| As a new type of molecular targets, microRNAs deserve a promising prospect to the diagnosis and treatment of non-small cell lung cancer (NSCLC). It has been discovered that a number of microRNAs directly participate in the occurrence and development of NSCLC, which are also correlated to the diagnosis, progress and prognosis of NSCLC. Recently, studies have been focused on the mechanism of microRNAs in malignant tumors. Researches have showed that miR-542-5p was down-regulated in neuroblastoma and miR-542-5p level was relatively correlated with the survival of neuroblastoma patients. In vivo, the over-expression of miR-542-5p could drive the function as a miRNA of "tumor suppressor", which could inhibit the proliferation, invasion and metastasis of neuroblastoma cells. So far, however, there is no report about the study of miR-542-5p in NSCLC patients. Therefore, the current thesis will analyze the relationship of the low expression of miR-542-5p and clinical pathological parameters in NSCLC. We will also predict the network of target genes, and then construct the animal model of chicken embryo chorioallantoic membrane (CAM) for NSCLC, as a result of exploring the elementary molecular mechanism of miR-542-5p in NSCLC with tumorigenesis and angiogenesis, so that to provide a good technical platform for further researches of the related biology of NSCLC.Purposes1. To detect the expression and clinical significance of miR-542-5p in NSCLC cell lines and tissues.2. To predict the target genes of miR-542-5p and perform the analysis of signaling pathway based on bioinformatics.3. To investigate the function and potential mechanism of miR-542-5p in NSCLC with tumorigenesis and angiogenesis in CAM.Methods1. The expression of miR-542-5p in NSCLCA total of 125 NSCLC tissue samples (101 cases of lung adenocarcinoma (LUAD) and 23 cases of lung squamous cell carcinoma (LUSC))were collected. The expression level of miR-542-5p in tissues was validated by real time quantitative polymerase chain reaction (RT-qPCR). We also analyzed the correlation of miR-542-5p with the clinicopathological characteristics of NSCLC and discussed the preliminary mechanism of this microRNA in lung cancer.2. The network analysis of prospective target genes of miR-542-5p2.1 Target genes of miR-542-5p were predicted by the softwares of miRWalk, Microt4, miRanda, mirbridge, miRDB, miRMap, miRNAMap, Pictar2, PITA, RNA22, RNAhybrid, and TargetScan. The software DAVID, was used to conduct GO enrichment analysis, KEGG pathway analysis and PANTHER analysis.2.2 The network chart of GO analysis was drawn with the software of Cytoscape_v3.3.0. The hub genes were extracted from the network chart of target genes drafted on the website (http://string-db.org/).2.3 The enrichment analysis of 452 target genes was utilized by Molecular Signatures Database (MSigDB) from the software of GSEA. Base on the data of NCI-60 cell lines derived from National Cancer Institute, the target genes were annotated by the form of gene expression profile and showed the trend of reciprocal expression in the defined function group. 3. The effects of miR-542-5p in NSCLC CAM.3.1 Four lung cancer cell lines, including H460, A549, H1299 and PC9, were chosen to screen the expression of miR-542-5p by RT-qPCR and one cell line with the lowest expression among them was selected for transfection.3.2 The lung cell line of H460 was selected to transfect after the establishment of the lentiviral vectors of LV-hsa-miR-542.3.3 The development of NSCLC in human patients was simulated in NSCLC CAM. In this model, the tumor size was measured. The ratio of the area of irregular vascular at the opening window was calculated by Image-Pro Plus software. The microscopic sections with HE staining and immunohistochemistry of Epidermal Growth Factor Receptor(EGFR), Vascular Endothelial Growth Factor(VEGF), D2-40 were investigated from Paraffin-embedded grafting tumor tissues. Thus the influence of miR-542-5p on tumorigenesis and angiogenesis of NSCLC could be studied and its possible