| Objective:Rhabdomyosarcoma（RMS）originates from primitive mesodermal mesenchymal tissue that differentiates into the RMS muscle.RMS is an abnormally differentiated,highly malignant soft tissue sarcoma which is the most common soft tissue malignancy in children and adolescents.The incidence of RMS is highest in children and adolescents under 20 years of age,with 4.5 cases per million,accounting for 50%of soft tissue sarcomas in children and adolescents.RMS also occurs in adults and accounts for 1%of adult solid malignancies.The World Health Organization（WHO）classifies RMS into four subtypes based on histological features:embryonal RMS（ERMS）,alveolar RMS（ARMS）,spindle cell/sclerosing RMS（SSRMS）,and pleomorphic RMS（pleomorphic RMS,PRMS）,the most common being ERMS and ARMS.ERMS occurs mostly in children under 10 years of age and accounts for 70%of all RMS,usually in the orbit,head,neck and genitourinary system of infants and children,with an overall 5-year survival rate of about 70%.ARMS is most common in adolescents and adults,usually occurs in the extremities and trunk,accounts for 20%of all RMS,is more malignant than ERMS,and has an overall 5-year survival rate of approximately 25%-30%.The current treatment for RMS is a combination of local and systemic treatments such as surgery,radiotherapy,chemotherapy,and immunotherapy.However,poor selectivity of radiotherapy and chemotherapy often produce various acute and chronic toxic side effects,which seriously affect the quality of life and treatment outcome of patients,so there is a need to find new and more effective treatments to improve the prognosis of RMS patients.Dimethylaminomicheliolide(DMAMCL,C17H27NO3)is a sesquiterpene lactone compound that releases micheliolide（MCL）slowly and persistently in vivo.DMAMCL has high oral bioavailability,high water solubility and stability,and has a strong ability to penetrate the blood-brain barrier.DMAMCL is a multi-target drug,which can act on NF-κB,PD-L1,ROS/MAPK,AKT/m TOR,AEBP1/PI3K/AKT,TNFα,Cyclin D1,BAX,Bim and other signaling pathways or molecules related to proliferation,migration,apoptosis,autophagy and metabolism to exert effective anti-inflammatory and anti-tumor effects.A previous study by our group found that DMAMCL exerts proliferation-inhibiting and apoptosis-inducing effects on rhabdomyosarcoma both in vitro and in vivo.To further clarify the mechanism of action of DMAMCL inhibition in rhabdomyosarcoma,the transcriptome of DMAMCL-acting rhabdomyosarcoma cells was sequenced and Nodal modulator2（NOMO2）with significantly up-regulated expression was screened in this study.NOMO is a type I transmembrane protein,and humans have three highly similar NOMO proteins,NOMO1,NOMO2 and NOMO3.The sequences of the genes encoding these three proteins are highly similar,and the three proteins are closely related and may have similar functions.NOMO mediates a variety of biological processes and plays an important positive regulatory role in embryonic development,cartilage development,cardiomyocyte differentiation and bone formation in zebrafish.Gene sequencing analysis revealed missense mutations in NOMO2 in mouse lung cancer transplant tumors with acquired resistance to EGFR tyrosine kinase inhibitors;NOMO2 gene amplification in circulating free DNA of patients with recurrent breast cancer;and increased copy number of NOMO2 in circulating tumor DNA of neoadjuvant chemo-resistant ovarian cancer patients prior to neoadjuvant chemotherapy.Except for the preliminary functional study that down-regulation of NOMO2 expression in lung cancer cells can increase the mortality of radiotherapy-resistant cells,in-depth functional studies on NOMO2 in tumorigenesis and development have not been reported yet.In this study,we investigated the role and possible mechanism of NOMO2 in the inhibition of RMS growth by DMAMCL through the regulation of its expression level in RMS cells.Methods:In the first part of this study,transcriptome sequencing was performed on RH30 cell line samples（three samples