Identification of Cerebrospinal Fluid MicroRNAs Associated With Leptomeningeal Metastasis From Lung Adenocarcinoma

Abstract

Background: Leptomeningeal metastasis (LM) has frequently been observed in patients with lung adenocarcinoma. So far, its diagnosis and disease course monitoring are still extremely difficult. Moreover, there is no effective treatment regimen for LM due to a lack knowledge on the molecular mechanism of LM. This study aimed to identify LM-related cerebrospinal fluid (CSF) miRNAs, which have potential value for diagnosing and monitoring LM and exploring the molecular mechanism. Methods: CSF miRNAs were screened and verified by microarray analysis and quantitative real-time PCR (qRT-PCR) in LM patients with lung adenocarcinoma and non-LM controls, and the diagnostic performance of candidate miRNAs was evaluated. Then, candidate miRNAs in matched CSF samples from LM patients at diagnosis, after initial therapy, at relapse, and after salvage therapy, were analyzed to assess the relationship between CSF miRNAs and LM disease course. The effect of candidate miRNAs on proliferation, invasion, and migration of lung adenocarcinoma cell lines was assessed. The targeted genes of the candidate miRNA were predicted by TargetScan, miRDB, and miRTarbase online analysis tools. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze the functional categories of predicted target genes. Results: CSF miR-7975, miR-7977, and miR-7641 were screened and verified to be statistically significantly up-regulated in LM patients compared to non-LM controls. The three miRNAs, when combined, exhibited optimal diagnostic performance. Longitudinal data of CSF miR-7975 and miR-7977 correlated well with clinical courses of LM. Overexpression of miR-7977 promoted proliferation, migration, and invasion of lung adenocarcinoma cells. Moreover, 385 targeted genes of miR-7977 were predicted and were involved in various pathways related to cancer metastasis. Conclusions: This study offers insights for future research of CSF miRNAs as robust tools for diagnosing and monitoring LM. It also reveals a novel pathway for exploration of underlying mechanisms of LM.

Keywords: bioinformatic analysis; cerebrospinal fluid; leptomeningeal metastasis; lung adenocarcinoma; microRNA profiling.

Copyright © 2020 Pan, Yang, He, Gao, Jiang, Chen and Zhao.

Figures

Figure 1

Differential expression of miRNAs in CSF from discover cohort. (A) The overlapping miRNAs differentially expressed between two comparison models (LMs, n = 10; BMs, n = 5; NCs, n = 5); (B) Heatmap of differentially expressed CSF miRNAs analyzed by microarray between LMs (n = 10) and non-LMs controls (BMs, n = 5; NCs, n = 5); (C) Heatmap of differentially expressed miRNAs analyzed by microarray in paired CSF samples from 6 LM patients at diagnosis and after initial LM-directed therapy. Fold change ≥2 or ≤0.5 was regarded as statistically significant. CSF, cerebrospinal fluid; LMs, leptomeningeal metastases; BMs, brain metastases; NCs, non-cancers.

Figure 2

Validation of candidate CSF miRNAs and evaluation of their diagnostic performance in validation cohort. RELs of CSF miR-7975 (A), miR-7977 (B), miR-7641 (C), and miR-4800-5p (D) in 68 LM patients vs. 48 non-LM controls (BMs, n = 43; NCs, n = 5) were detected by qRT-PCR. Then ROC curve analysis with RELs of CSF miRNAs including miR-7975 (E), miR-7977 (F), miR-7641 (G), and the combined three CSF miRNAs (H), was performed for discrimination of LMs and non-LM controls. RELs of miRNAs (y-axis) are normalized to cel-miR-39. The black horizontal lines represent median REL values with SEM. A value of P < 0.05 was regarded as statistically significant. RELs, relative expression levels; LMs, leptomeningeal metastases; BMs, brain metastases; NCs, non-cancers; SEM, standard error of mean; AUC, area under curve.

Figure 3

Identification of specific CSF miRNAs expression through the course of LM. RELs of miRNA were compared between matched CSF samples from 22 LM patients at diagnosis and after initial LM-directed therapy. Compared with the levels at diagnosis, CSF miR-7975 (A) and miR-7977 (B) were significantly down regulated in LM patients after initial efficacious therapy, while CSF miR-7641 (C) was not. Then miR-7975 and miR-7977 in sequential CSF samples from 8 LM patients collected at four time points: at diagnosis, after initial therapy, at relapse and after salvage therapy were analyzed. Longitudinal REL data of both CSF miR-7975 (D) and miR-7977 (E) in 6 out of 8 LM patients correlated well with the clinical courses of disease, decreasing after initial therapy, rising during relapse, and again returning to lower levels after salvage therapy. Lines, matched samples. RELs of miRNAs (y-axis) are normalized to cel-miR-39. A value of P < 0.05 was regarded as statistically significant. RELs, relative expression levels; LM, leptomeningeal metastasis.

Figure 4

The role of miR-7977 in regulating lung adenocarcinoma cell proliferation, migration, and invasion. After transfection with miRNA mimics and negative control at final concentrations of 50 nM for 24 h: (A) the expression levels of miR-7975 and miR-7977 in NCI-H1650 and A549 cells were detected using qRT-PCR; (B) the proliferation assay of NCI-H1650 and A549 cells was performed using CCK-8 kit at three time points: 12, 24, and 48 h; (C) wounds were made in NCI-H1650 and A549 cells layer using a sterile micropipette tip, and the width of the wound gap was viewed under a microscope and photographed at 0, 9, and 24 h after wounding; (D) the invasion assay of NCI-H1650 and A549 cells was performed using Matrigel-coated transwell chambers at another 48 h of incubation. The data were expressed as mean ± SD. Each experiment was performed at least three times independently on different days. *P < 0.05, **P < 0.0001. mimic NC, mimic negative control; SD, standard deviation.

Figure 5

The targeted genes of miR-7977 prediction and function anlysis. (A) The overlapping targeted genes were predicted using TargetScan, miRDB and miRTarbase online analysis tools; (B) The enriched GO annotation for target genes of miR-7977 including biological process, cellular component and molecular function; (C) The enriched KEGG pathways for target genes of miR-7977. The gene count ≥ 3 were set as the cut-off criteria.