Original Article Mir-140-5P Inhibits Proliferation, Invasion, and Promote

Int J Clin Exp Med 2019;12(12):13244-13255 www.ijcem.com /ISSN:1940-5901/IJCEM0099718

Original Article miR-140-5p inhibits proliferation, invasion, and promote apoptosis of non-small cell lung cancer cells through targeted inhibition of Wnt signaling pathway

Xiaobo Zhu1*, Hongyan Deng2*, Yueqiu Zhang3, Shijie Liu4

1Department of Oncology, The Second People Hospital of Dezhou, Dezhou, Shandong Province, China; 2Department of Oncology, Dezhou Hospital of Traditional Chinese Medicine, Dezhou, Shandong Province, China; 3Department of Respiration, Laoling People’s Hospital, Dezhou, Shandong Province, China; 4Department of Emergency, Yantai Municipal Laiyang Central Hospital, Yantai, Shandong Province, China. *Equal contributors and co-first authors. Received July 16, 2019; Accepted November 6, 2019; Epub December 15, 2019; Published December 30, 2019

Abstract: Objective: To investigate the regulation mechanism of miR-140-5p on proliferation, invasion and apoptosis of non-small cell lung cancer (NSCLC) cells through Wnt signaling pathway. Methods: NSCLC cell line H1975 was subjected to cell culture, grouping and transfection. The cells were divided into five groups: Blank group, NC group, miR-140-5p mimic group, pcDNA3.1-Wnt1 group and miR-140-5p mimic + pcDNA3.1-Wnt1 group. The targeting relationship between miR-140-5p and Wnt1 was confirmed using dual luciferase reporter assay. The mRNA and protein expression levels of Wnt1, β-catenin, TCF-4, PCNA, E-cadherin, N-cadherin, Bcl-2 and Bax in each group were detected using quantitative real-time PCR (qRT-PCR) and Western blot. Cell viability of each group was determined using MTT assay. Transwell assay was used to detect the invasive ability and flow cytometry was used to detect cell cycle and apoptosis in each group. Results: Compared with the normal human lung epithelial cell line Beas-2B, the expression of Wnt1 was significantly up-regulated in lung cancer cell lines (P<0.05), with the highest expression in H1975 cells. miR-140-5p specifically inhibited Wnt1 expression. There were no significant differences between the blank group and the NC group regarding all measured indices (all P>0.05). Compared with the blank group, the mRNA and protein expression levels of Wnt1, β-catenin, TCF-4, PCNA, N-cadherin and Bcl-2 in the miR-140-5p mimic group significantly decreased (all P<0.05), and the expression of E-cadherin and Bax significantly increased in the miR-140-5p mimics group (both P<0.05); meanwhile, the cell viability and invasion ability decreased, and the cell apoptosis increased in the miR-140-5p mimic group compared with the blank group (all P<0.05); moreover, the proportion of cells in G1 phase increased and the proportion of cells in S phase decreased significantly in the miR-140-5p mimic group compared with the blank group (both P<0.05). Compared with the blank group, the mRNA and protein expression levels of Wnt1, β-catenin, TCF-4, PCNA, N-cadherin and Bcl-2 in the pcDNA3.1-Wnt1 group significantly increased (all P<0.05), and the expression of E-cadherin and Bax significantly decreased in the pcDNA3.1-Wnt1 group (both P<0.05); meanwhile, the cell viability and invasion ability increased, and the cell apoptosis decreased in the pcDNA3.1-Wnt1 group compared with the blank group (all P<0.05); moreover, the proportion of cells in G1 phase decreased and the proportion of cells in S phase increased significantly in the pcDNA3.1-Wnt1 group compared with the blank group (both P<0.05). There are no differences between the miR-140-5p mimic + pcDNA3.1-Wnt1 group and blank group regarding cell invasion ability and cell apoptosis rate (both P>0.05). Conclusion: Overexpression of miR-140-5p inhibits the activation of Wnt signaling pathway by downregulating its target gene Wnt1, thereby inhibiting the proliferation, invasion, and promoting the apoptosis of NSCLC cells.

