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Tumour Marker Prognostic Study

Mir-556-3p Inhibits SqCLC via NUAK1

Authors:

Yini Cai,

Department of Oncology, The Second Affiliated Hospital of Nanchang University; Jiangxi Key Laboratory of Clinical and Translational Cancer Research, 1 Minde Road, Nanchang, Jiangxi, CN
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Ming Fang,

Department of Yangxin People’s Hospital of Hubei Province, 81 Ruxue Road, Xingguo Town, Yangxin County, Huangshi, Hubei, CN
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GongJi Yao,

Department of Oncology, The Second Affiliated Hospital of Nanchang University; Jiangxi Key Laboratory of Clinical and Translational Cancer Research, 1 Minde Road, Nanchang, Jiangxi, CN
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Lingmin Liao,

Department of Oncology, The Second Affiliated Hospital of Nanchang University; Jiangxi Key Laboratory of Clinical and Translational Cancer Research, 1 Minde Road, Nanchang, Jiangxi, CN
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Long Huang

Department of Oncology, The Second Affiliated Hospital of Nanchang University; Jiangxi Key Laboratory of Clinical and Translational Cancer Research, 1 Minde Road, Nanchang, Jiangxi, CN
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Abstract

Background: MicroRNAs (miRNAs) play a key role in the development of lung cancer; however, there has been little research on the role of miR-556-3p. The present study focused on the function and mechanism of miR-556-3p in lung squamous cell carcinoma (SqCLC).

Methods: The Cancer Genome Atlas (TCGA) database was searched to analyze the expression of miR-556-3p in SqCLC, and its relationship with prognosis and survival. Quantitative real-time reverse transcription PCR was used to detect the expression difference of miR-556-3p in normal lung epithelial cells (BEAS-2B) and SqCLC cells (H226 and SK-MES-1). The proliferation, migration, and invasion of H226 and SK-MES-1 cells were detected by Cell Counting Kit-8 (CCK-8), wound healing, and Transwell assays, respectively, after transfection with miR-556-3p-mimics. The changes in the levels of key proteins in the epithelial-mesenchyme transition (EMT) signaling pathway were detected using western blotting. Bioinformatic analyses predicted the target gene of miR-556-3p, which was verified using luciferase reporter gene assays. The role of miR-556-3p in SqCLC was verified in a nude mouse tumorigenesis model.

Results: Analysis of 523 patients with SqCLC in the TCGA database showed that the overall survival (OS) of patients with high miR-556-3p expression was significantly better than that of patients with low miR-556-3p expression (P < 0.05). Compared with that in BEAS-2B cells, miR-556-3p expression was downregulated in H226 and SK-MES-1. After transfection with a miR-556-3p-mimic, CCK-8 analysis showed that the proliferation rate of H226 and SK-MES-1 cells decreased by 66.6% and 60.1% compared with that of the control group. Wound healing assays showed that the migration ability of H226 and SK-MES-1 overexpressing miR-556-3p cells decreased significantly compared with the control group (P < 0.05). Transwell assays showed that the invasion ability of H226 and SK-MES-1 cells overexpressing miR-556-3p was also significantly reduced compared with that the control group (P < 0.05). Western blotting showed that E-Cadherin levels were significantly upregulated after transfection of H226 and SK-MES-1 cells with the miR-556-3p-mimic, while N-cadherin and Vimentin levels were significantly downregulated. Luciferase reporter assays showed that miR-556-3p could bind to the promoter region ofNUAK1(encoding NUAK family kinase 1), and the exogenous expression ofNUAK1could reverse the migration and invasion inhibition by miR-556-3p on H226 and SK-MES-1 cells.In vivoexperiments showed that miR-556-3p also inhibited tumor growth.

Conclusions: MiR-556-3p inhibits EMT by targetingNUAK1, thus inhibiting the migration and invasion of SqCLC. Thus, miR-556-3p might be developed as a target to treat SqCLC.

How to Cite: Cai Y, Fang M, Yao G, Liao L, Huang L. Mir-556-3p Inhibits SqCLC via NUAK1. International Journal of Surgery: Oncology. 2022;7(1):30–45. DOI: http://doi.org/10.29337/ijsonco.138
  Published on 09 May 2022
 Accepted on 23 Apr 2022            Submitted on 31 Mar 2022

1. Introduction

Lung cancer is the main cause of cancer death [1]. Lung cancer can be divided into small cell lung cancer and non-small cell lung cancer, of which the latter accounts for more than 85% of cases [2]. Non-small cell lung cancer can be divided into lung adenocarcinoma, lung squamous cell carcinoma (SqCLC) and large cell carcinoma [2, 3], of which SqCLC accounts for about 30% of cases [4]. The underlying molecular mechanism of carcinogenesis remains unclear. Therefore, it is important to carry out mechanistic research on SqCLC.

