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. 2019 Jul 23;39(7):BSR20181434.
doi: 10.1042/BSR20181434. Print 2019 Jul 31.

MicroRNA-140-5p inhibits cell proliferation, migration and promotes cell apoptosis in gastric cancer through the negative regulation of THY1-mediated Notch signaling

Kun Wu  1   2 , Jun Zou  1   2 , Chao Lin  2 , Zhi-Gang Jie  3
Affiliations

MicroRNA-140-5p inhibits cell proliferation, migration and promotes cell apoptosis in gastric cancer through the negative regulation of THY1-mediated Notch signaling

Kun Wu et al. Biosci Rep. .

Retraction in

Expression of concern in

Abstract

Studies have highlighted the importance of microRNAs (miRs) in the development of various cancers, including gastric cancer (GC), a commonly occurring malignancy, accompanied by high recurrence and metastasis rate. The aim of the current study was to investigate the role of miR-140-5p in GC. Microarray expression profiles were initially employed to screen the differentially expressed gene related to GC, and the miR regulating the gene was predicted accordingly. The data obtained indicated that thymus cell antigen 1 (THY1) was differentially expressed in GC and confirmed to be a target gene of miR-140-5p. Poorly expressed miR-140-5p and highly expressed THY1 were observed in the GC tissues. SGC-7901 cells were treated with miR-140-5p mimic/inhibitor, siRNA against THY1 and siRNA against Notch1 in order to determine their regulatory roles in GC cell activities. The relationship of miR-140-5p, THY1 and the Notch signaling pathway was subsequently identified. Moreover, cell proliferation, migration, invasion and apoptosis were determined using 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), wound-healing, transwell assay and flow cytometry, respectively. The overexpression of miR-140-5p and silencing of THY1 resulted in a diminished expression of the Notch signaling pathway-related proteins, as well as inhibited proliferation, migration and invasion of GC cells, enhanced expression of pro-apoptotic proteins in addition to elevated apoptosis rate. Taken together, the present study suggests that miR-140-5p directly targets and negatively regulates THY1 expression and inhibits activation of the Notch signaling pathway, whereby the up-regulation of miR-140-5p inhibits development of GC, highlighting the promise of miR-140-5p as a potential target for GC treatment.

Keywords: Gastric cancer; Migration; Notch signaling pathway; Proliferation; THY1; microRNA-140-5p.

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Conflict of interest statement

The present study was performed with the approval of the Clinic Ethics Committee of The First Affiliated Hospital of Nanchang University. All participating patients signed informed consent documentation prior to the study.

The authors declare that there are no competing interests associated with the manuscript.