per group for control and 5μM DMAMCL treated groups）.Transcriptome sequencing results were analyzed for m RNA expression differences,GO and KEGG enrichment.Validation of sequencing results in three RMS cell lines（ARMS typed cell lines RH28 and RH30,ERMS typed cell line RD）by RT-PCR.The changes of NOMO2 expression levels in DMAMCL-treated RMS cells and transplanted tumor tissues of RMS mice were detected by RT-PCR and Western Blot at the m RNA and protein levels,respectively.Correlation analysis of different expression levels of NOMO2 with the prognosis of RMS patients was performed using the R2 database.The second part of this study was conducted accordingly by transfecting NOMO2si RNA or overexpression plasmids in RH28,RH30 and RD cells to silence or overexpress the expression level of NOMO2 and by applying the TGF-βpathway inhibitor ITD-1 to block the TGF-βsignaling pathway.Under different treatment conditions,the Incu Cyte Zoom Live Cell Imaging Analysis System was used to observe the changes in cell fusion rate to indirectly reflect cell proliferation.Application of CCK-8 method or clone formation assay to detect lower cell proliferation.Cell cycle detection by flow cytometry after application of propidium iodide（PI）staining.Detection of apoptosis by flow cytometry after application of AnnexinⅤ-FITC/PI staining.Application of YOYO-1 staining to detect cell death.Western Blot was applied to detect the protein expression levels of NOMO2,Cyclin D1,CDK4,CDK6,PARP,cleaved-PARP,p-Smad2/3,and T-Smad2/3.Transplanted tumors were established by subcutaneous inoculation of RMS cells in mice,and then tumor tissues were collected after administration of DMAMCL treatment for m RNA and protein assays of related molecules.The results of the study were statistically analyzed by Graph Pad Prism 8,and the difference was statistically significant at P<0.05.Results:Part I:Transcriptomic sequencing of DMAMCL-acting rhabdomyosarcoma cells and screening of potential target molecules1.Analysis of gene expression differences by transcriptome sequencing:Through transcriptome sequencing of the RH30 cell line treated with DMAMCL（5μM）,a total of1373 m RNAs with significant differences in expression were found,including 685up-regulated m RNAs and 688 down-regulated m RNAs.2.GO enrichment analysis of differential genes:（1）The results of differential gene GO enrichment analysis were reflected in three aspects:biological processes（BP）,cellular components（CC）,and molecular functions（MF）.In terms of biological processes,differentially expressed genes were mainly enriched in the regulation of metabolic processes,regulation of gene expression,system development,regulation of response to stimulus,regulation of signaling,regulation of cell differentiation,etc.In terms of cellular components,differentially expressed genes were mainly enriched in the cell part,organelles,nuclear part,lumen,membrane-bounded vesicle,etc.In terms of molecular functions,the differential genes were mainly enriched in the binding of organic cyclic compounds,nucleic acids,protein,cation,carbohydrate derivative,G protein-coupled receptor binding and protein kinase and protein dimer activity etc.（2）GO enrichment analysis for genes with large difference fold:The functions of these genes are mainly involved in biological processes such as growth,development,metabolism,molecular binding,signaling,immunity,adhesion,macromolecular complexes,transport activity,and catalytic activity.3.KEGG enrichment analysis of differential genes:The differential genes were enriched in signaling pathways such as phospholipase D,MAPK,c AMP,Notch,Wnt,insulin,T and B cells receptor,in addition to RNA transport,adherens junction,mismatch repair,insulin resistance,dorsal-ventral axis formation,alanine-aspartate-glutamate metabolism,and regulation of actin cytoskeleton.4.Validation of sequencing results:Three cell lines,RH28,RH30 and RD,were treated with DMAMCL（5μM）,and the expression levels of the top 15 up-regulated and down-regulated m RNA in the