MicroRNAs (miRNAs) not only regulate the proliferation, apoptosis, invasion, and migration of lung cancer cells, but also play an important role in the diagnosis, treatment, and prognosis of lung cancer, especially SqCLC [5]. MiRNAs are short non-coding RNAs of about 22 nucleotides that mediate gene silencing by guiding the Argonaute protein to target sites in the 3’ untranslated region (UTR) of mRNAs [6]. They are widely associated with the pathogenesis of many human diseases, including cancer [7]. Their relative stability, small size, and significant regulation of gene expression are the unique characteristics of miRNAs, which distinguish them from mRNAs and could be identified as biomarkers and potential therapeutic targets in the future [5].

Among miRNAs, there are relatively few studies on mir-556. Like other miRNAs, because of the different processing of the 5’ and 3’ end arms of the precursor, mir-556-3p and mir-556-5p play different regulatory roles in different tumors, and the results of some studies are controversial and even contradictory. Upregulation of mir-556-5p can promote the occurrence of prostate cancer [8]. By contrast, it plays an inhibitory role in meningeal carcinoma and breast cancer [9, 10]. In atherosclerosis, mir-556-5p upregulation can reduce the damage to human umbilical vein endothelial cells induced by oxidized low-density lipoprotein [11]. Mir-556-3p upregulated migration and invasion enhancer 1 (MIEN1) promotes cell proliferation and the epithelial-mesenchyme transition (EMT) process in hepatocellular carcinoma [12]. In a clinical study at the Sun Yat-sen Memorial Hospital, mir-556-5p was negatively regulated by circHERC4 and played a role in inhibiting metastasis in rectal cancer [13]. However, other research came to different conclusions research. Lei Lei et al. believed that mir-556 might be an oncogene that downregulates the expression of chromosome 8 open reading frame 48 (C8orf48) and promotes the occurrence of colorectal cancer [14]; however, that study did not emphasize the expression form of mir-556. Nevertheless mir-556-5p overexpression inhibited scortosis and thereby inhibited allergic rhinitis. There were also studies on the drug resistance of mir-556-5p in non-small cell lung cancer, in which mir-556-3p upregulation showed cisplatin sensitization and tumor suppressive effects on lung cancer cells [15]. In addition, mir-556-5p downregulation improved the sensitivity of cisplatin through cell pyrophosis [16]. Previous studies have provided direct or indirect evidence that mir-556 is closely related to the diagnosis and prognosis of tumors; however, the regulation and effect of mir-556 on SqCLC is unclear. In this study, bioinformatic analysis and basic experiments were combined to perform a comprehensive analysis of the role and mechanism of mir-556-3p in SqCLC.

2. Materials and Methods

2.1 Data processing and miRNA determination

Raw sequence data and clinical information were downloaded from The Cancer Genome Atlas (TCGA) database (https://cancergenome.nih.gov/). The inclusion criteria were as follows: 1. Samples with miRNA sequencing data and clinical information 2. Samples with prognostic information. A total of 523 samples were included in this study, including 478 SqCLC tissues and 45 matched normal tissues. MiRNAs sequencing data were processed using the R language package. Differential expression of miRNAs in lung cancer and normal lung tissues was analyzed using the limma software package in R, and the fold change (FC) of individual miRNAs expression levels were calculated, a change in the log2 | FC | > 2.0 and P < 0.05 identified significantly differentially expressed miRNAs. The differentially expressed miRNAs were normalized after log2 transformation. Kaplan–Meier curves and the log-rank method were used to evaluate the prognostic value of the differentially expressed miRNAs. Mir-556-3p was differentially expressed and significantly associated with overall survival (OS) was thus further investigated as a possible prognostic miRNAs.

2.2 Cell lines and cell culture

Human BEAS-2B cells were purchased from Genio Bio (Guangzhou, China), human NCIH226 and SK-MES-1 cells were purchased from Procell Life Science & Technology (Wuhan, China), and the 293T cell line was purchased from the Bank of the Chinese Academy of Sciences (Shanghai, China). SK-MES-1 cells were cultured in minimal essential medium (Procell Life Science & Technology) at 37°C and 5% CO2; NCI-H226 cells were cultured in Roswell Park Memorial Institute 1640 medium (Gibco, Grand Island, NY, USA); and 293T cells were cultured in Dulbecco’s modified Eagle’s medium, all of which were supplemented with 10% fetal bovine serum. BEAS-2B cells were cultured in LHC medium (Gibco).

