Roquin1 inhibits cell growth by inducing G1-S phase cell cycle arrest in breast cancer cells
To determine Roquin1 function in breast cancer progression, Roquin1/GFP fusion protein was expressed in breast cancer cells MCF7 and MDA-MB-468, respectively, and identified by Western blot (Fig. 2a). When Roquin1 overexpression, we noticed that the proliferation (Fig. 2b, c) and activity (Fig. 2d, e) of breast cancer cells were considerably reduced. Similar results were also found in human lung cancer cell A549 and human liver cancer cell HepG2 with Roquin1 overexpression (Additional file 1: Figure S2A-2D). To determine whether Roquin1 inhibited cell proliferation by affected tumor cell cycle progression, we evaluated the effect of Roquin1 overexpression on cell cycle by flow cytometry (FCM). The G1 phase percentage of breast tumor cells was significantly increased in Roquin1-overexpressing cancer cells compared with the controls. Meanwhile, a significant decrease in S phase percentage was detected after Roquin1 overexpression. Similar results were also found in A549 and HepG2 cells with Roquin1 overexpression (Additional file 1: Figure S2G-2J). However, the G2 phase percentage of cells did not change consistently among tumor cells which might be due to different cell types (Fig. 2f, g; Additional file 1: Figure S2I-2J). These findings suggested that Roquin1 was able to induce the 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 breast tumor cells (Fig. 2h, i), and A549, HepG2 cells (Additional file 1: Figure S2K-2L). To determine whether Roquin1 induced apoptosis in breast tumor cells, cleaved caspase3 and PARP1, two key apoptosis indicators, were detected by Western blotting. Roquin1 was not able to cause significant cleavages of pro-caspase3 and pro-PARP1 in breast tumor cells, although a slightly cleaved PARP1 was detected in MDA-MB-468 cells 72 h after Roquin1 overexpression (Fig. 2j, k), indicating Roquin1 did not cause cell apoptosis in breast tumor cells. This detection was consistent with the observation that no obvious cell death occurred after Roquin1 overexpression during cell culture. Collectively, these data clearly demonstrate that Roquin1 induces G1/S cell cycle arrest of breast tumor cells.
Roquin1 selectively inhibited the mRNA expression of cell cycle–promoting genes through 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 common downregulated and 7067 genes were common upregulated in two breast tumor cells (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 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 were 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). And, 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, four downregulated cell cycle–promoting genes (i.e., CCND1, CCNE1, CDK6, and MCM2) and three upregulated cell cycle–inhibiting genes (i.e., p21, p27, and Rb1) were measured by real-time PCR. The mRNA expression of four cell cycle–promoting genes was reduced in a time-dependent manner by Roquin1 in breast cancer cells (Fig. 3d, e). Also, the protein levels of CCNE1 and MCM2 were downregulated by Roquin1 over time (Fig. 3f, g). However, the upregulated cell cycle–inhibiting genes without 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. In agreement with the overexpression results, the cell cycle–promoting genes were upregulated in Roquin1San/San MEF cells (Additional file 1: Figure S3F) , which further strengthened our findings. Taken together, these results indicate that Roquin1 regulates cell cycle pathway by inhibiting the mRNA expression of cell cycle-promoting genes.
As an RBP, we next examined whether Roquin1 binding to these cell cycle–promoting gene mRNAs. An RNA pull-down assay was performed with an anti-GFP antibody in Roquin1/GFP-expressing MDA-MB-468 cells, followed by detecting bound mRNAs by RT-PCR. Four cell cycle–promoting genes were amplified by PCR, whereas the GAPDH and 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 cell cycle–inhibiting genes. To determine if the mRNA binding was mediated through 3’UTR, we cloned the 3’UTRs of CCNE1, CCND1, CDK6 (part), and MCM2 downstream of the luciferase gene as previously described , and then co-transfected these reporters with Roquin1 expression vector and its empty vector into HEK293 cells, followed by the measurement of luciferase activity. As shown in Fig. 3J, Roquin1 significantly inhibited luciferase activities of all four 3’UTR reporters compared with 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 destabilized the mRNAs of cell cycle–promoting genes via the ROQ domain
We speculated that Roquin1 might reduce cell cycle–promoting gene mRNAs through destabilizing the mRNAs. To confirm that, Roquin1/GFP was expressed in MDA-MB-468 cells and then de novo mRNA synthesis 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 about 2-fold in Roquin1-overexpressing cells compared with cells expressing 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 expression through the mRNA stability.
