分泌蛋白YKL-40增强肿瘤细胞的上皮间质样转化

目的:分泌糖蛋白YKL-40在多种晚期肿瘤病人的血液中显著升高,提示YKL-40蛋白的血浓度是肿瘤恶变的生物标志物。本课题研究YKL-40重组蛋白和过表达YKL-40肿瘤细胞对肿瘤细胞的上皮间质样转化的作用。方法:构建YKL-40过表达的纤维状乳腺癌细胞系MDA-MB-231和非纤维状结肠癌细胞系HCT-116,观察细胞形态学变化,收集细胞和细胞培养液用于Western Blot(WB)检测YKL-40和上皮间质转化标记蛋白Vimentin和N-cadherin。观察重组蛋白YKL-40对原代MDA-MB-231细胞在无血清条件下的细胞存活影响;另外,用细胞存活试剂盒检测YKL-40过表达HCT-116细胞在无血清的培养液中细胞存活情况。最后,用细胞侵袭试验检测YKL-40过表达MDA-MB-231细胞的侵袭力,并用WB和Zymography来测定细胞分泌MMP9蛋白的表达和酶活性。结果:YKL-40过表达增强MDA-MB-231细胞的形态向上皮间质样转化,并显著提高Vimentin、N-cadherin蛋白的表达,但对HCT-116细胞无法诱导上皮间质样转化。在无血清培养基培养条件下,YKL-40可以增强两种细胞的存活能力,并且YKL-40过表达的MDA-MB-231细胞增强了细胞的侵袭能力,促进了MMP9蛋白表达和蛋白活性。结论:YKL-40可以增加肿瘤细胞的存活力,增强纤维状细胞向上皮间质样转化;并且,YKL-40增加MMP9蛋白表达和活性,增强细胞侵袭力。YKL-40是间质样肿瘤细胞EMT的增强子,此发现为抑制肿瘤恶变提供新靶点。

Secreted Protein YKL-40 Enhances Epithelial Mesenchymal Transition of Tumor Cells in Vitro

Objective:A large body of evidence has demonstrated that serum levels of secreted protein YKL-40(CHI3L1)were elevated in a broad type of human advanced cancers,suggesting that serum levels of YKL-40 serve as a cancer biomarker for the malignancy.The current study is to investigate the effects of recombinant protein YKL-40 and YKL-40 over-expressed by tumor cells on tumor epithelial mesenchymal transition(EMT).Methods:Two cancer lines breast cancer cell line MDA-MB-231 that displays a fibroblastic phenotype and colon cancer cell line HCT-116 that does not exhibit the fibroblastic morphology were selected to enforce expression of ectopic YKL-40.After both cell lines were established to stably express YKL-40,the cell phenotype was observed.EMT markers vimentin and N-cadherin proteins were analyzed by Western Blot.The cell cultured media were collected to test YKL-40 levels.Control and HCT-116 cells expressing ectopic YKL-40 were used to test cell survival in the presence of serum free medium for 72 h via a live and dead kit.Concomitantly,recombinant protein YKL-40 was added to parental MDA-MB-231 cells to observe cell survival in the serum-free medium for 72h.MDA-MB-231 cells expressing YKL-40 were employed to examine cell invasion and cultured media were exploited to test MMP9 expression via WB and enzymatic activity via zymography.Results:MDA-MB-231 cells expressing YKL-40 displayed a more mesenchymal phenotype than control cells;whereas HCT-116 cells expressing YKL-40 showed subtle induction of a fibroblastic phenotype.MDA-MB-231 cells expressing YKL-40,but not HCT-116 cells expressing YKL-40,revealed elevated expression of vimentin and N-cadherin.YKL-40 or YKL-40-expressing tumor cells induced tumor cell survival.YKL-40-expressing MDA-MB-231 cells increased invasion and the expression and enzymatic activity of MMP9.Conclusion:YKL-40 has the ability to increase tumor cell survival and enhance EMT,pointing to YKL-40 as a therapeutic target for cancer treatment.