LncRNA LOC441178 Overexpression Inhibits The Proliferation And Migration Of Esophageal Carcinoma Cells Via Methylation Of MiR-182

Objective: This article explores the role of lncRNA LOC441178 overexpression in EC,and provides new ideas for the diagnosis and treatment of esophageal cancer.Methods: The human esophageal squamous cell carcinoma(ESCC)cell lines TE-9 and Eca-109 were transfected to establish an ESCC cell line(TE-9 and Eca-109)with stable LOC441178 overexpression,Real-time quantitative polymerase chain reaction(RT-qPCR)was used to analyze the expression of LOC441178 in TE-9 and Eca-109 to verify the transfection effect,thereby confirming the successful construction of the LOC441178 stable overexpression cell line.The proliferation ability of TE-9 and Eca-109 cells transfected with LOC441178-OE was analyzed by CCK-8 experiment;5-Ethynyl-2′-deoxyuridine(EdU)assays test positive cells were used to evaluate cell proliferation after transfection.Flow cytometry assay was used to evaluate cell apoptosis at 72 hours after transfection of TE-9 and Eca-109 cells with LOC441178 plasmids in the absence or presence of miR-182 agomir-OE.The cell migration ability was measured by the transwell migration assay at 24 hours after transfection.At the same time,the cell migration ability was measured by the Wound healing assay at 0 and 48 hours after transfection.TE-9 and Eca-109 cells were infected with LOC441178-OE plasmids for 72 h.RT-qPCR was used to detect the levels of miR-21,miR-182,miR-141,miR-424 and miR-543 in TE-9 and Eca-109 cells.In addition,methylation-specific PCR(MSP)and bisulfite sequencing PCR(PSP)were used to detect the DNA methylation status of the miR-182 promoter in ESCC cells overexpressing LOC441178.TE-9 and Eca-109 cells were infected with LOC441178-OE plasmids in the absence or presence of miR-182 agomir.Expression levels of p-Akt,p-FOXO3 a,FOXO3a and cleaved caspase 3 in TE-9 cells were detected with Western blotting.The relative expression of p-Akt in TE-9 cells was quantified via normalization to Akt.The relative expressions of p-FOXOA3 a,FOXO3a and cleaved caspase 3 in TE-9 cells were quantified via normalization to β-actin.Animals were randomized into two groups: control and LOC441178-OE groups.TE-9cells stably expressing LOC441178-OE cells were injected subcutaneously into the left flank of nude mice.Tumor volume was monitored every week with a caliper.After 28 days of treatment,mice were sacrificed,and the entire tumors were weighed.Expression levels of p-Akt,FOXO3 a,cleaved caspase 3 in tumor tissues were detected with Western blotting.The relative expression of p-Akt in tumor tissues was quantified via normalization to Akt.The relative expressions of p-FOXO3 a,FOXO3a and cleaved caspase 3 in tumor tissues were quantified via normalization to β-actin.LOC441178 level in tumor tissues was analyzed by RT-qPCR.All statistical analyses were performed using Graph Pad Prism software(version 7.0,La Jolla,CA,USA).P<0.05 were considered statistically significant.Results:1.The level of LOC441178 was significantly upregulated in TE-9 and Eca-109 cells following transfection with LOC441178-OE plasmids,it showed that we established ESCC cell lines(TE-9 and Eca-109)with LOC441178 stable overexpression.2.CCK-8 assay indicated that overexpression of LOC441178 markedly suppressed the viability of TE-9 and Eca-109 cells,compared with the vector-ctrl group.Moreover,upregulation of LOC441178 notably inhibited proliferation of TE-9 and Eca-109 cells,as determined using EdU assays.3.Overexpression of LOC441178 notably downregulated the level of miR-182 in TE-9and Eca-109 cells,while no significant changes were observed in the levels of miR-21,miR-141,miR-424 and miR-543 in these conditions in TE-9 and Eca-109 cells.4.Overexpression of LOC441178 in TE-9 and Eca-109 cells significantly decreased DNA methylation levels in the promoter region of miR-182.This data was confirmed by bisulfite sequencing PCR5.Flow cytometry assay indicated that overexpression of LOC441178 markedly induced apoptosis of TE-9 and Eca-109 cells,and that effect was reversed in the presence of miR-182 agomir.These data indicated that overexpression of LOC441178 could induce apoptosis of ESCC cells through inhibition of miR-182.6.Transwell migration and wound healing assays showed that overexpression of LOC441178 notably inhibited the migration ability of TE-9 cells,and this phenomenon was reversed following transfection with miR-182 agomir.hese results suggested that overexpression of LOC441178 could inhibit the migration abilities of ESCC cells through inhibition of miR-182.7.Western blot showed that overexpression of LOC441178 decreased phosphorylation of Akt(p-Akt)and phosphorylation of FOXO3a(p-FOXO3a)and increased expressions of FOXO3 a and cleaved caspase 3 in TE-9 cells;however,these changes were reversed by miR-182 overexpression.These results illustrated that overexpression of LOC441178 could inhibit the growth of ESCC cells via regulating the miR-182/Akt FOXO3 a axis.8.To investigate the role of LOC441178 in regulating the tumor growth of ESCC in vivo,TE-9 and Eca-109 subcutaneous xenograft models were established.overexpression of LOC441178 significantly inhibited the tumor volume and tumor weight of TE-9subcutaneous xenografts,compared with control group.In addition,overexpression of LOC441178 obviously decreased p-Akt and p-FOXO3 a and increased the expressions of FOXO3 a and cleaved caspase 3 in tumor tissues,compared with control group.hese data revealed that overexpression of LOC441178 could inhibit tumorigenesis of ESCC subcutaneous xenografts in vivo.Conclusion:1.LOC441178 may serve as a tumor-suppressive role in ESCC.2.LOC441178 might inhibit the growth of ESCC cells via suppressing miR-182 expression.3.LOC441178 could inhibit the expression of miR-182 in ESCC cells via DNA methylation.4.Overexpression of LOC441178 induced apoptosis and inhibited migration in ESCC cells via inhibiting miR-182/Akt/FOXO3 a pathway.