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

Jefri M, Huang YN, Huang WC, Tai CS, Chen WL - BMC Cancer (2015)

Bottom Line: 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.Furthermore, determined by the PrognoScan database analysis, patients with high expression levels of YKL-40 were found with poor prognosis.In this study,YKL-40 was demonstrated to regulate EMT marker expressions such as Twist, Snail, Slug, N-cadherin, Vimentin, and E-cadherin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, 1001 University Road, Hsinchu, Taiwan, 300, ROC. malvinjefri@hotmail.com.

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.

No MeSH data available.


Related in: MedlinePlus

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
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Fig3: 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

Mentions: To investigate the effect of YKL-40 on cancer cell migration/invasion, YKL-40 knockdown in CL1-5 cells and YKL-40 overexpression in CL1-1 cells were constructed. After establishing the stable knockdown and overexpression cell lines, YKL-40 gene was transfected to re-overexpress in YKL-40 knockdown CL1-5 cells and re-knockdown of the CL1-1, YKL-40 overexpressed-cells. The YKL-40 expression by RT-PCR and Western blot in our cell lines are shown in Fig. 4a and b. The YKL-40 mRNA expression was substantially attenuated in YKL-40 knockdowned-cell and overexpression-reknockdown cell, when compared with cells without YKL-40 knockdown or the vector control. The YKL-40 gene was down-regulate to 6.9 % in the YKL-40 knockdowned CL 1–5 and had 35 %/22 % decrease in invasion/migration, when compared with the controls (Fig. 3a, b). The YKL-40 gene was up-regulated to 825 % in YKL-40 overexpression CL 1–1, which exhibited a 370 %/448 % increase of invasion/migration when compared with the controls (Fig. 3c, d). The YKL-40 overexpression-reknockdown CL1-1 cell was demonstrated to reverse its invasion/migration ability (Fig. 3a and b). Similarly, the YKL-40 knockdowned CL1-5 cell exhibited decreased ability of invasion/migration, when compared with the CL1-5 wild type. The invasion/migration ability of YKL-40 knockdowned CL1-5 cell was reversed when YKL-40 was re-overexpressed (Fig. 3c and d). Furthermore, the metastatic abilities of these cells were also confirmed in vivo. Four CL1-1 cell lines (WT, vector only, YKL-40 overexpression, and YKL-40 knockdown-reoverexpression) were injected into nude mice for 6 weeks and the number of metastatic lung tumor nodules were determined. The number of tumor nodules of the YKL-40 overexpression cells were significantly higher than those of the other groups (Fig. 3e). Overall, the results showed that an increase of YKL-40 expression can significantly enhance cancer migration/invasion and vice versa. This suggest that YKL-40 expression is associated with of NSCLC cells migration/invasion.Fig. 3


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

Jefri M, Huang YN, Huang WC, Tai CS, Chen WL - BMC Cancer (2015)

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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig3: 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
Mentions: To investigate the effect of YKL-40 on cancer cell migration/invasion, YKL-40 knockdown in CL1-5 cells and YKL-40 overexpression in CL1-1 cells were constructed. After establishing the stable knockdown and overexpression cell lines, YKL-40 gene was transfected to re-overexpress in YKL-40 knockdown CL1-5 cells and re-knockdown of the CL1-1, YKL-40 overexpressed-cells. The YKL-40 expression by RT-PCR and Western blot in our cell lines are shown in Fig. 4a and b. The YKL-40 mRNA expression was substantially attenuated in YKL-40 knockdowned-cell and overexpression-reknockdown cell, when compared with cells without YKL-40 knockdown or the vector control. The YKL-40 gene was down-regulate to 6.9 % in the YKL-40 knockdowned CL 1–5 and had 35 %/22 % decrease in invasion/migration, when compared with the controls (Fig. 3a, b). The YKL-40 gene was up-regulated to 825 % in YKL-40 overexpression CL 1–1, which exhibited a 370 %/448 % increase of invasion/migration when compared with the controls (Fig. 3c, d). The YKL-40 overexpression-reknockdown CL1-1 cell was demonstrated to reverse its invasion/migration ability (Fig. 3a and b). Similarly, the YKL-40 knockdowned CL1-5 cell exhibited decreased ability of invasion/migration, when compared with the CL1-5 wild type. The invasion/migration ability of YKL-40 knockdowned CL1-5 cell was reversed when YKL-40 was re-overexpressed (Fig. 3c and d). Furthermore, the metastatic abilities of these cells were also confirmed in vivo. Four CL1-1 cell lines (WT, vector only, YKL-40 overexpression, and YKL-40 knockdown-reoverexpression) were injected into nude mice for 6 weeks and the number of metastatic lung tumor nodules were determined. The number of tumor nodules of the YKL-40 overexpression cells were significantly higher than those of the other groups (Fig. 3e). Overall, the results showed that an increase of YKL-40 expression can significantly enhance cancer migration/invasion and vice versa. This suggest that YKL-40 expression is associated with of NSCLC cells migration/invasion.Fig. 3

Bottom Line: 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.Furthermore, determined by the PrognoScan database analysis, patients with high expression levels of YKL-40 were found with poor prognosis.In this study,YKL-40 was demonstrated to regulate EMT marker expressions such as Twist, Snail, Slug, N-cadherin, Vimentin, and E-cadherin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, 1001 University Road, Hsinchu, Taiwan, 300, ROC. malvinjefri@hotmail.com.

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.

No MeSH data available.


Related in: MedlinePlus