2.3 Quantitative real-time reverse transcription PCR (qRT-PCR) and transfection

The TRIzol Reagent (Invitrogen, Waltham, MA, USA) was used to extract RNA from each group, and Mir-X™miRNA QRT-PCR SYBR (TransGen Biotech, Beijing, China) reverse transcription of the RNA was carried out to produce cDNA. TB Green Premix Ex Taq II Kit (Takara Shiga, Japan) was used for the quantitative real-time PCR step, using the cDNA as the template. GAPDH (encoding glyceraldehyde-3-phosphate dehydrogenase) was used as the reference gene. MiRNAs were extracted using EasyScript one-step gDNA Removal and cDNA Synthesis SuperMix kits (TransGen Biotech) and reverse transcribed, using U6 as the reference gene. NCI-h226 and SK-MES-1 cells were transfected with the mir-556-3p mimic mimic-negative control (NC; Ribobio, Shanghai, China). The mir-556-3p mimic and mimicNC were detected using qRT-PCR.

2.4 Plasmids

Mir-556-3p overexpression plasmid, the NUAK1 (encoding NUAK family kinase 1) overexpression plasmid and NUAK1-mutated (MUT) plasmid were all were constructed by GeneChem Co. Ltd (Shanghai, China), using the GV272 vector and XbaI/XbaI enzyme digestion.

2.5 Cell survival assays

According to the instructions of the Cell Counting Kit-8 (CCK-8) reagent (Biyuntian Biotechnology Co., LTD, Shanghai, China), 10 µL per well of CCK-8 was added to each group of cells in culture medium in a 96-well plate, and fully mixed to ensure color uniformity in the well. The absorbance of each well was measured at 450 nm using a microplate reader after 4 h of culture in the dark.

2.6 Wound healing assay

Cell suspension was prepared and 2 mL of the mixed cell suspension was inoculated into a 6-well plate. After full mixing, the cell suspension was placed in an incubator with 5% CO2 at 37°C for overnight culture. On the second day, the cells had achieved 100% confluent growth. Two parallel wound lines were made on the surface of the confluent cell layer using a 1 mL pipette tip. The excess cells were washed off using phosphate-buffered saline three times, and serum-free medium was added to the different groups. An inverted microscope was used to observe and photograph the wounds at 0, 6, 12, and 24 h, until the cells in the blank group healed.

2.7 Transwell assays

100 µL (105 cells/ml) of cell suspension of each group was added into 100 µL of serum-free medium, mixed, and added into the upper chambers of the Transwell apparatus in a 24-well plate, separately. 500 µL of the corresponding medium containing with 20% fetal bovine serum was added into the lower chamber of the Transwell apparatus. The chambers were removed after 24 h, fixed with 4% paraformaldehyde for 15 min, and stained with crystal violet for 10 min. The cells in each field were photographed and counted in the different groups. In another experiment, the Transwell upper chamber gel and agglutination gel were prepared with melted Matrigel stock solution and precooled serum-free medium at 1:3. 100 µL (105 cells/mL) of cell suspension was taken from each group, mixed with 100 µL of serum-free and then added into Transwell chambers, separately. The rest of the method was the same as that detailed above.

2.8 Western blotting

NCI-H226 and SK-MES-1 cells were lysed in sodium dodecyl sulfate buffer (1% sodium dodecyl sulfate, 10 mM HEPES, pH 7.0, 2 mM MgCl2, 20 U/mL universal nuclease) to collect the supernatant containing the protein. The total protein concentration of cells was determined using a bicinchoninic acid assay kit (Elabscience, Wuhan, China). Then, 10 µL of each protein sample was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane was incubated with 5% skim milk powder with shaking for 1 h on a decolorizing shaker. Antibodies recognizing GAPDH (1:10,000, Abcam, Cambridge, MA, AB179467), E-cadherin (1:500, Abcam, AB15148), N-cadherin (1:10,000, Abcam, AB76011), Vimentin (1:1000, Abcam, AB20346added and incubated overnight separately. Secondary antibodies comprising horseradish peroxidase-Goat anti Rabbit (1:3000, Elabscience, E-AB-1037) or horseradish peroxidaseGoat anti Mouse (1:5000, Abcam, AB6789) were added and incubated at room temperature for 1 h. The freshly prepared ECL mixed solution (Bio-Rad, Hercules, CA, USA) was dropped onto the protein side of the membrane for luminescence detection. The optical densities of the immunoreactive protein bands were analyzed using the AlphaEaseFC software processing system (ProteinSimple, San Jose, CA, USA), and the ratio between each group and GAPDH was calculated and recorded as the relative gray level.

2.9 Target gene prediction

The target genes of prognostic miRNAs were predicted using Gene, TargetScan (http://www.targetscan.org/), miRDB (http://www.mirdb.org/miRDB/) and miRTarBase (http://mirtarbase.mbc.nctu.edu.tw) analysis tools. To further enhance the bioinformatic analysis reliability, overlapping target genes were identified using a Venn diagram. Three target genes (NR2C2, NUAK1, and G3BP2) were identified for further experimental study. The relative expression levels of NUAK1 mRNA in H226-mimic and SK-MES-1-mimic were detected by qRT-PCR with GAPDH as reference gene.

