Function And Mechanism Study Of Long Non-coding RNA GCMA In Gastric Cancer Metastasis

Background:Gastric cancer(GC)is one of the most common human malignancies,with a poor prognosis and a high mortality rate worldwide.Although the development of gastroscopy in recent years has greatly improved the diagnosis of early GC and further prolonged the survival of patients with early GC,patients with advanced GC are still lack of effective treatments and their survival is poor.The main cause of disease recurrence and poor prognosis of patients with advanced GC is tumor invasion and metastasis.Tumor metastasis is a complex process involving multiple stages and multiple regulatory factors.However,the underlying molecular mechanism is still not clear.In-depth exploration of the molecular mechanism of GC invasion and metastasis will aid the discovery of effective therapeutic targets and further improve the prognosis of patients with GC.Long non-coding RNA(lncRNA)(>200nt),a subtype of non-coding RNA(ncRNA),lacks recognizable open reading frame(ORF)and protein-coding abilities.Researches have shown that lncRNA plays an important role in carcinogenesis and cancer progression.LncRNA exerts profound biological functions through a variety of regulatory paradigms including induction of chromatin remodeling,transcriptional control and post-transcriptional processing.Despite of the rapid progress of researches on lncRNA,the function of most lncRNA is still unclear and the reports of IncRNA in GC metastasis are few.In the present study,based on the analysis of lncRNA microarray,IncRNA GCMA was identified as a metastasis-related IncRNA in GC.Then the expression of GCMA was detected in independent GC samples and the relationship between the expression of GCMA and clinicopathological parameters was analyzed.The upstream regulation modes of GCMA overexpression in GC was explored at the transcriptional level.The effect of GCMA on GC cell proliferation,invasion and metastasis was investigated via the subsequent in vivo and in vitro experiments.Further efforts were made to identify miRNAs that directly bind with GCMA and the downstream proteins.And the underlying mechanism of GCMA in regulating GC progression was revealed from the view of IncRNA-miRNA-targets networks.Methods:In this study,the high-throughput lncRNA microarray was used to screen IncRNAs that were significantly associated with GC metastasis.Real-time quantitative PCR(RT-qPCR)was used to detect the expression of GCMA in GC samples and GC cell lines.And the correlation between the expression of GCMA and clinicopathological parameters(age,sex,tumor size,depth of invasion,differentiation and LNM)was further analyzed.The bioinformatics databases were used for precise genetic analysis of GCMA including the chromosome location,nucleotide length and protein-coding ability.The cytological localization of GCMA was determined by fluorescence in situ hybridization(FISH)assay and the stability of GCMA in GC cells was detected by actinomycin D assay.To identify the proximal promoter region of GCMA,we constructed the truncated vectors containing four promoter regions of GCMA and performed luciferase assays.The bioinformatics websites were used to predict the putative transcription factors binding with the proximal promoter region of GCMA.Luciferase assays,RT-qPCR and chromatin immunoprecipitation(ChIP)assays were conducted to confirm the effect of transcription factors on the expression of GCMA.The effects of GCMA on the biological activities of GC cells were examined by in vitro Transwell assays,MTS and EdU cell proliferation assays and apoptosis assays.GCMA stable-knockdown cell lines were constructed by transfecting lentivirus packaged GCMA shRNA(LU-shGCMA)to GC cells.The GCMA stable-knockdown cells were then injected into nude mice subcutaneously and through the tail veins in order to determine the effect of GCMA on the growth,invasive and metastatic abilities of GC cells in vivo.The bioinformatics websites were used to predict putative miRNAs that could bind with GCMA.And their interactions were verified by RNA immunoprecipitation(RIP)assays and luciferase assays.Methylation specific PCR(MSP)assays were performed to detect the methylation levels of miRNA promoters in GC cell lines and clinical samples,The bioinformatics websites were used to predict the target genes of miRNAs.And the regulatory effect of miRNAs on their target genes was determined by luciferase assays and western-blot assays.Immunohistochemical(IHC)assay was used to detect the protein expression of miRNA targets in GC tissues.Correlation analysis of GCMA?miRNA and target genes expression in clinical