LINC01133 as ceRNA inhibits gastric cancer progression by sponging miR-106a-3p to regulate APC expression and the Wnt/β-catenin pathway

Abstract

Background: Gastric cancer (GC) is a common malignancy and frequent cause of cancer-related death. Long non-coding RNAs (lncRNAs) have emerged as important regulators and tissue-specific biomarkers of multiple cancers, including GC. Recent evidence has indicated that the novel lncRNA LINC01133 plays an important role in cancer progression and metastasis. However, its function and molecular mechanism in GC remain largely unknown.

Methods: LINC01133 expression was detected in 200 GC and matched non-cancerous tissues by quantitative reverse transcription PCR. Gain- and loss-of-function experiments were conducted to investigate the biological functions of LINC01133 both in vitro and in vivo. Insights into the underlying mechanisms of competitive endogenous RNAs (ceRNAs) were determined by bioinformatics analysis, dual-luciferase reporter assays, quantitative PCR arrays, TOPFlash/FOPFlash reporter assay, luciferase assay, and rescue experiments.

Results: LINC01133 was downregulated in GC tissues and cell lines, and its low expression positively correlated with GC progression and metastasis. Functionally, LINC01133 depletion promoted cell proliferation, migration, and the epithelial-mesenchymal transition (EMT) in GC cells, whereas LINC01133 overexpression resulted in the opposite effects both in vitro and in vivo. Bioinformatics analysis and luciferase assays revealed that miR-106a-3p was a direct target of LINC01133, which functioned as a ceRNA in regulating GC metastasis. Mechanistic analysis demonstrated that miR-106a-3p specifically targeted the adenomatous polyposis coli (APC) gene, and LINC01133/miR-106a-3p suppressed the EMT and metastasis by inactivating the Wnt/β-catenin pathway in an APC-dependent manner.

Conclusions: Our findings suggest that reduced expression of LINC01133 is associated with aggressive tumor phenotypes and poor patient outcomes in GC. LINC01133 inhibits GC progression and metastasis by acting as a ceRNA for miR-106a-3p to regulate APC expression and the Wnt/β-catenin pathway, suggesting that LINC01133 may serve as a potential prognostic biomarker and anti-metastatic therapeutic target for GC.

Keywords: APC; Gastric cancer; LINC01133; Progression; Wnt/β-catenin pathway; ceRNA; miR-106a-3p.

Fig. 1

LINC01133 is downregulated in GC and negatively correlates with poor prognosis in GC patients. a. Relative expression of LINC01133 detected by qRT-PCR in 200 paired GC cancer tissues and matched normal tissues. Results are presented as Δ cycle threshold (ΔCt) in tumor tissues relative to normal tissues. b qRT-PCR analysis of the relative expression of LINC01133 in nine GC cell lines and the immortalized gastric normal mucosal cell line GES-1. c, d Relative expression of LINC01133 in GC with different TNM stage and different numbers of positive regional lymph nodes. e, f Kaplan-Meier plots of the OS and PFS of GC patients with high (n = 100) and low (n = 100) levels of LINC01133. Data are presented as the mean ± SD

Fig. 2

Reduced expression of LINC01133 promotes proliferation and migration and induces the EMT in GC cells. a qRT-PCR was conducted to verify the relative expression of LINC01133 in AGS and BGC-823 cells transfected with two independent shRNAs targeting LINC01133. b CCK-8 assay of AGS and BGC-823 cells after knockdown of LINC01133. c, d Representative results of the colony formation and transwell assays of AGS and BGC-823 cells after shLINC01133–1 or shLINC01133–2 transfection. e Representative images of IF micrographs of the subcellular localization and expression of E-cadherin (green) and vimentin (red). Nuclei were counterstained with DAPI (blue). Scale bars represent 50 μm. For all quantitative results, the data are presented as the mean ± SD from three independent experiments. ^*P <  0.05 and ^**P <  0.01

Fig. 3

LINC01133 inhibits proliferation, migration and EMT in vitro and in vivo. a Relative expression of LINC01133 confirmed by qRT-PCR in SGC-7901 and HGC-27 cells with LINC01133 overexpression. b, c Proliferation and migration assays of SGC-7901 and HGC-27 cells with LINC01133 overexpression by the CCK-8 assay (b), and transwell assay (c). d Immunoblot assay to evaluate the expression of vimentin, E-cadherin, and N-cadherin proteins in AGS and BGC-823 cells after LINC01133 knockdown or in SGC-7901 cells after LINC01133 overexpression. The reference protein GAPDH was used as an internal control. Numbers shows the ratio of target protein/GAPDH (arbitrary unit). e The right armpit was injected with SGC-7901 cells transfected with LINC01133 expression vector or empty vector in upper panel. Representative images of xenograft tumors are indicated in the bottom panel. f, g Tumor volume and weight of the xenograft in LINC01133 overexpression groups and control group. h Left panel: representative images of lung metastatic nodules of LINC01133 overexpression groups and control group are indicated by blue arrows. Representative hematoxylin and eosin (H&E) staining results of corresponding lung metastatic nodules are shown in right panel. i Statistical analysis of numbers of metastatic nodules in the lung. Error bars: mean ± SD from three independent experiments. ^*P <  0.05 and ^**P <  0.01