2.10 Luciferase assay

The 3’ UTR mutant or wild-type 3’ UTR and mir-556-3p-mimic or mimic-NC were cotransfected into 293 T cells and the luciferase activity was monitored. The relative expression of luciferase in the mimic-NC group was set to 1 to obtain the relative Luciferase expression. Then, the UTR-NC group was set to 1, and the relative expression levels of luciferase in the UTR wild-type group and UTR mutant group were compared under the action of the same miRNAs.

2.11 In vivo experiments

H226 and SK-MES-1 cells were cultured in a 37°C CO2 incubator, separately. When the density was suitable, that were plated into a 10 cm dish and transfected with an agomir (miRNAs mimic) and agomir-NC, respectively. After culture for 36 h, the cells were digested with trypsin. Nude mice at 4–5 weeks old were injected with cells (107) via the caudal vein. All mice were purchased from Wuhan Zolabao Company (Hubei, China). The animals were housed at an ambient temperature of 22 ±1°C, relative humidity of 50 ± 1%, and light/dark cycle of 12/12 h. Specific pathogen-free mice were fed for 8 weeks and sacrificed. The primary tumor was resected and immunohistochemical staining and IHC staining were performed on the excised tumor tissue. All the above operations were carried out in accordance with institutional guidelines and according to national and international laws and policies.

2.12 Hematoxylin and eosin (H&E) and Immunohistochemistry

Lung tissue sections were stained with H&E and the morphology of lung tissue was observed. Lung tissue sections were sent to Pinuofei Biological technology Company (Wuhan, China) for dehydration and embedding. Briefly, primary anti-E-cadherin (1:500, ProteinTech, Rosemont, IL, USA, 60330-1-IG), N-cadherin (1:800, Servicebio, Wuhan, China, GB11135) and Vimentin (1:2000, ProteinTech, 60330-1-IG) antibodies were added in proportion to phosphate-buffered saline at 4°C and incubated overnight. Horseradish peroxidase-Goat anti-rabbit (DAKO, at Agilent, Santa Clara, CA, USA K5007) was added and incubated at room temperature for 50 minutes. A histochemical kit comprising 3, 3’-diaminobenzidine as the chromogenic agent (1:100, DAKO, K5007) was used. Three 200 fields were randomly selected for each section in each group, and the positive cumulative optical density (IOD) of each image was obtained by analyzing each image to calculate the average optical density. The analysis software used was Image-pro pus (6.0 Media Cybernetics, Ins, Rockville, MD, USA).

2.13 REMARK criteria

The work has been reported in line with the REMARK criteria [17].

2.14 Statistical analysis

Numerical data were expressed as the mean ± standard deviation (SD). SPSS 16.0 software (IBM Corp., Armonk, NY, USA) was used for all statistical analyses. Student’s T test was used to determine statistical differences between the two groups. P < 0.05 was considered statistically significant.

3. Results

3.1 Mir-556-3p shows low expression in SqCLC

A total of 523 samples were obtained from the TCGA database, including 478 SqCLC tissues and 45 normal lung tissues, and 177 differentially expressed miRNAs were screened out (Figure 1A). Through univariate regression analysis, Kaplan–Meier curve analyses, and log-rank tests, 12 miRNAs with P values less than 0.05 were obtained, among which mir-556-3p had the lowest P value (Figure 1B). Moreover, the 5-year or 10-year survival rate of patients with high mir-556-3p expression was significantly better than that of patients with low mir-556-3p expression (Figure 1C). The expression of mir-556-3p in normal bronchial epithelial cells (BEAS-2B) and two kinds of SqCLC cell lines (H226, SK-MES-1) was detected. The results showed that the expression level of mir-556-3p in H226 and SK-MES-1 cell lines was significantly lower than that in BEAS-2B cells (Figure 1D).

MiR-556-3p is downregulated in SqCLC
Figure 1 

MiR-556-3p is downregulated in SqCLC. (A) Volcano plot revealing a total of 46 miRNAs (red and green) that were differentially expressed between 478 SqCLC and 45 normal samples (cutoff criteria: fold change >2 and adjust Pvalue (FDR) < 0.05). The red and green spots represent upregulated and downregulated miRNAs, respectively. (B) Prognostic survival analysis of miRNAs with P < 0.05. (C) Kaplan–Meier survival curves of the ISCC cohort from the TCGA with different miR-556-3p expression levels. miRNAs, microRNAs; LSCC, Lung squamous cell carcinoma; DEMs, differentially expressed miRNAs. (D) The expression of miR-556-3p in different kinds of SqCLC cells (H226 and SK-MES-1 cells) and in a normal human bronchial epithelial cell line (BEAS-2B). The expression in the normal group was set as 1. * P < 0.05.