samples,luciferase assays,in vitro and in vivo rescue experiments and western-blot assays further confirmed the effect of the GCMA-miRNA-targets regulatory network on GC metastasis.Results:(1)LncRNA GCMA is a metastatic-related IncRNA in gastric cancerDifferentially expressed lncRNAs were analyzed using IncRNA microarray(GEO database:GSE72307)involving five GC samples with lymph node metastasis(LNM)and five without LNM.Analysis of microarray data revealed that IncRNA GCMA(Gastric Cancer metastasis associated IncRNA),was one of the significantly upregulated lncRNAs in GC tissues with LNM compared with those without LNM,RT-qPCR showed that GCMA was significantly upregulated in 53 GC tissues with LNM compared with 19 cases without LNM.What's more,GC cell lines,had higher GCMA expression than human gastric epithelial cell line and the expression of GCMA was correlated with LNM status.The receiver operating characteristic(ROC)curve suggested that GCMA could be used to distinguish patients with or without LNM.We also analyzed the relationship between expression of GCMA and clinicopathological features and found that high expression of GCMA was remarkably correlated with positive LNM.The Kaplan-Meier analysis and log-rank test revealed that GC patients with high expression of GCMA tended to have a reduction in overall survival(OS)and disease-free survival(DFS).Ensembl and NCBI databases showed that lncRNA GCMA,a 2198bp-long intergenic IncRNA,was located in chromosome 21.CPAT bioinformatics software predicted that GCMA did not have protein-coding ability.FISH assay showed that GCMA was located both in cytoplasm and nucleus in GC cells.Then the actinomycin D assay showed that GCMA was relatively stable in GC cells(2)SP1 activates lncRNA GCMA transcription in GC cellsThe promoter sequence of human GCMA gene was obtained from the Ensembl Genome Browser.To identify the proximal promoter region of GCMA,four regions(-746bp?-515bp?-253bp?-125bp)upstream of the transcription start site(TSS)were constructed to the pGL3-basic vector.Luciferase assays showed that the upstream 125bp promoter region was the proximal region for GCMA transcription.PROMO and JASPAR websites predicted that many transcription factors including CEBP-?,E2F1 and SP1 could bind with the proximal promoter region of GCMA.Luciferase assays and mutation experiments,RT-qPCR and ChIP assays further confirmed that SP1 could directly bind to the promoter region of GCMA,activate its transcription and thus upregulate the expression of GCMA in GC cells consequently.(3)GCMA promotes GC cell migration,invasion and growth in vitroTo explore the effect of GCMA on the biological activities of GC cells,GCMA was overexpressed and knocked down by transfecting GCMA overexpression plasmid and small interfering RNA(siRNA)targeting GCMA.Transwell assays showed that overexpression of GCMA significantly increased the migratory and invasive abilities of GC cells,whereas knockdown of GCMA dramatically inhibited the migratory and invasive abilities.MTS and EdU assays indicated that overexpression of GCMA promoted proliferation,while knockdown of GCMA inhibited growth capabilities of GC cells.However,cell apoptosis assays suggested that GCMA did not influence the apoptotic activity of GC cells.(4)GCMA promotes GC growth,invasion,and metastasis in vivoTo further investigate the role of GCMA on the biological behaviors of GC cells in an in vivo system,we first transfected GC cells with lentivirus containing GCMA shRNA(LV-shGCMA)and used puromycin to obtain stable GCMA-knockdown cells.RT-qPCR showed that the expression of GCMA was significantly lower in the LV-shGCMA group compared to the control(LV-NC).Next,we established the hematogenous metastasis model by injecting GCMA-knockdown MKN45 cells to the tail vein of the nude mice.As expected,in vivo imaging and hematoxylin and eosin(HE)staining showed that knockdown of GCMA significantly decreased lung metastatic ability of GC cells.To observe the effect of GCMA on tumor growth and local invasion,GCMA-knockdown cells were subcutaneously injected into the nude mice.Compared with the control group,the growth of the xenograft tumors in the LV-shGCMA group was significantly slower and were non-invasive or well-encapsulated.