Lung cancer is the main cause of cancer death . 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 . 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 . 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 . 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 . They are widely associated with the pathogenesis of many human diseases, including cancer . 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 .
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 . 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 . Mir-556-3p upregulated migration and invasion enhancer 1 (MIEN1) promotes cell proliferation and the epithelial-mesenchyme transition (EMT) process in hepatocellular carcinoma . 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 . 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 ; 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 . In addition, mir-556-5p downregulation improved the sensitivity of cisplatin through cell pyrophosis . 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.
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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
The work has been reported in line with the REMARK criteria .
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.
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-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.
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).
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).
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.
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.
Lung cancer is still the leading cause of cancer death in worldwide, with a 5-year survival rate of about 20% . MiRNAs have been proven to be related to the pathogenesis, diagnosis, and prognosis of lung cancer . For example, mir-200 b, miRNA-148a , mir-196b-5p , mir-451A , and miRNA-300  regulate the migration and invasion of non-small cell lung cancer cells. MicroRNA-218  and microRNA-423-3p  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 . By contrast, other studies have found that miRNA-556-3p can promote the proliferation, migration, and invasion of bladder cancer cells  or the proliferation of esophageal cancer cells . 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 . 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 . 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 , 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 . A high expression level of NUAK1 is associated with the poor prognosis of ovarian cancer , nasopharyngeal cancer , pancreatic cancer  and non-small cell lung cancer , 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 . 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 . 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 . 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 . In contrast, miRNA-4262 inhibits the EMT of cervical cancer cells by targeting ZBTB33 (encoding zinc finger and BTB domain containing 33) . 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.
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].
The authors have no competing interests to declare.
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.
Not commissioned, externally peer-reviewed by IJ SONCOLOGY.
Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin, 2021; 71: 209–249. DOI: https://doi.org/10.3322/caac.21660
Socinski MA, Obasaju C, Gandara D, Hirsch FR, Bonomi P, Bunn P, Kim ES, Langer CJ, Natale RB, Novello S, et al. Clinicopathologic Features of Advanced Squamous NSCLC. J Thorac Oncol, 2016; 11: 1411–1422. DOI: https://doi.org/10.1016/j.jtho.2016.05.024
Justilien V, Walsh MP, Ali SA, Thompson EA, Murray NR, Fields AP. The PRKCI and SOX2 oncogenes are coamplified and cooperate to activate Hedgehog signaling in lung squamous cell carcinoma. Cancer Cell, 2014; 25: 139–151. DOI: https://doi.org/10.1016/j.ccr.2014.01.008
Ettinger DS, Wood DE, Akerley W, Bazhenova LA, Borghaei H, Camidge DR, Cheney RT, Chirieac LR, D’Amico TA, Demmy TL, et al. Non-Small Cell Lung Cancer, Version 6.2015. J Natl Compr Canc Netw, 2015; 13: 515–524. DOI: https://doi.org/10.6004/jnccn.2015.0071