Keywords: miR-140-5p, Wnt1 gene, Wnt signaling pathway, non-small cell lung cancer, proliferation, apoptosis, invasion

Introduction (NSCLC) is the most common one, accounting for more than 80% of lung cancer patients [1-3]. Lung cancer is one of the most common and At present, the clinical treatment of NSCLC life-threatening cancers in the world. Among all mainly relies on radiotherapy, chemotherapy types of lung cancer, non-small cell lung cancer and targeted therapy; however, none of those miR-140-5p inhibits Wnt signaling pathway in NSCLC methods has achieved satisfactory outcome Materials and methods [4-6]. Therefore, exploring and identifying the pathogenesis and regulation network of NSC- Dual luciferase reporter assay LC are crucial for improving the therapeutic effects. The bioinformatics analysis was performed using online software http://www.targetscan. Current research indicates that Wnt signaling org, and dual luciferase reporter assay was per- pathway regulates multiple life activities. In the formed to verify whether Wnt1 is a target gene field of oncology, Wnt signaling pathway is con- of miR-140-5p. The 3’UTR region of the Wnt1 sidered to be mainly involved in tumor occur- gene was cloned and inserted downstream of rence, development, invasion, apoptosis, etc. the firefly gene of pmirGLO vector (Promega, [7-10]. The overexpression of Wnt signaling USA), which was labeled as Wt-Wnt1. The mutated 3’UTR of Wnt1 gene was inserted pathway significantly increases the occurrence downstream of the firefly gene of pmirGLO vec- of NSCLC; moreover, the expression of Wnt sig- tor and labeled as Mut-Wnt1. miR-140-5p naling pathway is found to be up-regulated in mimic and miR-140-5p mimic negative control NSCLC. Previous studies have confirmed that (NC) were co-transfected into 293T cells with the overexpression of Wnt1 could promote luciferase reporter vector, and the luciferase tumor growth, invasion, and inhibit tumor apop- activity was detected 48 hours after tran- tosis [11-13]. In the classic Wnt signaling path- sfection. way theory, the activation of Wnt signaling pathway promotes the recruitment of β-catenin, Cell culture which is transferred to intracellular signals and in turn affects tumor proliferation, invasion, The normal human lung epithelial cell line Beas- and apoptosis [14-16]. MicroRNA is a research 2B, and 4 lung cancer cell lines NCI-H292, hotspot in recent years, especially in the field of A549, H1299 and H1975 (ATCC, China) were oncology. In an organism, the same microRNA purchased and cultured. Cells were cultured in can act on multiple target genes, and multiple RPMI1640 medium (Gibco, USA) containing microRNAs can also act on the same target 10% fetal bovine serum (Gibco, USA), 50 U/mL gene [17, 18]. According to the literature, multi- penicillin (Gibco, USA), and 100 μg/mL strepto- ple microRNAs, such as miR-148a-3p, miR- mycin (Gibco, USA). Cells were placed in an 513b, miR-142-3p, etc. can affect tumor cell incubator (Besun Medical Devices Co., Ltd., activities including proliferation, migration and China) at 37°C and 5% CO2. The medium was invasion. The upstream microRNAs of the Wnt changed every 2 days and cells were passaged signaling pathway have not been fully defined every 3-4 days. Cells in good condition and log- [19-21]. Through bioinformatics analysis we arithmic growth phase were selected for tr- found that there is a putative binding site ansfection. between miR-140-5p and Wnt1. In addition, Cell grouping and transfection multiple previous studies demonstrated that miR-140-5p could inhibit the proliferation and The H1975 cells in logarithmic growth phase epithelial-mesenchymal transition of NSCLC were seeded in a 6-well culture plate at a den- [22, 23]. However, there is no literature to con- sity of 1*105 cells/well. DMEM medium confirm that miR-140-5p regulates the Wnt signal- taining no serum or antibiotic was used for cell ing pathway. Therefore, we hypothesized that culture one day before the transfection. The miR-140-5p can inhibit the proliferation, inva- cells were divided into 5 groups: Blank group sion, and apoptosis of NSCLC cells by inhibiting (no treatment), NC group (transfected with 50 the Wnt signaling pathway. nM miR-140-5p mimic NC), miR-140-5p mimics group (transfected with 50 nM miR-140-5p In this study, we used NSCLC cell line H1975 to mimic), pcDNA3.1-Wnt1 group (transfected explore the relationship between miR-140-5p with 4 μg pcDNA3.1-Wnt1), and miR-140-5p and Wnt signaling, in an effort to reveal the mimic + pcDNA3.1-Wnt1 group (co-transfected effects of miR-140-5p and the Wnt signaling with 50 nM miR-140-5p mimic and 4 μg pc- pathway on the proliferation, invasion, and DNA3.1-Wnt1). Transfection was performed in apoptosis of NSCLC cells. accordance with Lipofectamine 2000 instruc-