sequencing results were detected by RT-PCR.Among the first 15 up-regulated m RNAs in the sequencing results,except SYTL2 in RH28 cell line and GNAS,GNPDA1 and TGFβ1I1 in RD cell line,the expression of the remaining m RNAs was up-regulated in the DMAMCL-treated groups of the three cell lines,which was consistent with the sequencing results.Among the top 15 down-regulated m RNAs in the sequencing results,except for ST13 and SLC9A6 in RH28 cell line and CTSB,RIF1,RBM33,SLC9A6 and MLH1 in RD cell line,the expression of the remaining m RNAs was down-regulated in the DMAMCL treatment groups of the three cell lines,which was consistent with the sequencing results.Among them,NOMO2 showed a consistent and significant trend in the three cell lines.In vitro,the protein expression levels of NOMO2were significantly increased in RMS cells after treatment with DMAMCL（5μM）compared with the control group.In vivo,the m RNA and protein expression levels of NOMO2 in transplanted tumor tissues of RMS mice treated with DMAMCL（25 mg/kg once daily by gavage for 21 days）were elevated compared with the control group.5.Prognostic correlation analysis of NOMO family and RMS:Survival analysis using the R2 database revealed that the higher the expression levels of NOMO2 and NOMO3,the longer the overall survival time of RMS patients,while the lower the expression level of NOMO1,the shorter the overall survival time of RMS patients.Part II:Study of the effects and mechanisms on the inhibition of growth via upregulating NOMO2 by the DMAMCL in Rhabdomyosarcoma1.Down-regulation of NOMO2 expression in RMS cells attenuated the inhibitory effect of DMAMCL on cell proliferation.RH28,RH30 and RD cells were first transfected with NOMO2 si RNA to down-regulate its expression level,and then treated with different concentrations of DMAMCL（5μM,7.5μM,10μM）for 48h.The changes of cell fusion rate and proliferation rate were examined.The results showed that under the same DMAMCL treatment conditions,the cell fusion rate and proliferation rate of NOMO2 si RNA transfected group were significantly higher compared with control si RNA transfected group,and the difference was statistically significant.Among them,the cell fusion rate increased by 9.03%,15.42%and 19.52%,and the cell proliferation rate increased by16.10%,16.96%and 22.76%after treatment with different concentrations of DMAMCL（5μM,7.5μM and 10μM）in RH28 cells,respectively.The cell fusion rate was increased by 18.09%,21.10%and 19.91%,and the cell proliferation rate was increased by 12.11%,34.02%and 43.38%,respectively,in RH30 cells.The cell fusion rate was increased by12.06%,14.93%and 24.13%,and the cell proliferation rate was increased by 10.13%,10.05%and 25.86%,respectively,in RD cells.2.Down-regulation of NOMO2 expression in RMS cells inhibits DMAMCL-induced apoptosis.RH28,RH30 and RD cells were first transfected with NOMO2 si RNA to down-regulate its expression level,and then treated with DMAMCL（10μM）for 48h to detect apoptotic changes.The results showed that in RH28,RH30 and RD cells,the apoptosis rate increased by 23.3%,32.03%and 25.54%in control si RNA-transfected group cells treated with DMAMCL（10μM）,respectively,and the differences were statistically significant.Under the same DMAMCL treatment conditions,the apoptosis rate of NOMO2 si RNA transfected group was reduced by 12.11%,10.36%and 20.36%in RH28,RH30 and RD cells,respectively,compared with the control si RNA transfected group,and the differences were all statistically significant.In all three cell lines,the control si RNA transfected group showed a decrease in PARP protein expression level and an increase in cleaved-PARP protein expression level after DMAMCL treatment.Under the conditions of DMAMCL treatment,compared with the control si RNA transfected group,the PARP protein expression levels were all increased in the NOMO2 si RNA transfected group,while the cleaved-PARP protein expression levels were all significantly decreased.The down-regulation of NOMO2 expression blocked the DMAMCL-induced