Iqbal MA, Arora S, Prakasam G, Calin GA, Syed MA. MicroRNA in lung cancer: role, mechanisms, pathways and therapeutic relevance. Mol Aspects Med., 2019; 70: 3–20. DOI: https://doi.org/10.1016/j.mam.2018.07.003
Gebert LFR, MacRae IJ. Regulation of microRNA function in animals. Nat Rev Mol Cell Biol, 2019; 20: 21–37. DOI: https://doi.org/10.1038/s41580-018-0045-7
Rupaimoole R, Slack FJ. MicroRNA therapeutics: towards a new era for the management of cancer and other diseases. Nat Rev Drug Discov, 2017; 16: 203–222. DOI: https://doi.org/10.1038/nrd.2016.246
Zhao W, Cao L, Zeng S, Qin H, Men T. Upregulation of miR-556-5p promoted prostate cancer cell proliferation by suppressing PPP2R2A expression. Biomed Pharmacother, 2015; 75: 142–147. DOI: https://doi.org/10.1016/j.biopha.2015.07.015
Zhang Y, Yu R, Li Q, Li Y, Xuan T, Cao S, Zheng J. SNHG1/miR-556-5p/TCF12 feedback loop enhances the tumorigenesis of meningioma through Wnt signaling pathway. J Cell Biochem, 2020; 121: 1880–1889. DOI: https://doi.org/10.1002/jcb.29423
Hu F, Chen X, Gao J, Shen Y, Yang J. CircDIP2C ameliorates oxidized low-density lipoprotein-induced cell dysfunction by binding to miR-556-5p to induce TET2 in human umbilical vein endothelial cells. Vascul Pharmacol, 2021; 139: 106887. DOI: https://doi.org/10.1016/j.vph.2021.106887
Liu Y, Wang X, Li H, Guan E, Luo K. Propofol Ameliorates the Proliferation and Epithelial-Mesenchymal Transition of Hepatoma Carcinoma Cells via Non-Coding RNA Activated by DNA Damage (NORAD)/microRNA (miR)-556-3p/Migration and Invasion Enhancer 1 (MIEN1) Axis. J Environ Pathol Toxicol Oncol, 2021; 40: 87–97. DOI: https://doi.org/10.1615/JEnvironPatholToxicolOncol.2021039471
He J, Chu Z, Lai W, Lan Q, Zeng Y, Lu D, Jin S, Xu H, Su P, Yin D, et al. Circular RNA circHERC4 as a novel oncogenic driver to promote tumor metastasis via the miR-556-5p/CTBP2/E-cadherin axis in colorectal cancer. J Hematol Oncol, 2021; 14: 194. DOI: https://doi.org/10.1186/s13045-021-01210-2
Lei L, An G, Zhu Z, Liu S, Fu Y, Zeng X, Cao Q, Yan B. C8orf48 inhibits the tumorigenesis of colorectal cancer by regulating the MAPK signaling pathway. Life Sci, 2021; 266: 118872. DOI: https://doi.org/10.1016/j.lfs.2020.118872
Wu Z, Gong Q, Yu Y, Zhu J, Li W. Knockdown of circ-ABCB10 promotes sensitivity of lung cancer cells to cisplatin via miR-556-3p/AK4 axis. BMC Pulm Med, 2020; 20: 10. DOI: https://doi.org/10.1186/s12890-019-1035-z
Shi F, Zhang L, Liu X, Wang Y. Knock-down of microRNA miR-556-5p increases cisplatin-sensitivity in non-small cell lung cancer (NSCLC) via activating NLR family pyrin domain containing 3 (NLRP3)-mediated pyroptotic cell death. Bioengineered, 2021; 12: 6332–6342. DOI: https://doi.org/10.1080/21655979.2021.1971502
Altman DG, McShane LM, Sauerbrei W, Taube SE. Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK): Explanation and Elaboration. PLoS Med, 2012; 9(5): e1001216. DOI: https://doi.org/10.1371/journal.pmed.1001216
Wu KL, Tsai YM, Lien CT, Kuo PL, Hung AJ. The Roles of MicroRNA in Lung Cancer. Int J Mol Sci, 2019; 20. DOI: https://doi.org/10.3390/ijms20071611
Xiao P, Liu W, Zhou H. [Retracted] miR200b inhibits migration and invasion in nonsmall cell lung cancer cells via targeting FSCN1. Mol Med Rep, 2021; 24. DOI: https://doi.org/10.3892/mmr.2021.12218
Chen Y, Min L, Ren C, Xu X, Yang J, Sun X, Wang T, Wang F, Sun C, Zhang X. miRNA-148a serves as a prognostic factor and suppresses migration and invasion through Wnt1 in non-small cell lung cancer. PLoS One, 2017; 12: e0171751. DOI: https://doi.org/10.1371/journal.pone.0171751
Liang G, Meng W, Huang X, Zhu W, Yin C, Wang C, Fassan M, Yu Y, Kudo M, Xiao S, et al. miR-196b-5p-mediated downregulation of TSPAN12 and GATA6 promotes tumor progression in non-small cell lung cancer. Proc Natl Acad Sci U S A, 2020; 117: 4347–4357. DOI: https://doi.org/10.1073/pnas.1917531117
Yang Y, Ding L, Hu Q, Xia J, Sun J, Wang X, Xiong H, Gurbani D, Li L, Liu Y, Liu A. MicroRNA-218 functions as a tumor suppressor in lung cancer by targeting IL-6/STAT3 and negatively correlates with poor prognosis. Mol Cancer, 2017; 16: 141. DOI: https://doi.org/10.1186/s12943-017-0710-z
