YKL-40 regulated epithelial-mesenchymal transition and migration/invasion enhancement in non-small cell lung cancer

Abstract

Background: YKL-40 is a secreted inflammatory protein that its overexpression has been reported to correlate with poor outcome of various malignant diseases, especially in cancer. However, the function of this protein is still unclear.

Methods: The clinical prognosis of non-small cell lung cancers (NSCLC) patients and their clinical YKL-40 expressions were obtained from the Prognoscan database. The expressions of YKL-40 in patient samples were determined by Western Blotting assay. YKL-40 gene knockdown and overexpression were performed on NSCLC cancer cells (CL1-1 and CL1-5). The cells were investigated for their epithelial-mesenchymal transition (EMT) markers gene modulation through Western Blotting and RT-PCR. Further cell metastatic abilities were assessed by transwell migration and invasion assay.

Result: In this study, YKL-40 was observed to be highly expressed in NSCLC specimens. Furthermore, determined by the PrognoScan database analysis, patients with high expression levels of YKL-40 were found with poor prognosis. In the in vitro study, different characteristics of NSCLC cell lines (CL1-1, H23, H838, CL1-5, and H2009) were used as study models, where YKL-40 expression levels were determined to correlate with the phenotypic characteristics of cancer metastasis. In this study,YKL-40 was demonstrated to regulate EMT marker expressions such as Twist, Snail, Slug, N-cadherin, Vimentin, and E-cadherin. The protein's affects in cancer cell migration and invasion were also observed in YKL-40 overexpression or knock down NSCLC cell lines.

Conclusion: All of results from this study suggest that YKL-40 is a major factor in NSCLC metastasis. Thus, YKL-40 may serve as therapeutic targets for NSCLC patients in the future.

Fig. 1

a Western Blot analysis comparison of YKL-40 gene expressions in normal lung tissues and non-small cell lung cancer (NSCLC). b YKL-40 immunohistochemistry staining comparison between normal lung tissue and lung tumor tissue (200 x magnification). c YKL-40 expression comparison analysis between normal lung cell and lung adenocarcinoma (Luad) & Lung squamous carcinoma (Lusc), *, P < 0.05, **, P < 0.005 (d) The correlation between YKL40 expression and NSCLC patient prognosis. Comparison of YKL40 expression and patient prognosis was examined using the Prognoscan database

Fig. 2

The effect of YKL-40 expression level on the invasion and migration abilities of the NSCLC cell lines. a The five NSCLC cell lines were divided into low metastasis (CL1-1, H23) and high metastasis (H838, CL1-5, H2009) groups by invasion and migration assay. b The YKL-40 expression level analysis by reverse transcription polymerase chain reaction (RT-PCR) and Western blot: indicating that the low and high metastasis groups expressed reduced and increased levels of YKL-40 mRNA and protein, respectively

Fig. 3

a and b CL1-1 cells (I) were transfected with the green fluorescent protein (GFP) expression plasmid (pEGFP-C1) plasmid, encoding empty vector (II) or full-length YKL-40 (III). After stable expression, cells were knockdowned via shRNA against human YKL-40 (IV). Four cells were subjected to Matrigel invasion assay (a) and transwell migration assay (b). c and d CL1-5 cells (I) were transfected with shRNA vector control (II) or shRNA against human YKL-40 (III). The cells were then co-transfected with the DsRed plasmid encoding YKL-40 (IV). The ability of these four cells to invade through Matrigel (c) or to migrate (d) was assayed. Mean ± transwell assay was determined by three independent experiments; statistical significance was measured using one way ANOVA, *, P < 0.05, **, P < 0.01, ***, P < 0.001. e in vivo study of each transfected cell lines’ migration ability. Each transfected cell lines were injected into nude mice for 6 weeks before lung harvest and lung nodules number determination. This experiment was conducted in triplicates for each group

Fig. 4

The epithelial–mesenchymal transition (EMT) marker expression regulation in modulated YKL-40 gene expression NSCLC. a EMT markers expression analysis of YKL-40 overexpressed CL1-1 cell (I) and YKL-40 reknockdown of YKL-40 overexpressed CL1-1 cell, as well as b EMT markers expression analysis of YKL-40 knockdowned CL1-5 cell (I) and YKL-40 re-overexpression of YKL-40 knockdowned CL1-1 cell using RT-PCR and Western Blot analysis. Mean ± transwell assay were determined by three independent experiments; statistical significance was measured using one way ANOVA, *, P < 0.05, **, P < 0.01, ***, P < 0.001

Fig. 5

The schematic representation of EMT markers regulation by YKL-40, resulting in the induction of EMT