Long non-coding RNA LINC01314 represses cell migration, invasion, and angiogenesis in gastric cancer via the Wnt/β-catenin signaling pathway by down-regulating KLK4

Abstract

Background: In recent years, gastric cancer (GC) has become a major cause of mortality among various malignancies worldwide with high incidence rates. Long non-coding RNA (lncRNAs) may serve as oncogenes and tumor suppressors in cancers. Therefore, we investigated the effect of LINC01314 on the development of GC cells in relation to the Wnt/β-catenin signaling pathway.

Methods: Microarray data analysis was conducted to screen GC-related differentially expressed lncRNAs, followed by determination of the binding interaction between LINC01314 and kallikrein 4 (KLK4). Human GC cell line SGC-7901 was treated with over-expressed or silenced LINC01314 or KLK4 to investigate the mechanism LINC01314 affecting GC cellular activities. The levels of KLK4, Wnt-1, β-catenin, cyclin D1, N-cadherin and E-cadherin were measured, and cell invasion and migration were evaluated. Next, the tumor weight, micro-vessel density (MVD) and the expression of VEGF-C and VEGFR-3 in transplanted tumors were measured.

Results: LINC01314 was poorly expressed in GC cells and KLK4 was revealed to be a direct target gene of LINC01314. Overexpressed LINC01314 or silencing of KLK4 led to inhibited GC cell migration and invasion, corresponding to decreased Wnt-1, β-catenin, cyclin D1 and N-cadherin while increased E-cadherin. Also, in response to over-expression of LINC01314 or silencing of KLK4, tumor weight and the MVD of transplanted tumors were reduced and angiogenesis was suppressed, which was indicated by down-regulated positive expression of VEGF-C and VEGFR-3.

Conclusion: The findings indicated that over-expression of LINC01314 down-regulated KLK4 to inhibit the activation of the Wnt/β-catenin signaling pathway, thus suppressing migration, invasion, and angiogenesis in GC cells, which provides new insight for the treatment of GC.

Keywords: Angiogenesis; Gastric cancer; Kallikrein 4; Long non-coding RNA LINC01314; Wnt/β-catenin signaling pathway.

Fig. 1

LINC01314 was identified to target the KLK4 gene. a Heat map of differentially expressed lncRNAs associated with GC. The abscissa indicates the sample number, and the ordinate represents the differentially expressed gene. The top right histogram is the color gradation. Each rectangle corresponds to a sample expression value. Red indicates high expression and green indicates low expression. b Binding sites between LINC01314 and KLK4-3′UTR predicted using the biological prediction website. c Luciferase activity detected using dual-luciferase reporter gene assay. *p < 0.05, compared with the NC group. GC gastric cancer, KLK4 kallikrein 4, lncRNA long non-coding RNA, UTR untranslated region, Wt wild type, Mut mutant

Fig. 2

The mRNA levels of KLK4, Wnt-1, β-catenin, cyclin D1 and N-cadherin were decreased while those of E-cadherin increased with elevation of LINC01314; *p < 0.05 vs. the blank group; ^#p < 0.05 vs. the OE-LINC01314 group; RT-qPCR, reverse transcription quantitative polymerase chain reaction. KLK4 kallikrein 4

Fig. 3

Over-expressed LINC0134 could inhibit the activation the Wnt/β-catenin signaling pathway by inhibiting the expression of KLK4. a The protein levels of KLK4, Wnt-1, β-catenin, cyclin D1, N-cadherin and E-cadherin detected by Western blot analysis; b the grey value of KLK4, Wnt-1, β-catenin, cyclin D1, N-cadherin, E-cadherin, and GAPDH determined by Western blot analysis; *p < 0.05 vs. the blank group; ^#p < 0.05 vs. the OE-LINC01314 group. NC negative control, KLK4 kallikrein 4, GAPDH glyceraldehyde-3-phosphate dehydrogenase

Fig. 4

Over-expressed LINC01314 inhibited cell invasion in GC. a The cells penetrating through the matrigel stained with crystal violet (×200). b statistical results of the number of the cells penetrating through the matrigel; *p < 0.05 vs. the blank group; ^#p < 0.05 vs. the OE-LINC01314 group. NC negative control, GC gastric cancer

Fig. 5

Over-expressed LINC01314 repressed cell migration in GC. a Wound-healing condition of GC cells at the 0th and 24th h; b histogram of migration ability of GC cells; *p < 0.05 vs. the blank group; ^#p < 0.05 vs. the OE-LINC01314 group. NC negative control, GC gastric cancer

Fig. 6

Over-expressed LINC01314 regulated factors related to migration and invasion in GC cells by down-regulating the expression of KLK4. a Immunofluorescence staining images of N-cadherin and E-cadherin after treatment; b the positive expression rate of N-cadherin and E-cadherin after treatment; *p < 0.05 vs. the blank group; ^#p < 0.05 vs. the siRNA group. NC negative control, KLK4 kallikrein 4

Fig. 7

Over-expressed LINC01314 or down-regulated KLK4 suppressed tumor growth in nude mice. a Representative tumors excised from the nude mice; b the tumor weight of mice; *p < 0.05 vs. the blank group; ^#p < 0.05 vs. the OE-LINC01314 group. NC negative control, GC gastric cancer, KLK4 kallikrein 4

Fig. 8

Elevated LINC01314 or depleted KLK4 decreased the MVD of tumor in nude mice (×400). a Immunohistochemical staining of CD34; b the MVD of mice; *p < 0.05 vs. the blank group; ^#p < 0.05 vs. the OE-LINC01314 group. NC negative control, GC gastric cancer, MVD micro-vessel density, KLK4 kallikrein 4, CD34 cluster of differentiation 34

Fig. 9

Over-expressed LINC01314 or KLK4 silencing repressed angiogenesis of GC. a Immunohistochemical staining of VEGF-C and VEGFR-3; b the positive expression rate of VEGF-C and VEGFR-3; *p < 0.05 vs. the blank group; ^#p < 0.05 vs. the OE-LINC01314 group. NC negative control, GC gastric cancer, KLK4 kallikrein 4, VEGF vascular endothelial growth factor, VEGFR vascular endothelial growth factor receptor