Wang R, Li G, Zhuang G, Sun S, Song Z. Overexpression of microRNA-423-3p indicates poor prognosis and promotes cell proliferation, migration, and invasion of lung cancer. Diagn Pathol, 2019; 14: 53. DOI: https://doi.org/10.1186/s13000-019-0831-3
Feng C, Sun P, Hu J, Feng H, Li M, Liu G, Pan Y, Feng Y, Xu Y, Feng K, Feng Y. miRNA-556-3p promotes human bladder cancer proliferation, migration and invasion by negatively regulating DAB2IP expression. Int J Oncol, 2017; 50: 2101–2112. DOI: https://doi.org/10.3892/ijo.2017.3969
Shah MY, Ferrajoli A, Sood AK, Lopez-Berestein G, Calin GA. microRNA Therapeutics in Cancer – An Emerging Concept. EBioMedicine, 2016; 12: 34–42. DOI: https://doi.org/10.1016/j.ebiom.2016.09.017
Zhong S, Golpon H, Zardo P, Borlak J. miRNAs in lung cancer. A systematic review identifies predictive and prognostic miRNA candidates for precision medicine in lung cancer. Transl Res, 2021; 230: 164–196. DOI: https://doi.org/10.1016/j.trsl.2020.11.012
Suzuki A, Kusakai G, Kishimoto A, Lu J, Ogura T, Lavin MF, Esumi H. Identification of a novel protein kinase mediating Akt survival signaling to the ATM protein. J Biol Chem, 2003; 278: 48–53. DOI: https://doi.org/10.1074/jbc.M206025200
Lizcano JM, Goransson O, Toth R, Deak M, Morrice NA, Boudeau J, Hawley SA, Udd L, Makela TP, Hardie DG, Alessi DR. LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. EMBO J, 2004; 23: 833–843. DOI: https://doi.org/10.1038/sj.emboj.7600110
Cossa G, Roeschert I, Prinz F, Baluapuri A, Vidal RS, Schulein-Volk C, Chang YC, Ade CP, Mastrobuoni G, Girard C, et al. Localized inhibition of protein phosphatase 1 by NUAK1 promotes spliceosome activity and reveals a MYC-sensitive feedback control of transcription. Mol Cell, 2021; 81: 2495. DOI: https://doi.org/10.1016/j.molcel.2021.05.013
Port J, Muthalagu N, Raja M, Ceteci F, Monteverde T, Kruspig B, Hedley A, Kalna G, Lilla S, Neilson L, et al. Colorectal Tumors Require NUAK1 for Protection from Oxidative Stress. Cancer Discov, 2018; 8: 632–647. DOI: https://doi.org/10.1158/2159-8290.CD-17-0533
Zhao MM, Ge LY, Yang LF, Zheng HX, Chen G, Wu LZ, Shi SM, Wang N, Hang YP. LncRNA NEAT1/miR-204/NUAK1 Axis is a Potential Therapeutic Target for Non-Small Cell Lung Cancer. Cancer Manag Res, 2020; 12: 13357–13368. DOI: https://doi.org/10.2147/CMAR.S277524
Xu T, Zhang J, Chen W, Pan S, Zhi X, Wen L, Zhou Y, Chen BW, Qiu J, Zhang Y, et al. ARK5 promotes doxorubicin resistance in hepatocellular carcinoma via epithelial-mesenchymal transition. Cancer Lett, 2016; 377: 140–148. DOI: https://doi.org/10.1016/j.canlet.2016.04.026
Shi L, Zhang B, Sun X, Lu S, Liu Z, Liu Y, Li H, Wang L, Wang X, Zhao C. MiR-204 inhibits human NSCLC metastasis through suppression of NUAK1. Br J Cancer 2014; 111: 2316–2327. DOI: https://doi.org/10.1038/bjc.2014.580
Humbert N, Navaratnam N, Augert A, Da Costa M, Martien S, Wang J, Martinez D, Abbadie C, Carling D, de Launoit Y, et al. Regulation of ploidy and senescence by the AMPK-related kinase NUAK1. EMBO J, 2010; 29: 376–386. DOI: https://doi.org/10.1038/emboj.2009.342
Phippen NT, Bateman NW, Wang G, Conrads KA, Ao W, Teng PN, Litzi TA, Oliver J, Maxwell GL, Hamilton CA, et al. NUAK1 (ARK5) Is Associated with Poor Prognosis in Ovarian Cancer. Front Oncol, 2016; 6: 213. DOI: https://doi.org/10.3389/fonc.2016.00213
Liu J, Tang G, Huang H, Li H, Zhang P, Xu L. Expression level of NUAK1 in human nasopharyngeal carcinoma and its prognostic significance. Eur Arch Otorhinolaryngol, 2018; 275: 2563–2573. DOI: https://doi.org/10.1007/s00405-018-5095-0
Xu H, Mao J, Yang X, Chen F, Song Z, Fei J, Chen W, Zhong Z, Wang X. AMPactivated protein kinase family member 5 is an independent prognostic indicator of pancreatic adenocarcinoma: A study based on The Cancer Genome Atlas. Mol Med Rep, 2020; 22: 4329–4339. DOI: https://doi.org/10.3892/mmr.2020.11504
Chen P, Li K, Liang Y, Li L, Zhu X. High NUAK1 expression correlates with poor prognosis and involved in NSCLC cells migration and invasion. Exp Lung Res, 2013; 39: 9–17. DOI: https://doi.org/10.3109/01902148.2012.744115
Wang X, Song Z, Chen F, Yang X, Wu B, Xie S, Zheng X, Cai Y, Chen W, Zhong Z. AMPK-related kinase 5 (ARK5) enhances gemcitabine resistance in pancreatic carcinoma by inducing epithelial-mesenchymal transition. Am J Transl Res, 2018; 10: 4095–4106.
