Guanine nucleotide-binding protein subunit beta-2-like 1, a new Annexin A7 interacting protein Yue Du 1 , Jinyi Meng 1 , Yuhong Huang, Jun Wu, Bo Wang, Mohammed M. Ibrahim, Jianwu Tang ⇑ Key Laboratory of Tumor Metastasis of Liaoning Province, Department of Pathology, Dalian Medical University, Dalian 116044, China article info Article history: Received 16 January 2014 Available online 1 February 2014 Keywords: Guanine nucleotide-binding protein subunit beta-2-like 1 (Gnb2l1) Receptor of activated protein kinase C 1 (RACK1) Annexin A7 (ANXA7) Lymph node metastasis (LNM) abstract We report for the first time that Guanine nucleotide-binding protein subunit beta-2-like 1 (RACK1) formed a complex with Annexin A7. Hca-F and Hca-P are a pair of syngeneic mouse hepatocarcinoma cell lines established and maintained in our laboratory. Our previous study showed that both Annexin A7 and RACK1 were expressed higher in Hca-F (lymph node metastasis >70%) than Hca-P (lymph node metasta- sis <30%). Suppression of Annexin A7 expression in Hca-F cells induced decreased migration and invasion ability. In this study, knockdown of RACK1 by RNA interference (RNAi) had the same impact on metas- tasis potential of Hca-F cells as Annexin A7 down-regulation. Furthermore, by co-immunoprecipitation and double immunofluorescence confocal imaging, we found that RACK1 was in complex with Annexin A7 in control cells, but not in the RACK1-down-regulated cells, indicating the abolishment of RACK1- Annexin A7 interaction in Hca-F cells by RACK1 RNAi. Taken together, these results suggest that RACK1-Annexin A7 interaction may be one of the means by which RACK1 and Annexin A7 influence the metastasis potential of mouse hepatocarcinoma cells in vitro. Ó 2014 Elsevier Inc. All rights reserved. 1. Introduction For carcinoma, lymphatic metastasis is a common phenome- non leading to poor prognosis. Metastasis results from altera- tions of multiple genes and signaling pathways which remain to be characterized. Our series of studies aimed at identifying the signaling pathways and potential protein molecules in- volved in tumor progression and metastasis. In our previous studies, we choose a pair of syngeneic mouse hepatocarcinoma cell lines, Hca-F and Hca-P, which were established and main- tained in our laboratory. When inoculated subcutaneously in Chinese 615 mice, Hca-P showed a low of LNM rate (<30%), whereas Hca-F showed a high LNM rate (>70%). Sharing the same genetic background, these two cell lines are ideal experi- mental subjects for revealing potential biomarkers related to tumor malignancy [1,2]. Suppressive subtracted hybridization technique, gene chip and quantitative proteomics techniques were used to identify differentially expressed genes and pro- teins between the two cell lines [3–5]. Guanine nucleotide- binding protein subunit beta-2-like 1 (gene symbol: Gnb2l1), together with Annexin A7, Jun-N-termnal kinase, et al., were expressed higher in Hca-F than Hca-P at both mRNA and protein levels [5–7]. Guanine nucleotide-binding protein subunit beta-2-like 1 was alternatively named Receptor of activated protein kinase C 1 (RACK1). Although RACK1 was early identified as an anchoring pro- tein for protein kinase C (PKC) [8], actually it recruits and binds a variety of signaling molecules such as Integrins, Proto-oncogene tyrosine-protein kinase Src (pp60c-src), Focal adhesion kinase (FAK), et al. [9–11]. Acting as a scaffold protein, RACK1 integrates signals from diverse pathways and contributes to cancer progress. The expression of RACK1 is altered in many cancers. Elevated RACK1 level in pulmonary adenocarcinoma is associated with poor prognosis [12]. In contrast, other reports indicate that RACK1 level is decreased in breast cancer [13]. Several studies have demon- strated that RACK1 may influence migration and proliferation of tumors [14–16], but its underlying mechanisms in hepatocarci- noma remain to be explored further. Therefore, in the current study, we down-regulated RACK1 in mouse hepatocarcinoma cell line Hca-F by RNA interference (RNAi) and assessed its effect on the malignant behaviors. What’s more, association between Annexin A7 and RACK1 were explored through Ingenuity Pathway Analysis (IPA) software and protein–protein interaction experi- ments. These findings offer new perspectives for understanding the way by which RACK1 and Annexin A7 influence malignant behaviors of tumors. http://dx.doi.org/10.1016/j.bbrc.2014.01.119 0006-291X/Ó 2014 Elsevier Inc. All rights reserved. ⇑ Corresponding author. Address: Dalian Medical University, Department of Pathology, 9-Western Section, Lvshun Southern Street, 116044 Dalian, Liaoning, China. E-mail address: jianwutdlmedu@163.com (J. Tang). 1 These authors contributed equally to this work. Biochemical and Biophysical Research Communications 445 (2014) 58–63 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc