Sohlh1 Modulates The Stemness And Differentiation Of Glioma Stem-like Cells By Up-regulating SFRP1

Background:Glioma is the most common fatal malignancy of the central nervous system,accounting for about 80%malignancies of the malignant primary brain tumor.Glioma can occur at any age,which is characterized by developing rapidly,aggressive,and a poor prognosis.In recent years,although progress has been made in the treatment of glioma in terms of radiotherapy,surgery,chemotherapy and targeted therapy,the results are still unsatisfactory.Recurrence after surgical resection is still a major problem,leading to treatment failure.Studies have shown that glioma stem-like cells are the main cause of glioma recurrence.Therefore,the study of the role and mechanism of glioma stem-like cells in the occurrence and development of glioma can provide a new and effective therapeutic target for glioma.There are a small number of highly tumorigenic glioma stem-like cells(GSLCs)in glioma.GSLCs have the potential for self-renewal,immortal proliferation,and multilineage differentiation,which play a key role in tumor recurrence,radio-resistance,and chemotherapy resistance.Therefore,targeting GSLCs may become a new therapeutic approach to improve the prognosis of glioma patients.Sohlh1 is one of the important members of the basic helix-loop-helix(bHLH)transcription factor family that combines with a conserved E-box sequence in the promoter region of target genes and are involved in a variety of biological behaviors including cell proliferation,differentiation,and apoptosis.Our previous experimental results had demonstrated that Sohlhl is a novel tumor suppressor and that the proliferation,migration and invasion of glioma cells can be inhibited,but its role in GSLCs remains unclear.Our previous results showed that the expression of Sohlhl was markedly decreased in GSLCs,which suggests that Sohlhl may play a role in the sternness maintenance and differentiation of GSLCs.Wnt/β-catenin signaling pathway is essential in embryogenesis,nervous system evolution,cell proliferation and sternness maintenance of cancer stem-like cells.Its abnormal activation is strongly associated with the occurrence and progression of a variety of tumors.Upon activation of the classical Wnt signaling pathway,Wnt proteins bind to cell membrane surface receptors,which causes the accumulation of β-catenin in the cytoplasm and its translocation to the nucleus.Then β-catenin is combined with LEF/TCF transcription factors and the expression of downstream target genes is promoted,such as C-myc and Cyclin D1,which enhances the self-renewal of GSLCs.Secreted Frizzled-related proteinl(SFRP1),an extracellular protein,functions as an antagonist of Wnt/β-catenin signaling.SFRP1 can competitively bind to Wnt ligands,preventing the formation of Wnt-FZD-LRP5/6 complex and inhibiting Wnt/β-catenin signaling.SFRP1 has been confirmed as a tumor suppressor in glioma,however the regulation mechanism of SFRP1 expression in GSLCs remains unknown.Preliminary studies in the laboratory had confirmed that Sohlh1 suppresses tumor cell invasion,migration and proliferation through repressing Wnt/β-catenin signaling pathway.Therefore,we presumed that Sohlh1 may inhibit the activation of Wnt signaling pathway by regulating the expression of SFRP1,thereby regulating the sternness and differentiation of GSLCs.Methods:(1)To explore the effects of Sohlh1 on the stemness of GSLCsU87MG and U251 human glioma cells were cultured in DMEM with 10%FBS.Lentivirus was transfected into the cells as well as Sohlh1 overexpression and Sohlh1 knockdown stable cell lines were prepared.GSLCs in U87MG and U251 cell lines were enriched in low adhesion dishes with serum-free medium.Mammosphere formation assay,CCK-8,immunofluorescent staining,and FACS assay were performed to analyze the effects of Sohlh1 on the stemness of GSLCs.In addition,we used q-PCR and Western-blot assays to detect the impact that Sohlh1 had on the expression of GSLC markers,such as Nestin,CD 133,CD44,SOX2,OCT4.(2)To explore the effects of Sohlh1 on the differentiation of GSLCsThe experiments of induced differentiation of stem-like cells were performed to study the effect of Sohlh1 on the differentiation of GSLCs.The effect of Sohlh1 on the expression of MAP2,GFAP and MBP were detected by q-PCR,Western-blot and immunofluorescence.(3)To explore the role of Sohlh1 on GSLCs-driven tumor growth in vivoSohlh1 overexpressing U87MG-Luc GSLCs and its control cells were injected intracranially in nude mice using a brain stereotaxic instrument(1×106/mice).Nude mice were subjected to bioluminescent imaging after three weeks of injection and observed the effect of Sohlh1 on intracranial tumor growth.Brain tumor tissues were taken from nude mice,and the expression of stemness-and differentiation-related markers in the tumor tissues were analyzed by immunohistochemistry,immunofluorescence,q-PCR and Western-blot assays.