Targeting the SMO oncogene by miR-326 inhibits glioma biological behaviors and stemness
Targeting the SMO oncogene by miR-326 inhibits glioma biological behaviors and stemness
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miR-326 靶向 SMO 癌基因可抑制神经胶质瘤的生物学行为和干性。
DOI：
10.1093/neuonc/nou217
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发表时间：
2015-02-01
期刊：
影响因子：
15.9
通讯作者：
Jiang, Chuanlu
中科院分区：
文献类型：
--
作者：
Du, Wenzhong;Liu, Xing;Jiang, Chuanlu
背景很少有研究将microRNAs（miRNAs）与hedgehog（Hh）通路联系起来。在这里，我们研究了用miR-326靶向smoothened（SMO）是否会影响胶质瘤的生物学行为和干度。为了研究SMO和miR-326在胶质瘤标本和细胞系中的表达，我们利用定量实时（qRT）-PCR、Western印迹、免疫组织化学和荧光原位杂交。荧光素酶报告基因检测用于验证SMO和miR-326之间的关系。我们使用膜联蛋白-V标记进行细胞计数试剂盒-8，transwell和流式细胞术测定，以检测用针对SMO或miR-326的siRNA转染后的变化。利用qRT-PCR、神经球形成和免疫荧光检测U251肿瘤干细胞中自我更新和干细胞性的修饰。使用U251植入颅内模型来研究miR-326对肿瘤体积和SMO抑制功效的影响。SMO在胶质瘤中表达上调，并与肿瘤分级和生存期相关。抑制SMO可抑制胶质瘤细胞的生物学行为。SMO表达与miR-326呈负相关，并被鉴定为miR-326的新的直接靶点。miR-326过表达不仅抑制SMO及其下游基因，而且降低Hh通路的活性。此外，miR-326过表达降低了U251肿瘤干细胞的自我更新和干性，并部分促进了分化。反过来，Hh的抑制部分提高了miR-326的表达。miR-326转染可降低颅内致瘤性，且部分通过降低SMO表达介导。这项工作提示了miR-326/SMO轴的可能分子机制，这可能是胶质瘤的潜在替代治疗途径。
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 U251tumor 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.