The suppressive role of miR-542-5p in NSCLC: the evidence from clinical data and in vivo validation using a chick chorioallantoic membrane model

Background Non-small cell lung cancer (NSCLC) has led to the highest cancer-related mortality for decades. To enhance the efficiency of early diagnosis and therapy, more efforts are urgently needed to reveal the origins of NSCLC. In this study, we explored the effect of miR-542-5p in NSCLC with clinical samples and in vivo models and further explored the prospective function of miR-542-5p though bioinformatics methods. Methods A total of 125 NSCLC tissue samples were collected, and the expression of miR-542-5p was detected by qRT-PCR. The relationship between miR-542-5p level and clinicopathological features was analyzed. The effect of miR-542-5p on survival time was also explored with K-M survival curves and Cox’s regression. The effect of miR-542-5p on the tumorigenesis of NSCLC was verified with a chick chorioallantoic membrane (CAM) model. The potential target genes were predicted by bioinformatics tools, and relevant pathways were analyzed by GO and KEGG. Several hub genes were validated by Proteinatlas. Results The expression of miR-542-5p was down-regulated in NSCLC tissues, and consistent results were also found in the subgroups of adenocarcinoma and squamous cell carcinoma. Down-regulation of miR-542-5p was found to be connected with advanced TNM stage, vascular invasion, lymphatic metastasis and EGFR. Survival analyses showed that patients with lower miR-542-5p levels had markedly poorer prognosis. Both tumor growth and angiogenesis were significantly suppressed by miR-542-5p mimic in the CAM model. The potential 457 target genes of miR-542-5p were enriched in several key cancer-related pathways, such as morphine addiction and the cAMP signaling pathway from KEGG. Interestingly, six genes (GABBR1, PDE4B, PDE4C, ADCY6, ADCY1 and GIPR) from the cAMP signaling pathway were confirmed to be overexpressed in NSCLCs tissues. Conclusions This evidence suggests that miR-542-5p is a potential tumor-suppressed miRNA in NSCLC, which has the potential to act as a diagnostic and therapeutic target of NSCLC.

Background Non-small cell lung cancer (NSCLC) is the most frequent type of lung cancer with high mortality worldwide [1]. Lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) are the major subtypes of NSCLC, composing approximately 40% and 30% of NSCLC, respectively [2]. Like most malignancies, the patients with NSCLC who received diagnoses at an early stage achieved higher five-year survival rates, compared to patients whose diagnoses were made at an advanced stage [3]. However, only a minority of NSCLC patients received early diagnosis because of the lack of significant symptoms in early stages [3]. Currently, therapeutic measures for advanced NSCLC patients are still limited. Although the study of molecular targeted therapies is progressing, including EGFR and ALK-targeted therapies in lung adenocarcinoma, which have had a successful beginning, they are efficient in just 20% of patients [4]. Given these results, high-performance biological markers are critically needed to find and diagnose NSCLC in early stages, to prevent NSCLC from advancing, and to help advanced patients achieve a better prognosis.
MicroRNAs (miRNAs, miRs) are a type of non-coding RNA with a short (less than 22 nucleotides), single stranded nucleotide chain. MiRNAs can regulate the generation of proteins by binding to the untranslated region of messenger RNAs, using complementary base pairing. Through this mechanism, miRNAs can regulate the differentiation, proliferation and apoptosis of cells [5]. Many studies have found that the dysregulation of miRNAs correlates with diseases, including lung cancer [6,7]. In NSCLC, hundreds of dysregulated miRNAs have been detected from high-throughput experiments [8,9]. However, the clinicopathological significance and related mechanisms of dysregulated miRNAs in NSCLC remain largely unclear. In preliminary studies, we found that miR-30a [10], miR-193a-3p and miR-133a-3p were down-regulated in NSCLC tissues [11,12], and all of them have an effect on survival time of patients.
In the current study, we explored the expression of miR-542-5p in NSCLC tissues, assessed the relationship between miR-542-5p and clinicopathological parameters, and verified the function of miR-542-5p on NSCLC in vivo. Furthermore, the potential mechanism of miR-542-5p action on NSCLC was predicted by bioinformatics methods.