13245 Int J Clin Exp Med 2019;12(12):13244-13255 miR-140-5p inhibits Wnt signaling pathway in NSCLC

Table 1. qRT-PCR primer sequences CT (target gene) - CT (GAPDH or U6), ΔΔCt = ΔCt (experimental Gene Sequence group) - ΔCt (control group), with U6 as internal reference for miR-140-5p and GAPDH as internal Wnt1 F: 5’-ATTTGGTTCGGTTCTTCTTCAG-3’ reference for other genes. All primers are listed R: 5’-CTTATGAGGGTGTGTAGTGCGA-3’ in Table 1. miR-140-5p F: 5’-AGAGTTGCACCTTCAGATT-3’ R: 5’-TGGAGGGGTCCTGCAG-3’ Western blot β-catenin F: 5’-TCTGCACACGTGAGTGTGGCA-3’ R: 5’-TAAACACACGCCTTCGGTTCA-3’ At 48 h after transfection, cells were harvested and total protein was extracted from all groups TCF-4 F: 5’-CTAGCCTCTCCACTAGCCC-3’ using RIPA lysate (Solarbio, China) containing R: 5’-TTGCTTCAGGGTGTCTTCGTC-3’ PMSF. Protein concentration was determined E-cadherin F: 5’-CTCTGTGTATCAGGCTCG-3’ using BCA kit (Thermo Fisher Scientific, USA), R: 5’-TAGACTGAGGGCCTCTCT-3’ and the concentration was adjusted with deion- N-cadherin F: 5’-CCAGTAGCTACGTATGGAC-3’ ized water. Samples mixed with loading buffer R: 5’-CGTCATCACATAGTGGCAT-3’ were boiled for 10 min. A total of 30 µg of each Bcl-2 F: 5’-GTCTTCGCTGCGGAGATCAT-3’ sample was loaded into the gel and subject to R: 5’-CATTCCGATATACGCTGGGAC-3’ electrophoresis for 2 h at 80 V. Protein was then wet transferred onto PVDF membrane Bax F: 5’-CATATAACCCCGTCAACGCAG-3’ (Millipore, USA) at 110 V for 2 h. The PVDF R: 5’-GCAGCCGCCACAAACATAC-3’ membrane was blocked in 5% skim milk at 4°C GAPDH F: 5’-GGGCTGCTTAACTCTGTTGT-3’ for 2 h. After washing with TBST, the membrane R: 5’-GCAGTTCTAGTGTTACGG-3’ was incubated with primary rabbit anti-human U6 F: 5’-CTCGGACTCAGCCTGCA-3’ antibody Wnt1 (1:1,000, Abcam, UK), β-catenin R: 5’-TCAATATTACCGAGCTGCGT-3’ (1:5,000, Abcam, UK), TCF-4 (1:1,000, Abcam, UK), PCNA (1:1,000, Abcam, UK), E-cadherin (1:500, Abcam, UK), N-cadherin (1:1,000, tion manual. At 6 h after transfection, culture Abcam, UK), Bax (1:1,000, Abcam, UK), Bcl-2 medium was switched to RPMI1640 medium (1:1,000, Abcam, UK), GAPDH (1:2,000, Abcam, (Gibco, USA) containing 10% fetal bovine ser- UK), respectively, at 4°C overnight. The mem- um. At 48 h after transfection, cells were har- brane was rinsed with TBST 3 times 10 min vested for subsequent experiments. each time. Then the membrane was incubated with HRP-conjugated goat anti-rabbit IgG Quantitative real-time PCR (qRT-PCR) (1:5,000, Beijing Zhongshan Biotechnology Co., Ltd., China). After rinsing with TBST, the mem- Total RNA was extracted from all groups of cells brane was placed on a glass plate. A medium using Trizol (Thermo Fisher Scientific, USA). amount of solution A and solution B from RNA was reverse transcribed into cDNA using Fluorescence Detection Kit (Ameshame, UK) PrimeScriptTM RT reagent kit with gDNA Eraser were mixed in a dark room and applied onto the kit (TaKaRa, Japan). The reaction mixture was membrane. The membrane was exposed and prepared using SYBR® Premix Ex TaqTM II kit photographed using Bio-Rad image analysis (Xingzhi Biotechnology Co., Ltd., China). The system (BIO-RAD, USA). Relative protein expres- reaction mixture contained the following com- sion was calculated by comparing the grey ponents: 25 μL SYBR® Premix Ex TaqTM II (2×), 2 value of each sample with the grey value of μL upstream primer, 2 μL downstream primer, l GAPDH using Image J software. μL ROX Reference Dye (50×), 4 μL DNA tem- MTT assay plate, and 16 μL ddH2O. qRT-PCR was per- ® formed using ABI PRISM 7300 system (Kunke At 48 h after transfection, cells were harvested Instrument Equipment Co., Ltd., China). The and seeded in a 96-well culture plate at a den- reaction conditions were as follows: pre-dena- sity of 3-6*103 cells/well. The volume of each turation at 95°C for 10 min, denaturation at well is 100 µL, and each group has 6 replicate 95°C for 15 s, annealing at 60°C for 30 s; the wells. At 24 h, 48 h, and 72 h, a volume of 20 cycle was repeated 32 times followed by exten- μL MTT (Gibco, USA) solution at a concentration sion at 72°C for 1 min. Relative expression of 5 mg/mL was added into each well and the level was calculated using the formula: ΔCt = plate was incubated for 4 h in dark room. Then

