The Function And Mechanism Of Long Non-coding RNA-AC142119.1 In Neuroblastoma

Objective To explore the epigenetic mechanism of the abnormally high expression of MYCN in neuroblastoma from epigenetic perspective and screen out the long non-coding RNAs(LncRNAs)that are related to MYCN expression.To verify the biological function of LncRNA-AC142119.1 in vivo and in vitro and clarify the mechanism of which LncRNA-AC142119.1 regulating MYCN expression.Methods LncRNA expression microarray was used to screen out the differentially expressed LncRNAs in MYCN-amplified and non-amplified NB cell lines.The LncRNA closest to the MYCN gene position and with largest fold change was chosen using bioinformatics,RACE assay was used to verify the sequence of LncRNA-AC142119.1 in NB,and its coding ability was predicted based on the sequence.The location of LncRNA-AC142119.1 in cells was analyzed using q RT-PCR and FISH assay.56 NB tissues were collected from the Children’s Hospital of Chongqing Medical University,the expression of LncRNA-AC142119.1and MYCN in NB tissue were detected using q RT-PCR.We constructed the LncRNA-AC142119.1 overexpression lentivirus and LncRNA-AC142119.1 knockdown LNA Gapme Rs,the RNA levels of LncRNA-AC142119.1 and MYCN were detected by q RT-PCR and the protein level of MYCN was detected by Western blot after overexpression or knockdown of LncRNA-AC142119.1 in NB cell lines.The effect of LncRNA-AC142119.1 on cell proliferation was detected by CCK-8 assay,the effect of LncRNA-AC142119.1 on the cell cycle distribution was detected by flow cytometry assay,and the effect of LncRNA-AC142119.1on the xenograft model was detected by animal experiments.RNA pull-down-MS was used to detect the histone methyltransferases that can binding to LncRNA-AC142119.1,and the specific sequence of which LncRNA-AC142119.1 binding to WDR5 was verified by RNA pull-down-WB.The binding of WDR5 to LncRNA-AC142119.1 was confirmed by RIP assay.The effect of LncRNA-AC142119.1 on the H3K4me3 level in MYCN promoter region was analyzed by Ch IP-PCR.Results The differentially expressed LncRNAs in MYCN-amplified and non-amplified NB cell lines were screened by LncRNA expression microarray,and found that LncRNA-AC142119.1 was close to MYCN gene position and had the largest fold change.RACE assay confirmed that the full length of LncRNA-AC142119.1 was 924 bp,and bioinformatics analysis showed that LncRNA-AC142119.1 is a non-coding RNA.q RT-PCR and FISH experiments showed that LncRNA-AC142119.1 was localized in the nucleus.The expression of MYCN was positively correlated with LncRNA-AC142119.1 in NB tissues.The expression of MYCN was increased after overexpression of LncRNA-AC142119.1 in the NB cell line,and vice versa.CCK-8 and cell cycle assay showed that LncRNA-AC142119.1 can promote the proliferation of NB cells and enable NB cells to pass through the G1/S checkpoint.Animal experiments showed that LncRNA-AC142119.1 can significantly promote the growth of NB cells in vivo.The result of RNA pull-down-MS showed that LncRNA-AC142119.1 can bind to WDR5,and the truncation experiment showed that the main site of LncRNA-AC142119.1 binding to WDR5 is1-400 bp.The RIP experiment further confirmed that WDR5 can bind to LncRNA-AC142119.1.The result of Ch IP-PCR showed that LncRNA-AC142119.1 enhanced the H3K4me3 activity in MYCN promoter region.Conclusions LncRNA-AC142119.1 may combine with WDR5 to increase the H3K4me3 level in MYCN promoter region,which in turn promotes MYCN transcriptional activation and expression,promotes NB cells to pass the G1/S cell cycle checkpoint,and ultimately leads to the malignant proliferation of NB.