Roquin1 protein contains a RING finger, a ROQ domain, a zinc finger (ZF), and a proline-rich domain (PRD), of which the ROQ domain is involved in the destabilization of mRNAs . To determine whether the ROQ domain is also responsible for the cell cycle–promoting mRNAs decay, a series of truncated Roquin1 mutants, including aa 1-441 containing RING, ROQ, and ZF domains, aa 441–1133 containing PRD domain, and aa 174–326 containing ROQ domain, were generated (Fig. 4e) and identified by Western blot analysis (Fig. 4f). Then, the mutants were co-transfected 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 4 h, WT, mutants aa 1-441 and aa174-326 suppressed the mRNA expression of four cell cycle–promoting genes and the luciferase activities of their 3’UTR reporters, but not mutant aa 441–1133, which was also consistent with the 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, which also signified 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. Roquin1 was reduced approximately 65% and 74% by #1shRNA and #2shRNA, respectively (Fig. 5a). Although Roquin1 is lowly expressed in breast tumors, the knockdown of Roquin1 considerably promoted the proliferation and activities of breast tumor cells (Fig. 5b, c), and increased the mRNA expression of cell cycle–promoting genes (Fig. 5d). These results were also consistent with previous data in Roquin1san/san MEF cells . 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 gene transcripts. As expected, reduced Roquin1 significantly prolonged the half-lives of the indicated cell cycle–promoting mRNAs (Fig. 5e-h). Furthermore, we also found reduced percentage of G1 phase cells and increased S phase cell percentage in MDA-MB-231 cells after knocking down Roquin1 (Fig. 5i; Additional file 1: Figure S5B). To confirm whether the cell cycle–promoting genes were involved in Roquin1-induced cell cycle arrest, CCNE1 and MCM2 were knocked down by shRNA lentivirus in Roquin1 knockdown MDA-MB-231 cells. Figure 5J showed that these shRNAs effectively knocked down CCNE1 and MCM2 expression. Upon co-knockdown of Roquin1 and CCNE1/MCM2, cell proliferation was closed to the scramble control compared to Roquin1 knockdown alone (Fig. 5k). Additionally, the percentage of G1 phase cells was significantly increased compared with the group of 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 through 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, respectively, which could form a similar stem–loop structure (Additional file 1: Figure S6A-6D) using the 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 co-transfected into HEK293 cells, followed by luciferase activity measurement. Roquin1 could significantly inhibit the luciferase activity of full-length CCNE1 and MCM2 3’UTR, 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 stem–loop secondary conformation for mRNA degradation, two 3’UTR mutant reporters of CCNE1 and MCM2 were generated, of which the stem–loop structure of mutant1 was deleted by replacing two or four nucleotides and mutant2 retained the stem–loop structure after replacing four nucleotides (Fig. 6e). Subsequently, the two reporters were co-transfected with Roquin1 for measuring luciferase activity. Deletion of the stem–loop structure in the 3’UTRs of CCNE1 and MCM2 (mutant1) allowed them completely to resistant to Roquin1 inhibition, while the mutant2 that kept 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 if Roquin1 physically bound to the stem–loop in the 3’UTRs of CCNE1 and MCM2, we performed 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 Roquin1/GFP fusion protein. Then, streptdavidin-coated magnetic beads were used for the pull-down assay, followed by Western blot detection with anti-GFP antibody. Roquin1/GFP fusion protein was pulled down by wild-type and mutant2 probes, but cannot 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. Roquin1/GFP fusion protein was expressed in MDA-MB-468 cells, the protein-RNA complex was pulled down by GFP antibody-coated beads, after the bound mRNAs 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 pull-down group, but not in the group using isotype IgG (Fig. 6h), indicating that the binding of Roquin1 to the 3'UTRs of cell cycle–promoting mRNAs inside breast tumor cells. Overall, these data demonstrate that Roquin1 recognized and bound to the stem–loop structure in the 3′UTRs of cell cycle–promoting genes for degradation.