13246 Int J Clin Exp Med 2019;12(12):13244-13255 miR-140-5p inhibits Wnt signaling pathway in NSCLC

100 μL of DMSO was added to each well, and re-suspended with RPMI1640 medium and the absorbance value (OD) of each well at 570 adjusted to a density of 1*105 cells/mL. A vol- nm was obtained using a microplate reader ume of 200 μL cell suspension was added to (Beijing Noah Instrument Co., Ltd., China). The the upper chamber and 600 μL RPMI1640 cell viability curve was plotted with the time medium containing 20% fetal bovine serum points as the abscissa and the OD values as (Gibco, USA) was added to the lower chamber. the ordinate. After 24 h the Transwell insert was removed and cells on the side surface of the chamber Flow cytometry was wiped out with cotton swab. Cells were fixed with 4% paraformaldehyde (Beijing Lea- Cell cycle: Cells were harvested 48 h after gene Bio, China) for 15 min, then stained with transfection. Cells were washed with PBS 3 0.5% crystal violet solution (Beijing Solarbio, times and centrifuged at 3,000 r/min for 20 China) for 15 min. The chamber was rinsed with min. The supernatant was discarded and the 5 PBS 3 times and observed with an inverted cell density was adjusted to 1*10 /mL with microscope. Five random fields (200×) were PBS. Then 1 mL 75% ethanol pre-cooled at selected for photographing and counting cells -20°C was added and the cells were fixed at that have migrated through the membrane. 4°C for 1 h. Cells were centrifuged at 1,500 r/ min for 5 min and washed with PBS twice. Then Statistical analysis 100 μL Rnase A (Thermo Fisher, USA) was added and the cells were placed in 37°C water All data were analyzed with SPSS21.0 soft- bath for 30 min. A volume of 400 μL PI (Sigma, ware. Quantitative values were expressed as USA) was added and the cells were placed in mean ± standard deviation. One-way ANOVA dark room at 4°C for 30 min. Cell cycle was follow by Tukey post-hoc test was used to com- measured with a flow cytometer (Beckman- pare the differences between groups. A P value Coulter, USA) by detecting the red fluorescence less than 0.05 was considered statistically at a wavelength of 488 nm. significant.