(4)To explore whether Sohlh1 inactivates Wnt/β-catenin signaling via SFRP1In GSLCs and brain xenograft tumor tissues,the effects of Sohlh1 on the expression of SFRP1 and the downstream target genes of the Wnt/β-catenin signaling pathway,including C-myc,Cyclin D1,MMP2 and MMP9 were detected by q-PCR and Western-blot assays.Whether Sohlh1 blocked the activation of Wnt/β-catenin signaling was investigated by TOP flash/FOP flash luciferase report assay.Whether Sohlh1 directly regulated the expression of SFRP1 was investigated by ChIP and luciferase reporter assays.SFRP1 overexpression and knockdown plasmids were transfected into GSLCs to explore whether SFRP1 mediated the regulation of Sohlh1 on the stemness and differentiation of GSLCs.(5)To explore the correlation between Sohlh1 and Nestin,SFRP1 in clinical glioma samplesUsing glioma tissue chip,the correlation between Sohlh1 and Nestin,SFRP1 in glioma tissues was analyzed by immunohistochemical staining.Results:(1)Sohlh1 represses the stemness of GSLCsThe results of q-PCR and Western-blot experiments showed that overexpression of Sohlh1 suppressed GSLC markers expression,Nestin,CD133,CD44,SOX2,OCT4,while knockdown of Sohlh1 increased the expression of these genes at mRNA and protein levels(P<0.01).The results of mammosphere formation experiments showed that overexpression of Sohlh1 significantly reduced the size and number of GSLC spheres(P<0.001),while knockdown of Sohlh1 increased the size and number of GSLC spheres(P<0.01).The results of CCK-8 assay showed that Sohlh1 could significantly reduce the activity of GSLCs,while knockdown of Sohlh1 enhance their activity.Immunofluorescence staining revealed that overexpression of Sohlh1 decreased the expression of GSLC markers,Nestin and CD 133,while knockdown of Sohlh1 augmented the expression of Nestin and CD133 in GSLC spheres.FACS analysis results showed that overexpression of Sohlh1 significantly decreased the proportion of Nestin and CD133 double-positive GSLCs(P<0.001),while knockdown of Sohlh1 increasing the proportion of Nestin and CD133 double-positive GSLCs(P<0.01).(2)Sohlh1 enhances the differentiation of GSLCsIn the medium containing 1%serum,stem cell differentiation experiments indicated that GSLC spheres in the Sohlh1 group were remarkably more differentiated than those in the control group,while the differentiation of GSLC spheres in the Sohlh1 knockdown group was less than that in the control group.q-PCR,Western-blot and immunofluorescence staining results demonstrated that Sohlh1 overexpression upregulated the expression of GSLC differentiation markers MAP2,GFAP and MBP,while knockdown of Sohlh1 had the opposite results(P<0.01).(3)Sohlh1 suppresses the growth of intracranial xenografts from GSLCs in vivoBioluminescent imaging and HE staining results indicated that overexpression of Sohlh1 markedly suppressed the growth of intracranial tumors in nude mice.The results of immunohistochemistry,immunofluorescence staining,q-PCR and Western-blot experiments revealed that overexpression of Sohlh1 inhibited the expression of Nestin,CD133,CD44,SOX2 and OCT4,and enhanced the expression of MAP2,GFAP and MBP at mRNA and protein levels(P<0.01).(4)Sohlh1 inactivates Wnt/β-catenin signaling via SFRP1The results of q-PCR and Western-blot experiments showed that Sohlh1 increased the level of SFRP1 expression and decreased the expression of the downstream target genes of the Wnt/β-catenin signaling pathway,including C-myc,Cyclin D1,MMP2 and MMP9,while Sohlh1 deficiency obtained the opposite results.Analysis of the TOP flash/FOP flash luciferase reporter indicated that Sohlh1 inhibited the activation of the Wnt/β-catenin signaling pathway,and knockdown of Sohlh1 promoted the activation of the Wnt/β-catenin signaling pathway(P<0.05).ChIP and luciferase report assays showed that Sohlh1 could bind to the promoter of SFRP1 and directly regulate the transcription of SFRP1.After Sohlh1 overexpression/knockdown GSLCs were transfected with SFRP1 overexpression/shRNA plasmids respectively,the results of q-PCR and Western-blot experiments showed that SFRP1 could partially block the repressive of Sohlh1 on the expression of GSLC markers and downstream target genes of the Wnt/β-catenin signaling pathway,and enhance the differentiation of GSLCs.(5)In clinical samples,Sohlh1 was negatively correlated with the expression of Nestin and positively correlated with the expression of SFRP1The immunohistochemical staining results of glioma clinical sample chip demonstrated that Sohlh1 was negatively correlated with Nestin experssion and positively correlated with SFRP1 expression(P<0.001).Conclusion:(1)Sohlh1 represses the GSLC stemness and promotes the GSLC differentiation.(2)Sohlh1 upregulates the expression of SFRP1 to inhibit the activation of Wnt/βcatenin signaling pathway in GSLCs.