decrease in PARP protein expression and the increase in cleaved-PARP protein expression.3.Down-regulation of NOMO2 expression in RMS cells inhibits DMAMCL-induced G1 phase block in RMS cells.NOMO2 si RNA was first transfected in RH28,RH30 and RD cells to down-regulate their expression levels,and then DMAMCL（5μM）was given for 48h to detect changes in cell cycle and cell cycle-related protein expression.The results showed that in RH28,RH30 and RD cells,the proportion of G1-phase cells in control si RNA transfected group cells treated with DMAMCL increased by 12.57%,11.54%and 6.37%,respectively,and the differences were all statistically significant when compared with the group without DMAMCL treatment.Under the same DMAMCL treatment conditions,the proportion of G1-phase cells in the NOMO2 si RNA transfected group was reduced by 7.62%,7.96%and 2.53%in RH28,RH30 and RD cells,respectively,compared with the control si RNA transfected group,all with statistically significant differences.The protein expression levels of Cyclin D1,CDK4 and CDK6 were reduced in the control si RNA transfected group cells after DMAMCL treatment.Under the same DMAMCL treatment conditions,the protein expression levels of Cyclin D1,CDK4,and CDK6 in the NOMO2si RNA-transfected cells were elevated in all three RMS cell lines compared with the control si RNA-transfected group.That is,down-regulation of NOMO2 expression blocked the reduction of Cyclin D1,CDK4 and CDK6 protein expression levels induced by DMAMCL（5μM）.4.Up-regulation of NOMO2 expression in RMS cells inhibits cell proliferation.NOMO2 overexpression plasmids were transfected in RH28,RH30 and RD cells to up-regulate their expression levels,and changes in cell fusion and proliferation rates were examined after 7 days of continuous observation,and changes in clonogenic ability were examined after 14 days of continuous observation.The results showed that compared with the control group,the cell fusion rate was reduced by 24.84%,49.75%and 39.27%in RH28,RH30 and RD cells,respectively,and the cell proliferation rate was reduced by32.14%,39.07%and 45.94%,respectively;the number of clone formation was reduced by 44.58%,66.17%and 66.77%,respectively,in the NOMO2 overexpression group.The differences were all statistically significant.5.Upregulation of NOMO2 expression in RMS cells can cause G1 phase cell block.NOMO2 overexpression plasmids were transfected in RH28,RH30 and RD cells to upregulate their expression levels and detect changes in cell cycle and cell cycle-related protein expression.The results showed that the proportion of G1-phase cells in RH28,RH30 and RD cells with NOMO2 overexpression 2d group was 4.16%,5.47%and6.38%higher than the control group,respectively;the proportion of G1-phase cells in NOMO2 overexpression 5d group was 4.10%,4.95%and 6.16%higher than the control group,respectively,and the differences were statistically significant.The protein expression levels of Cyclin D1,CDK4,and CDK6 were reduced in the NOMO2overexpression group cells of all three cell lines compared with the control group.6.Upregulation of NOMO2 expression in RMS cells could not cause cell death.NOMO2 overexpression plasmids were transfected in RH28,RH30 and RD cells to upregulate their expression levels,and cell death was detected.The results showed that there was no significant change in the proportion of apoptotic cells or overall dead cells in the NOMO2 overexpression group compared with the control group,regardless of whether apoptosis was detected using flow cytometry or overall cell death was detected by YOYO1 staining.The protein expression levels of the apoptotic molecules PARP and cleaved-PARP also did not show significant changes.7.DMAMCL reduces phosphorylation levels of Smad2 and Smad3,key molecules of the TGF-βsignaling pathway,in vitro and in vivo.Cells or tumor tissues were collected after treatment of RMS cells with DMAMCL or treatment of mice inoculated with RMS transplanted tumors with DMAMCL,and changes in the expression levels of