(5)GCMA functions as a ceRNA for miR-124 and miR-34a in GCNext,we explored the underlying regulatory pattern of GCMA on GC progression.After GCMA was knocked down,FISH assay showed that the cytoplasmic localization of GCMA was significantly reduced,while the nuclear localization seemed not changed,which reflected that the cytoplasmic GCMA may participate in the post-transcriptional modification of the oncogenes/tumor suppressor genes in GC.RIP assay showed that compared with anti-IgG,there was an obvious enrichment of GCMA immunoprecipitated by anti-Ago2 antibody,indicating that Ago2 may mediate the interaction between GCMA and miRNAs.Segallab and RegRNA websites predicted the putative miRNAs that combine with GCMA.Based on intersection of two websites and relative reports,19 miRNAs with tumor suppressive activity were selected.Luciferase assays and mutation experiments indicated that miR-124 and miR-34a could directly bind with GCMA through respective binding sites.MSP assays showed that the promoters of miR-124 and miR-34a were partially methylated in GC cell lines and GC tissues,indicating that the two miRNAs were partially epigenetically silenced and could be potentially absorbed by GCMA.Targetscan and miRanda websites were used to predict the target genes of miR-124 and miR-34a.Considering the function of GCMA on GC metastasis and relative reports,slug was selected to be the target gene for miR-124 and snail for miR-34a.Luciferase assays,mutation experiments and western-blot assays showed that miR-124 and miR-34a could bind with the 3'UTRs of slug and snail respectively and reduced the protein expression of slug and snail.The rescue experiments ofluciferase assays and western-blot assays revealed that GCMA could function as a ceRNA to competitively absorb miR-124 and miR-34a,thus preventing the two miRNAs binding to the 3'UTRs of slug and snail and resulting in the de-repression of slug and snail.In addition,the correlation of GCMA,miRNAs and target genes were analyzed in clinical GC samples.As expected,GCMA was negatively correlated with miR-124 and miR-34a,miR-124 and miR-34a were negatively correlated with slug and snail expression,respectively.And GCMA was negatively correlated with slug and snail expression.(6)GCMA promotes GC progression through a ceRNA pattern in vitro and in vivoTo explore whether GCMA exerts biological functions via a ceRNA pattern,a rescue experiment was performed.As expected,the decreased migratory,invasive and proliferatory abilities induced by knockdown of GCMA was regained by co-transfecting inhibitor-124 and inhibitor-34a and then abolished by introducing slug and snail shRNA in GC cells.Moreover,in the in vivo experiment,the intervention of antagomir-124 and antagomir-34a significantly abolished the decreased tumor growth in LV-shGCMA tumors,which could also be reversed by knockdown of slug and snail.In addition,western-blot assays of the xenograft tumors showed that antagomir-124 and antagomir-34a recovered the decreased protein expression of slug and snail in the shGCMA tumors,which could be reduced by slug and snail knockdown,respectively.These results demonstrated that miR-124 and slug,miR-34a and snail mediated the promotion function of GCMA on GC cell invasion,migration and growth in vitro and in vivo.(7)GCMA acts as a ceRNA to promote EMT in GC.GC cells became elongated and spindle-shaped,resembling morphology of the mesenchymal cells 48h after transfection with GCMA-overexpression plasmid.Western-blot assays indicated that overexpression of GCMA promoted EMT by reducing the expression of epithelial markers ZO-1 and E-cadherin and upregulating the expression of mesenchymal markers ?-catenin and vimentin.Whereas knockdown of GCMA inhibited EMT by increasing the expression of ZO-1 and E-cadherin and decreasing the expression of ?-catenin and vimentin.Subsequently,the rescue experiments showed that the inhibition of EMT induced by miR-124 and miR-34a could be reversed by overexpression of GCMA in GC cells and these reversions were abolished by knockdown of slug and snail.Consistently,in shGCMA cells,the inhibitory effect on EMT was eliminated by transfection of inhibitor-124 and inhibitor-34a and regained by knockdown of slug and snail.Furthermore,we explored the regulatory mechanism of GCMA in vivo.Compared with the control group,knockdown of GCMA in the xenograft tumors in the LV-shGCMA group significantly upregulated epithelial markers ZO-1 and E-cadherin and reduced mesenchymal markers ?-catenin and vimentin.These results showed that GCMA-miR-124-slug and GCMA-miR-124-snail networks regulated EMT and promoted the invasion and metastasis in GC.Conclusions:LncRNA GCMA was significantly upregulated in GC tissues with LNM,High expression of GCMA was correlated with the poor prognosis of GC patients.Transcription factor SP1 directly activated GCMA transcription and upregulated the expression of GCMA in GC.Mechanically,GCMA acted as a ceRNA to competitively absorb miR-124 and miR-34a and upregulate slug and snail,therefore promoting EMT and tumor invasion and metastasis in GC.