Study On The Effect And Mechanism Of Capsaicin On Osteosarcoma And Osteosarcoma Stem Cells

Background and aim: Osteosarcoma(OS)is the most common primary malignant bone cancer and the second leading cause of cancer death in children and adolescents.Distant metastasis is the difficulty in the treatment of OS.The occurrence of metastasis is an important factor affecting the 5-year survival of OS patients.The theory of cancer stem cell(CSC)explains the great inhibition of OS.It is considered to be the key point of recurrence,metastasis and drug-resistance.Therefore,it is necessary to further understand the mechanism of OS and OS-CSC development,and to find a new reliable drug for the treatment.The anti-tumor effect of phytochemical is gradually recognized.At the same time,due to the rapid development of transcriptomics,it has been found that phytochemical can be used as the regulator of long non-codingRNA(lncRNA)to inhibit the occurrence and development of tumors.In previous studies,our team demonstrated that low concentration(100u M)of capsaicin inhibited the proliferation of OS and increased its sensitivity to cisplatin.Therefore,the purpose of this study is to explore its transcriptome regulation mechanism,and continue to explore the correlation between the effect of capsaicin on metastasis of OS and the effect of capsaicin on OS stemness,so as to enrich the theoretical basis of capsaicin as a potential drug for the treatment of OS.Methods: 1)LncRNA + mRNA high-throughput sequencing was performed in 3 samples of treatment group(HOS treated with 100 u M capsaicin for 24h)and 3 samples of control group(HOS treated without capsaicin);2)Download the GSE126209 dataset from the open database GEO,which contains 11 pairs of pairedRNA sequencing samples of OS carcinoma and paracancer;3)Download the transcriptional sequencing data of osteosarcoma in TARGET-OS database;4)Using principal component analysis(PCA),weighted gene co-expression network analysis(WGCNA)and online database star Base,the OS-related competitive endogenousRNA(ceRNA)network of lncRNA-miRNA-mRNA and lncRNA-mRNA pairs were constructed;5)Through intersection analysis,the ceRNA network and lncRNA-mRNA pair of the effect of capsaicin on OS was obtained;6)Survival analysis was used to observe the relationship between OS survival and theRNAs regulated by capsaicin;7)The migration and invasion abilities of capsaicin against osteosarcoma cells,HOS and MG63,were detected by wound healing experiment and Transwell invasion experiment;8)Model genes of capsaicin inhibiting OS metastasis were screened by LASSO regression;9)The stemness changes of OS treated with low concentration of capsaicin were calculated by mRNAsi;10)Single sample gene enrichment analysis(ss GSEA)was used to observe the changes of "Cancer Hallmark" in OS treated with capsaicin;11)The effect of capsaicin on OS stemness was observed by enriching OS-CSCs from HOS with ultra-low adhesion serum-free tumor stem cells balls experiment;12)RT-q PCR was used to verify the inhibitory effect of capsaicin at different concentrations on dry factors SOX2 and EZH2 in OS-CSCs;13)To observe the change of migration ability of OS-CSCs after stemness inhibited by capsaicin;14)Correlation analysis was conducted to evaluate the relationship between OS metastasis and OS stemness.Results: 1)Five lncRNA-mRNA pairs of OS inhibited by capsaicin were screened through trans action;2)From 1116 OS-related lncRNA-miRNA-miRNAs,33 ceRNA networks composed of 3 lncRNAs,2 miRNAs and 17 mRNAs were screened out as the key ceRNA networks for the treatment of OS by capsaicin;3)MIR14HG(lncRNA),miR-196a-5p(miRNA)and WDR33(mRNA)are independent prognostic factors for the survival of OS,which were the molecules in the ceRNA network of capsaicin treatment;4)Low concentration of capsaicin can inhibit the migration and invasion of osteosarcoma;5)Low concentration of capsaicin can inhibit the metastasis of OS through 4 lncRNAs and 21 mRNAs;6)Capsaicin can inhibit the stemness of OS in a dose-dependent manner;7)Capsaicin reduced the stemness of OS mainly by inhibiting the expression of SOX2 and EZH2;8)The inhibition of OS stemness by capsaicin is closely related to the inhibition of OS metastasis by capsaicin;9)Capsaicin can inhibit the migration of OS-CSCs.Conclusions: Low concentration of capsaicin could regulate the expression of non-codingRNA and mRNA in OS,which inhibit OS through the mechanism of ceRNA and lncRNA-mRNA.Low concentration of capsaicin can inhibit the metastasis and stemness of OS,and can effectively inhibit the migration of OS-CSCs.Meanwhile,capsaicin’s inhibition of stemness is related to capsaicin’s regulation of lncRNA and mRNA in OS metastasis.In conclusion,capsaicin is a potential therapeutic agent that can inhibit OS and reduce its metastasis and stemness.