| In recent years,accumulating studies have documented the aberrant expression of long non-coding RNAs(lnc RNAs)in a variety of tumors including hepatocellular carcinoma,breast cancer and the nervous system malignancies,prompting essential roles of these lnc RNAs in cancer initiation,progression and metastasis.Many lnc RNA can regulate the transcription of messenger RNA(m RNA).Lnc RNA is also involved in biological processes exemplified by X chromatin inactivation,genomic imprinting,cell differentiation,apoptosis and the maintenance of pluripotency.It is also established that lnc RNA is involved in the development of the central nervous system.Lnc RNA can recruit chromatin-modifying complexes,can be combined with some of the key transcription factor,can regulate gene expression at transcriptional and post transcriptional level,and can function as a competitive endogenous RNA(ce RNA)to sequester the counteract the role of micro RNAs.Therefore,the diversity of lnc RNA function providesnovel insights into the mechanisms underlying carcinogenesis and hopefully implies new opportunities for clinical treatment.Hedgehog subtype medulloblastoma(MB)is characterized by the abnormallyactivation of the Hedgehog signaling pathway.Nkx2.2-AS1 is identified by our previous gene microarray analysis as a lnc RNA significantly downregulated in mouse MB cells.It is transcribed from the antisense strand of the gene loci for the transcriptional factor Nkx2.2,which functions as a tumor suppressor in neoplasms such as Ewing’s sarcoma.Despite the impressive advances in lnc RNA studies,it remains unclear whether lnc RNAs play a regulatory role in MB pathogenesis.It deserves investigation whether individual lnc RNAs are involved in MB development,whether lnc RNAsaffect the malignant behaviors of MB cells,and whether lnc RNAs are regulated by the Hedgehog pathway transcriptional factors,Gli 1 to 3.【Aims】To determine whether Gli2 can affect the expression of Nkx2.2-AS1;to measure the expression and potential correlation of Nkx2.2-AS1 and Nkx2.2;to explore the role of both genes in the proliferation,apoptosis,and migration of the MB cell line,Daoy,which will hopefully provide a rationale for early diagnosis and treatment of MB.【Methods】1.Gli2 transcriptional factors were knocked down to determine its regulatory role on Nkx2.2-AS1 expression in MB cells.2.The expression of Nkx2.2-AS1 and Nkx2.2 in MB and normal cerebella was investigated via in situ hybridization and immunohistochemical staining.3.The construct for Nkx2.2-AS1 overexpression was generated,MB cells stably overexpressing Nkx2.2-AS1 was obtained,cell proliferation was evaluated via plate colony formation and CCK-8 assays,cell migration capability was measured via Transwell experiment,and cell cycle and apoptosis were monitored by flow cytometry.4.Nkx2.2 was knockdown in Daoy cells followed by the evaluation of its effect on cell behaviors as aforementioned.5.q RT-PCR and Western blot were used to investigate the influence of Nkx2.2-AS1 overexpression on Nkx2.2 m RNA and protein.6.Cells overexpressing Nkx2.2-AS1 were subject to Nkx2.2 knockdown,followed by CCK-8 assay for cell proliferation.【Results】1.q RT-PCR results showed that after Gli2 knockdown,the level of Nkx2.2-AS1increased.2.In situ hybridization and immunohistochemical staining showed that the expression of Nkx2.2-AS1 and Nkx2.2 in medulloblastoma is significantly lower than that of normal tissue.3.Plate clone formation experiment and CCK-8 assay showed that overexpression of Nkx2.2-AS1 can inhibit the proliferation of the neuroblastoma cell line,Daoy,whereas knockdown of Nkx2.2 can promote the proliferation of Daoy cells.4.Flow cytometry showed that overexpression of Nkx2.2-AS1 can promote the apoptosis of Daoy cells,and knockdown of Nkx2.2 inhibits the apoptosis of Daoy cells.5.Transwell assay shows that overexpression of Nkx2.2-AS1 can inhibit the migration of Daoy cells;6.Scratch optical experiments show that Nkx2.2 does not affect the migration ability of Daoy cell.7.Western blot and q RT-PCR results showed that overexpression of Nkx2.2-AS1 causes slightly increased Nkx2.2 in both m RNA and protein levels.8.CCK-8 assay showed that the knockdown of Nkx2.2 in Nkx2.2-AS1 overexpressing Daoy cells had no effect on the proliferation of Daoy cells.【Conclusion】1.The downregulation of Nkx2.2-AS1 in MB cells is mediated by the key Hedgehog pathway transcriptional factor Gli2.2.Nkx2.2-AS1 and Nkx2.2 suppresses the proliferation and facilitate the apoptosis of MB cell line,Daoy.3.Nkx2.2-AS1 inhibits the migration of Daoy cells.4.Nkx2.2-AS1 plays a suppressive role in MB in a manner largely independent of Nkx2.2. |
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