Fig. 4

LINC01133 inhibited GC metastasis by directly targeting miR-106a-3p. a Volcano plot of different targeted miRNAs of LINC01133 on AGS cells transfected with shLINC01133–2 compared with cells transfected with scramble sequence (NC). Red marks, > 2-fold change (log2) and P <  0.05 (−log10); Green marks, < − 2-fold change (log2) and P <  0.05 (−log10). b qRT-PCR assay further confirmed the expression levels of five upregulated candidate miRNAs in 30 paired GC cancer tissues and matched normal tissues. c, d Relative expression of miR-106a-3p in AGS and BGC-823 cells transfected with miR-106a-3p mimic or inhibitor c or cells after transfection with shLINC01133 or scramble sequence (d). e Relative expression of LINC01133 in AGS and BGC-823 cells transfected with miR-106a-3p mimic or inhibitor. f Relative luciferase activities of wild type (WT) and mutated (Mut) LINC01133 reporter plasmid in human embryonic kidney (HEK) 293FT cells co-transfected with miR-106a-3p mimic. g Correlation analysis between LINC01133 and miR-106a-3p expression in 200 GC tumor tissues. h Transwell assay following low expression of LINC01133 and/or inhibition of miR-106a-3p. Representative images (left panel) and quantifications (right panel) were shown. Error bars: mean ± SD. n.s, not significant, ^*P <  0.05 and ^**P <  0.01

Fig. 5

APC is a direct target of miR-106a-3p in GC metastasis. a Different expression of metastasis-related genes were examined in AGS cells after miR-106a-3p inhibitor or negative control transfection using a human tumor metastasis PCR array. Red marks, > 2-fold change (log2) and P <  0.05 (−log10); Green mark, < − 2-fold change (log2) and P <  0.05 (−log10). b The expression levels of eight upregulated candidate mRNAs confirmed by qRT-PCR in 30 paired GC cancer tissues and matched normal tissues. c Luciferase reporter assay in HEK-293FT cells co-transfected with wide type (WT) or mutated (Mut) APC 3’-UTR reporter vector and miR-106a-3p mimic or inhibitor. d Relative expression of APC mRNA in AGS cells with LINC01133 knockdown or SGC-7901 cells with LINC01133 overexpression. e qRT-PCR was conducted to evaluate the mRNA expression of APC gene in AGS cells following reduced expression of LINC01133 and/or inhibition of miR-106a-3p. f qRT-PCR was performed to access the mRNA expression of APC gene in AGS cells following ectopic expression of miR-106a-3p and/or pCMV-APC expression vector lacking the 3’-UTR. g Representative images and quantification of cell migration in AGS cells after transfection miR-106a-3p mimic and/or pCMV-APC lacking the 3’-UTR. Error bars: mean ± SD, n = 3. ^*P <  0.05 and ^**P < 0.01

Fig. 6

LINC01133 attenuates the EMT and metastatic abilities of GC cells through APC/Wnt signaling pathway. a Identification of 93 commonly changed targeted mRNAs of miR-106a-3p from the four publicly profile datasets (TargetScan, microT-CDS, TargetMiner, and mirDIP). Different color areas represented different datasets. The cross areas meant the commonly changed mRNAs of miR-106a-3p. b GO analysis and significant enriched GO terms of 93 commonly changed targeted mRNAs in gastric cancer on their biological process (BP). The statistically significant results were defined with -log10 (P value) > 1.30 as the cut-off criterion. c, d Dual luciferase assay demonstrating the effect on TOP/FOP reporter activity in HEK-293FT cells, AGS cells transfected with shLINC01133 vector or SGC-7901 cells with LINC01133 overexpression. Results were normalized to a Renilla transfection control. e Dual luciferase assay showing the effect on TOP/FOP reporter activity in AGS cells following reduced expression of LINC01133 and/or inhibition of miR-106a-3p. f Immunoblot assay of E-cadherin, vimentin, N-cadherin, APC, and total and nuclear β-catenin proteins in AGS cells transfected with shLINC01133–2 and/or miR-106a-3p inhibitor. Numbers showed quantification of relative protein amount. GAPDH was used as an internal control. Lamin B1 was used as an endogenous control for the cell nuclear fraction. g Schematic diagram of the regulatory mechanism of LINC01133/miR-106a-3p/APC axis in the inhibition of GC proliferation and metastasis. Error bars: mean ± SD, n = 3. ^*P < 0.05 versus corresponding control