Bioinformatics analysis of differentially expressed miRNAs in non-small cell lung cancer

Abstract

Objective: Non-small cell lung cancer (NSCLC) contains 85% of lung cancer. Lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) are the largest NSCLC subgroups. The aim of the study was to investigate the underlying mechanism in developing more effective subtype-specific molecular therapeutic procedures.

Methods: A total of 876 specimens were used in this study: 494 LUAD tissues (ie, 449 LUAD tissues and 45 matched normal tissues) and 382 LUSC tissues (ie, 337 LUSC tissues and 45 matched normal tissues). The miRNA sequencing data were processed using R. The differential expressed miRNAs between lung cancer and normal tissues were analyzed using the limma package in R. Gene expression, Western blotting, hematoxylin and eosin staining, and luciferase assay were used to test LUAD and LUSC.

Results: LUAD and LUSC appear sharply distinct at molecular and pathological level. Let-7a-5p, miR-338, miR-375, miR-217, miR-627, miR-140, miR-147b, miR-138-2, miR-584, and miR-197 are top 10 relevant miRNAs and CLDN3, DSG3, KRT17, TMEM125, KRT5, NKX2-1, KRT7, ABCC5, KRAS, and PLCG2 are top 10 relevant genes in NSCLC. At the same time, the miRNAs expression levels were also quite different between the two groups. Among the differential expressed miRNAs, let-7a-5p was significantly down-regulated in LUAD while miR-338 was markedly down-regulated in LUSC. Bioinformatics analyses appeared that let-7a-5p directly targets high-molecular weight keratin 5 (KRT5) which were shown to be a strong risk factor for LUAD. And NK2 homeobox 1(NKX2-1) which was associated with tumor progression in LUSC was identified as a target gene of miR-338.

Conclusions: Distinct profile of miRNAs can take a part in the development of LUAD and LUSC and thus could serve as a subtype-specific molecular therapeutic target to protect against LUAD and LUSC.

Keywords: biomarker; lung adenocarcinoma; lung squamous cell carcinoma; miRNAs; non-small cell lung cancer.

FIGURE 1

Transcriptomic and histopathologic differences between LUAD and LUSC tissues. A, Normalized expression of LUAD‐selected genes in LUAD, LUSC, and normal lung tissues. B, Normalized expression of LUSC‐selected genes in LUAD, LUSC, and normal lung tissues. C, Representative images from hematoxylin and eosin‐stained sections of LUAD, LUSC, and normal lung tissues. Scale bar: 200 μm. The data represent the mean ± SEM. *P < .05; **P < .001; ***P < .0001; (Student's t test)

FIGURE 2

The differentially expressed miRNAs between LUAD and normal tissues. A, In the volcano plot, the red dots represent up/down‐regulated miRNAs, and black dots represent normally expressed miRNAs. B, Volcano plot graph of miRNAs in LUSC and normal lung tissues. C, Venn diagram represents miRNAs whose expression was different among LUAD, LUSC, and normal lung tissues. A‐I, Relative levels of miR‐217 (A), miR‐331 (B), miR‐627 (C), miR‐877 (D), miR‐378 (E), miR‐584 (F), miR‐140 (G), miR‐197 (H), and miR‐375 (I). The data represent the mean ± SEM. *P < .05; **P < .001; ***P < .0001; (Student's t test)

FIGURE 3

Let‐7a‐5p may prevent LUAD development by inhibiting KRT5. A, KRT5 protein levels in LUAD, LUSC, and normal lung tissues. B, Putative miRNA target sites of let‐7a‐5p within the 3′‐UTR of KRT5. C, Bioinformatic prediction of let‐7a‐5p target sites and free energy values within the 3′‐UTR of the human KRT5 gene. D, Relative expression level of let‐7a‐5p in LUAD, LUSC, and normal lung tissues. E, Pearson's correlation scatter plot of the fold changes of let‐7a‐5p and KRT5 protein in human LUAD tissue pairs. The data represent the mean ± SEM. *P < .05; **P < .001; ***P < .0001; (Student's t test). F, Relative luciferase activity in HEK293T cells transfected with plasmid reporter constructs containing the 3′‐UTR or mutated 3′‐UTR of KRT5, co‐transfected with mimic‐let‐7a‐5p. G, H, KRT5 mRNA (G) and protein (H) levels in HEK293T cells transfected with let‐7a‐5p mimic. I, KRT5 protein levels in HEK293T cells transfected with let‐7a‐5p antisense oligonucleotide. J, miRNA‐target interactions and functional associations using network‐based visual analysis

FIGURE 4

MiR‐338 may prevent LUSC development by inhibiting NKX2‐1. A, NKX2‐1 protein levels in LUSC, LUSC, and normal lung tissues. B, Putative miRNA target sites of miR‐338 within the 3′‐UTR of NKX2‐1. C, Bioinformatic prediction of miR‐338 target sites and free energy values within the 3′‐UTR of the human NKX2‐1 gene. D, Relative expression level of miR‐338 in LUSC, LUSC, and normal lung tissues. E, Pearson's correlation scatter plot of the fold changes of miR‐338 and NKX2‐1 protein in human LUSC tissue pairs. The data represent the mean ± SEM. *P < .05; **P < .001; ***P < .0001; (Student's t test). F, Relative luciferase activity in HEK293T cells transfected with plasmid reporter constructs containing the 3′‐UTR or mutated 3′‐UTR of NKX2‐1, co‐transfected with mimic‐miR‐338. G, H, NKX2‐1 mRNA (G) and protein (H) levels in HEK293T cells transfected with miR‐338 mimic. I, NKX2‐1 protein levels in HEK293T cells transfected with miR‐338 antisense oligonucleotide. J, miRNA‐target interactions and functional associations using network‐based visual analysis