Tissue samples
The tissue samples fixed in this study were from 125 lung cancer patients who underwent surgeries at the First Affiliated Hospital of Guangxi Medical University between January 2012 and February 2014. All tissues were obtained before any cancer-related therapy was carried out. Adjacent noncancerous tissues were obtained from at least two centimeters away from the edge of the tumor node. All samples were prepared in the form of formalin-fixed and paraffin-embedded (FFPE). The included lung cancer tissues were divided into 101 LUAD, 23 LUSC and 1 large cell lung cancer. Among 125 included patients, 75 were males, and 50 were females. There were 57 younger (<60 years) patients and 68 older (>60 years) patients. The subgroups were divided based on clinicopathological parameters, such as tumor size, smoking history, and vascular invasion, which are displayed in Table 1. Among 125 patients, 57 LUAD patients were followed-up  The construction and packaging of miR-542-5p lentivirus vector, as well as lentivirus infection, were performed by Shanghai Genechem Co. Ltd., and stored at −80°C.

Cell transfection
Among four types of NSCLC cells, the H460 cell line showed the lowest level of miR-542-5p, so we chose H460 to conduct the following experiments. Blank control, empty vector control (H460-Lv-vector) and experimental (H460-Lv-miR-542) groups were designed. H460 cells were seeded and infected with lentivirus in 6-well plates at an MOI of 100 and selected by puromycin. The green fluorescence expressed in cells served as a marker to measure the infection efficiency.

Chick chorioallantoic membrane model
The chick chorioallantoic membrane (CAM) model was used to explore the effect of miR-542-5p on tumor growth and angiogenesis of NSCLC cells. After being obtained from local hatchery, fertilized eggs were incubated in an incubator at 37°C and 80% humidity and rotated every 5 h until day 7. On the 8th day of fertilization, air space and embryo were checked under the egg candler, and the head of the embryo and large vessels in the CAM were marked by pencil. To separate the chorioallantoic membrane from the inner shell membrane, a tiny hole (φ = 0.1 cm) in the middle of the air space was made by a mini electric grinder, and an aurilave was used to draw out the gas. A 1.5 cm 2 window was made in the shell where large vessels in the CAM were marked. A silicone ring (φ = 0.5 cm, height = 0.3 cm) was put at the crossing of large vessels, and cells were then transferred into the silicone ring; the quantity of cells was 10 × 10 6 in each egg. The eggs were randomly grouped into blank control, empty vector control (H460-Lv-vector) and experimental (H460-Lv-miR-542) groups. Biological informatics analysis of potential target genes of miR-542-5p The target genes of miR-542-5p were predicted by 13 platforms (miRWalk, Microt4, miRanda, mirbridge, miRDB, miRMap, miRNAMap, Pictar2, PITA, RNA22, RNAhybrid, Targetscan and mirTarbase). Genes copredicted on at least five platforms were collected. To explore the potential function of miR-542-5p in NSCLC, the target genes were analyzed by Gene Ontology (GO) analysis, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in Gorilla (http://cbl-gorilla.cs.technion.ac.il/) and DAVID (https://david.ncifcrf.gov/). String (http://www.stringdb.org/) was used to analyze the connections among genes and draw connected figures. The protein expression data from Proteinatlas (http://www.proteinatlas.org/) was checked for the validation of several potential target genes of miR-542-5p in NSCLCs.

Statistical analysis
The medium value of miR-542-5p was used to divide the NSCLC patients into low expression and high Fig. 3 Kaplan-Meier survival curves in 57 patients with lung adenocarcinoma with different miR-542-5p levels expression groups. Student's t test was selected to assess the different expression levels of miR-542-5p between NSCLC and adjacent lung tissues, trials and control groups. Spearman method was used to assess the relationship between miR-542-5p level and clinical pathological parameters. Kaplan-Meier (KM) method, log rank test as well as Cox's regression were performed in the survival analyses. Adjusted hazard ratios (HRs) were calculated. p < 0.05 was considered to be statistically significant. The data were calculated by SPSS 22.0, and figures were constructed with Graphpad Prism 5.0.