Cell apoptosis: At 48 h after transfection, cells Results were digested with trypsin containing no EDTA (Thermo Fisher, USA) and collected into a flow Wnt1 mRNA expression was different between cytometry tube. The tube was centrifuged at normal lung epithelial cell line and lung cancer 3,000 r/min for 30 min and the supernatant cell lines was discarded. Cells were washed with cold PBS 3 times then centrifuged at 3,000 r/min The expression of Wnt1 gene in normal human for 15 min. The supernatant was discarded. lung epithelial cell line and lung cancer cell The Annexin-V-FITC/PI staining solution was lines was detected by qRT-PCR. The results made by mixing HEPES buffer (Thermo Fisher, showed that Wnt1 expression was significantly USA), Annexin-V-FITC and PI in a ratio of 50:1:2. up-regulated in lung cancer cell lines compared Cells were mixed with 100 μL Annexin-V-FITC/PI with normal human lung epithelial cell line staining solution and let sit in room tempera- Beas-2B (all P<0.05), with the highest expres- ture for 15 min. After adding 1 mL HEPES buf- sion in H1975 cells. See Figure 1. fer, cell apoptosis was measured in a flow cytometer at a wavelength of 488 nm. Cell miR-140-5p specifically inhibited the expres- apoptosis rate was determined by analyzing sion of Wnt1 10,000 cells using CELLquest software. Bioinformatics analysis via online software Transwell invasion assay microrna.org (http://www.targetscan.org) pre- dicted a putative binding site between miR- A Transwell insert (Shanghai Kelton Bio, China) 140-5p and Wnt1 (Figure 2A). Dual luciferase was placed in a 24-well plate. The upper sur- reporter assay showed luciferase activity sig- face of the bottom membrane was coated with nificantly decreased in 293T cells co-transfect- 1:8 Matrigel (Shanghai Qianchen Biotechnology, ed with miR-140-5p mimic and Wt-Wnt1 (all China) and air-dried at room temperature. Cells P<0.05), indicating that miR-140-5p specifical- were digested and rinsed with PBS twice, then ly inhibited the expression of Wnt1 (Figure 2B).

13247 Int J Clin Exp Med 2019;12(12):13244-13255 miR-140-5p inhibits Wnt signaling pathway in NSCLC

group (both P>0.05). Compared with the blank group, the expression of PCNA, N-cadherin and Bcl-2 decreased, but the expression of E-cadherin and Bax increased both in mRNA and protein level in the miR-140-5p mimic group (all P<0.05). In contrast, the expression of PCNA, N-cadherin and Bcl-2 increased, but the expression of E-cadherin and Bax decreased in the pcDNA3.1-Wnt group (all P<0.05).

miR-140-5p overexpression inhibited the viability of H1975 cells; Wnt1 overexpression reversed the effects of miR-140-5p

