Roquin1 expression is reduced in breast cancer patients and is associated with poor survival
Roquin1 expression was first analyzed in a breast tumor database (www.oncomine.org). Low levels of Roquin1 were found in various breast cancers, including breast carcinoma, invasive ductal breast carcinoma, and invasive mixed breast carcinoma, although a moderately high level was found in invasive lobular breast cancer tissues (Fig. 1a; Additional file 1: Figure S1A). Experimentally, Roquin1 mRNA expression was significantly reduced in breast tumors compared with normal tissues (Fig. 1b). Roquin1 protein expression was also lower in four randomly selected pairs of breast cancer tissues than in their surrounding normal tissues (Fig. 1c). Moreover, Roquin1 expression was significantly repressed at both the protein (Fig. 1d) and mRNA (Fig. 1e) levels in several human breast cancer cell lines compared with normal mammary gland epithelial cells. Notably, by surveying Roquin1 expression across a gene array dataset , we found that low Roquin1 expression in human breast tumor samples was strongly associated with poor overall survival and relapse-free survival of patients (Fig. 1f and 1g). Furthermore, the levels of Roquin1 in breast tumors were associated with patient survival in the luminal A, luminal B, and basal-like subsets (Fig. 1h-j). Although no significant correlation was found between Roquin1 expression and patient survival in the HER2+ subsets, a similar trend in three other subsets was found (Fig. 1k). These results suggested that Roquin1 was important for the prognosis of patients with breast cancer. In addition, we found that Roquin1 was suppressed in other types of human cancers, including lung cancer, ovarian cancer, gastric cancer, and bladder carcinoma (Additional file 1: Figure S1B-1E). Roquin1 expression levels were also significantly correlated with the prognosis of patients with these cancers and liver cancer (Additional file 1: Figure S1F-1J), indicating Roquin1 might be clinically predictive for multiple cancers.
Roquin1 inhibits cell growth by inducing G1/S phase cell cycle arrest in cancer cells
For analysis of Roquin1 function in breast cancer progression, the Roquin1/GFP fusion protein was expressed in MCF7 and MDA-MB-468 cells and identified by Western blotting (Fig. 2a). When Roquin1 was overexpressed, we found that the proliferation (Fig. 2b, c) and activity (Fig. 2d, e) of the tumor cells were substantially reduced. Similar results were also found in A549 and HepG2 with Roquin1 overexpression (Additional file 1: Figure S2A-2D). To determine whether Roquin1 inhibited cell proliferation by affecting tumor cell cycle progression, we evaluated the effect of Roquin1 overexpression on the cell cycle by flow cytometry (FCM). The G1 phase percentage of breast tumor cells was significantly increased in the Roquin1-overexpressing cancer cells compared with the controls. Moreover, a significant decrease in S phase percentage was detected after Roquin1 overexpression (Fig. 2f, g; Additional file 1: Figure S2E-2F). Similar results were also found in the A549 and HepG2 cells overexpressing Roquin1 (Additional file 1: Figure S2G-2J). However, the percentage of cells in G2 phase cells did not change consistently among the tumor cells, which might be due to different cell types. These findings suggested that Roquin1 could induce G1/S cell cycle arrest in breast tumor cells. Indeed, the protein levels of p21, a typical cell cycle inhibitor, were induced by Roquin1 in tumor cells (Fig. 2h; Additional file 1: Figure S2K-2L). To determine whether Roquin1 induced apoptosis in breast tumor cells, we detected cleaved caspase3 and PARP1, two key apoptotic indicators, by Western blotting. Roquin1 could not induce significant cleavage of pro-caspase3 and pro-PARP1 in breast tumor cells, although cleaved PARP1 was detected in MDA-MB-468 cells 72 h after Roquin1 overexpression (Fig. 2i). Our FACS data also showed that Roquin1 did not cause cell apoptosis in breast tumor cells (Fig. 2j). Collectively, these data clearly demonstrated that Roquin1 induces G1/S cell cycle arrest in breast tumor cells.