Results
The expression of miR-542-5p in NSCLC tissues MiR-542-5p showed evidently lower expression in NSCLC when compared to adjacent normal lung tissues, and consistent results were also found in the subgroups of LUAD and LUSC (Fig. 1a-c). The diagnostic value was assessed by ROC curves. The AUC values were 0.859, 0.876 and 0.769 in NSCLC, LUAD and LUSC, respectively (Fig. 1d-f ).

In vivo study using the CAM model
MiR-542-5p was also detected by RT-qPCR in NSCLC cell lines H460, PC9, H1299 and A549. Among the four different cell lines, the lowest level of miR-542-5p was found in H460, and it was selected for transfection with a miR-542-5p mimic to unveil the function of miR-542-5p in NSCLC. The efficiency of lentiviral transfection was higher than 90%. In CAM, the tumor size was assessed after cells were transplanted and harvested on day 5. Compared to the blank and negative control groups, the trial group showed smaller tumor size, and angiogenesis was suppressed (Fig. 4). The expression levels of EGFR, VEGF and D2-40 in the trial group were weaker, when compared to the blank control group (Fig. 5).
Potential target genes of miR-542-5p and functional annotation analysis A total of 457 target genes of miR-542-5p were predicted by the 12 platforms mentioned above. Close connections were found among the genes listed in Fig. 6. These genes were analyzed by GO and KEGG pathways. In the CC of GO analysis, the genes were significantly enriched in the plasma membrane and extrinsic components of the cytoplasmic side of the plasma membrane (Fig. 7a). In MF, non-membrane spanning protein tyrosine kinase activity, ATP binding and other binding items were significantly enriched by genes (Fig.  7b). In BP, the adenylate cyclase-activating G-protein coupled receptor signaling pathway was the most significantly enriched pathway (Fig. 7c). The most significantly enriched KEGG pathway was morphine addiction; and cancer-related mechanisms, such as the cAMP signaling pathway and the Hippo signaling pathway, were also significantly enriched in the 457 potential target genes of miR-542-5p (Table 2, Fig. 8). The genes from cAMP signaling pathway were selected for further protein expression validation in NSCLC tissues by using data from Proteinatlas. Six genes showed particularly stronger expression pattern in lung cancer tissues, as compared to normal lungs (Figs. 9,10,11,12,13 and 14), which demonstrated that these six genes (GABBR1, PDE4B, PDE4C, ADCY6, ADCY1 and GIPR) had more likelihood of being direct targets of miR-542-5p in lung cancers. However, this hypothesis still needs in vitro and in vivo verification.