3.2 Mir-556-3p inhibits the proliferation of SqCLC cells

Mir-556-3p-mimic was transfected into H226 and SK-MES-1 cells, and cell transfected with the mimic-NC were used as the controls. The mir-556-3p expression level was detected 36 h after transfection. The relative expression levels of mir-556-3p in H226 and SK-MES-1 cells were 736.22 and 970.18 times higher than those in NC group, respectively, confirming successful mir556-3p transfection and overexpression (Figure 2A-B). CCK8 assays showed that the cell proliferation rate of H226 and SK-MES-1 cells decreased by 66.6% and 60.1%, respectively, after transfection with mir-556-3p-mimic (Figure 2C-D). Thus, mir-556-3p effectively inhibited the proliferation of SqCLC.

MiR-556-3p suppresses the growth of SqCLC cells
Figure 2 

MiR-556-3p suppresses the growth of SqCLC cells. (A, B) qRT-PCR analysis of miR-556-3p expression in H226 and SK-MES-1 cells after transfection with miR-556-3p mimic or mimic-NC. The expression in the NC group was set as 1. (C, D) CCK8 analysis of the proliferation of H226 and SK-MES-1 cells treated as in (A) or (B). (*** P < 0.0001).

3.3 Mir-556-3p inhibited the invasion and migration of SqCLC cells

To investigate the role of mir-556-3p in SqCLC cells, the effects of mir-556-3p on the invasion and migration of H226 and SK-MES-1 cells were detected after transfection with mir-556-3p-mimic. At 24 h after wounding, the healing of the mir-556-3p-mimic group was significantly worse than that of mimic-NC group (Figure 3A-B). Similarly, in a Transwell experiment, the invasion and migration ability of mimic-NC group were significantly better than those of the mir-556-3p-mimic group. After 24 h, the numbers of H226 and SK-MES-1 cells that passed through the membrane in the mimic-NC group of were 1.47 times and 1.65 times higher, respectively, than those in the mir-556-3p-mimic group. In the migration experiment, the numbers of H226 and SK-MES-1 cells that passed through the membrane in the mimic-NC group were 1.45 and 1.23 times higher, respectively, than those in the mir-556-3p-mimic group (Figure 3C-D). The levels of E-cadherin, N-cadherin, and Vimentin in the two SqCLC cell lines were significantly different. The results confirmed that the levels of Ecadherin in the mir-556-3p-mimic group were significantly higher than those in the NC group. N-cadherin and Vimentin levels in the in mir-556-3p-mimic group were significantly lower than those in NC group. In conclusion, upregulation of mir-556-3p inhibited the invasion and migration of cells, and this effect of mir-556-3p acted through the EMT process (Figure 3E-F).

MiR-556-3p suppresses the invasion and migration of SqCLC cells
Figure 3 

MiR-556-3p suppresses the invasion and migration of SqCLC cells. (A) When the cells filled the petri dish, the cells were wounded, and the wound was photographed at 0, 6, 12, 24 h (view 200 μm). (B) The migration distances were measured in three independent experiments. (C) The migration or invasion of H226 and SK-MES-1 cells after transfection with mir-556-3p/NC was measured and cells were stained with crystal violet (view 100 μm). (D) The number of cells per view was counted and the experiment was repeated three times. (E) Western blotting was performed after transfecting cells with miR-556-3p mimics or mimic-NC, and E-Cadherin, NCadherin, and Vimentin protein levels were detected in three independent experiments. (F) After western blotting AlphaEaseFC was used to analyze the optical density of the immunoreactive protein bands, which were compared with the ratio of miR-556-3p/NC and GAPDH levels. (* P < 0.05, ** P < 0.001, *** P < 0.0001).

3.4 Mir-556-3p inhibits NUAK1 expression

Target genes downstream of mir-556-3p were searched for in the Gene, TargetScan, miRDB, and miRTarBase databases. A Venn diagram was constructed, which showed that three genes overlapped in the analyses of the four databases, namely NR2C2, NUAK1, and G3BP2, which might be the mir-556-3p target genes (Figure 4A). Then we detected the expression level of NUAK1 mRNA in different SqCLC cells. The relative expression level of NUAK1 in the mir-556-3p mimic group was significantly lower than that in the NC group, suggesting that mir-556-3p targeted NUAK1 (Figure 4B). The luciferase reporter gene assay showed that mir-556-3p binds to the 3’ UTR of the NUAK1 mRNA, resulting in degradation of the target mRNA or inhibition of protein synthesis. We constructed the 3’ UTR region of the NUAK1 attached to the luciferase reporter gene (Figure 4C). Next, a luciferase assay showed that mir-556-3p inhibited the luciferase activity of the wild-type 3’ UTR construct compared with 3’ UTR-NC-miRNA (P < 0.05), indicating that the binding of mir-556-3p to the NUAK1 3’ UTR inhibits its expression; however, mir-556-3p did not affect the luciferase activity of the mutant 3’ UTR (with a mutated miRNA binding site), indicating that inhibition of luciferase activity by mir-556-3p was abolished using the mutant NUAK1 3’ UTR (Figure 4D).

