Targeting the SMO oncogene by miR-326 inhibits glioma biological behaviors and stemness

Abstract

Background: Few studies have associated microRNAs (miRNAs) with the hedgehog (Hh) pathway. Here, we investigated whether targeting smoothened (SMO) with miR-326 would affect glioma biological behavior and stemness.

Methods: To investigate the expression of SMO and miR-326 in glioma specimens and cell lines, we utilized quantitative real-time (qRT)-PCR, Western blot, immunohistochemistry, and fluorescence in situ hybridization. The luciferase reporter assay was used to verify the relationship between SMO and miR-326. We performed cell counting kit-8, transwell, and flow cytometric assays using annexin-V labeling to detect changes after transfection with siRNA against SMO or miR-326. qRT-PCR assays, neurosphere formation, and immunofluorescence were utilized to detect the modification of self-renewal and stemness in U251 tumor stem cells. A U251-implanted intracranial model was used to study the effect of miR-326 on tumor volume and SMO suppression efficacy.

Results: SMO was upregulated in gliomas and was associated with tumor grade and survival period. SMO inhibition suppressed the biological behaviors of glioma cells. SMO expression was inversely correlated with miR-326 and was identified as a novel direct target of miR-326. miR-326 overexpression not only repressed SMO and downstream genes but also decreased the activity of the Hh pathway. Moreover, miR-326 overexpression decreased self-renewal and stemness and partially prompted differentiation in U251 tumor stem cells. In turn, the inhibition of Hh partially elevated miR-326 expression. Intracranial tumorigenicity induced by the transfection of miR-326 was reduced and was partially mediated by the decreased SMO expression.

Conclusions: This work suggests a possible molecular mechanism of the miR- 326/SMO axis, which can be a potential alternative therapeutic pathway for gliomas.

Keywords: SMO; glioma; hedgehog; miRNA; stemness.

Fig. 1.

SMO expression in glioma specimens and cell lines correlated with poor survival. (A) SMO expression in glioma specimens and normal brain tissues as assessed by qRT-PCR. (B) SMO expression in different grade gliomas between peritumor tissues and tumor tissues by Western blot assay. (C) qRT-PCR analysis showed T98G, SHG44, U251, and U251-TSC glioma cells expressed higher levels of SMO than the Olig cell line. The data represent mean ± SE of 3 replicates (*P < .05). (D) Kaplan–Meier survival curves indicating cumulative survival as a function of time for those patients with SMO high expression versus low expression. The patients with high SMO expression experienced a significantly worse outcome (*P < .05).

Fig. 2.

SMO knockdown suppressed the biological behaviors of glioma cells, and SMO was a direct target of miR-326. (A) Representative cartogram showing the cell proliferation in glioma cells and tumor stem cells regulated by SMO knockdown. (B) Representative images of in vitro transwell assays of U251 and T98G after transfection with siSMO or scramble RNA. (C) The Annexin V-PI assay reveals increased apoptosis in U251, T98G, and U251-TSC cells following siSMO treatment. The data represent mean ± SE of 3 replicates (*P < .05). (D) Diagram of the seed sequence of miR-326 matched the 3′UTRs of the SMO gene and the design of wild or mutant SMO 3′UTRs containing reporter constructs. (E) Western blot for SMO expression 48 hours after transfection with miR-Scr or miR-326. (F) Luciferase reporter assays in glioma cells after cotransfection of cells with wild-type or mutant 3′UTR SMO and miRNA. The data represent the fold change in the expression (mean ± SE) of 3 replicates.

Fig. 3.

SMO expression was inversely correlated with miR-326 expression in glioma. (A) The expression of SMO and miR-326 in glioma specimens was assessed by IHC and FISH (scale bars, 20 µm). (B) Spearman's correlation analysis was used to determine the correlation between the expression-level scores of SMO and miR-326 (Spearman's correlation analysis, r = −0.74654, P < .001). (C) qRT-PCR analysis showed that T98G, U87, U251, SHG44, and U251-TSC glioma cells expressed low levels of miR-326 compared with Olig. The data represent mean ± SE of 3 replicates (*P < .05). (D) A plot of the linear inverse correlation between the expression of SMO and miR-326 expression in U87, T98, U251, SHG44, and U251-TSC cells (P < .05, linear correlation r = −0.9007).

Fig. 4.

Upregulation of miR-326 affected the Hh signaling pathway and rescued the protumor effects of SMO. (A) A plasmid containing the entire SMO coding sequence without its 3′-UTR fragment was transfected into miR-326 or miR-Scr overexpressing cells followed by cotransfection of the reporter containing 8 directly repeated copies of a consensus GLI binding site (8×-GLI) downstream of the luciferase gene to determine the Hh pathway transcriptional activity. The data represent the mean ± SE of 3 replicates (*P < .05). (B) and (C) The overexpression of miR-326 significantly decreased the GLI1 expression, as shown by qRT-PCR with or without N-Shh compared with the miR-Scr-treated group. (D) The Western blot assay indicated that glioma cells transfected with miR-326 efficiently restrained the protein expression of GLI1, N-myc, and CyclinD1 compared with the miR-Scr-treated group. GAPDH was used as a control. (E) and (F) SMO expression levels in U251 cells transfected with SMO and/or miR-326 were assessed by Western blot. Representative cartograms showing the relative expression of the SMO protein between different groups (*P < .05). (G), (H), and (I) Representative cartograms showing that proliferation, invasion and apoptosis are regulated by miR-326 and/or SMO.

Fig. 5.

Upregulation of miR-326 regulated the ability of self-renewal and stemness and prompted differentiation in glioma stem cells. (A) miR-326 upregulation significantly decreased the percentage of neurosphere formation with or without SMO overexpression. (B) and (C) The upregulation of miR-326 decreased Bmi-1, Oct-4, Sox-2, Nanog, and Shh expression, as demonstrated by qRT–PCR and Western blot assays. (D) and (E) The overexpression of miR-326 partly decreased nestin expression and increased the GFAP expression in U251-TSC cells, as determined by immunofluorescence (scale bars, 50 µm). (F) Inhibition of the Hh pathway by antagonists that bind to SMO (cyclopamine,10uM) elevated the miR-326 expression in glioma cells. The data represent mean ± SE of 3 replicates (*P < .05).

Fig. 6.

Overexpression of miR-326 inhibited tumor growth in vivo and prolonged survival. (A) Luminescence imaging for miR-326–treated U251-luc tumors versus scramble-treated controls. (B) H&E-stained coronal brain sections showing representative tumor xenografts. (C) miR-326 and SMO expression in an intracranial graft assessed by RT-PCR and Western blot. (D) SMO and GLI1 expression after transfecting miR-326 in tumor sections following IHC analysis (*P < .05; the bars represent 25 μm). (E) Kaplan-Meier survival curves indicating that mice transfected with miR-326 showed a significantly better outcome than the miR-Scr-treated group (*P < .05).