Discussion
In this study, miR-542-5p was found to be predominantly down-regulated in NSCLC tissues, which had a negative effect on the prognosis of NSCLC patients. Exploration of the effect of miR-542-5p on NSCLC with a CAM model confirmed the suppressive function of miR-542-5p on cell growth and angiogenesis of NSCLC. Furthermore, the mechanism of miR-542-5p predicted by bioinformatical approaches suggested valuable pathways that might relate to tumorigenesis and tumor development. Many dysregulated miRNAs have been shown to relate to tumorigenesis or development in numerous cancers. In published studies, miR-542-5p was described as a tumor-suppressed miRNA in endometrial carcinosarcoma [14], neuroblastoma [15] and rectal cancer [16]. However, in several cancers, miR-542-5p Fig. 11 Validation of the protein expression of PDE4C in NSCLCs. PDE4C protein was detected by the antibody of HPA048975. a, b: pneumocytes in normal lungs with no staining. c, d: high staining in LUAD. Immunohistochemistry, ×100 was regarded as a cancer promotor. For example, in osteosarcoma, miR-542-5p was overexpressed in cancer tissues and linked with poor prognosis [17]. In lung cancer, the effect of miR-542-5p was reported in only one publication [18]. In the study of Yamaguchi et al. [18], miR-542-5p was found to be inversely expressed with EGFR in lung cancer tissues when tested by immunohistochemistry, which was consistent with our study. In addition, the expression of VEGF and D2-40 in NSCLC tissues were also tested in current study, and both of them were supported the idea that miR-542-5p can suppress angiogenesis in NSCLC. In vitro, miR-542-5p was reported to suppress the proliferation of the lung cancer cell line A549, which was supported by our CAM assays. In the study of Yamaguchi et al. [18], because of the lack of normal lung tissues to compare with, the relative level of miR-542-5p in lung cancer tissues was not clear. To supplement this research, the differential expression of miR-542-5p between NSCLC and adjacent normal lung tissues was tested in the current study, and the results suggested that miR-542-5p was notably down-regulated in NSCLC tissues. It is also interesting to find that the lower miR-542-5p level in NSCLC could predict the poorer prognosis with the adjust HR being 0.948 (p = 0.003), which further confirms that miR-542-5p acts as a tumor-suppressive miRNA in the pathogenesis and progression of NSCLC, and higher level of miR-542-5p level could act as a protective indicator of NSCLC. However, this finding needs to be verified with larger sample size.
Although many miRNAs have been affirmed as tumor suppressors or promoters in NSCLC, the functional mechanism of miRNAs in NSCLC was still unclear. In the current study, we explored the potential target genes of miR-542-5p using 13 programs with different algorithms, then analyzed target genes by functional annotation. The enriched results of GO analysis and KEGG pathways suggested that most potential target genes are significantly related to message transfer. The trio of enriched KEGG pathways, the cAMP signaling pathway, the Hippo signaling pathway, and other cancer-related pathways, hinted at the probable mechanism of miR-542-5p in cancers. Cyclic adenosine monophosphate (cAMP), as a second messenger, can regulate cellular responses by activated effectors [19]. The most famous effector of cAMP is the cAMP-dependent protein kinase (PKA). Shaikh et al. [20] found that prevention of the activity of PKA could suppress the hypoxia-mediated epithelial-mesenchymal transition (EMT), which is involved in invasion and migration in human lung cancer cells. cAMP signaling can also down-regulate p300, which is a transcriptional coactivator, through Epac and p38 MARK [21]. The Hippo signaling pathway was another significant enriched KEGG pathway found in our analysis. It has been reported to regulate the proliferation and apoptosis of cells, mediated by transcription coactivators like yes-associated protein (YAP) [22]. Several studies of the Hippo signaling pathway in NSCLC have been published [23][24][25]. You et al. [23] found that Hippo/YAP signaling was inhibited after knockdown of ERK1/2. In breast cancer cells, Zhang et al. [26] found that the Hippo signaling pathway has an effect on EMT. EMT is also a vital process in NSCLC, which is developed from normal lung epithelial cells [27]. Wnt signaling was reported to relate to Hippo signaling [28], and it was notable that two members of the Wnt family (Wnt10A and Wnt7B) were predicted as target genes of miR-542-5p in the current study and were enriched in the Hippo signaling pathway. In our functional annotation of genes, the cAMP and Hippo signaling pathways were enriched in 15 and 12 genes, respectively. To test the predicting power and validate the potential target genes of miR-542-5p in NSCLC, the protein level of all the genes involved in the cAMP pathway were checked in Proteinatlas. Interestingly, six genes (GABBR1, PDE4B, PDE4C, ADCY6, ADCY1 and GIPR) were confirmed to be overexpressed in NSCLCs tissues. These six genes have greater possibility to be real target genes of miR-542-5p in NSCLCs. This signaling pathway might play an integral part in the potential mechanism of miR-542-5p in NSCLC. Fig. 13 Validation of the protein expression of ADCY1 in NSCLCs. ADCY1 protein was detected by the antibody of CAB018364. a, b: pneumocytes in normal lungs with no staining, macrophages with medium staining. c: high staining in LUAD. d: high staining in LUSC. Immunohistochemistry, ×100