| Objective: To investigate the role of lncRNA H19 as a competitive endogenous RNA(ceRNA)in the regulation of miR-340-3p and thus promoting the expression of target gene YWHAZ and its induction of breast cancer progression as well as molecular mechanism.Methods:(1)Significantly differentially expressed lncRNAs and miRNAs were screened by microarray analysis;(2)qRT-PCR assays were performed to detect the differential expression of lncRNA H19 and miR-340-3p in MCF-7 and MCF-7/PR cells;(3)lncRNA H19 interference fragments and lentivirus overexpression vectors were constructed respectively and effect on miR-340-3p expression were detected as well as miR-340-3p effect on lncRNA H19 expression were detected;(4)CCK-8,Wound healing,Transwell and Flow cytometry analysis were used to detect the effect of lncRNA H19 as well as miR-340-3p on the proliferation,migration and apoptotic capacity of breast cancer cells;(5)Subcellular fractionation assay was conducted to determine that lncRNA H19 was enriched in the cytoplasm and nucleus of breast cancer cells;(6)Bioinformatics software and dual-luciferase reporter assays were performed to analysis the structural complementarity between lncRNA H19 and miR-340-3p as well as between miR-340-3p and target gene;(7)qRT-PCR and Western blot assays were used to examine the effect of lncRNA H19 combined with miR-340-3p on the expression of target gene;(8)Wound healing and Transwell assay were used to detect the effect of lncRNA H19 combined with miR-340-3p on the migration of breast cancer cells;(9)qRT-PCR and Western blot assays were used to detect the EMT related mRNA and protein levels by lncRNA H19 combined with miR-340-3p in breast cancer cells.(10)qRT-PCR and Western blot assays were used to detect the EMT and Wnt/?-catenin signaling pathway related protein levels by miR-340-3p combined with YWHAZ in breast cancer cells.(11)Wound healing and Transwell assay were used to detect the effect of miR-340-3p combined with YWHAZ on the migration of breast cancer cells.Results:(1)Compared with MCF-7 cell line,the expression of lncRNA H19 was up-regulated but miR-340-3p was down-regulated in MCF-7/PR cells;(2)The expression of miR-340-3p was up-regulated and down-regulated after interfering or overexpression of lncRNA H19,but lncRNA H19 expression had no significance after transfection of miR-340-3p mimics or inhibitor;(3)Interfering with lncRNA H19 was able to suppress proliferation,migration but promote apoptosis of MCF-7/PR cells,in addition,the overexpression of lncRNA H19 has the opposite effects;(4)lncRNA H19 was mainly expressed in the cytoplasm of MCF-7 and MCF-7/PR cells;(5)The luciferase activity of the transfected miR-340-3p mimics and wild-type lncRNA H19 vectors were weakened,and the luciferase activity of the transfected miR-340-3p mimics and mutant-type lncRNA H19 vectors were insignificant;(6)Bioinformatics software predicts that YWHAZ might be a target gene for miR-340-3p and YWHAZ was down-regulated in MCF-7/PR cells compared with MCF-7 parental cells,the expression of YWHAZ was down-regulated or up-regulated after transfection with miR-340-3p mimics or inhibitor by qRT-PCR and Western blot assays;(7)the luciferase activity of the transfected miR-340-3p mimics and wild-type YWHAZ vectors were weakened,and the luciferase activity of the transfected miR-340-3p mimics and mutant-type YWHAZ vectors were insignificant;(8)Interfering with lncRNA H19 in combination with miR-340-3p inhibitor was able to reverse the down-regulation of YWHAZ results of interfering with lncRNA H19 alone,in addition,the overexpression of lncRNA H19 in combination with miR-340-3p mimics has the opposite effects;(9)Interfering with lncRNA H19 in combination with miR-340-3p mimics and overexpression of lncRNA H19 in combination with miR-340-3p inhibitor,the expression level of YWHAZ was more significant than the transfection group alone;(10)Interfering with lncRNA H19 in combination with miR-340-3p inhibitor was able to promote migration of MCF-7/PR cells compared to interfer with lncRNA H19 alone,in addition,the overexpression of lncRNA H19 in combination with miR-340-3p mimics has the opposite effects;(11)Downregulation/Overexpression of lncRNA H19 combined with miR-340-3p mimics/inhibitor affected the EMT phenotype in breast cancer cells;(12)Interfering with YWHAZ in combination with miR-340-3p inhibitor affected the migration,EMT phenotype and Wnt/?-cateninin signaling pathway related protein expression of breast cancer cells.Conclusion:(1)LncRNA H19 is an oncogene in breast cancer MCF-7 cell line;(2)LncRNA H19 might sever as a ceRNA to spong miR-340-3p to regulate the expression level of target gene YWHAZ;(3)LncRNA H19 promotes the proliferation and migration of breast cancer cells,suppresses apoptosis of breast cancer cells,and promotes the EMT by modulating miR-340-3p. |
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