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Transgelin Interacts with PARP1 in Human Colon Cancer Cells

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Transgelin Interacts with PARP1 in Human Colon Cancer Cells Lew et al. Cancer Cell Int (2020) 20:366 https://doi.org/10.1186/s12935-020-01461-y Cancer Cell International PRIMARY RESEARCH Open Access Transgelin interacts with PARP1 in human colon cancer cells Zhen‑xian Lew1,2,3†, Hui‑min Zhou4†, Yuan‑yuan Fang5, Zhen Ye1,2, Wa Zhong1,2, Xin‑yi Yang6, Zhong Yu1,2, Dan‑yu Chen1,2, Si‑min Luo1,2, Li‑fei Chen7 and Ying Lin1,2* Abstract Background: Transgelin, an actin‑binding protein, is associated with cytoskeleton remodeling. Findings from our previous studies demonstrated that transgelin was up‑regulated in node‑positive colorectal cancer (CRC) ver‑ sus node‑negative disease. Over‑expression of TAGLN afected the expression of 256 downstream transcripts and increased the metastatic potential of colon cancer cells in vitro and in vivo. This study aims to explore the mechanisms through which transgelin participates in the metastasis of colon cancer cells. Methods: Immunofuorescence and immunoblotting analysis were used to determine the cellular localization of endogenous and exogenous transgelin in colon cancer cells. Co‑immunoprecipitation and subsequently high‑ performance liquid chromatography/tandem mass spectrometry were performed to identify the proteins that were potentially interacting with transgelin. The 256 downstream transcripts regulated by transgelin were analyzed with bioinformatics methods to discriminate the specifc key genes and signaling pathways. The Gene‑Cloud of Biotech‑ nology Information (GCBI) tools were used to predict the potential transcription factors (TFs) for the key genes. The predicted TFs corresponded to the proteins identifed to interact with transgelin. The interaction between transgelin and the TFs was verifed by co‑immunoprecipitation and immunofuorescence. Results: Transgelin was found to localize in both the cytoplasm and nucleus of the colon cancer cells. Approximately 297 proteins were identifed to interact with transgelin. The overexpression of TAGLN led to the diferential expression of 184 downstream genes. Network topology analysis discriminated seven key genes, including CALM1, MYO1F, NCK- IPSD, PLK4, RAC1, WAS and WIPF1, which are mostly involved in the Rho signaling pathway. Poly (ADP‑ribose) polymer‑ ase‑1 (PARP1) was predicted as the unique TF for the key genes and concurrently corresponded to the DNA‑binding proteins potentially interacting with transgelin. The interaction between PARP1 and transgelin in human RKO colon cancer cells was further validated by immunoprecipitation and immunofuorescence assays. Conclusions: Our results suggest that transgelin binds to PARP1 and regulates the expression of downstream key genes, which are mainly involved in the Rho signaling pathway, and thus participates in the metastasis of colon cancer. Keywords: Transgelin, PARP1, Colon cancer, Rho signaling, Bioinformatics Background *Correspondence: [email protected] Colorectal cancer (CRC) is a frequent malignant tumor †Zhen‑xian Lew and Hui‑min Zhou contributed equally to this work in the gastrointestinal tract worldwide. In 2018, it was 1 Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat‑sen Memorial Hospital, Sun Yat‑sen ranked the third in terms of incidence (1.8 million new University, No. 107 West Yanjiang Road, Guangzhou 510120, Guangdong, cases) and second in terms of mortality rate (881,000 China deaths) [1]. Although mechanisms of CRC tumorigen- Full list of author information is available at the end of the article esis and metastasis have been extensively studied, high © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Lew et al. Cancer Cell Int (2020) 20:366 Page 2 of 12 mortality rates are still reported, especially in patients transcriptional regulator. However, the role of transgelin with advanced disease [1]. in colon cancer metastasis remains unknown. Tumor metastasis, the spread of cancer cells from the Herein, we verifed the nuclear localization of transge- primary tumor to other areas of the body, is a complex lin in diferent colon cancer cell lines. Approximately 297 process associated with remodeling of the cytoskeleton. proteins that are potentially interacting with transgelin Te intracellular cytoskeleton