mechanism could be further discussed.. Results1. The expression of miR-542-5p in NSCLC patients.1.1 In NSCLC patients (n=125), the expression of miR-542-5p in tumor tissue was significantly lower than that in adjacent tissues (1.952±1.507 vs 4.568±1.993, t=-11.703, P<0.001). Additionally, the expression of miR-542-5p in tumor tissue of LUAD (n=101) was significantly lower than that in adjacent tissues (2.355±1.647 vs 4.682±2.056, t=-11.183, P<0.001), and the expression of miR-542-5p in tumor tissue of LUSC was also significantly lower than that in adjacent tissues (2.355±1.647 vs 4.103±1.851; t=-3.383, P=0.002). The ROC curve was used to identify the diagnostic value of miR-542-5p expression levels in lung cancer. The AUC of miR-542-5p expression in NSCLC patients was 0.799 (95%CI 0.721～0.877, P<0.001). AUCs was also included in the LUAD group, as 0.832 (95%CI 0.753～0.911, P<0.001) for this group.1.2 Among the 125 NSCLC patients, the relative miR-542-5p expression levels in stage â…¢-â…£ patients was significantly lower than that of patients at stage â… and â…¡ (1.292±1.101 vs 2.822±1.536, t=6.488, P<0.001). In addition, miR-542-5p expression in patients with vascular invasion was significantly lower than that in patients without vascular invasion (1.475±1.305 vs 2.138±1.545, t=2.247, P<0.001). Also, miR-542-5p expression in patients with lymph node metastasis was significantly lower than that in patients without lymph node metastasis (1.504±1.266 vs 2.506±1.604, t=3.905, P<0.001). Spearman’s correlation analyses showed that the expression of miR-542-5p was negatively correlated with TNM stage (r=-0.505, P<0.001), lymph node metastasis (r=-0.332, P<0.001), and vascular invasion (r=-0.199, P=0.026).1.3 Similar results were noted in the subgroup of LUAD. In 101 LUAD cases, the expression of miR-542-5p in stage III-IV patients was significantly lower than that of patients at stage I and II (1.142±0.935 vs 2.810±1.516, t=6.417, P<0.001). miR-542-5p expression in patients with vascular invasion was significantly lower than that in patients without vascular invasion (1.375±1.30 vs 2.087±1.497, t=2.286, P=0.024). And miR-542-5p expression in patients with lymph node metastasis was significantly lower than that in patients without lymph node metastasis (1.333±1.095 vs 2.085±1.085, t=-2.477, P =0.027). Additionally, the expression of miR-542-5p was higher in patients who smoke than that of patients without smoking (3.065±1.542 vs 2.535±1.616, t=4.265, P<0.001). Spearman’s correlation analyses showed that the expression of miR-542-5p was negatively correlated with TNM stage (r=-0.564, P<0.001),. lymph node metastasis (r=-0.408, P<0.001), and vascular invasion (r=-0.224, P=0.024).1.4 In 23 LUSC cases, there was no significant association between the expression of miR-542-5p and clinical parameters.1.5 In tumor tissues of NSCLC patients, the relative expression of miR-542-5p in patients with high EGFR protein expression was significantly lower than that of patients with low EGFR protein expression (0.739±0.407 vs 3.049±1.194, t=7.753, P<0.001). Spearman’s correlation analyses showed that the expression of miR-542-5p was negatively correlated with EGFR protein expression (r=-0.723, P<0.001). While in the groups of LUAD and LUSC, the expression of miR-542-5p in patients with high EGFR protein expression was significantly lower than that of patients with low EGFR protein expression (0.836±0.373 vs 3.180±0.952, t=10.098, P<0.001, LUAD; 0.436±0.345 vs 2.888±1.448; t=6.543, P<0.001, LUSC). Spearman’s correlation analyses also showed that the expression of miR-542-5p was negatively correlated with EGFR protein expression in LUAD (r=-0.818, P<0.001) and LUSC (r=-0.828, P<0.001).1.6 Base on the median expression level of miR-542-5p in NSCLC patients (3.260±2.197), we divided the patients into two groups with high expression of miR-542-5p and low expression of miR-542-5p (4.568±1.993 vs 1.953±1.507). The results of Kaplan-Meier curve survival analyses showed that NSCLC patients with low miR-542-5p expression (n=50,11.274±1.387 months) had a significantly poorer prognosis than those patients with high miR-542-5p expression (n=7,35.714±3.469 months) (t=-6.219, P<0.001).2. The network analysis of the target genes of miR-542-5p2.1 A total of 57233 candidate target genes were screened by using 12 bioinformatics databases. Then, a total of 452 target genes of miR-542-5p were selected under the condition of six or more databases containing the genes and subjected to GO and pathway analyses.2.2 The 452 predicted target genes of miR-542-5p were inputted into the DAVID database for GO gene clustering enrichment analyses, with 147 annotations identified in biological processes (BP),15 annotations in cellular component (CC) and 37 annotations in molecular function (MF) (P<0.05). The top three of the most significant pathways in biological process (GOTERM BP FAT) were as follows, GO:0006357-regulation of transcription from RNA polymerase II promoter (P=0.002); GO:0008104-protein localization (P=0.002); GO:0016055-Wnt receptor signaling pathway (P=0.002). The top three of the most significant pathways in cellular component (GOTERM_CC_FAT) were as follows, GO:0043005-neuron projection (P=0.003); GO:0045202～synapse (P<0.001); GO:0042995-cell projection (P<0.001). The top three of the most significant pathways in molecular function (GOTERM MF FAT) were as follows, GO:0008022-protein C-terminus binding (P=0.003); GO:0030528-transcription regulator activity (P=0.017); GO:0043621-protein self-association (P=0.037).2.3 The 452 predicted target genes of miR-542-5p were inputted into the DAVID database for KEGG pathway enrichment analyses. And 37 annotations were identified in KEGG pathway database. The top three of the most significant pathways in KEGG were as follows, hsa04114:Oocyte meiosis (P=0.01); hsa05414:Dilated cardiomyopathy (P=0.012); hsa04916: Melanogenesis (P=0.018).2.4 The 452 predicted target genes of miR-542-5p were inputted into the DAVID database for KEGG pathway enrichment analyses. And 12 annotations were identified in PANTHER. The top three of the most significant pathways in PANTHER were as follows, P05731:GABA-B receptor II signaling (P=0.001); P00039:Metabotropic glutamate receptor group III pathway (P=0.013); P00033:Insulin/IGF pathway-protein kinase B signaling cascade (P=0.024).2.5 The 452 predicted target genes of miR-542-5p were also inputted into the STRING database for GO enrichment analyses, with 121 annotations identified in biological processes (BP),20 annotations in cellular component (CC), one annotations in molecular function (MF) and seven annotations in KEGG (P<0.05). The top three of the most significant pathways in biological process (GOTERM BP FAT) were as follows, nervous system development (GO:0007399), nervous (GO:0022008) and cell differentiation (GO:0030154). The top three of the most significant pathways in cellular component (GOTERM CC FAT) were as follows, cell projection, (GO:0042995), neuron projection (GO:0043005), neuron part (GO:0097458). The significant pathway in molecular function (GOTERM MF FAT) was protein blind (GO:0005515). The top three of the most significant pathways in KEGG were as follows, Morphine addiction, Cocaine addiction, Adrenergic signaling in cardiomyocytes.The 21 hub genes (RMT8, ADAMTS8, MASP1, HOXC9, ARTN, NPDC1, CASC3, NCDN, ARPC4, PGPEP1, TNNI3K, SLC24A3, EPS8L2, FARSA, PDDC1, PLEKHM1, UNKL, PRDM9, ISYNA, BCL2L1) were screened by using the network analysis and the score was ranked by STRING database.2.6 Compendia expression profiles of miR-542-5p target genes adapted from NCI-60 cell line showed that multiple target genes including NSD1, ELF4, HOXA9, ABL1, ABL2, NOTCH1, TCF3, SEPT6, NUP214 and CRTC1 were up-regulated in lung cancer cell lines of HOP92, HOP62, NCI-H226, NCI-H23, NCI-H322, NCI-460 and EKVX.3. The effects and elementary mechanism of miR-542-5p in NSCLC CAM.3.1 The lowest expression level of miR-542-5p was noted in H460 cell line as detected by RT-qPCR, thus H460 was selected for the further construction of NSCLC CAM.3.2 The best