Stable knockdown of Kif5b in MDCK cells leads to epithelialemesenchymal transition Ju Cui a, b, * , Guoxiang Jin b , Bin Yu b , Zai Wang b, c , Raozhou Lin b , Jian-Dong Huang b, d, * a The Key Laboratory of Geriatrics, Beijing Hospital & Beijing Institute of Geriatrics, Ministry of Health, Beijing, China b Department of Biochemistry, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China c Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing, China d The Centre for Synthetic Biology Engineering Research, Shenzhen Institutes of Advanced Technology, Shenzhen, China article info Article history: Received 28 April 2015 Available online 20 May 2015 Keywords: Kif5b MDCK cell Epithelialemesenchymal transtion abstract Polarization of epithelial cells requires vectorial sorting and transport of polarity proteins to apical or basolateral domains. Kif5b is the mouse homologue of the human ubiquitous Kinesin Heavy Chain (uKHC). To investigate the function of Kif5b in epithelial cells, we examined the phenotypes of Kif5b- deficient MDCK cells. Stable knockdown of Kif5b in MDCK cells resulted in reduced cell proliferation rate, profound changes in cell morphology, loss of epithelial cell marker, and gain of mesenchymal marker, as well as increased cell migration, invasion, and tumorigenesis abilities. E-cadherin and NMMIIA could interact with Kif5b in polarized MDCK cells, and their expression levels were decreased in Kif5b- deficient MDCK cells. Overexpression of E-cadherin and NMMIIA in Kif5b depleted MDCK cells could decrease mesenchymal marker expression and cell migration ability. These results indicate that stable knockdown of Kif5b in MDCK cells can lead to epithelialemesenchymal transition, which is mediated by defective E-cadherin and NMMIIA expression. © 2015 Elsevier Inc. All rights reserved. 1. Introduction The polarity is a fundamental feature of many eukaryotic cells. In a study of Caenorhabditis elegans development, it was found that centrosomes or associated microtubules provide an essential cue for the specification of the body polarity axis [1]. Microtubules act as structural components and participate in the regulation of many cellular activities, including cell division and intracellular trans- portation [2]. Therefore, microtubules and their associated motor proteins (kinesin and dynein) are indispensable for delivering proteins related to polarity establishment and maintenance. Using polarized transport assay in permeabilized MDCK cells, Lafont et al. [3] found that both apical and basolateral transport require microtubule motors and that cytosolic kinesin is responsible for basolateral transport, while both dynein and kinesin are involved in apical transport. Several kinesin family proteins have been reported to be involved in apical transport in epithelial cells. KIF3A has been shown to mediate the interaction of the polarity protein Par3/Par6/ aPKC complex with microtubules in polarized MDCK cells [4]. KIFC3, a minus end-directed microtubule motor protein, is identi- fied and characterized for apical transport of annexin XIIIb- associated vesicles [5]. Post-Golgi transport of p75 to apical mem- brane in polarized MDCK cells is mediated by Kif5b (the heavy chain of Kinesin-1) [6]. Kinesin-1 also participates in the basolateral trafficking of Na þ -K þ ATPase in alveolar epithelial cells [7]. Furthermore, p120 catenin forms a complex with Kif5b to facilitate the transport of N-cadherin-catenin complexes to adhesion junc- tions in HeLa cells [8], indicating that Kif5b might be involved in E- cadherin transport in epithelial cells. Moreover, in colonic epithelial cells, Kif5b is localized at intact and internalized apical junctions and can mediate the disassembly and internalization of adhesion junctions and tight junctions upon Ca 2þ depletion [9]. In the adult mouse kidney, Kif5b is selectively and asymmetrically expressed in the basolateral domain of epithelial cells in the thick ascending limbs and distal convoluted tubules, and knockout of Kif5b leads to depolarization of Na þ -K þ ATPase [10]. Against this background, and as a first step in obtaining clues to the functions of Kif5b in epithelial cells, we have employed RNAi technology to knockdown of Kif5b in MDCK cells and examined the phenotypes of Kif5b-deficient cells. * Corresponding authors. The Key Laboratory of Geriatrics, Beijing Hospital & Beijing Institute of Geriatrics, Ministry of Health, Beijing, China. Fax: þ86 10 65132982. E-mail addresses: juzi.cui@gmail.com (J. Cui), jdhuang@hku.hk (J.-D. Huang). Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc http://dx.doi.org/10.1016/j.bbrc.2015.05.045 0006-291X/© 2015 Elsevier Inc. All rights reserved. Biochemical and Biophysical Research Communications 463 (2015) 123e129