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. 2014 Apr 30;5(8):2318-29.
doi: 10.18632/oncotarget.1913.

Overexpression of lncRNA H19 enhances carcinogenesis and metastasis of gastric cancer

Hao Li  1 , Beiqin Yu, Jianfang Li, Liping Su, Min Yan, Zhenggang Zhu, Bingya Liu
Affiliations

Overexpression of lncRNA H19 enhances carcinogenesis and metastasis of gastric cancer

Hao Li et al. Oncotarget. .

Abstract

Long non-coding RNAs (lncRNAs) play key roles in the progression and metastasis of some carcinomas. We previously showed that the expression of lncRNA H19 (H19) was higher in gastric cancer (GC) tissues than that in paired noncanerous tissues. However, the underlying mechanisms remain unclear. In this study, H19/miR-675 knockdown models in the MKN45 cell line and ectopic expression models in the SGC7901 cell line were established, and a co-expression network of H19 was generated to identify target genes by RIP and DLR. The results showed that overexpression of H19 promoted the features of GC including proliferation, migration, invasion and metastasis. An H19 co-expression network identified ISM1 as a binding protein of H19, and its expression was positively correlated with that of H19. CALN1 was identified as a target gene of miR-675 and its expression was negatively correlated with that of miR-675. H19 and MiR-675 function in a similar manner. However, H19 RNA actively binds to ISM1 and miR-675 targets CALN1. These differences suggest that H19 plays other roles besides encoding miR-675 in GC. Our results suggest that the effect of H19 in GC is mediated by the direct upregulation of ISM1 and the indirect suppression of CALN1 expression via miR-675.

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Figures

Fig1
Fig1. H19 and miR-675 are systematically up-regulated in gastric cancer and H19 has prognostic value for survival
A) Heat map representing unsupervised hierarchical clustering of 48 lncRNAs expression values of a panel of 32 gastric cancers relative to paired noncancerous tissues. Each column represents the paired tissue samples. Each row indicates 48 candidate lncRNAs based on the 3 limitations at the 1st screening. H19 is one such transcript (absolute fold change=6.6, P=0.0000361). B) Relative expression of H19 in 74 paired gastric cancer tumor tissues and noncancerous normal tissues. Statistical difference was analyzed by Wilcoxon signed–rank test (P=0.017). The relative expression of miR-675 showed the same pattern (data not shown). The H19 expression level was measured by quantitative reverse transcription-PCR. C) Kaplan-Meier curves for overall survival of the 74 patients with gastric cancer by H19 expression in tumor tissues (P=0.036). D) Positive correlation of H19 and miR-675 expression in 74 gastric cancer tissues.
Fig2
Fig2. H19 and miR-675 promote cell proliferation of gastric cancer cells
A) Relative expression of H19 and miR-675 in human gastric cancer cell lines and human immortalized gastric epithelial cell line (GES-1) measured by quantitative reverse transcription-PCR. B) Cell proliferation was measured by CCK-8 assay. MKN45 cells were knock-down H19 or miR-675 expression by siRNA respectively (left panel). SGC7901 cells were transfected with H19 or miR-675 as well as H19 with 1st exon depletion (H19mu) (right panel). The CCK-8 assay was performed every 24h for 5 days and the results were means of sextuplicate.
Fig3
Fig3. H19/miR-675 promotes migration and invasion of MKN45 or SGC7901 cells based on transwell and wound-healing assay
A) Representative photographs of migratory cells on the membrane (magnification, 100x). B) Representative photographs of invasion cells on the membrane (magnification, 100x). The right panel of each row in A and B is the average cell number of triplicate (*, **, *** p<0.05). MKN45 cells were knock-down H19 or miR-675 expression (upper panel). SGC7901 cells were transfected with H19 or miR-675 as well as H19 with 1st exon depletion (H19mu) (lower panel). C)The rate of migration was measured by quantifying the total distance from the edge of the wound toward the other side (distance between two lines) for 2 or 3 days.MKN45 cells were knock-down H19 or miR-675 expression (left panel). SGC7901 cells were transfected with H19 or miR-675 as well as H19 with 1st exon depletion (H19mu) (right panel).
Fig4
Fig4. H19 or miR-675 enhances tumor growth and metastasis in vivo
A) Photographs of tumors derived from SGC7901 cells six weeks after transfected with H19, miR-675 and H19mu, respectively. B) The tumor growth curves of SGC7901 ectopic expression subgroups showed the same pattern as cell proliferation assay. C) Average weight of tumors derived from SGC7901 cells six weeks after transfected with H19, miR-675 and H19mu, respectively. D) Average number of peritoneal metastatic nodules in H19/mioR-675 expression knock-down of MKN45 cells (left panel) and H19/miR-675 and H19mu ectopic expression of SGC7901 cells (right panel).
Fig5
Fig5. Co-expression network of H19 and the identification of the H19 binding protein
A)Photograph of the H19 co-expression network was built on the data from microarray and identified by the Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) database (up-regulated: full lines, down-regulated: dotted lines). B) The expression of H19 in the RBP Immunoprecipitation by RIP assay measured by quantitative reverse transcription-PCR. C) The ISM1 protein level in H19/miR-675 knock-down of MKN45 cells and ectopic expression of SGC7901 cells.
Fig6
Fig6. miR-675 targets CALN1 expression
A) Relative luciferase activity of CALN1construct with miR-NC was significantly increased, however, the changes in the luciferase activity of WIT1 and RUX1 were not observed. B) The CALN1 protein level in H19/miR-675 knock-down of MKN45 cells and ectopic expression of SGC7901 cells.
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