| Objective To identify the prognosis value and correlations with clinical characteristics of Heparan Sulfate Proteoglycan 2(HSPG2)in MDS and AML/MDS,we detected expression levels of HSPG2 in primary bone marrow of these patients.Meanwhile,this study investigated the effects of knockdown of HSPG2 on the proliferation,apoptosis and cell cycle of AML/MDS cell lines in vitro,and initially explore the mechanisms of its effects on apoptosis,proliferation related signaling pathways.In addition,the effect of Chidamide(CDM)on the growth of HSPG2 knockdown AML/MDS cells was investigated,and the potential mechanisms will be explored using network pharmacology.Material and Methods1.The primary bone marrow specimens and related clinical data were obtained from MDS and AML/MDS patients or negative controls who were diagnosed between 2018 June with 2021 December.2.The mRNA and protein expression levels of HSPG2 in these specimens was detected by RT-qPCR and Western blotting.3.To explore the correlation between the differences in HSPG2 expression and clinical characteristics of MDS and AML/MDS patients with different prognostic risks,different disease stages,and different treatment regressions by ROC curve analysis,and survival analysis.4.The mRNA expression of HSPG2 in different cell lines in vitro was detected by RT-qPCR,and the cell with the highest expression of HSPG2 was selected as the AML/MDS cell model for follow-up study.5.Furthermore,the successful construction of cell modle with knockdown of HSPG2 by CRISPR/Cas9 technology was verified by Sanger sequencing and WB assay.6.The effects of AML/MDS cells with HSPG2 knockdown or combined with CDM on the proliferation were detected by CCK8 assay.7.The effects of apoptosis and cell cycle were detected by flow cytometry assays.8.To initially explore the potential mechanism of HSPG2 affecting proliferative function,the protein expression of apoptosis-related key proteins(Caspase3,Cleaved Caspase3,Cleaved Caspase9 and Bax)were detected by WB,and the mRNA expression of key effector molecules(LATS1,LATS2,YAP1,TAZ)of proliferation-related Hippo signaling pathway were detected by RT-qPCR.9.The preliminary investigation of the potential mechanism of action of CDM combined with knockdown of HSPG2 on the proliferative function of AML/MDS using a network pharmacology strategy.Results1.In this study,bone marrow specimens were collected from 65 patients,including 20 newly diagnosed MDS patients(including 7LR-MDS cases and 13 HR-MDS cases),12 newly diagnosed AML/MDS patients,10 AML patients with CR status,3 AML patients with relapsed,and 20 negative control patients,.2.Compared with negative controls,HSPG2 mRNA expression in bone marrow specimens of newly diagnosed HR-MDS patients were increased(P < 0.05),while HSPG2 expression levels were higher in AML/MDS group compared with HR-MDS(P < 0.05).In addition,HSPG2 transcript levels significantly decreased in AML/MDS with CR groups(P <0.01),but increased again after relapse(P < 0.05).These suggested that HSPG2 might be involved in the conversion process of MDS to AML/MDS.3.ROC curve analysis of expression of HSPG2 in patients with MDS or AML/MDS suggested that the AUC value was 0.8542(P < 0.01)and the optimal cutoff value was 9.26 based on the Jorden index.According to the optimal cutoff value,all patients were divided into high and low HSPG2 expression groups.Analysis with clinical characteristics revealed that patients with high expression of HSPG2 might be associated with higher blast cell count,lower platelet count and shorter OS and LFS(P < 0.01).4.SKM-1 cell line had the highest HSPG2 mRNA expression levels relative to the other four cell lines detected by RT-qPCR assay(P < 0.01).Subsequently,CRISPR/Cas9-based lentiviral-mediated knockdown of HSPG2 in SKM-1 cells was verified by Sanger sequencing,which demonstrated mutations in the target sg RNA sequence at the DNA level,and WB assay,which revealed a decrease in HSPG2 protein levels,suggesting that HSPG2 knockdown cell model(SKM-1-KD)have been successfully constructed.5.Compared with the two control groups,the proliferation rate of SKM-1 cells with HSPG2 knockdown by gradually decreased at 48 h and72h detected by CCK8 assay(P < 0.05).In addtion,flow cytometric assays revealed a significant decrease in late and total apoptotic rates in HSPG2 knockdown cells(P < 0.01),but no significant difference in cell cycle assays.6.Further detection by WB revealed that knockdown of HSPG2 resulted in increased expression of the pro-apoptotic proteins(Bax,Cleaved Caspase3 and Cleaved Caspase9)(P < 0.05).Meanwhile,RT-qPCR assay detected the mRNA expression of key molecules of proliferation-related Hippo pathway,and found that mRNA expression of LATS1 were significantly increased(P < 0.05),while the mRNA expression of YAP1 and TAZ were decreased(P < 0.05).However,there was no significant difference in the mRNA expression of LATS2 in the KD group compared with the VC group.7.The CCK8 assay revealed that CDM monotherapy might inhibit the proliferation of SKM-1 cells in a concentration-and time-dependent manner,and the IC50 was 2.913 μmol/l and 1.138 μmol/l at 48 and 72 h,respectively.Further study revealed that targeted knockdown of HSPG2 combined with CDM intervention in SKM-1 cells showed a significantly time-dependent higher cell inhibition rate,compared with the respective single-intervention groups(P < 0.01).8.Further network pharmacological analysis revealed that the synergistic effect between HSPG2 knockdown and CDM might mediate several biological processes and multiple signaling pathways,which provides a theoretical basis for subsequent experiments to explore the specific mechanism.ConclusionIn conclusion,this study is the first report to identify HSPG2 overexpression in patients with MDS and AML/MDS,and its expression level is closely related to clinical outcomes such as prognostic risk and post-treatment disease status of patients.HSPG2 Highexpression might be associated with worse OS and LFS,which has potential to be a poor prognostic biomarker for MDS.In addition,SKM-1 cell model based on CRISPR/Cas9 mediated HSPG2 knockdown was successfully constructed in vitro.And cell experiments revealed that HSPG2 knockdown might cause inhibiting SKM-1 growth,and promoting apoptosis by mediating proliferation-related Hippo signaling pathway and inducing apoptosis-related pathway.Besides,further investigation revealed that targeted knockdown of HSPG2 could enhance the synergistic growth inhibition of SKM-1 cells by Chidamide,and the network pharmacology results suggested that anti-tumor effects of this combination might be mediated through multiple signaling pathways,which provided a theoretical basis for further investigation of the mechanism of HSPG2 in the transformation of MDS to AML/MDS and the potential of targeted therapy. |
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