.Assessment of the sequence utilizing the JASPAR database showed a presumed p53-binding site on the promoter zone.To validate the p53 binding site,two different p GL3-based luciferase reporter plasmids containing wild type binding site(BS)and binding site mutant(BSM)were successfully constructed and transfected into 293 T cells.The data showed that overexpression of the p53 gene strongly inhibited the activity of the luciferase reporter gene harboring BS but not BSM.Subsequent Chromatin immunoprecipitation(Ch IP)assay further confirmed the above results.These data showed that the p53 protein binds to the SNHG15 promoter.Part3 SNHG15 diminishes cisplatin-induced apoptosis and ROS accumulation in osteosarcomaTo explore the functions of SNHG15 in OS cells,we overexpressed SNHG15 in U2 OS and 143 B cells.The cells were then treated with gradual increase in cisplatin concentration.Western blot,flow cytometry and CCK-8 confirmed that the overexpression of SNHG15 inhibited cisplatin-induced osteosarcoma cell apoptosis and ROS accumulation,and promoted cell proliferation.To further confirm,we silenced the expression of SNHG15 with si RNA and found that the apoptosis of osteosarcoma cells induced by cisplatin was enhanced.Finally,to verify whether p53 promotes osteosarcoma cells apoptosis induced by cisplatin through inhibiting SNHG15,we knocked down both p53 and SNHG15 and treated them with cisplatin.The results showed that the knockdown of p53 significantly reduced cisplatin-induced apoptosis,and this reduction was reversed by the knockdown of SNHG15.Taken together,these data demonstrated that SNHG15 is a key regulator in the OS chemoresistance and mediates the effect of p53 on cisplatin-induced apoptosis.Part4 SNHG15 suppresses cisplatin-induced apoptosis by acting as a ce RNA of miR-335-3p1)SNHG15 inhibits the expression of miR-335-3pInvestigations have indicated that SNHG15 can inhibit the expression of miRNA by serving as a competitive endogenous RNA(ce RNA).We use bioinformatics analysis to predict that its downstream targets are miR-7158-5p and miR-335-3p,respectively.After dual luciferase reporter gene experiments,we screened out miR-335-3p for follow-up experiments.Subsequently,SNHG15 containing miR-335-3p wild-type and mutant-type binding sites were inserted into the p SICHECK-2 reporter plasmid,and then transfected into U2 OS cells.The overexpression of miR-335-3p significantly inhibited luciferase activity,confirming that SNHG15 inhibits the expression of miR-335-3p.2)SNHG15 inhibits cisplatin-induced apoptosis by suppressing the expression of miR-335-3pTo study the role of miR-335-3p in cisplatin-induced apoptosis,we transfected miR-335-3p mimics into U2 OS cells and noticed that high level of miR-335-3p can enhance cisplatin-induced apoptosis,while the influence of miR-335-3p inhibitor was opposite.Subsequently,overexpression of miR-335-3p and SNHG15 at the same time,we found that the depressed results of SNHG15 on cisplatin-induced apoptosis was reversed by the overexpression of miR-335-3p,confirming that SNHG15 inhibits the expression of miR-335-3p by inhibiting cisplatin-induced apoptosis of osteosarcoma cells.Part5 SNHG15 decreases cisplatin-induced apoptosis and ROS accumulation via the modulation of the p53/ SNHG15/miR-335-3p/ZNF32 axis1)miR-335-3p inhibits the expression of ZNF32We predicted the downstream target gene of miR-335-3p through bioinformatics analysis,and screened out the downstream target gene related to chemotherapy resistance and oxidative stress as ZNF32.Transfecting miR-335-3p mimics into U2 OS and 143 B cells,we found that the expression of ZNF32 was inhibited;and miR-335-3p inhibitor could enhance the expression of ZNF32.Dual luciferase reporter genes showed that ZNF32 3’UTR containing wild-type binding sites can match miR-335-3p and significantly reduce luciferase activity,confirming that miR-335-3p inhibits the expression of ZNF32.2)cisplatin-induced apoptosis and ROS accumulation are regulated by p53/SNHG15/miR-335-3p/ZNF32 axisKnockdown of SNHG15 in U2 OS and 143 B cells,western blot results showed that the expression of ZNF32 was reduced,and this inhibitory effect on ZNF32 was reversed by miR-335-3p inhibitor.Subsequently,miR-335-3p was overexpressed in U2 OS cells stably knocked out of p53.Western blot showed that silencing p53 can enhance the expression of ZNF32,and this enhancement was reversed by the overexpression of miR-335-3p.These data demonstrated that the regulation of SNHG15 or p53 on the ZNF32 expression was mediated by miR-335-3p.Finally,we studied ZNF32 in the chemotherapy resistance of osteosarcoma,and the results showed that ZNF32 can inhibit cisplatin-induced osteosarcoma cell apoptosis and ROS accumulation.Taken together,cisplatin-induced apoptosis and ROS accumulation are regulated by p53/ SNHG15/miR-335-3p/ZNF32 axis.ConclusionIn the present study,we found that lnc RNA SNHG15 negatively regulated by p53,inhibited cisplatin-induced osteosarcoma cell apoptosis and ROS accumulation through the miR-335-3p/ZNF32 signal axis in further in vitro molecular mechanism experiments.The research results of this topic added new targets for the research of osteosarcoma in chemotherapy resistance,and provided new ideas for the clinical treatment of osteosarcoma.