| Objective:To investigate the expression of long non-coding RNA(lncRNA)HOXD-AS2 in gastric cancer(GC)and its role in the occurrence and development of GC,and its effect on the biological behavior and specific regulatory mechanisms of GC cells.Methods:(1)According to the previous chip results,HOXD-AS2 with large expression difference was selected in the chip.Then,real-time fluorescence quantitative PCR(RT-qPCR)was used to verify the expression of HOXD-AS2 in 79 cases of GC tissues and their corresponding adjacent tissues,GC cell lines and normal gastric mucosal cells.(2)The relationship between the expression level of HOXD-AS2 and the clinicopathological characteristics of GC patients was analyzed and its prognostic value was evaluated.(3)RT-qPCR and Western-blot(WB)were used to detect the expression of HOXD8 in GC tissues and cells.The online database was used to predict the co-expressed gene HOXD8 related to HOXD-AS2 and its relationship with the prognosis of GC patients.(4)The relationship between the expression of HOXD-AS2 and HOXD8 was confirmed by circlncRNAnet prediction and spearman correlation analysis.(5)Matrigel Transwell and wound healing assays were used to confirm the effect of overexpression of HOXD-AS2 on the ability of GC cells to invasion and migration.(6)In vitro experiment,after overexpressing HOXD-AS2,the changes in the target genes HOXD8 and PI3K/Akt signaling pathway levels were observed to investigate the role and regulatory mechanism of HOXD-AS2 in GC invasion and migration.Result:(1)The expression of HOXD-AS2 was reduced in GC tissues and cells.(2)Low expression of HOXD-AS2 was associated with lymph node metastasis and TNM stage in patients with GC.HOXD-AS2 expression level could be used as an independent risk factor for GC invasion and migration.(3)Kaplan Meier Plotter database survival analysis results showed that GC patients with low expression of HOXD-AS2 had shorter survival time and worse prognosis.(4)The expression level of HOXD8 mRNA in GC tissues is significantly lower than that in adjacent tissues,and in GC cells is significantly lower than normal gastric mucosal cells.HOXD8 expression level is positively correlated with HOXD-AS2 expression level,suggesting that HOXD8 is most likely a HOXD-AS2 target regulatory gene.(5)After using a plasmid-based pc-HOXD-AS2 overexpressHOXD-AS2,the ability of GC cells SGC-7901 and SNU-1 invasion and migration were significantly inhibited.(6)At the same time,the levels of HOXD8 and p53 proteins were significantly upregulated in SGC-7901 and SNU-1 GC cells,and the expression levels of key proteins PI3K,Akt and MDM2 in the PI3K/Akt signaling pathway were suppressed.(7)After treatment of GC cells SGC-7901 and SNU-1 with a specific PI3K/Akt signaling pathway inhibitor LY294002,the expression levels of HOXD-AS2 and HOXD8 were significantly increased.Conclusion:(1)HOXD-AS2 was low expressed in GC tissues and GC cell lines,and was positively correlated with GC lymphatic metastasis and TNM stage.Survival analysis showed that patients with low HOXD-AS2 expression had shorter survival times and worse prognosis.The results suggested that HOXD-AS2 may be involved in the regulation of GC invasion and migration.(2)HOXD8 was low expressed in GC tissues and GC cell lines.The expression level of HOXD-AS2 is positively correlated with the expression level of HOXD8 mRNA.Overexpression of HOXD-AS2 can increase the level of HOXD8 protein,suggesting that HOXD8 is a target regulatory gene of HOXD-AS2.(3)Overexpression of HOXD-AS2 can inhibit the activity of the PI3K/Akt signaling pathway,which in turn can increase the expression of HOXD-AS2 and HOXD8 protein after specifically inhibiting the PI3K/Akt signaling pathway,and both can inhibit the ability of GC cells to invade and metastasize.The results suggest that HOXD-AS2 is very likely to inhibit the invasion and metastasis of GC cells by targeting the HOXD8 gene and inhibiting the PI3K/Akt signaling pathway activity... |
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