Identification of the DE-miRNAs as well as the potentially targeted genes
LUAD and normal tissue miRNAs expression profile was obtained from TCGA-LUAD. The microarray data had 283 Stage I LUAD tissues, 123 Stage II LUAD tissues, 84 Stage III LUAD tissues, 24 Stage IV LUAD tissues and 54 normal tissues. There are 147 DE-miRNAs were obtained between Stage I/II LUAD and normal tissues and 151 DE-miRNAs were obtained between Stage III/IV LUAD and normal tissues. Overall 123 miRNAs with differed expression levels were identified, including 41 down-regulated DE-miRNAs and 82 up-regulated DE-miRNAs (Fig. 1a, Additional file 1). In order to show the significantly differential distribution of the 123 DE-miRNAs, a heat map of the above identified miRNAs was drown using data profile TCGA as a reference (Fig. 1b). Additionally, 1716 target genes for identified miRNAs were achieved based on the miRWalk2.0 database, among which 1202 genes were for the up-regulated miRNAs while 514 ones were for the down-regulated miRNAs.
Enrichment analyses of DEGs
The GO functional enrichment analysis of the targeted genes was performed with the DAVID database, in which three functional groups were included (i.e. molecular function/MF, cell composition/CC, biological processes/BP) (Fig. 2). Among the target genes of the up-regulated miRNAs, the top 3 enriched BPs were positive regulation of RNA polymerase II promoter transcription, RNA polymerase II promoter transcription and negative regulation of RNA polymerase II promoter transcription, and the most enriched CCs were nucleoplasm, nucleus and cytoplasm, while the most enriched MFs included protein binding, transcriptional activator activity, RNA polymerase II core promoter proximal region sequence-specific binding and RNA polymerase II core promoter proximal region sequence-specific DNA binding (Fig. 2a and b). Similarly, among the target genes of the down-regulated miRNAs, the top 3 enriched BPs were positive regulation of RNA polymerase II promoter transcription, positive regulation of transcription, DNA-templated and osteoblast differentiation, and the mostly enriched CCs were nucleus, cytoplasm and cell-cell adheres junction, while the most enriched MFs included protein binding, RNA polymerase II core promoter proximal region sequence-specific DNA binding and transcriptional activator activity, RNA polymerase II core promoter proximal region sequence-specific binding (Fig. 2c and d). All of the above results showed that majority of the DEGs were significantly enriched in RNA polymerase II promoter, cell part and binding.
Signaling pathway enrichment analysis
According to the KEGG pathway enrichment analysis, the potentially targeted genes of the up-regulated DE-miRNAs included FoxO signaling pathway, AMPK signaling pathway, transcriptional misregulation in cancer, pathways in cancer, adherens junction, axon guidance, PI3K-Akt signaling pathway, MAPK signaling pathway and regulation of actin cytoskeleton (Fig. 3a), whereas with respect to the down-regulated DE-miRNAs, the enriched KEGG pathways included adherens junction, ubiquitin mediated proteolysis, signaling pathways that regulate stem cell pluripotency, proteoglycans in cancer, pathways in cancer, hippo signaling pathway, SCLC and microRNAs in cancer (Fig. 3b).
Analyzing target genes in LUAD using PPI network and modular
According to the STRING database, great deal of interaction was observed among the identified target genes. A total of 1008 target genes (693 up-regulated and 315 down-regulated genes) of the 1716 key genes were selected to construct the PPI network. For the purpose of better display, the screeening was conducted respectively for the 15 hub nodes of which the DE-miRNAs were up-regulated or down-regulated to the most extent. Finally, the up-regulated nodes with the filtering of degree >= 37 were selected, including LRRK2, EP300, MAPK14, CDC42, HDAC2, RAC1, PIKFYVE, SIRT1, NFKB1, KAT2B, NRAS, RB1, ASH1L, UBE2D1 and HUWE1 (Fig. 4a), among which the LRRK2 demonstrated the most significant degree (degree = 93). Similarly, the down-regulated nodes with the filtering of degree >= 18 were selected, including NOTCH1, BCL2, FOS, MYB, ASH1L, UBE2D1, FBXW7, IGF1R, CUL3, WNT3A, SOCS1, PIKFYVE, HUWE1, INSR and UBE4A (Fig. 4b), among which the NOTCH1 was the most significant (degree = 40).
Validation of the DE-miRNAs by using the TCGA LUAD dataset
In order to verify our results, among the 123 DE-miRNAs identified in our study, 22 miRNAs were also found to be significant according to the downloaded LUAD TCGA dataset (e.g. mir-31, mir-196b, mir-133b, mir-215, mir-548v and mir-328) (p < 0.05) (Fig. 5 and Fig. 6).
Exploration of significant pathways associated with OS correlated microRNAs
In order to explore the pathogenesis of microRNA in LUAD, we selected two OS associated microRNAs, mir-9 and mir-486, which with the greatest significance in expression, and then explored their carcinogenic mechanism. By retrieving the mirwalk2.0 database, we found that two miRNAs were associated with multiple pathways. Moreover, some significant target genes were also obtained and showed in Fig. 7.