MicroRNA-29c mediates initiation of gastric carcinogenesis by directly targeting ITGB1

Abstract

Objective: Gastric cancer (GC) remains difficult to cure due to heterogeneity in a clinical challenge and the molecular mechanisms underlying this disease are complex and not completely understood. Accumulating evidence suggests that microRNAs (miRNAs) play an important role in GC, but the role of specific miRNAs involved in this disease remains elusive. We performed next generation sequencing (NGS)-based whole-transcriptome profiling to discover GC-specific miRNAs, followed by functional validation of results.

Design: NGS-based miRNA profiles were generated in matched pairs of GCs and adjacent normal mucosa (NM). Quantitative RT-PCR validation of miR-29c expression was performed in 274 gastric tissues, which included two cohorts of matched GC and NM specimens. Functional validation of miR-29c and its gene targets was undertaken in cell lines, as well as K19-C2mE and K19-Wnt1/C2mE transgenic mice.

Results: NGS analysis revealed four GC-specific miRNAs. Among these, miR-29c expression was significantly decreased in GC versus NM tissues (p<0.001). Ectopic expression of miR-29c mimics in GC cell lines resulted in reduced proliferation, adhesion, invasion and migration. High miR-29c expression suppressed xenograft tumour growth in nude mice. Direct interaction between miR-29c and its newly discovered target, ITGB1, was identified in cell lines and transgenic mice. MiR-29c expression demonstrated a stepwise decrease in wild type hyperplasia-dysplasia cascade in transgenic mice models of GC.

Conclusions: MiR-29c acts as a tumour suppressor in GC by directly targeting ITGB1. Loss of miR-29c expression is an early event in the initiation of gastric carcinogenesis and may serve as a diagnostic and therapeutic biomarker for patients with GC.

Figure 1. Discovery and validation of miR-29c transcriptome in GC tissues by next-generation sequencing (NGS)

(A) Differential expression of 26 GC-specific miRNAs between paired gastric cancer (GC) and normal mucosa tissues (NM) in NGS analysis. (B) Identification of 4 shared miRNAs (miR-29c, miR-135b, miR-148a, and miR-204) between our data set (NGS profile) and two other data sets (Profiles #1 and 2). (C) Expression status of miR-29c in an independent validation cohort of 24 pairs of matching GC and NM tissues. ***p<0.0001, paired t-test

Figure 2. In vitro functional analysis of miR-29c

(A) Expression of miR-29c in GC cell lines was analyzed by real-time TaqMan PCR. MTT cell proliferation assay following transfection with either (B) miR-29c mimic or (C) anti miR-29c inhibitor. (D) Cell adhesion assay, in which adherence ability of GC cell to fibronectin. (E) Cell invasion assays using Matrigel-coated transwell membrane. (F) Wound-healing assay. Cell monolayers were scratched with a pipette tip and images were taken 0 h and 48 h after wound formation. *p<0.05, **p<0.01, ***p<0.001, t test.

Figure 3. In vivo GC tumorigenesis analysis in the xenograft nude mouse models

(A) Up-regulation of miR-29c expression inhibited tumor growth in the xenograft nude mouse model. Control, SNU-601 parental cell line; Vector, SNU-601 cell line transfected with empty vector; miR-29c, SNU-601 cell line transfected with miR-29c expression vector (B) Treatment of miR-29c suppressed xenograft nude mouse tumor. Either miR-29c mimic (miR-29c) or negative control (NC) were intratumorally injected using liposome at 31, 37, and 43 days after SNU-601 cells implantation. Tumor volumes represented as means ± SD. **p<0.01, ***p<0.001, two-way ANOVA test.

Figure 4. Prediction and validation of miR-29c target gene in GC cell line

(A) 135 target genes which have miR-29c seed sites were predicted via 4 different miRNA target prediction tools (miRanda, TargetScan, PicTar, and PITA). (B) Overexpression of miR-29c suppressed mRNA (qRT-PCR) and protein expression (Western blotting) of ITGB1. (C) Immunocytochemistry for ITGB1 showed low ITGB1 fluorescence intensity following miR-29c treatment. (D-E) Luciferase reporter assays revealed direct binding of miR-29c to the wild-type (WT), but not the mutant (Mut) sequences within the 3’UTR regions of TGB1. NC, transfection of negative control; miR-29c, transfection of miR-29c mimic; FL, firefly luciferase; RL, Renilla luciferase. *p<0.05, t test

Figure 5. In vitro functional analysis and expression of ITGB1 in GC

(A) Treatment of si-ITGB1 diminished mRNA (qRT-PCR) and protein expression (Western blotting) of ITGB1 in GC cell line. Down-regulation of ITGB1 by transfection with si-ITGB1 suppressed GC cell (B) adherence ability to fibronectin, (C) invasion ability into matrigel-coated transwell membrane, and (D) cell migration ability in a scratch wound healing assay. (E) Expression status of ITGB1 in human GC tissues (GC) and corresponding normal mucosa tissues (NM). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (F) Correlation analysis between miR-29c and target gene (ITGB1) expression in GC tissues. Red line represents linear regression line.*p<0.05, **p<0.01, ***p<0.001, t test.

Figure 6. Clinical relevance and in situ hybridization (ISH) expression of miR-29c in GC tissues

(A) Expression status of miR-29c in pairs of matching GC and NM tissues. (left panel, intestinal type GC; right panel, diffuse type GC; ***p<0.001, t test) (B) In situ hybridization analysis of miR-29c in matching GC and NM tissues. (Positive control, U6 snRNA, purple color; Negative control, scrambled miRNA control, pink color; Inserts show higher magnifications)

Figure 7. Functional analysis of miR-29c in gastritis (K19-C2mE mice) and gastric tumor (Gan mice) transgenic mice models

Gastritis in K19-C2mE mice and gastric tumors in K19-Wnt1/C2mE mice. (A) Macroscopic photographs and (B) H&E staining of the glandular stomach of the wild-type (left), K19-C2mE (middle), and Gan (right) mice. White arrows in middle panel of (A) indicate gastric hyperplasia lesions. White arrows in middle panel of (A) indicate gastric dysplastic gastric tumors. (C) Immunohistochemistry (IHC) staining for macrophages using F4/80 antibody in the wild-type mouse stomach (left), K19-C2mE (middle), and Gan (right) mice. (D) Expression status of miR-29c (left) and ITGB1 (middle), and correlation analysis (right) between miR-29c and target gene (ITGB1) expression in transgenic mice models. Red line represents linear regression line.*p<0.05, **p<0.01, ***p<0.001, one-way ANOVA test.