requires a high degree were identifed, of which 23 were DNA-binding proteins. of functional integration and coordination of actin Over-expression of TAGLN afected the expression lev- (microflament), microtubules and intermediate fla- els of 184 genes. Seven key genes that mainly involved ments [2–5]. However, abnormal expression of related in the Rho signaling pathway were also identifed. By genes or efector proteins may lead to the activation analyzing the promoter regions of these key genes, poly of various signaling pathways, thus promoting tumor (ADP-ribose) polymerase-1 (PARP1), a DNA-bind- metastasis [2–5]. ing protein, was predicted to be the transcription fac- Transgelin (also known as 22 kDa actin-binding pro- tor (TF) of these genes. PARP1 was also among the 23 tein, protein WS3-10 or smooth muscle protein 22 alpha) DNA-binding proteins that were perceived to interact is an actin-binding protein with a molecular weight of with transgelin. Te interaction between transgelin and 23 kDa and consists of 201 amino acids [6]. It is encoded PARP1 was further verifed by immunoprecipitation and by the TAGLN gene and composes of an N-terminal immunofuorescence. calmodulin homologous (CH) domain and a C-termi- nal calmodulin-like (CLIK) domain, which is closely Materials and methods related to actin binding activity [6]. Transgelin is broadly Cell lines expressed in the vascular and visceral smooth muscle and Te human CRC cell lines, including RKO, SW480, is an early marker of smooth muscle diferentiation [7]. HCT116, and LOVO were obtained from the Stem Cell Furthermore, transgelin is associated with the remode- Bank, Chinese Academy of Sciences (Shanghai, China). ling of the actin cytoskeleton and promotes the migration Cells were cultured in a minimum Eagle’s medium and invasion of cancer stem cells [8–10]. (MEM, Gibco, USA), Mccoy’s 5A medium (Gibco, Recent studies have shown that besides the involve- USA) and Roswell Park Memorial Institute (RPMI) 1640 ment in the regulation of actin nucleation, cellulose cap- medium (Gibco, USA) with 10% fetal bovine serum ping, fragmentation, actin monomer binding and other (Gibco, USA). Cells were then cultured and incubated at functions in the cytoplasm, actin-binding proteins are 37 °C with 5% CO2. also involved in the formation of transcription complexes [11]. Transgelin has been shown to be a poor prognostic Immunofuorescence factor associated with advanced CRC [12] and it also pro- Localization of endogenous transgelin in RKO, SW480, motes transforming growth factor β (TGF β)-dependent HCT116 and LOVO cell lines and the expression of tumor growth and migration [13]. Moreover, results from PARP1 in RKO cells were determined by immunofuo- in vitro experiments and a xenograft metastatic mouse rescence. Te primary antibody (anti-transgelin, 1:500, model suggest that transgelin may be a promising thera- Abcam, USA; anti-PARP1, 1:500, Cell signaling tech- peutic target for treating bladder cancer metastasis [14]. nology, USA), secondary antibody (Alexa Flour 594 Terefore, we believe that transgelin may serve as a bio- goat anti-rabbit IgG, Alexa Flour 488 goat anti-rabbit marker for tumor metastasis. IgG, 1:500, Invitrogen, USA), and the VECTASHIELD In our previous study, transgelin was up-regulated in mounting medium (Vector Laboratories, USA)) with the node-positive CRC versus node-negative disease 4′, 6-diamidino-2-phenylindole (DAPI) were used. Te [15]. While the up-regulation of transgelin promoted immunofuorescence images were taken and preserved the metastasis of colon cancer cells, down-regulation under the laser scanning confocal microscope using a substantially decreased the ability of cell invasion and 63 × oil-immersion objective lens (Carl Zeiss, USA). metastasis [9, 10, 15]. In addition, gene expression pro- fling showed that over-expression of TAGLN afected Transfection the expression of the 256 downstream transcripts, which Te SW480 and RKO cells were cultured in 12-well plates were closely related to cell morphology, migration and and transfected with pcDNA6/myc-His B-TAGLN-fag invasion [9]. We also found that transgelin localized in and pcDNA6/myc-His B-fag plasmids (Takara, Japan). both the cytoplasm and nucleus of the cultured CRC Te RKO cells were transfected with pENTER-TAGLN- cells and afected the expression
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