Roquin1 selectively inhibits the mRNA expression of cell cycle–promoting genes by targeting 3'UTRs
Next, we identified the genes affected by Roquin1 using RNA-seq in Roquin1-overexpressing MCF7 and MDA-MB-468 cells. Venn diagrams showed that 6556 genes were commonly downregulated and 7067 genes were commonly upregulated in two breast tumor cell lines (Additional file 1: Figure S3A). We further focused on the expression of cell cycle–related genes. Interestingly, the genes that promote cell cycle progression, including G1/S transition, G2/M transition, S phase transition, and M phase transition, were suppressed, whereas the genes inhibiting the cell cycle (p21 and Rb1) were enhanced by Roquin1 in MCF7 (Fig. 3a) and MDA-MB-468 cells (Additional file 1: Figure S3B). Similar trends were also found in A549 and HepG2 cells (Additional file 1: Figure S3C-3D), indicating that Roquin1 could regulate the expression of cell cycle-related genes in tumor cells. Detailed RNA-seq data are summarized in Additional file 3: Table S1. Moreover, the ‘cell cycle’ pathway was the first of the top ten signaling pathways significantly enriched in the KEGG pathway analysis of downregulated genes (Fig. 3b). The cell cycle–related terms ‘cell division’ and ‘mitotic nuclear division’ were enriched in the Gene Ontology (GO) analysis of downregulated genes (Fig. 3c). These computational analyses further supported our experimental findings. To validate the RNA-seq data, we measured four downregulated cell cycle–promoting genes (CCND1, CCNE1, CDK6, and MCM2) and three upregulated cell cycle–inhibiting genes (p21, p27, and Rb1) by real-time PCR. The mRNA expression of the four cell cycle–promoting genes was reduced in a time-dependent manner by Roquin1 in tumor cells (Fig. 3d, e). Additionally, the protein levels of CCNE1 and MCM2 were downregulated by Roquin1 over time (Fig. 3f, g). However, the upregulated cell cycle–inhibiting genes did not exhibit time-dependent changes (Additional file 1: Figure S3E). Notably, no time-dependent changes in the protein levels of p21 were observed in breast tumor cells (Fig. 2h, i). These results confirmed our RNA-seq data. Consistent with the overexpression results, the cell cycle–promoting genes were upregulated in the Roquin1San/San MEF cells (Additional file 1: Figure S3F) , which further strengthened our findings. Taken together, these results indicate that Roquin1 regulates the cell cycle pathway by inhibiting the mRNA expression of cell cycle-promoting genes.
We next examined whether Roquin1 binds to the mRNAs of these cell cycle–promoting genes as an RBP. An RNA pull-down assay was performed with an anti-GFP antibody in the Roquin1/GFP-expressing MDA-MB-468 cells, followed by detection of the bound mRNAs by RT-PCR. The four cell cycle–promoting genes were amplified by PCR, whereas GAPDH and the cell cycle–inhibiting mRNAs were not amplified (Fig. 3h, i). TNFα was used as a positive control. These results indicated that Roquin1 selectively bound to the cell cycle–promoting genes but not the cell cycle–inhibiting genes. To determine whether mRNA binding was mediated through the 3'UTR, we cloned the 3'UTRs of CCNE1, CCND1, CDK6 (part), and MCM2 downstream of the luciferase gene as previously described  and then cotransfected these reporters with the Roquin1 expression vector and its empty vector into HEK293 cells, followed by the measurement of luciferase activity. As shown in Figure 3J, Roquin1 significantly inhibited the luciferase activities of all four 3'UTR reporters compared with those of the cells transfected with control vector. The β-actin 3'UTR was used as a negative control. Collectively, these results suggested that Roquin1 specifically suppressed the mRNA expression of cell cycle–promoting genes by targeting their 3'UTRs.
Roquin1 destabilizes the mRNAs of cell cycle–promoting genes via the ROQ domain
We speculated that Roquin1 might reduce cell cycle–promoting genes by destabilizing their mRNAs. For confirmation of this hypothesis, Roquin1/GFP was expressed in MDA-MB-468 cells and then de novo mRNA synthesis was blocked using ActD (5 μg/mL) and DRB (5 μg/mL), followed by the measurement of the remaining mRNAs at different time points. The half-lives of indicated cell cycle–promoting mRNAs were shortened approximately 2-fold in Roquin1-overexpressing cells compared with the cells expressing the empty vector (Fig. 4a-d), while the half-lives of cell cycle–inhibiting mRNAs (including p21, Rb1, and p27) were barely affected by Roquin1 (Additional file 1: Figure S4A-4C), demonstrating that Roquin1 indeed inhibits cell cycle–promoting genes through mRNA stability.
