| Objective Many previous studies have demonstrated that lnc RNA HOTAIR was involved in the occurrence and development of gastric cancer via regulating miRNAs and its target genes expression levels.However,the role of miR-1277-5p in gastric cancer has not been clearly elucidated.Thus,in the present study,we investigated the whether miR-1277-5p was participated in the occurrence and development of gastric cancer and if so,its underlying mechanisms.Methods 1.High expressed lnc RNAs and genes was screened through bioinformatics methods,and Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway,String analysis and Kaplan Meier plotter was used to identified a target lnc RNA and gene.Moreover,miR-1277-5p was regarded as a common target miRNA of identified lnc RNA and gene.To demonstrate the relationship between miR-1277-5p and its upstream lnc RNA,quantitative real-time PCR(q RT-PCR)was performed to detect the expression level of miR-1277-5p in response to overpression or knockdown of its upstream lnc RNA.2.To explore the role of the lnc RNA/ miR-1277-5p /target gene in gastric cancer,Western blot and q RT-PCR were used to determine the expression levels of the target gene in gastric cancer cells and gastric cancer tissues,and two gastric cancer cell lines highly expressed target gene were selected for the following experiments.In addition,we used a Cell Counting Kit-8(CCK-8)to detect the effect of target gene on gastriccancer cells proliferation.3.MiR-1277-5p overexpressed and low-level expressed gastric cancer cell models was constructed by transient transfection,and MTT,would healing,transwell migration and invasion assay was applied to investigate the changes in the proliferation,migration,invasion and motility of gastric cancer cells.4.Dual-luciferase reporter gene assay was used to validate whether the miR-1277-5p could bind to its upstream lnc RNA and target gene.Furthermore,the alteration of target gene in response to overexpression or knockdown of upstream lnc RNA and miR-1277-5p was determined by Western blot and q RT-PCR to demonstrate the relationship among upstream lnc RNA,miR-1277-5p and target gene.Upstream lnc RNA overexpressed and low-level expressed gastric cancer cell models was constructed by transient transfection,and MTT,would healing,transwell migration and invasion assay was applied to investigate its effects on the proliferation,migration,invasion and motility of gastric cancer cells.Results 1.Bioinformatics analysis indicated that lnc RNA HOTAIR and COL5A1 were highly expressed in gastric cancer tissues,and high expressions of lnc RNA HOTAIR and COL5A1 were negatively related to overall survival(OS)of gastric cancer patients.MiR-1277-5p,a common miRNA of lnc RNA HOTAIR and COL5A1,was negatively regulated by lnc RNA HOTAIR.Therefore,the lnc RNA HOTAIR/miR-1277-5p/ COL5A1 axis was established in gastric cancer.2.The results of Western blot and q RT-PCR found that COL5A1 was overexpressed in gastric cancer cells(SGC-7901,MGC-803,AGS,HGC-27 and MKN-45)comparing with normal gastric mucosa cells(GES-1),and the expression level of COL5A1 in gastric cancer tissues was higher than that in adjacent tissues.Besides,knockdown of COL5A1 inhibited gastric cancer cells proliferation.3.In vitro experiments suggested that overexpression of miR-1277-5p could inhibited the proliferation,migration,invasion and motility of gastric cancer cells(AGS and HGC27 cell lines),and knocrdown of miR-1277-5p had the reverse effects.4.Dual-luciferase reporter gene assay revealed that miR-1277-5p could bind to lnc RNA HOTAIR and COL5A1.In addition,overexpression or knockdown of lnc RNA HOTAIR was able to promote or inhibit the expression of COL5A1,respectively.Conversely,overexpression or knockdown of miR-1277-5p could inhibit or promote the expression of COL5A1.Additionally,In vitro experiments demonstrated that overexpression of lnc RNA HOTAIR could promoted the proliferation,migration,invasion and motility of gastric cancer cells(AGS and HGC27 cell lines),and knocrdown of lnc RNA HOTAIR had the reverse effects.Conclusions 1.MiR-1277-5p negatively regulated the proliferation,migration,invasion and motility of gastric cancer cells.2.COL5A1 was a target gene of miR-1277-5p and lnc RNA HOTAIR was an upstream lnc RNA of miR-1277-5p.Lnc RNA HOTAIR promoted the expression of COL5A1 via interacting with miR-1277-5p and further promoted the proliferation,migration,invasion and motility of gastric cancer cells.3.The lnc RNA HOTAIR/miR-1277-5p/COL5A1 axis was involved in the occurrence and development of gastric cancer. |
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