phosphorylated Smad2 and Smad3（p-Smad2 and p-Smad3）or total Smad2 and Smad3（T-Smad2 and T-Smad3）proteins in the cells or tumor tissues were detected by applying Western Blot.In vitro,the expression levels of p-Smad2 and p-Smad3 were significantly lower in the DMAMCL-treated RMS cells compared with the control group,while the expression levels of T-Smad2 and T-Smad3were not significantly different compared with the control group.In vivo,the expression levels of p-Smad2 and p-Smad3 were significantly lower in the DMAMCL-treated RMS transplant tumors compared with the control group,while the expression levels of T-Smad2 and T-Smad3 were not significantly different compared with the control group.8.TGF-βsignaling pathway inhibitor ITD-1 inhibits RMS cell proliferation.RMS cells were treated with different concentrations of TGF-βsignaling pathway inhibitor ITD-1（8μM,10μM,15μM,20μM）,and the cell fusion rate was observed continuously,and the change of cell fusion rate at 72h was analyzed.The results showed that compared with the control group,the fusion rate of RH30 cells decreased by 9.89%,17.68%,27.19%and 48.87%after treatment with different concentrations of ITD-1（8μM,10μM,15μM and 20μM）;while the fusion rate of RD cells decreased by 4.01%,9.79%,17.63%and 29.86%,respectively,all showing concentration dependent reduction,and the differences were statistically significant.9.Increased inhibition of RMS cell growth by DMAMCL in combination with ITD-1,an inhibitor of the TGF-βsignaling pathway.RMS cells were treated with DMAMCL（5μM）and ITD-1（10μM）separately or in combination,and the cell fusion rate was observed continuously,and the changes of cell fusion rate and proliferation rate at 72h were analyzed.The results showed that in RH30cells,the fusion and proliferation rates of cells in the combination drug group were reduced by 24.10%and 17.86%compared with the DAMCL-treated group,and by38.24%and 31.05%compared with the ITD-1 group;in RD cells,the fusion and proliferation rates of cells in the combination drug group were reduced by 16.14%and10.03%compared with the DMAMCL-treated group;and by 27.28%and 26.67%compared with the ITD-1 group by 27.28%and 26.67%,and the differences were statistically significant.10.NOMO2 mediates DMAMCL-induced reduction of Smad2 and Smad3phosphorylation levels.NOMO2 si RNA was transfected in RMS cells to down-regulate its expression level and DMAMCL（5μM）was administered for 48h.Western Blot was performed to detect changes in p-Smad2/3 protein expression.The results showed that the protein expression levels of p-Smad2 and p-Smad3 were reduced in RMS cells after DMAMCL treatment,and down-regulation of NOMO2 expression could increase the protein expression levels of p-Smad2 and p-Smad3;suggesting that the down-regulation of NOMO2 expression level could attenuate the inhibitory effect of DMAMCL on Smad2 and Smad3phosphorylation.After overexpression of NOMO2 in RMS cells,the levels of Smad2 and Smad3 phosphorylation in the cells were reduced compared to the control,similar to the regulation of phosphorylated Smad2 and Smad3 by DMAMCL.Conclusion:1.The transcriptome sequencing of DMAMCL-treated RMS cells provided more new targets and ideas for the study of DMAMCL mechanism of action,which enriched the theoretical basis of DMAMCL research.2.DMAMCL elevates the expression level of NOMO2 in RMS cells in vivo and in vitro.3.Down-regulation of NOMO2 expression levels in RMS cells blocked the effects of DMAMCL in inhibiting cell proliferation,inducing cell G1 phase arrest and promoting apoptosis.4.Overexpression of NOMO2 in RMS cells inhibited cell proliferation and induced cellular G1 phase arrest,but did not cause significant cell death.5.DMAMCL reduces Smad2 and Smad3 phosphorylation levels in vivo and in vitro,thus blocking the TGF-βsignaling pathway and thereby inhibiting RMS growth.6.DMAMCL regulates the TGF-βsignaling pathway through NOMO2 to exert its effect on inhibiting the growth of RMS cells. |
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