Identification of retinoid acid induced 16 as a novel androgen receptor target in prostate cancer cells

https://doi.org/10.1016/j.mce.2020.110745Get rights and content

Highlights

  • RAI16 expression is positively related to AR in prostate cancer cells.

  • RAI16 expression could be increased by R1881 in LNCaP(AD) and C4-2 cells.

  • AR binds to RAI16 promoter region.

  • RAI16 is involved in AR stimulation on cell growth.

Abstract

Background

Retinoid acid induced 16 (RAI16) was reported to enhance tumorigenesis in hepatocellular carcinoma (HCC). The androgen receptor (AR) is a nuclear hormone receptor that functions as a critical oncogene in several cancer progressions. However, whether RAI16 is a candidate AR target gene that may involve in prostate cancer progression was unclear.

Materials & methods

RAI16 expression was detected in prostate cancer cells with or without the AR agonist R1881 treatment by quantitative RT-PCR and Western blot. Direct AR binding to the RAI16 promoter was tested using AR chromatin immunoprecipitation (ChIP) and luciferase assay. Cell viability and colony formation assays in response to R1881 were analyzed in cells with RAI16 knockdown by specific siRNA.

Results

The expression of RAI16 was high in LNCaP(AI), LNCaP(AD), C4-2 expressing AR, but low in Du145 and Pc-3 cells without AR expressing. In addition, the expression of RAI16 could be induced by 10 nM R1881 treatment LNCaP(AD) and C4-2 cells, but inhibited by AR specific siRNA treatment. Furthermore, AR binds directly to ARE3 (−2003~-1982bp) of RAI16 promoter region by ChIP and luciferase assay. RAI16 knockdown inhibited the enhancement of cell viability and colony formation of AR stimulation.

Conclusions

We demonstrate for the first time that RAI16 is a direct target gene of AR. RAI16 may involved in cell growth of prostate cancer cells in response to AR signaling.

Introduction

Retinoic acid induced protein 16 (RAI16) belongs to a family of retinoic acid induced proteins (RAI). In the past decades, RAI1, RAI2, RAI3, RAI14 and RAI17 have been reported successively to play roles in cell physiology or pathology (Vilboux et al., 2011; Walpole et al., 1999; Nagahata et al., 2005; Vendrov et al., 2006; Kuhl et al., 2008). However, the functional studies on RAI16 are limited. Human RAI16 gene located in at chromosome 8p21.3, including 17 exons which code a 743aa protein (NP_073586.5). RAI16 is conserved in human, rat, mouse and other sapiens by homology analysis, which indicated that RAI16 might play important roles in basic cell functions. Previously, our group firstly reported that RAI16 enhanced tumorigenesis in hepatocellular carcinoma (HCC) and could serve as biomarker for HCC diagnosis (Wang et al., 2012). Furthermore, we identified RAI16 as a novel protein kinase A anchoring protein (AKAP), which regulated HSP70 associated anti-apoptosis signaling (Ding et al., 2015). Recently, in order to explore the other possible roles of RAI16 in physiological or pathological progression of cell in further, RAI16 knockout (RAI16−/−) mouse model was generated with CRISPR/Cas9 strategy (Xu et al., 2019). We demonstrated that RAI16−/− mice were highly susceptible to dextran sodium sulfate (DSS) induced colitis and colitis associated colorectal cancer (CAC) and showed greater production of pro-inflammatory and tumor promoting factors, which suggests an important role of RAI16 in the pathogenesis of colitis and CAC (Xu et al., 2019).

Androgen Receptor (AR) belongs to a family of nuclear transcription factors which mediate the regulating actions of steroid hormones. Cytoplasmic AR, while bound with androgens, translocates to the nucleus and binds to the androgen response elements (AREs) on the promoter region of target genes, regulating the transcription of these target genes (Lonergan and Tindall, 2011). AR is necessary for the function, survival, and differentiation of normal cells (Chmelar et al., 2007; Niu et al., 2008), and AR signaling is also critical for the development and progression of various cancers (Wadosky and Koochekpour, 2017; Tan et al., 2015; Feng and He, 2019; Chen et al., 2019). Recent studies have identified many candidate target genes of AR (Prescott et al., 2007; Jariwala et al., 2007; Allioli et al., 2011; Makkonen et al., 2008; Mou et al., 2013; Rizza et al., 2014; Naderi, 2017).

Here we identify RAI16 as a novel AR target gene which is involved in cell growth of AR-signaling in prostate cancer cells. We show that the mRNA and protein expression of RAI16 were induced by in vitro androgenic stimulation, RAI16 is a direct androgen regulated target gene. We also identified a functional ARE in the promoter region of RAI16 gene. Finally, functional studies revealed that RAI16 is involved in cell growth of AR-signaling in prostate cancer cells and therefore has a potential role in prostate cancer process.

Section snippets

Cell cultures

Human prostate cancer cells LNCaP(AI) (Shi et al., 2004), LNCaP(AD), C4-2, Du145 and Pc-3 were cultured in Dulbecco's modified Eagle medium (DMEM)-F12 (phenol red-free, Cat. No: 21041025; Thermo Fisher, USA) supplemented with 5% charcoal stripped fetal bovine serum (FBS) (Cat. No: 12676-029; Thermo Fisher), 1% glutamine (Cat. No: 25030081; Thermo Fisher) and 1 mg/ml penicillin/streptomycin (Cat. No: 15240096; Thermo Fisher) at 37 °C in a humidified atmosphere with 5% CO2.

Plasmids

The DNA fragments

Expression of RAI16 is positively correlated with AR in prostate cancer cell lines

The mRNA expressions of RAI16 and AR were analyzed in various prostate cell lines. The mRNA expression of RAI16 was higher in LNCaP(androgen dependent, AD) and C4-2 cells which expresses higher AR than that in LNCaP(androgen independent, AI) cells which expresses lower AR or Du145 and Pc-3 cells which doesn't express AR (Fig. 1A). Consistently, the protein expression of RAI16 was higher in LNCaP(AI), LNCaP(AD) and C4-2 cells than that in Du145 and Pc-3 cells (Fig. 1B). In addition, the

Discussion

The steroid nuclear receptor AR plays a crucial role in both androgen-dependent prostate cancer development and its progression to an advanced hormone-refractory stage (Wang et al., 2009). The precise identification of AR target genes and their molecular mechanisms of regulation by androgens should provide new therapeutic targets and early markers of tumor progression (Eacker et al., 2007; Zhou et al., 2011). The comparative analysis of RAI16 expression in several prostatic cell lines showed

Funding

This study was supported by National Key R&D Program of China (2018YFC1200603).

Authors’ contributions

Ding CL: performed biochemistry experiments. Qian CL: performed cell molecular experiments. Qi ZT: design the study and revise the manuscript. Wang W: design the study, analyze the data and draft the manuscript.

Declaration of competing interest

The authors disclose no potential conflict of interests.

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