MiR-556-3p directly targets NUAK1
Figure 4 

MiR-556-3p directly targets NUAK1. (A) Venn diagram of overlapping target miRNAs from ‘TargetScan’ ‘miRDB’ ‘miRTarBase’ and ‘Gene’ analyses. (B) The mRNA expression of NUAK1 in different kinds of SqCLC cells (H226, SK-MES-1) after transfection with miR-556-3p mimic or mimic-NC. (C) The putative miR-556-3p binding site in the NUAK1 3’UTR. (D) Luciferase activity was analyzed after co-transfection with either miR-556-3p-mimics or the negative control wildtype plasmid or the mutant plasmid in 293T cells. TRAF6 was used as a positive control. (* P < 0.05, *** P < 0.0001).

3.5 NUAK1 promotes proliferation, invasion, and migration of SqCLC cells

In the wound healing assay, the healing degree of mir-556-3p-mimic + NUAK1 group was significantly better than that of the other two groups (Figure 5A-B). Through a CCK8 assay, the proliferation of mir-556-3p-mimic + vector group was significantly reduced compared with that of the mimic NC + Vector group, while the proliferation of the mir-556-3p-mimic + NUAK1 group was significantly higher than that of the other two groups (Figure 5C). Similarly, in both H226 and SK-MES-1 cells, the number of cells that passed through the Transwell membrane after 24 h in the mir-556-3p mimic-NC + Vector group was significantly lower than that in mimic-NC + Vector group, which further proved that mir-556-3p inhibited cell invasion. In contrast, the number of cells that passed through the Transwell membrane in the, mir-556-3p mimic + NUAK1 group was significantly higher than that of the other two groups. Cell migration experiments showed similar results (Figure 5D-E). Therefore, mir-556-3p, targets and regulates NUAK1, and NUAK1 promotes the proliferation of lung squamous cell carcinoma cells. Similarly, in H226 cells, the level of the E-cadherin protein correlated negatively with the expression level of NUAK1, while in the mir-556-3p + NUAK1 group, the levels of N-cadherin and Vimentin decreased. The same results were obtained in SK-MES-1 cells (Figure 5F). However, there was no statistically significant difference, possibly due to the insufficient reversal effect of NUAK1. We concluded that NUAK1 is a direct functional target of mir-556-3p, and via EMT, NUAK1 promotes the proliferation, invasion, and migration of SqCLC cells.

NUAK1 is a target of miR-556-3p
Figure 5 

NUAK1 is a target of miR-556-3p. (A) Proliferation of H226 and SK-MES-1 cells was detected by CCK8 assays after transfection with miR-556-3p mimics/NC/NUAK1. (B) When the cells filled the petri dish, the cells were wounded, and the wound was photographed at 0, 6, 12, 24 h (view 200 μm). (C) The migration distances were measured in three independent experiments. The migration of the NUAK1 group was set as 1. (D) The metastasis or invasion of H226 and SK-MES-1 cells after transfection with miR-556-3p/NC/NUAK1 was measured and the cells were stained with crystal violet (view 100 μm). (E) The number of cells per view was counted and the experiment was repeated three times. (F) Western blotting was performed after transfecting cells with miR-556-3p mimics or mimic-NC or NUAK1, and E-Cadherin, N-Cadherin, and Vimentin protein levels were detected in three independent experiments. (* P < 0.05, ** P < 0.001, *** P < 0.0001).

3.6 Mir-556-3p inhibits tumor growth in vivo

According to H&E staining, the lung tissue structure of the mir-556-3p agomir group was abnormal, with some atrophied alveoli and thickening of alveolar walls, lung parenchyma, and a large number of neutrophils and red blood cells being observed, and no obvious cell nodules were found in the tissues. In the agomir-NC group, cell nodules, partial alveolar wall thickening, alveolar atrophy and collapse, and alveolar wall thickening were observed in the lung tissues, with mild parenchyma and neutrophils and red blood cells. The agomir-NC group significantly contributed to tumor formation compared with the agomir group (Figure 6A). In addition, the protein levels of E-cadherin, N-Cadherin, and Vimentin in the agomir group were analyzed by immunohistochemistry, and the expression of E-Cadherin in the agomir group was significantly higher than that in the agomir-NC group. In contrast, E-cadherin and Vimentin levels were significantly higher in the agomir-NC group than in the agomir group (Figure 6B-C). This result was consistent with the in vivo experimental results, suggesting that mir-556-3p could inhibited the proliferation, invasion, and migration of SqCLC in vivo and in vitro, and inhibited the effect of EMT.