Lu S, Niu N, Guo H, Tang J, Guo W, Liu Z, Shi L, Sun T, Zhou F, Li H, et al. ARK5 promotes glioma cell invasion, and its elevated expression is correlated with poor clinical outcome. Eur J Cancer, 2013; 49: 752–763. DOI: https://doi.org/10.1016/j.ejca.2012.09.018
Kusakai G, Suzuki A, Ogura T, Miyamoto S, Ochiai A, Kaminishi M, Esumi H. ARK5 expression in colorectal cancer and its implications for tumor progression. Am J Pathol, 2004; 164: 987–995. DOI: https://doi.org/10.1016/S0002-9440(10)63186-0
Chang XZ, Yu J, Liu HY, Dong RH, Cao XC. ARK5 is associated with the invasive and metastatic potential of human breast cancer cells. J Cancer Res Clin Oncol, 2012; 138: 247–254. DOI: https://doi.org/10.1007/s00432-011-1102-1
Yang H, Wang X, Wang C, Yin F, Qu L, Shi C, Zhao J, Li S, Ji L, Peng W, et al. Optimization of WZ4003 as NUAK inhibitors against human colorectal cancer. Eur J Med Chem, 2021; 210: 113080. DOI: https://doi.org/10.1016/j.ejmech.2020.113080
Ye Z, Chen X, Chen X. ARK5 promotes invasion and migration in hepatocellular carcinoma cells by regulating epithelial-mesenchymal transition. Oncol Lett, 2018; 15: 1511–1516. DOI: https://doi.org/10.3892/ol.2017.7453
Xiong X, Sun D, Chai H, Shan W, Yu Y, Pu L, Cheng F. MiR-145 functions as a tumor suppressor targeting NUAK1 in human intrahepatic cholangiocarcinoma. Biochem Biophys Res Commun, 2015; 465: 262–269. DOI: https://doi.org/10.1016/j.bbrc.2015.08.013
Huang X, Lv W, Zhang JH, Lu DL. miR96 functions as a tumor suppressor gene by targeting NUAK1 in pancreatic cancer. Int J Mol Med, 2014; 34: 1599–1605. DOI: https://doi.org/10.3892/ijmm.2014.1940
Bell RE, Khaled M, Netanely D, Schubert S, Golan T, Buxbaum A, Janas MM, Postolsky B, Goldberg MS, Shamir R, Levy C. Transcription factor/microRNA axis blocks melanoma invasion program by miR-211 targeting NUAK1. J Invest Dermatol, 2014; 134: 441–451. DOI: https://doi.org/10.1038/jid.2013.340
Lim J, Thiery JP. Epithelial-mesenchymal transitions: insights from development. Development, 2012; 139: 3471–3486. DOI: https://doi.org/10.1242/dev.071209
Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest, 2009; 119: 1420–1428. DOI: https://doi.org/10.1172/JCI39104
Fischer KR, Durrans A, Lee S, Sheng J, Li F, Wong ST, Choi H, El Rayes T, Ryu S, Troeger J, et al: Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance. Nature, 2015; 527: 472–476. DOI: https://doi.org/10.1038/nature15748
Ye X, Weinberg RA. Epithelial-Mesenchymal Plasticity: A Central Regulator of Cancer Progression. Trends Cell Biol, 2015; 25: 675–686. DOI: https://doi.org/10.1016/j.tcb.2015.07.012
Zheng X, Carstens JL, Kim J, Scheible M, Kaye J, Sugimoto H, Wu CC, LeBleu VS, Kalluri R. Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer. Nature, 2015; 527: 525–530. DOI: https://doi.org/10.1038/nature16064
Du L, Pertsemlidis A. microRNAs and lung cancer: tumors and 22-mers. Cancer Metastasis Rev, 2010; 29: 109–122. DOI: https://doi.org/10.1007/s10555-010-9204-9
