| Objective: Gastric cancer(GC)is one of the most common malignant tumor in digestive system.There were approximately more than 1 million new cases and 0.8 million deaths from GC one year.At present,surgery combined with radiotherapy and chemotherapy is the primary curative treatment for the disease.However,GC is usually diagnosed in the progressing stage due to its atypical symptoms and lack of healthy checkup.Hence,there is an urgent need to improve the early diagnosis and improve the prognosis of GC patients by identifying novel targets and specific biomarker.The Encyclopedia of DNA Elements project has demonstrated that approximately 98% of the genome does not encode proteins and is thus regarded as non-coding RNA.Long non-coding RNAs(lncRNAs)are defined as a class of transcripts of more than 200 nucleotides without an open reading frame.In recent years,Studies on the role and mechanism of lncRNA in GC have become an academic hotspot.Accumulating articles have reported that lncRNAs participate in diverse neoplastic biological processes,including carcinogenesis,proliferation,metastasis and apoptosis.By analyzing the cancer genome atlas(TCGA)database,we identified a survival-related lncRNA,DLX6-AS1,which was upregulated in GC.DLX6-AS1 is a 1990 bp non-coding transcript located at chromosomal band 7q21.3 that has been reported to be overexpressed in hepatocellular cancer,osteosarcoma,pancreatic cancer,lung adenocarcinoma and ovarian cancer.Nevertheless,the carcinogenesis and regulatory mechanism of DLX6-AS1 in GC remains unknown.The research was aimed to clarify the level of DLX6-AS1 in GC tissues and cell lines,further reveal the role and potential mechanism of DLX6-AS1 through a serious of bioinformatics prediction and molecular biological experiments.At first,we detected the expression of DLX6-AS1 in GC and analyzed the relationship between expression and clinicopathological characteristics.Then,we verified the function of DLX6-AS1 in proliferation,invasion,migration and the epithelial-mesenchymal transition(EMT).Furthermore,DLX6-AS1 was able to competitively bind to miR-204-5p.At last,we further reveal the mechanism of DLX6-AS1 in GC including the target gene regulated by DLX6-AS1/miR-204-5p axis and transcriptional factor induced DLX6-AS1.Methods: Firstly,we downloaded all of the GC transcriptome profiles from TCGA database which including 375 GC tissues and 32 normal tissues.Then,we analyzed the expression of DLX6-AS1 in diverse tissues and the relationship with prognosis.We further performed qRT-PCR to detect the exression of DLX6-AS1 in 56 paired GC tissue and adjacent normal tissue,the relationship between expression and clinicopathological characteristics were analyzed.We transfected si-DLX6-AS1 into DLX6-AS1 upregulated GC cell lines SGC-7901 and AGS.DLX6-AS1 was efficiently knockdown and negative control group was constructed.CCK8,colony formation and transwell assays were performed to detect the proliferative and metastatic capacity of GC cell lines.We then conducted F-actin phalloidin staining to exam actin cytoskeleton remodeling.QRT-PCR and western blot assays were performed to detect EMT-related gene expression.Luciferase reporter assays were applied to verify whether miR-204-5p was targeted by DLX6-AS1.Furthermore,we performed cellular functional assays again to reveal the role of DLX6-AS1/miR-204-5p axis and effect on EMT of GC.We performed bioinformatics software to predict the gene regulated by miR-204-5p and verified the hypothesis by luciferase reporter assay.We then conducted cellular functional assays,detected EMT-related gene expression and actin cytoskeleton remodeling in transfected SGC-7901 and AGS cell lines.Based on JASPAR database prediction,the results of luciferase reporter assays and ChIP assays,we identified and revealed the transcriptional factor that promoting DLX6-AS1 expression.Results: 1.DLX6-AS1 is upregulated in GC.We collected and analyzed the RNA-seq data of 375 GCs and 32 normal tissues from TCGA database.The results showed