MiR-556-3p suppresses tumor growth in vivo
Figure 6 

MiR-556-3p suppresses tumor growth in vivo. (A) Tumors were stained with hematoxylin and eosin (H&E). (B) The E-Cadherin, N-Cadherin, and Vimentin protein levels was measured using immunohistochemistry. (C) Three 200-fold visual fields were randomly selected from each section in each group, and the positive cumulative optical density (IOD) value of each image was obtained through analysis and then the mean IOD was calculated (view 100 μm); (* P < 0.05, *** P < 0.0001).

4. Discussion

Lung cancer is still the leading cause of cancer death in worldwide, with a 5-year survival rate of about 20% [1]. MiRNAs have been proven to be related to the pathogenesis, diagnosis, and prognosis of lung cancer [18]. For example, mir-200 b[19], miRNA-148a [20], mir-196b-5p [21], mir-451A [22], and miRNA-300 [23] regulate the migration and invasion of non-small cell lung cancer cells. MicroRNA-218 [24] and microRNA-423-3p [25] are associated with poor prognosis of lung cancer. However, there have been few studies targeting mir-556-3p. In this study, mir-556-3p was observed to have low expression in lung cancer cells, and the wound-healing ability of the mir-556-3p mimic group was stronger than that of the mimic-NC group. Similarly, the invasion and migration ability of the mir-556-3p mimic group was stronger than of the mimic-NC group in Transwell experiments. Similar to our results, Wu et al. found that mir-556-3p could inhibit the proliferation and migration of lung cancer cells [15]. By contrast, other studies have found that miRNA-556-3p can promote the proliferation, migration, and invasion of bladder cancer cells [26] or the proliferation of esophageal cancer cells [27]. The phenomenon that miRNAs act as oncogenes in one cancer subtype but can induce tumor suppression in another might be caused by the diversity of miRNA target genes and their biological functions; therefore, the opposite regulation in different cancer types is reasonable [28, 29]. Consistent with the research results of Zhihui Wu et al., we also explored the inhibitory effect of Mir-556-3p on lung cancer in vitro experiments. However, they focused on the therapeutic effect of cisplatin-related therapy and emphasized the role of Mir-556-3p in the signaling pathway, without verification in vivo. It has been confirmed not only in vitro experiments, but also in vivo experiments. We believe that Mir-556-3p has an inhibitory effect on lung squamous cell carcinoma, and we are looking forward to whether Mir-556-3p can be used as a new biomarker for diagnosis and prognosis.

We found that the 5-year or 10-year OS of the mir-556-3p high expression group was better than that of mir-556-3p low expression group. Cell experiments showed that Mir-556-3p the inhibited invasion and migration of SqCLC cells. Subsequently, three possible Mir-556-3p target genes were identified: NR2C2, NUAK1, and G3BP2. To explore the potential mechanism of Mir-556-3p in SqCLC, in this study, Luciferase assays confirmed NUAK1 as a Mir-556-3p target gene. NUAK1 (also known as AMP-activated protein kinase family member 5 (ARK5)) is considered to be the fifth member of the human AMP-activated protein kinase (AMPK) family and is unique in the AMPK catalytic subunit family. This gene encodes a protein of 661 amino acids with a molecular weight of 74 kDa [29]. NUAK1 was first identified as one of 12 AMPK kinases that are homologous to the catalytic subunit of AMPK and are phosphorylated and activated by the major tumor suppressor liver kinase B1 (LKB1) [30, 31]. NUAK1 is mainly located in the nucleus, and is one of the main nuclear targeting subunits of S313 phosphorylated protein phosphatase-PNUTS [32]. One of the main physiological functions of NUAK1 is to protect cells from oxidative stress, which supports the survival of tumor cells in the harsh tumor microenvironment [33], and also participates in tumor invasion, metastasis, and drug resistance [34, 35, 36]. However, some studies have shown that NUAK1 has novel tumor suppressive function in LKB1-related signaling [37]. A high expression level of NUAK1 is associated with the poor prognosis of ovarian cancer [38], nasopharyngeal cancer [39], pancreatic cancer [40] and non-small cell lung cancer [41], as well as tumor drug resistance [35, 42]. NUAK1 has been reported to promote invasion and metastasis of cancer cells, including breast cancer, colorectal cancer, and glioma [43, 44, 45]. Recent studies have found that the optimized NUAK1/2 inhibitor, WZ4003 9Q, could effectively inhibit the growth of colorectal cancer and has good in vivo safety [46]. In this study, transfection of the NUAK1 overexpression plasmid reversed the inhibitory effect of mir-556-3p on invasion and migration of SqCLC cells. Similar to the results of this study, NUAK1 knockout inhibited EMT, migration, and invasion of prostate cancer and hepatocellular carcinoma cells [47, 48]. Similar to the results of this study, mir-145, mir-211, and mir-96 all inhibit the proliferation, invasion, and migration of cancer cells by targeting NUAK1 [49, 50, 51]. It is known that different miRNAs target the same genes and have the same effects, possibly because they act on the same signaling pathway.