Figures

Figure 1
Figure 1. THY1 and miR-140-5p are engaged in the progression of GC
(A–C) Heatmaps of differentially expressed genes. The transversal coordinates represent the sample number, the ordinate represents the name of the gene, the left dendrogram represents the gene expression clustering, and each small square in the graph is the level of a gene in one sample. The histogram of the upper right is the color order. (D) Venn analysis of the top 100 differentially expressed genes in three datasets, and the center area represents the intersection of three datasets. (E) Interaction analysis between differentially expressed genes and GC-related genes, each cycle stands for a gene and the line between cycles indicates the interaction between genes. The brighter the color of the circle indicates that the gene is at the core in the network map. The red font represents the GC-related genes obtained in the MalaCards database, and the black font represents the differentially expressed gene in GC. (F) The prediction results of the regulatory miRs of THY1, the left cycle is the prediction results of the mirDIP database, the right cycle indicates the top 150 miRs of the miRGator database, and the middle part indicated the intersection of the prediction results of the two datasets.
Figure 2
Figure 2. miR-140-5p is poorly expressed and THY1 is overexpressed in GC tissues
(A) Expression of miR-140-5p in GC tissues and paracancerous tissues detected by RT-qPCR. (B) mRNA expression of THY1 in GC tissues and paracancerous tissues detected by RT-qPCR. (C) Protein bands of THY1 in GC tissues and paracancerous tissues detected by Western blot analysis. (D) Quantitative analysis of THY1 protein expression in GC and paracancerous tissues. (E) Correlation analysis of miR-140-5p expression and the mRNA expression of THY1 in GC and paracancerous tissues; *, P<0.05.
Figure 3
Figure 3. SGC-7901 and MGC-803 cell lines are selected for subsequent experiments
(A) miR-140-5p expression measured by RT-qPCR. (B) mRNA expression of THY1 measured by RT-qPCR. (C,D) Protein expression of THY1 detected by Western blot analysis; *, P<0.05.
Figure 4
Figure 4. miR-140-5p specifically binds to THY1
(A) Binding sites of miR-140-5p and THY1 predicted by the online bioinformatics software, microrna.org. (B) Dual-luciferase reporter gene assay for confirmation of the targeting relationship between miR-140-5p and THY1; *, P<0.05 vs. the NC + THY1-wt group. The wt sequence of THY1 was 5′-CCUGACUUCUCCCCAACCACUU-3′ and mut sequence was 5′-CCUGACUUCUCCCCAAUUGCUU-3′.
Figure 5
Figure 5. miR-140-5p negatively regulates the expression of proteins in the Notch signaling pathway in SGC-7901 cells
(A) Expression of miR-140-5p in SGC-7901 cells after transfection detected by RT-qPCR. (B) mRNA expression of THY1 in SGC-7901 cells after transfection detected by RT-qPCR. (C) Protein expression of THY1 in SGC-7901 cells after transfection detected by Western blot analysis. (D) Gray value analysis of THY1 protein. (E) Expression of Notch signaling pathway-related proteins (NICD, HES1, HES5). (F) Gray value analysis of Notch signaling pathway-related proteins (NICD, HES1, HES5); *, P<0.05 vs. the control group, #, P<0.05 vs. the NC group; &, P<0.05 vs. the si-NC group; ∆, P<0.05 vs. the miR-140-5p inhibitor group.
Figure 6
Figure 6. miR-140-5p overexpression inhibits proliferation of SGC-7901 cells
*, P<0.05 vs. the control group.
Figure 7
Figure 7. miR-140-5p overexpression leads to the suppression of migration and invasion of SGC-7901 cells
(A) SGC-7901 cell migration detected by wound-healing. (B) SGC-7901 cell invasion detected by Transwell assay. (C) Statistical analysis of (A). (D) Statistical analysis of (B). (E) Expression of N-cadherin, vimentin, MMP-2, MMP-9, E-cadherin in SGC-7901 cells after transfection detected by RT-qPCR; *, P<0.05 vs. the control group; #, P<0.05 vs. the NC group; &, P<0.05 vs. the si-NC group; ∆, P<0.05 vs. the miR-140-5p inhibitor group.
Figure 8
Figure 8. miR-140-5p overexpression promotes apoptosis of SGC-7901 cells
(A) Apoptosis of SGC-7901 cells in each group by flow cytometry. (B) Histogram of apoptosis of each group; *, P<0.05 vs. the control group; #, P<0.05 vs. the NC group; &, P<0.05 vs. the si-NC group; ∆, P<0.05 vs. miR-140-5p inhibitor group.
Figure 9
Figure 9. miR-140-5p overexpression promotes expression of pro-apoptotic protein and suppresses expression of anti-apoptotic protein in SGC-7901 cells
(A) Apoptosis-related proteins expression in SGC-7901 cells detected by Western blot analysis. (B) Statistical analysis of (A). (C) The protein expression of cleaved PARP1 detected by Western blot analysis. (D) Statistical analysis of (C). *, P<0.05 vs. the control group; #, P<0.05 vs. the NC group; &, P<0.05 vs. the si-NC group; ∆, P<0.05 vs. the miR-140-5p inhibitor group.
Figure 10
Figure 10. miR-140-5p participates in GC cell proliferation, migration, invasion and apoptosis by mediating Notch signaling pathway via THY1
miR-140-5p is poorly expressed in GC and THY1 is overexpressed in GC. miR-140-5p down-regulates THY1 expression. THY1 gene activates the Notch signaling pathway, and results in the inhibition of caspase-3 and Bax expression and promotes the expression of Bcl-2, which ultimately affects the proliferation, migration, invasion and apoptosis of GC.
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