that DLX6-AS1 was overexpressed in GC tissues(P = 0.0004)and was significantly associated with poor prognosis of GC patients(P = 0.042).We performed qRT-PCR to analyze the expression level of DLX6-AS1 in 56 GC and corresponding normal tissues.The result of qRT-PCR was consistent with the data in TCGA database(P < 0.01).The higher expression level in patients was associated with T3/T4 invasion(P = 0.035)and distant metastasis(P = 0.018).Furthermore,DLX6-AS1 was upregulated in GC cell lines,and SGC-7901 and AGS expressed the highest levels.2.DLX6-AS1 promotes GC proliferation,migration,invasion,and EMT-related gene expression.DLX6-AS1 was knocked down in SGC-7901 and AGS cell lines,then cellular functional assays were performed.The results of CCK8 assay and colony formation assay revealed that DLX6-AS1 downregulation reduced cell growth ability(P < 0.01).We observed that siRNA transfection significantly inhibited the migration and invasion capacity of GC cells by transwell assays(P < 0.01).Knockdown of DLX6-AS1 inhibited N-cadherin(P < 0.05),MMP9(P < 0.05),and SLUG(P < 0.05)expression and promoted Ecadherin(P < 0.01)expression according to qRT-PCR and western blot results.Moreover,the distribution of both the actin cytoskeleton and of filopodia were significantly decreased when DLX6-AS1 was knocked down.3.DLX6-AS1 acts as a competing endogenous RNA(ceRNA)by directly binding miR-204-5p.We performed bioinformatics prediction to identify the target of DLX6-AS1,so miR-204-5p was selected for further study.The results of luciferase reporter assay verified the previous hypothesis(P < 0.05).MiR-204-5p was obviously reduced in GC cell lines and tumor tissues(P < 0.01),and its expression was negatively correlated with tumor size(P = 0.003),invasion(P = 0.035),distant metastasis(P < 0.001)and AJCC staging(P = 0.033).4.Tumor promotor effects of DLX6-AS1 are partially mediated by miR-204-5p.SGC-7901 and AGS cell lines were co-transfected si-RNAs and miRNA inhibitors for functional assays,the results indicated that miR-204-5p inhibitor partially rescued the antitumor function of si-DLX6-AS1.Moreover,miR-204-5p inhibitor regulated EMT-related markers expression at the transcriptional and translational level.The results of phalloidin staining further indicated that suppression of miR-204-5p facilitated remodeling of the actin cytoskeleton.5.OCT1 is a target gene of the DLX6-AS1/miR-204-5p axis.According to the prediction of miRTarBase and molecular biological experiments,OCT1 was verified as a target of miR-204-5p.There was a significant positive correlation between OCT1 and DLX6-AS1 expression(P = 3.22E-10)and a negative relationship between miR-204-5p and OCT1 expression(P = 1.87E-4).6.OCT1 is upregulated in GC and facilitates GC progression and the EMT in vitro.According to the results of qRT-PCR and western blot assays,OCT1 was upregulated in GC.OCT1 downregulation inhibited proliferation,migration and invasion of GC cells,and this effect could be partly reversed by the miR-204-5p inhibitor(P < 0.01).Furthermore,knockdown of OCT1 reduced MMP9 and SLUG expression(P < 0.05)and promoted E-cadherin expression(P < 0.01).7.DLX6-AS1 is activated by the transcription factor OCT1 in GC.According to bioinformatics prediction,luciferase reporter assay and ChIP assay,we located the binding sites in promoter that OCT1 activated DLX6-AS1 transcription.According to qRT-PCR assays in 56 GC tissues,there was a positive correlation between OCT1 and DLX6-AS1(P = 2.31E-8).Conclusions: 1.DLX6-AS1 was upregulated in GC tissues and cell lines.2.Upregulation of DLX6-AS1 was associated with invasion and distant metastasis.3.DLX6-AS1 promotes GC proliferation,migration,invasion,actin remodeling and EMT.4.DLX6-AS1 competitively binded to miR-204-5p for its biological function.5.OCT1 was regulated by DLX6-AS1/miR-204-5p.6.DLX6-AS1/miR-204-5p/OCT1 axis promoted GC progression and EMT.7.Upregulated DLX6-AS1 is activated by the transcription factor OCT1. |
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