Epithelial-mesenchymal transformation (EMT) is a highly dynamic and reversible process that is important in embryonic development, tissue repair, cancer progression, and chemotherapy resistance [52, 53, 54, 55, 56]. It is the core process of cancer metastasis and is usually observed in embryonic development but rarely in adult cells. EMT is characterized by Ecadherin-mediated loss of cell adhesion and increased cell motility, which promotes tumor invasion and metastasis [57]. SqCLC is one of the most aggressive malignancies because of its high metastasis rate, and it is also closely related to EMT. Studies showed that the invasion and migration ability of SqCLC cells was significantly increased after EMT enhancement [58, 59]. Autophagy-mediated degradation of E-cadherin promotes metastasis of SqCLC cells [60]. Decreased E-cadherin and increased Vimentin are independent predictors of poor prognosis in patients with SqCLC [61, 62]. Increasing evidence indicates that miRNAs play a key role in EMT and lung cancer metastasis [63, 64]. Both the mir-200 family and mir-1199-5p are EMT-promoting factors [65, 66, 67], and mir-182 promotes transforming growth factor beta (TGFβ)-induced EMT and cancer cell invasion [68]. In contrast, miRNA-4262 inhibits the EMT of cervical cancer cells by targeting ZBTB33 (encoding zinc finger and BTB domain containing 33) [69]. In vitro experiments of this study found that E-cadherin levels increased and N-cadherin and Vimentin levels decreased after transfection of mir-556-3p. However, mir-556-3p +NUAK1 co-transfection promoted EMT. Studies have shown that EMT is not a necessary prerequisite for metastasis [54, 56]. To this end, in vivo metastatic tumor experiments were conducted, which showed similar results to the in vitro experiments. These results suggested that the EMT process might play an important role in SqCLC metastasis. However, no obvious tumor formation was found in the construction of metastatic tumor model, which might be related to the short time of the experiment; however, the specific reasons need to be further studied. Indeed, the characteristics, specific regulatory signaling pathways, and prognostic effects of mir-556-3p, NUAK1, and EMT in patients with SqCLC require further research.

In conclusion, the results of the present study suggest that mir-556-3p inhibits EMT, invasion, and migration of SqCLC cells. Mechanistically, mir-556-3p inhibits SqCLC metastasis by directly targeting NUAK1. Mir-556-3p may function as a tumor suppressor gene and could be developed as a prognostic biomarker and therapeutic target for SqCLC. In the future, we may be able to develop specific prevention strategies for Mir-556-3p. Of course, we need to conduct further exploration based on clinical samples, which can be another direction of our future research with the rapid development of targeted therapy and immunotherapy. we believe that targeting molecular signaling pathways the study of the cancer genome will eventually lead to the introduction of new therapies into the clinic. Our new findings may provide new insights into the molecular changes in different tumor cell subpopulations, which may also provide new insights and hints for future individualized therapies based on the results of in vitro and in vivo experiments, regulation of Mir-556-3p expression can be considered as an appropriate strategy for the treatment of lung squamous cell carcinoma, and Mir-556-3p may become a novel diagnostic prognostic and individualized therapeutic prediction biomarker for lung squamous cell carcinoma.

Acknowledgements

This work was supported by the Key Research and Development Project of Jiangxi province [grant number 20192ACB70013, 20181ACG70011] and Science and Technology Innovation Outstanding Young talents training Program of Jiangxi Province [grant number 20192BCBL23023].

Competing Interests

The authors have no competing interests to declare.

Author Contributions

Long Huang and Ming Fang completed the project design. The operation experiment was performed by Ming Fang, Yini Cai, Gongji Yao, Lingmin Liao. Data analysis was done by Ming Fang, Yini Cai, Gongji Yao. Articles written by Yini Cai and Ming Fang. Data and article review by Long Huang.

Yini Cai and Ming Fang contributed equally to the manuscript.

Peer review

Not commissioned, externally peer-reviewed by IJ SONCOLOGY.

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