Xu L, Yan Y, Xue X, Li CG, Xu ZY, Chen HZ. Angiogenin elevates the invasive potential of squamous cell lung carcinoma cells through epithelialmesenchymal transition. Oncol Rep, 2016; 36: 2836–2842. DOI: https://doi.org/10.3892/or.2016.5107
Li B, Chen P, Wang JH, Li L, Gong JL, Yao H. Ferrerol overcomes the invasiveness of lung squamous cell carcinoma cells by regulating the expression of inducers of Epithelial Mesenchymal Transition. Microb Pathog, 2017; 112: 171–175. DOI: https://doi.org/10.1016/j.micpath.2017.09.048
Xu W, Chen B, Ke D, Chen X. TRIM29 mediates lung squamous cell carcinoma cell metastasis by regulating autophagic degradation of E-cadherin. Aging (Albany NY), 2020; 12: 13488–13501. DOI: https://doi.org/10.18632/aging.103451
Aruga N, Kijima H, Masuda R, Onozawa H, Yoshizawa T, Tanaka M, Inokuchi S, Iwazaki M. Epithelial-mesenchymal Transition (EMT) is Correlated with Patient’s Prognosis of Lung Squamous Cell Carcinoma. Tokai J Exp Clin Med, 2018; 43: 5–13.
Zhang H, Liu J, Yue D, Gao L, Wang D, Zhang H, Wang C. Clinical significance of E-cadherin, beta-catenin, vimentin and S100A4 expression in completely resected squamous cell lung carcinoma. J Clin Pathol, 2013; 66: 937–945. DOI: https://doi.org/10.1136/jclinpath-2013-201467
Zhang J, Ma L. MicroRNA control of epithelial-mesenchymal transition and metastasis. Cancer Metastasis Rev, 2012; 31: 653–662. DOI: https://doi.org/10.1007/s10555-012-9368-6
Legras A, Pecuchet N, Imbeaud S, Pallier K, Didelot A, Roussel H, Gibault L, Fabre E, Le Pimpec-Barthes F, Laurent-Puig P, Blons H. Epithelial-to-Mesenchymal Transition and MicroRNAs in Lung Cancer. Cancers (Basel), 2017; 9. DOI: https://doi.org/10.3390/cancers9080101
Diepenbruck M, Tiede S, Saxena M, Ivanek R, Kalathur RKR, Luond F, Meyer-Schaller N, Christofori G. miR-1199-5p and Zeb1 function in a double-negative feedback loop potentially coordinating EMT and tumour metastasis. Nat Commun, 2017; 8:1168. DOI: https://doi.org/10.1038/s41467-017-01197-w
Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, Farshid G, Vadas MA, Khew-Goodall Y, Goodall GJ. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol, 2008; 10:593–601. DOI: https://doi.org/10.1038/ncb1722
Burk U, Schubert J, Wellner U, Schmalhofer O, Vincan E, Spaderna S, Brabletz T. A reciprocal repression between ZEB1 and members of the miR-200 family promotes EMT and invasion in cancer cells. EMBO Rep, 2008; 9: 582–589. DOI: https://doi.org/10.1038/embor.2008.74
Yu J, Lei R, Zhuang X, Li X, Li G, Lev S, Segura MF, Zhang X, Hu G. MicroRNA-182 targets SMAD7 to potentiate TGFbeta-induced epithelial-mesenchymal transition and metastasis of cancer cells. Nat Commun, 2016; 7: 13884. DOI: https://doi.org/10.1038/ncomms13884
Feng, J. Upregulation of MicroRNA-4262 Targets Kaiso (ZBTB33) to Inhibit the Proliferation and EMT of Cervical Cancer Cells. Oncol Res, 2018; 26: 1215–1225. DOI: https://doi.org/10.3727/096504017X15021536183526