The NC group and the miR-140-5p mimic + Figure 1. Wnt1 mRNA expression in normal lung epi- pcDNA3.1-Wnt1 group showed no differences thelial cell line and lung cancer cell lines. *P<0.05, than the blank group in cell viability at all time compared with Beas-2B cell line. points by MTT assay (all P>0.05). However, at 48 h and 72 h, cell viability decreased in the miR-140-5p inhibited the expression of miR-140-5p mimic group and increased in the pcDNA3.1-Wnt1 group compared with the blank Wnt signaling pathway related genes Wnt1, group (all P<0.05). The miR-140-5p mimic + β-catenin, and TCF-4 pcDNA3.1-Wnt1 group had increased cell viabil- The expression level of Wnt pathway related ity over the miR-140-5p mimic group (P<0.05). genes Wnt1, β-catenin, and TCF-4 were detect- See Figure 5. ed by Western blot (Figure 3). There were no miR-140-5p overexpression inhibited the pro- significant differences between the blank liferation of H1975 cells; Wnt1 overexpression group and the NC group (P>0.05). The miR- reversed the effects of miR-140-5p 140-5p group and the miR-140-5p mimic + pcDNA3.1-Wnt1 group had significantly higher The NC group and the miR-140-5p mimic + miR-140-5p expression than other groups pcDNA3.1-Wnt1 group showed no differences (both P<0.05). Compared with the blank group, than the blank group in cell cycle by flow cytom- the expression of Wnt1, β-catenin, and TCF-4 etry (both P>0.05). Compared with the blank decreased in miR-140-5p mimic, but increased group, the ratio of G1 phase cells increased in pcDNA3.1-Wnt1 group (both P<0.05). There and the ratio of S phase cells decreased in the were no significant differences between the miR-140-5p mimic group (both P<0.05). While blank group and the miR-140-5p mimic + in the pcDNA3.1-Wnt1 group, the ratio of G1 pcDNA3.1-Wnt1 group regarding β-catenin and phase cells decreased and the ratio of S phase TCF-4 expression (both P>0.05). The results cells increased (both P<0.05). Compared with indicated that miR-140-5p inhibited the activa- the miR-140-5p mimics group, the miR-140-5p tion of Wnt signaling pathway by targeting mimic + pcDNA3.1-Wnt1 group had lower G1 Wnt1. phase ratio and higher S phase ratio (both P<0.05). See Figure 6. The expression of cell proliferation, cell invasion and apoptosis related genes were differ- miR-140-5p overexpression promoted apop- ent among all groups tosis of H1975 cells; Wnt1 overexpression reversed the effects of miR-140-5p The expression of cell proliferation related gene PCNA, cell invasion related gene N-cad- The NC group and the miR-140-5p mimic + herin and E-cadherin, and apoptosis related pcDNA3.1-Wnt1 group showed no differences gene Bcl-2 and Bax were detected with qRT- than the blank group in cell apoptosis by flow PCR and Western blot (Figure 4). The NC group cytometry (both P>0.05). Compared with the and the miR-140-5p mimic + pcDNA3.1-Wnt1 blank group, cell apoptosis rate increased in group showed no differences than the blank the miR-140-5p mimic group, and decreased in

13248 Int J Clin Exp Med 2019;12(12):13244-13255 miR-140-5p inhibits Wnt signaling pathway in NSCLC

Figure 2. Bioinformatics showed miR-140-5p specifically inhibited the expression of Wnt1. A: The putative binding site between miR-140-5p and Wnt1 3’-UTR; B: Relative luciferase activity; *P<0.05, compared with miR-140-5p mimic NC.

Figure 3. The expression of miR-140-5p, Wnt1, β-catenin and TCF-4 by qRT-PCR and Western blot. A: mRNA expression of miR-140-5p, Wnt1, β-catenin and TCF-4 by qRT-PCR; B, C: Protein expression of miR-140-5p, Wnt1, β-catenin and TCF-4 by Western blot; *P<0.05, compared with the blank group; #P<0.05, compared with the NC group; &P<0.05, compared with the miR-140-5p mimic group; @P<0.05, compared with the pcDNA3.1-Wnt1 group.

Figure 4. The expression of PCNA, N-cadherin, E-cadherin, Bcl-2, and Bax by qRT-PCR and Western blot. A: mRNA expression of PCNA, N-cadherin, E-cadherin, Bcl-2, and Bax by qRT-PCR; B, C: Protein expression of PCNA, N-cadherin, E-cadherin, Bcl-2, and Bax by Western blot; *P<0.05, compared with the blank group; #P<0.05, compared with the NC group; &P<0.05, compared with the miR-140-5p mimic group; @P<0.05, compared with the pcDNA3.1-Wnt1 group.

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retical basis for the development of more effective clinical treatment methods.