transfection concentration was tested:LV-hsa-mir-542 virus (virus 10 μl+ENIS 290 μl+ploy 1.25 μl+complete 2.2 ml) and empty carrier (virus 3 μl+ENIS 300 ul+ploy 1.25 μl+complete 2.2 ml). The qRT-PCR results showed that the miR-542-5p was up-regulated after being transfected with LV-hsa-miR-542, which indicated that the transfection was successful (P<0.001).3.3 The result of NSCLC CAM model:the cell lines in blank groups, negative groups and transfected groups were cultured in the 25ml petri dish. When the cells covered the dish, two good ones of each were selected and cultivated in the chicken embryo. The tumorigenesis and angiogenesis of the three groups from the first day to the fifth day were then observed. No" difference on first day was observed for tumor size. On the fifth day, compared to the blank group (30.13±0.06 mm3), the tumor size in CAM of the transfected group (4.34±0.07 mm3) was suppressed (t=543.260, P<0.001), but no significant difference (t=1.312, P=0.260) of negative group (30.09±0.07 mm3) was found compared to the blank control.3.4 Vasculogenesis:Vessels grew well and equally in all groups on the first day. On the fifth day of transfection, compared to the blank group (16.94±0.11), the area of vasculogenesis in CAM of the transfected group (8.33±0.08) was significantly suppressed (t=128.208, P<0.001), but no significant difference (t=-0.612, P=0.573) was observed between negative group (16.99±0.20) and blank control.3.5 HE section showed that local infiltration of the transfected group was not as obvious as the control groups. IHC showed the normal expression and comparative expression pattern of Epidermal Growth Factor Receptor (EGFR), vascular endothelial growth factor (VEGF), D2-40. The expression of EGFR (11.220±0.536 vs 6.680±0.536; t=13.399, P<0.001), VEGF (7.320±0.370 vs 4.320±0.421; t=11.971, P<0.001) and D2-40 (2.020±0.390 vs 1.500±0.255; t=2.496, P=0.037) in transfected group was significantly down-regulated on the fifth day of transfection.Conclusion1. The low expression of miR-542-5p in NSCLC tissues could inhibit the genesis and progression of tumors as well as the prognosis. miR-542-5p expression might have a moderately diagnostic value to lung cancer. Furthermore, the downregulation of miR-542-5p in NSCLC tissues could suppress the expression of EGFR protein. Therefore, miR-542-5p down-regulation in NSCLC indicates a new direction of the treatment for NSCLC patients.2. The molecular function, biological process and cellular structures of the potential target genes of miR-542-5p were analyzed by GO analysis. The protein pathway was analyzed by KEGG and PANTHER pathway. The most valuable genes among them were VEZT, KIT, CAMK2D, WEE2, PARVA, LTK, CLCN3.452 genes targeted by miR-542-5p were analyzed by the software, STRING. There was remarkable evidence that the potential target genes of miR-542-5p participate in the massages of nervous system. miR-542-5p also plays a vital role in the physiological processes of drug poisoning, like morphine. The current research also detected that the different expression level of multiple target genes of miR-542-5p targets annotated from NCI-60 cell line by compendia over-expression profiles. The analysis of potential targeting bioinformatics of miR-542-5p could lead to a theoretical foundation for researches into the molecular mechanisms of miR-542-5p with the occurrence and development of disease.3. The overexpression of miR-542-5p in CAM of NSCLC was likable to inhibit tumor growth and angiogenesis. It was verified that low expression of miR-542-5p could promote the progression and development of lung cancer. In vivo, tumors with the over-expression of miR-542-5p could suppress the expression of EGFR, VEGF and D2-40. It is speculated that miR-542-5p might participate in one of pathways regulating the expression of three proteins to inhibit tumorigenesis and angiogenesis in NSCLC. However, the specific molecular mechanism of miR-542-5p remains to be further studied. |
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