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. 2020 Jan;19(1):415-423.
doi: 10.3892/ol.2019.11102. Epub 2019 Nov 14.

miR-512-5p suppresses the progression of non-small cell lung cancer by targeting β-catenin

Zhexin Wang  1 , Xiaolei Zhu  2 , Tuo Zhang  1 , Feng Yao  1
Affiliations

miR-512-5p suppresses the progression of non-small cell lung cancer by targeting β-catenin

Zhexin Wang et al. Oncol Lett. 2020 Jan.

Abstract

The oncogenic protein β-catenin is regulated by microRNAs (miRs) in non-small cell lung cancer (NSCLC). miR-512-5p is downregulated in NSCLC compared with healthy tissues and exhibits a tumour-suppressive effect. To study whether miR-512-5p acts on β-catenin to exert its anticancer effect in NSCLC, miR-512-5p mimic and inhibitor were transfected into NSCLC A549 and H1975 cells. miR-512-5p mimic inhibited the invasion of NSCLC cells and increased apoptosis, which suggested an inhibitory effect of miR-512-5p in NSCLC progression in vitro. By contrast, transfection with the miR-512-5p inhibitor resulted in the opposite effects. A dual-luciferase assay demonstrated that miR-512-5p complementarily bound to the 3'-untranslated region of β-catenin. miR-512-5p mimic suppressed the transcription and translation of β-catenin and reduced the expression of the downstream oncogenes cyclin D1 and matrix metalloproteinases, leading to the inhibition of Wnt/β-catenin signalling and subsequent inhibition of NSCLC tumourigenesis in vitro. In conclusion, miR-512-5p may function as a tumour suppressor in NSCLC by inhibiting the Wnt/β-catenin pathway.

Keywords: Wnt/β-catenin pathway; microRNA-512-5p; non-small cell lung cancer.

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Figures

Figure 1.
Figure 1.
Expression of miR-512-5p and β-catenin in human NSCLC. Reverse transcription-quantitative PCR analysis was used to determine the levels of (A) miR-512-5p and (B) β-catenin in 30 pairs of non-tumourous and tumourous lung tissues from patients with NSCLC. NSCLC, non-small cell lung cancer; miR, microRNA.
Figure 2.
Figure 2.
miR-512-5p targets β-catenin in non-small cell lung cancer cells. (A) The sequence of β-catenin 3′-UTR targeted by miR-512-5p. (B) Dual-luciferase assay in A549 cells using the WT or mutant 3′-UTR of β-catenin. **P<0.01. miR, microRNA; UTR, untranslated region; WT, wild-type; mut, mutant; CTNNB1, β-catenin.
Figure 3.
Figure 3.
miR-512-5p functions in the tumourigenesis of NSCLC in vitro. (A) The mRNA levels of miR-512-5p in human NSCLC cell lines A549, H460, HCC827, H1975 and H157. Relative miR-512-5p expression in H1975 were set as ‘1’. Successful establishment of miR-512-5p (B) overexpression and (C) silencing in A549 cells analysed by RT-qPCR. Successful establishment of miR-512-5p (D) overexpression and (E) silencing in H1975 cells analysed by RT-qPCR. **P<0.01 vs. NC. NSCLC, non-small cell lung cancer; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; miR, microRNA.
Figure 4.
Figure 4.
miR-512-5p suppresses non-small cell lung cancer cell invasion. Cell invasive ability was analysed by Transwell assays 24 h post-transfection with the inhibitor NC, miR-512-5p mimics or miR-512-5p inhibitor. Representative images of crystal violet-stained (A) A549 and (C) H1975 lung cancer cells. Quantification of invasive (B) A549 and (D) H1975 lung cancer cells. Data are presented as the mean ± standard error of the mean. **P<0.01. NC, negative control; miR, microRNA.
Figure 5.
Figure 5.
miR-512-5p promotes early and late stage apoptosis in lung cancer cells. Flow cytometry analyses of apoptosis in (A) A549 and (C) H1975 lung cancer cells transfected with the inhibitor NC, miR-512-5p mimics or miR-512-5p inhibitor were performed. The respective proportions of cells in the early and late stages of apoptosis in transfected (B) A549 and (D) H1975 lung cancer cells were analysed. (E) Caspase activity was measured by DEVD rhodamine fluorescence in A549 cells transfected with the inhibitor NC, miR-512-5p mimics or miR-512-5p inhibitor. Data are presented as the mean ± SEM. *P<0.05, **P<0.01. PI, propidium iodide; NC, negative control; miR, microRNA; AFU, arbitrary fluorencence units.
Figure 6.
Figure 6.
miR-512-5p downregulates β-catenin, APC, cyclin D1 and MMP7 expression in the A549 cell line. At 48 h post-transfection with the NC, miR-512-5p mimic or the miR-512-5p inhibitor, the expression levels of (A) miR-512-5p and (B) β-catenin, APC, cyclin D1 and MMP7 in A549 cells was analysed by reverse transcription-quantitative PCR. (C) Western blot analyses were performed to evaluate the protein expression levels of β-catenin, APC, cyclin D1 and MMP7 in A549 cells transfected with the NC, miR-512-5p mimic or miR-512-5p inhibitor. Quantitative analysis of protein levels of (D) β-catenin, (E) cyclin D1, (F) MMP7 and (G) APC. *P<0.05, **P<0.01. miR, microRNA; NC, negative control; APC, adenomatosis polyposis coli; MMP7, matrix metallopeptidase 7.
Figure 7.
Figure 7.
Roles of miR-512-5p in the Wnt/β-catenin pathway. Immunohistochemical analysis of β-catenin expression in (A) adjacent normal lung tissues and (B) lung cancer tissues. (C) A model of miR-512-5p functions in lung cancer cells. NC, normal control; LC, lung cancer; miR, microRNA; MMP7, matrix metallopeptidase 7.
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