Evaluation of Exosomal miRNA in Blood as a Potential Diagnostic Biomarker for Human Non-Small Cell Lung Cancer

Abstract

BACKGROUND Tumor-derived exosomes have been used as diagnostic biomarkers to discriminate between tumor patients and healthy people. This study explored the roles of exosomal miRNAs in lung adenocarcinoma metastasis by microarray and developed a novel method for diagnosis of lung adenocarcinoma. MATERIAL AND METHODS Four lung adenocarcinoma patients' peripheral blood, including 2 metastasis and 2 N-metastasis, were used for exosomes miRNA microarray analysis. Exosomes were extracted by ultracentrifugation and identified by transmission electron microscopy. All the raw data were normalized by R software with limma packet. qRT-PCR was used to validate the microarray results. A549 cells were used to identify the functions of miR-4448. Western blot, qRT-PCR, RNAi, CCK8, and transwell invasion assay were used to verify the metastasis and proliferation abilities. RESULTS miR-4436a and miR-4687-5p were upregulated between the metastasis and N-metastasis group, while miR-22-3p, miR-3666, miR-4448, miR-4449, miR-6751-5p and miR-92a-3p were downregulated. miR-4448 was also downregulated between the metastasis and control group, whereas there was no significant difference between the N-metastasis group and control group. qRT-PCR confirmed the downregulation of miR-4448 in exosomes from lung adenocarcinoma patients compared with N-metastasis patients and healthy people. CCK8 and transwell invasion assay showed that A549 cells transfected with miR-4448 inhibitor had higher proliferation and metastasis ability. qRT-PCR and Western blot confirmed the high expression of MMP2 and MMP9 in A549 cells transfected with miR-4448 inhibitor. CONCLUSIONS miR-4448 can inhibit A549 cell proliferation and metastasis. miR-4448 in exosomes has the potential to serve as a diagnostic marker of patients with adenocarcinoma metastasis.

Figure 1

Identification of exosomes. (A) Transmission electron microscopy identified morphological characteristics of exosomes. All the extracellular vesicles were cup-shaped with diameters of nearly 100 nm. Scale bar: 100 nm. (B) Western blot shows the vesicles expressed CD9, CD63, and CD81.

Figure 2

Integrity of RNA and normalized intensity values. (A) Denaturing agarose gel electrophoresis of all RNA. (B) Light blue represents intensity values. (C) Deep blue represents normalized intensity values.

Figure 3

Volcano plot showed all the different expression miRNAs. The Pearson correlation was 0.9565 between N-metastasis and control group, 0.9776 between metastasis and control group, and 0.9651 metastasis and N-metastasis group. Red represents upregulated, green represents downregulated, and black means no significant change.

Figure 4

Expression of miR-4448 in exosomes among adenocarcinoma metastasis, no-Metastasis, and control. qRT-PCR showed that miR-4448 had significantly higher expression in N-metastasis (0.79±0.34) and control exosomes (1.02±0.36) compared with metastasis exosomes (0.18±0.21), but there was no significant difference between N-metastasis and control. * P<0.05.

Figure 5

miR-4448 inhibits A549 proliferation. A549 transfected with miR-4448 mimics showed less proliferation activity than in the control group at 2 h (1.21±0.11 vs. 1.45±0.09, P=0.043), 3 h (1.51±0.13 vs. 1.96±0.14, P=0.0151), 4 h (1.91±0.13 vs. 2.44±0.142, P=0.0086). However, A549 transfected with miR-4448 inhibitor showed enhanced proliferation activity compared to the control group at 2 h (1.71±0.12 vs. 1.45±0.09, P=0.0399), 3 h (2.44±0.13 vs. 1.96±0.14, P=0.0121), and 4 h (3.32±0.15 vs. 2.44±0.142, P=0.0018).

Figure 6

miR-4448 inhibits A549 migration. (A) Transwell invasion assay showed low expression of miR-4448 promoted migration. A549 transfected with miR-4448 inhibitor showed enhanced invasion activity (311.5±11.66) compared to normal A549 (211.7±10.51), P=0.0004. High expression of miR-4448 inhibited invasion (139.5±10.77), P=0.0011. * P<0.05. (B) qRT-PCR confirmed that high expression of MMP2 (3.57±0.68 vs. 1.09±0.43, P=0.0059) and MMP9 (4.22±0.89 vs. 1.11±0.45, P=0.0057) in A549 transfected with miR-4448 inhibitor. (C) Western blot analysis showed that high expression of MMP2 (3.82±0.72 vs. 1.05±0.23, P=0.0032) and MMP9 (4.35±0.79 vs. 1.06±0.25, P=0.0023) in A549 cells transfected with miR-4448 inhibitor.