The Roquin1 protein contains a RING finger, a ROQ domain, a zinc finger (ZF), and a proline-rich domain (PRD), and the ROQ domain is involved in the destabilization of mRNAs . To determine whether the ROQ domain is also responsible for cell cycle–promoting mRNA decay, we generated a series of truncated Roquin1 mutants, including aa (amino acid) 1-441 containing the RING, ROQ, and ZF domains, aa 441-1133 containing the PRD domain, and aa 174-326 containing the ROQ domain (Fig. 4e), and identified by Western blot analysis (Fig. 4f). Then, the mutants were cotransfected with wild-type (WT) Roquin1 as well as different 3'UTR reporters (Additional file 1: Figure S4D) into HEK293 cells. As shown in Fig. 4g and 4h, the WT and the mutants aa 1-441 and aa174-326, but not the mutant aa 441-1133, suppressed the mRNA expression of four cell cycle–promoting genes and the luciferase activities of their 3'UTR reporters, which was also consistent with a previous report . In addition, aa 174-326 significantly inhibited the proliferation (Fig. 4i) and cell cycle progression (Fig. 4j; Additional file 1: Figure S4E) of MDA-MB-468 cells, indicating that the ROQ domain in Roquin1 is essential for the induction of breast tumor cell cycle arrest.
Roquin1 knockdown stabilizes cell cycle–promoting gene transcripts and promotes tumor cell cycle progression
To further confirm the inductive effects of Roquin1 on tumor cell cycle arrest, we suppressed Roquin1 expression with two shRNAs in MDA-MB-231 cells, another triple-negative breast cancer cell line. Roquin1 was reduced by approximately 65% and 74% by #1shRNA and #2shRNA, respectively (Fig. 5a). Although Roquin1 is expressed at low levels in breast tumors, the knockdown of Roquin1 strongly promoted the proliferation and activities of breast tumor cells (Fig. 5b, c) and increased the mRNA expression of cell cycle–promoting genes (Fig. 5d). However, depletion of Roquin1 had no effect on the mRNA levels of p21, Rb1, and p27 (Additional file 1: Figure S5A), again suggesting that Roquin1 directly suppressed the mRNA expression of cell cycle–promoting genes. Next, we examined the effect of Roquin1 knockdown on the half-life of cell cycle–promoting genes. As expected, reduced Roquin1 significantly prolonged the half-lives of the indicated cell cycle–promoting mRNAs (Fig. 5e-h). Furthermore, we found a reduced percentage of G1 phase cells and an increased percentage of S phase MDA-MB-231 cells after Roquin1 knockdown (Fig. 5i; Additional file 1: Figure S5B). To confirm whether the cell cycle–promoting genes were involved in the Roquin1-induced cell cycle arrest, we knocked down CCNE1 and MCM2 by shRNA lentivirus in the Roquin1 knockdown MDA-MB-231 cells. Fig. 5J shows that these shRNAs effectively knocked down CCNE1 and MCM2 expression. Upon co-knockdown of Roquin1 and CCNE1/MCM2, cell proliferation was closed to that of the scramble control compared to Roquin1 knockdown alone (Fig. 5k). Additionally, the percentage of G1 phase cells was significantly increased compared with that of the group with Roquin1 knockdown alone, and the percentage of S phase cells significantly decreased (Fig. 5l). Collectively, these results confirmed that Roquin1 repression indeed promotes breast tumor cell cycle progression by stabilizing cell cycle-promoting genes.
Roquin1 binds to the stem–loop structure of cell cycle-promoting genes for degradation
Roquin1 is known to degrade target mRNAs by binding to the stem–loop structure . The 3'UTR sequences of four cell cycle–promoting genes were analyzed, and a conserved sequence was identified across species, which could form a similar stem–loop structure (Additional file 1: Figure S6A-6D) using RNAfold WebServer . To investigate the role of the stem–loop structure in Roquin1-mediated degradation of cell cycle–promoting mRNAs, we generated deletion constructs by deleting the sequences containing the stem–loop in the 3'UTRs of CCNE1 and MCM2 (Fig. 6a). Then, full-length and deletion reporters with Roquin1 were cotransfected into HEK293 cells, followed by measurement of luciferase activity. Roquin1 significantly inhibited the luciferase activity of the full-length CCNE1 and MCM2 3'UTRs but not the deletion mutant reporters (Fig. 6b). In addition, Roquin1 reduced the activities of the reporters containing human β-actin 3'UTR with CCNE1 or MCM2 stem–loop structures compared with that in the control group (Fig. 6c, d). These findings indicated that the stem-loop structure was pivotal for Roquin1-mediated cell cycle–promoting mRNAs decay.
