LncRNA CASC11 promoted gastric cancer cell proliferation, migration and invasion in vitro by regulating cell cycle pathway

Abstract

In this study, we aimed to investigate the effects of lncRNA CASC11 on gastric cancer (GC) cell progression through regulating miR-340-5p and cell cycle pathway. Expressions of lncRNA CASC11 in gastric cancer tissues and cell lines were determined by qRT-PCR. Differentially expressed lncRNAs, mRNAs and miRNAs were screened through microarray analysis. The relationship among CASC11, CDK1 and miR-340-5p was predicted by TargetScan and validated through dual luciferase reporter assay. Western blot assay examined the protein level of CDK1 and several cell cycle regulatory proteins. GO functional analysis and KEGG pathway analysis were used to predict the association between functions and related pathways. Cell proliferation was determined by CCK-8 assays. Cell apoptosis and cell cycle were detected by flow cytometry assay. CASC11 was highly expressed in GC tissues and cell lines. Knockdown of CASC11 inhibited GC cell proliferation, promoted cell apoptosis and blocked cell cycle. KEGG further indicated an enriched cell cycle pathway involving CDK1. QRT-PCR showed that miR-340-5p was down-regulated in GC cells tissues, while CDK1 was up-regulated. Furthermore, CASC11 acted as a sponge of miR-340-5p which directly targeted CDK1. Meanwhile, miR-340-5p overexpression promoted GC cell apoptosis and induced cell cycle arrest, while CDK1 overexpression inhibited cell apoptosis and accelerated cell cycle. Our study revealed the mechanism of CASC11/miR-340-5p/CDK1 network in GC cell line, and suggested that CASC11 was a novel facilitator that exerted a biological effect by activating the cell cycle signaling pathway. This finding provides a potential therapeutic target for GC.

Keywords: CDK1; Gastric cancer; LncRNA CASC11; cell cycle; miR-340-5p.

Figure 1.

CASC11 was overexpression in gastric cancer tissues. (a) LncRNA microarray analysis of 6 GC tissue samples and 6 corresponding adjacent normal tissues were performed. (b) Relative CASC11 expression in GC tissues compared with corresponding normal tissues (n = 6). CASC11 expression was examined by qRT-PCR analysis and normalized to GAPDH expression. The association between the expression CASC11 and (c) tumor size (d) lymph node metastasis and (e) TNM stage in GC tissues compared with corresponding normal tissues (n = 12). CASC11 expression was examined by qRT-PCR analysis and normalized to GAPDH expression. **P < 0.01. ***P < 0.001. Error bars represent the mean ± SD of triplicate experiments.

Figure 2.

CASC11-1 could promote proliferation and inhibit apoptosis of gastric cancer cells and accelerate cell cycle. (a) The relative CASC11 expression was detected in three GC cell lines (KATOⅢ, AZ521, MKN7) compared to normal gastric epithelial cell GES-1, CASC11 expression was examined by qRT-PCR analysis and normalized to GAPDH expression. (b) The CASC11 was silencing by two si-CASC11-1 and si-CASC11-2 in AZ521 or MKN7 cells. CASC11 expression was examined by qRT-PCR analysis and normalized to GAPDH expression. (c) Cell proliferation were detected by CCK-8 assays in AZ521 or MKN7 cells. (d) Apoptosis rates were verified by cell apoptosis assays in AZ521 and MKN7 cell lines. (e) Cell cycle was verified by cell cycle assays in AZ521 and MKN7 cell lines.

Figure 3.

Gene co-expression network between CASC11, miR-340-5p and CDK1. (a) Top 20 differentially expressed gene were detected through gene microarray analysis based on the limitation of |log₂ (Fold change)| >1 and adj.P.val <0.05. Genes in red indicates overexpression; those in green indicate reduced expression. (b) The correlation between CASC11 and CDK1 was predicted by Cytoscape. (c) Top 20 differentially expressed miRNA was detected through microarray analysis based on the limitation of |log₂ (Fold change)| >1 and adj.P.val <0.05. Gene in red indicate overexpression; those in green indicate reduced expression. *P < 0.05. **P < 0.01. ***P < 0.001 indicated statistical significance compared with si-NC group.