As a member of the microRNA family, miR-140- 5p regulates a variety of life activities. It was reported that miR-140-5p inhibited the proliferation and invasion of tumor cells as well as the occurrence of NSCLC [27, 28]. In this study, we cultured NSCLC cell line H1975 and transfected cells with miR-140-5p mimic to detect cell proliferation, invasion, and apoptosis in each group. The results showed that compared with the blank group, the cell proliferation and invasion ability of the miR-140-5p mimic group were significantly decreased, and the apoptosis rate was significantly increased; in addition, cell cycle was more hindered in the miR-140-5p Figure 5. Comparison of cell viability. *P<0.05, com- mimic group. The expression of PCNA, N-cad- # pared with the blank group; P<0.05, compared with herin, and Bcl-2 were higher, and the expres- the NC group; &P<0.05, compared with the miR- 140-5p mimic group; @P<0.05, compared with the sion of E-cadherin and Bax were lower in the pcDNA3.1-Wnt1 group. miR-140-5p mimic group than in the blank group. The above results indicated that overexpression of miR-140-5p could inhibit prolifera- the pcDNA3.1-Wnt1 group (both P<0.05). The tion and invasion, and promote apoptosis of miR-140-5p mimic + pcDNA3.1-Wnt1 group NSCLC cells. had significantly lower cell apoptosis rate than the miR-140-5p mimics group (P<0.05). See MicroRNAs primarily regulate life activities by Figure 7. targeting downstream genes [29, 30]. We found through bioinformatics that there is a putative miR-140-5p overexpression inhibited invasion binding site between miR-140-5p and Wnt1 potential of H1975 cells; Wnt1 overexpression 3’UTR. As a member of Wnt signaling pathway, reversed the effects of miR-140-5p when overexpressed, Wnt1 could lead to recruitment factors such as β-catenin and TCF- The NC group and the miR-140-5p mimic + 4, thus triggering the expression of downstream pcDNA3.1-Wnt1 group showed no differences translation related factors. As a result, tumor than the blank group in Transwell invasion cell proliferation and invasion was promoted assay (both P>0.05). Compared with the blank and apoptosis was inhibited [31, 32]. In this group, cell invasion potential decreased in the study, we transfected H1975 cells with pc- miR-140-5p mimic group, and increased in the DNA3.1-Wnt1 to verify whether the activation of pcDNA3.1-Wnt1 group (both P<0.05). The miR- Wnt signaling pathway would affect the prolif- 140-5p mimic + pcDNA3.1-Wnt1 group had sig- eration, invasion, apoptosis and cell cycle of nificantly higher invasion potential than the NSCLC. The results confirmed that after Wnt1 miR-140-5p mimic group (P<0.05). See Figure overexpression, the proliferation and invasion 8. ability of NSCLC was significantly enhanced, Discussion cell apoptosis was reduced, and cell cycle was also accelerated. To further explore the link NSCLC includes the most common types of between miR-140-5p and Wnt signaling path- lung cancer, and the number of NSCLC patients way, we performed a dual luciferase reporter accounts for more than 80% of all lung cancer assay to confirm that Wnt1 is a binding target of patients. The morbidity and mortality of pa- miR-140-5p; overexpression of miR-140-5p sig- tients with NSCLC are extremely high, which nificantly inhibited the expression of Wnt signal- poses a great threat to the patient’s life [24- ing pathway members Wnt1, β-catenin and 26]. Therefore, exploring the regulatory network TCF-4. To determine whether the overexpres- and pathogenesis of NSCLC will provide a theo- sion of Wnt1 would reverse the effects of miR-

13250 Int J Clin Exp Med 2019;12(12):13244-13255 miR-140-5p inhibits Wnt signaling pathway in NSCLC

Figure 6. Comparison of cell cycle. A: Cell cycle detected by flow cytometry; B: Cell cycle percentage; *P<0.05, compared with the blank group; #P<0.05, compared with the NC group; &P<0.05, compared with the miR-140-5p mimic group; @P<0.05, compared with the pcDNA3.1-Wnt1 group.

13251 Int J Clin Exp Med 2019;12(12):13244-13255 miR-140-5p inhibits Wnt signaling pathway in NSCLC

Figure 7. Comparison of cell apoptosis. A: Cell apoptosis detected by flow cytometry; B: Cell apoptosis rate;* P<0.05, compared with the blank group; #P<0.05, compared with the NC group; &P<0.05, compared with the miR-140-5p mimic group; @P<0.05, compared with the pcDNA3.1-Wnt1 group.

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Figure 8. Comparison of cell invasion. A: Pictures of Transwell invasion assay; B: Cell counts of each group; *P<0.05, compared with the blank group; #P<0.05, compared with the NC group; &P<0.05, compared with the miR-140-5p mimic group; @P<0.05, compared with the pcDNA3.1-Wnt1 group.

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