To determine the necessity of the stem–loop secondary conformation for mRNA degradation, we generated two 3'UTR mutant reporters of CCNE1 and MCM2; the stem–loop structure of mutant1 was deleted by replacing two or four nucleotides, and mutant2 retained the stem–loop structure after replacement of four nucleotides (Fig. 6e). Deletion of the stem–loop structure in the 3'UTRs of CCNE1 and MCM2 (mutant1) allowed them to be completely resistant to Roquin1 inhibition, while the mutant2 that maintained the stem–loop structure remained sensitive to Roquin1 suppression (Fig. 6f), indicating that the stem–loop structure in 3'UTRs was critical for cell cycle–promoting mRNAs decay. To further determine whether Roquin1 physically bound to the stem–loop in the 3'UTRs of CCNE1 and MCM2, we performed an RNA affinity binding assay with biotin-labeled RNA probes. Wild-type RNA probes and mutant probes with the stem–loop structure either disrupted (mutant1) or retained (mutant2) were incubated with lysates of MDA-MB-468 cells expressing the Roquin1/GFP fusion protein. Then, streptavidin-coated magnetic beads were used for the pulldown assay, followed by Western blot detection with an anti-GFP antibody. The Roquin1/GFP fusion protein was pulled down by wild-type and mutant2 probes but not by the stem–loop structure-deficient mutant1 probe (Fig. 6g), indicating that Roquin1 indeed interacted with the stem–loop structure of CCNE1 and MCM2 in vitro. Furthermore, a modified RNA immunoprecipitation-chromatin immunoprecipitation (RIP-ChIP) assay was performed to verify that Roquin1 could bind the stem–loop structure in vivo. The Roquin1/GFP fusion protein was expressed in MDA-MB-468 cells, and the protein-RNA complex was pulled down by GFP antibody-coated beads after the bound mRNAs were sonicated, followed by amplification of the stem–loop sequences by RT-PCR. As expected, the stem–loop sequences in the 3'UTRs of CCNE1 and MCM2 could be amplified in the GFP antibody pulldown group but not in the group using isotype IgG (Fig. 6h), indicating the binding of Roquin1 to the 3'UTRs of cell cycle–promoting mRNAs in breast tumor cells. Overall, these data demonstrated that Roquin1 recognized and bound to the stem–loop structure in the 3¢UTRs of cell cycle–promoting genes for degradation.
Roquin1 suppresses breast tumor growth and metastasis
To determine the inhibitory effect of Roquin1 on breast cancer progression in vivo, we inoculated MDA-MB-468/Roquin1-GFP cells (expressing the Roquin1/GFP fusion protein) and MDA-MB-468/GFP cells into the mammary gland fat pads of female nude mice. The growth and sizes of the tumors expressing the Roquin1/GFP fusion protein were significantly reduced compared with those of the control tumors (Fig. 7a, b). Roquin1/GFP fusion protein expression in tumors was confirmed by Western blot analysis (Additional file 1: Figure S7A). Moreover, a significant decrease in the number of metastatic foci (Fig. 7c) and metastatic white nodules (Additional file 1: Figure S7B) was observed in the lung tissues from Roquin1/GFP tumor-bearing mice. To avoid the impacts of the manual manipulation of gene expression and simulate the clinical treatment of breast cancer, we prepared adenoviruses expressing the Roquin1/GFP fusion gene and its control virus (expresses GFP) to treat the established MDA-MB-231 breast tumors in nude mice. When tumor mass reached approximately 5 mm in diameter, 1010 pfu of Roquin1/GFP adenovirus in 100 μL of PBS and the control adenovirus were injected every other day for five injections in total (Fig. 7d). Two days after injection, the tumors began to shrink and grew slowly, while the tumors treated with control adenovirus continued growing (Fig. 7e). At the end of the experiment, the sizes of tumors treated with the Roquin1/GFP adenovirus were significantly smaller than those in the control group (Additional file 1: Figure S7C). Tumor metastasis was also significantly suppressed by Roquin1 adenovirus treatment (Fig. 7f; Additional file 1: Figure S7D). Consistent with the in vitro results, the protein levels of CCNE1 and MCM2 were also reduced in the Roquin1 adenovirus-treated tumors (Fig. 7g), further confirming that Roquin1 suppressed the expression of cell cycle–promoting genes in vivo. Interestingly, the expression of CCNE1 and MCM2 was also significantly inhibited as Roquin1 increased in 1,006 human breast cancer samples (Fig. 7h, i) (Additional file 4: Table S2) (Oncolnc.org/). Notably, higher levels of CCNE1 and MCM2 negatively correlated with poor survival of patients with breast cancer (Fig. 7j, k). Conclusively, these findings strongly suggest that Roquin1 is a promising breast tumor suppressor and that the Roquin1-cell cycle-promoting gene axis might be considered a new therapeutic target for breast tumor treatment in the future.