Figure 4.

GO term enrichment analysis of DEGs. (a-b) The GO annotation analysis was used for functional studies of differential genes.

Figure 5.

KEGG pathway enrichment analyses of DEGs. (a-b) The KEGG pathway analysis was used for biochemistry pathways studies in all differential genes. (c) GSEAplot showed most genes of KEGG_cell_cycle signaling pathway were discovered in the region where genes were overexpressed in GC. (d) 20 differentially expressed genes were screened which were in the cell cycle signaling pathway, and based on the limitation of |log₂ (Fold change)| >1 and adj.P.val <0.05. Gene in red indicated overexpression; those in green indicate reduced expression.

Figure 6.

CDK1 was overexpression in gastric cancer tissues and cells, but miR-340-5p was opposite. (a-b) Relative CDK1 and miR-340-5p expression in GC tissues compared with corresponding normal tissues (n = 12). CDK1 and miR-340-5p expression were examined by qRT-PCR analysis and normalized to GAPDH expression. The association between the expression CDK1 and miR-340-5p and (c-d) tumor size (E-F) lymph node metastasis and (g-h) TNM stage in GC tissues compared with corresponding normal tissues (n = 12). CDK1 and miR-340-5p expression were examined by qRT-PCR analysis and normalized to GAPDH expression. (i-j) The relative CDK1 and miR-340-5p expressions were detected in three GC cell lines (KATOⅢ, AZ521, MKN7) compared to normal gastric epithelial cell GES-1, CDK1 and miR-340-5p expressions were examined by qRT-PCR analysis and normalized to GAPDH expression. **P < 0.01. ***P < 0.001 error bars represent the mean ±SD of triplicate experiments.

Figure 7.

MiR-340-5p was targeted at CASC11, while CDK1 was targeted at miR-340-5p. (a) CDK1 protein expression levels were measured through western blot experiment. (b) Bioinformatics analysis revealed that CDK1 and miR-340-5p, CASC11 and miR-340-5p had a binding site respectively via targetScan and miRanda database. (c) Dual-luciferase reporter assay showed that miR-340-5p reduced the intensity of fluorescence in GC cells transfected with CDK1, while had no effect on the CDK1-mut vector. (d) Dual-luciferase reporter assay showed that miR-340-5p reduced the intensity of fluorescence in GC cells transfected with CASC11-wt, while had no effect on the CASC11-mut vector. **P < 0.01 indicated statistical significance compared with miR-NC group.

Figure 8.

MiR-340-5p was targeted CASC11, while CDK1 was targeted miR-340-5p. (a) The relative expression level of miR-340-5p was determined by qRT-PCR in AZ521 and MKN7 cell lines. (b) CDK1 protein expression levels were detected by western blot in AZ521 and MKN7 cell lines. (c) The relative expression level of miR-340-5p was determined by qRT-PCR in AZ521 and MKN7 cell lines. (d) CDK1 protein expression levels were detected by western blot in AZ521 and MKN7 cell lines. (e) The relative expression level of CDK1 was determined by qRT-PCR in AZ521 and MKN7 cell lines. (f) CDK1 protein expression levels were detected by western blot in AZ521 and MKN7 cell lines.

Figure 9.

Overexpression of miR-340-5p could promote apoptosis of gastric cancer cells and accelerate cell cycle. Flow cytometry performed to detect the apoptosis ratio (the sum ratio of two parts on the right) with staining by Annexin V/PI of GC cells (a) AZ521 and (b) MKN7. Cell cycle was verified by cell cycle assays in AZ521 (c) and MKN7 (d). **P < 0.01 indicated statistical significance compared with blank group. Cell cycle regulatory proteins in AZ521 (e) and MKN7 (f) cells were detected by western blot.