Circ_DOCK1 regulates USP11 through miR-132-3p to control colorectal cancer progression

Background Circular RNAs (circRNAs) take part in colorectal cancer malignancies. CircRNA dedicator of cytokinesis 1 (circ_DOCK1) is involved in colorectal cancer progression, but the mechanism underlying this circRNA that takes part in colorectal cancer development remains largely undetermined. Methods Tumor and normal para-cancerous tissues were collected from 42 colorectal cancer patients. Human colorectal cancer cell lines (HCT116 and SW480) were used for the experiments in vitro. Circ_DOCK1, microRNA (miR)-132-3p, and ubiquitin-specific protease 11 (USP11) levels were measured through quantitative real-time polymerase chain reaction and Western blotting. Cell growth, metastasis, and apoptosis were investigated via colony formation, 5-ethynyl-2′-deoxyuridine (EdU) staining, MTT, flow cytometry, Western blotting, and transwell analyses. The target association was evaluated via dual-luciferase reporter analysis, RNA pull-down, and immunoprecipitation (RIP). Xenograft assay was performed using HCT116 cells. USP11 and Ki67 levels in tumor tissues were detected via immunohistochemistry. Results Circ_DOCK1 expression was enhanced in colorectal cancer tissues and cells. Silencing circ_DOCK1 repressed cell growth, migration, and invasion, and facilitated apoptosis. Circ_DOCK1 sponged miR-132-3p, and miR-132-3p silence mitigated the effect of circ_DOCK1 interference on cell growth, metastasis, and apoptosis. MiR-132-3p targeted USP11, and circ_DOCK1 could regulate USP11 level by miR-132-3p. MiR-132-3p suppressed cell growth, metastasis, and apoptosis, and USP11 attenuated these effects. Knockdown of circ_DOCK1 decreased colorectal cancer cell xenograft tumor growth. Conclusion Circ_DOCK1 interference suppressed cell growth and metastasis, and increased apoptosis of colorectal cancer via decreasing USP11 by increasing miR-132-3p. Supplementary Information The online version contains supplementary material available at 10.1186/s12957-021-02173-x.


Introduction
Colorectal cancer is a deadly cancer accounting for about 10% of all diagnosed cancers and cancer-related death worldwide [1]. The incidence has a decreasing trend in highly developed countries, while it is predictive to increase to 2-5 million new cases in 2035 [1]. About 20-30% patients are diagnosed at advanced stages, and 40-50% of those at early stages develop relapse [2]. The 5-year survival rate for patients at early stages is 90%, and the survival rate for those at advanced stages is 13.1% [3]. With the advance in the diagnosis and treatment, great improvement has been gained on the prognosis of patients with early colorectal cancer [4]. However, the survival of patients with the advanced malignancy remains low. Hence, there is a need to explore new mechanism for understanding colorectal cancer pathogenesis and find novel strategy for colorectal cancer therapy.
MiRNAs are small noncoding RNAs that are associated with the diagnosis, development, and therapy of colorectal cancer [13]. Multiple evidences suggest miR-132-3p can serve as a tumor suppressor via regulating various mRNAs in human tumors, like lung adenocarcinoma, retinoblastoma, and liver cancer [14][15][16]. Moreover, the downregulated miR-132-3p predicts the worse prognosis of colorectal cancer [17], and miR-132-3p suppresses colorectal cancer metastasis via regulating zincfinger E-box-binding homeobox-2 (ZEB2) [18]. A previous study suggests circ_DOCK1 (hsa_circ_0020394) can sponge with miR-132-3p in bladder carcinoma [10]. Yet no study reports miR-132-3p is associated with circ_ DOCK1 (hsa_circ_0020397)-mediated network in colorectal cancer. Protein ubiquitination is a classic posttranslational modification and involved in multiple cell processes [19,20]. The ubiquitination controls many processes to participate in the development of diseases, and modulating ubiquitination might be helpful for providing strategy for the therapy of cancers [21]. Ubiquitin-specific protease 11 (USP11) is an important deubiquitinase [22]. More importantly, the emerging evidence suggests that USP11 can promote the development of colorectal cancer by protecting protein phosphatase 1 via deubiquitination to activate mitogenactivated protein kinase pathway [23]. We performed the bioinformatic analysis to predict the potential interaction between miR-132-3p and circ_DOCK1 or USP11. Hence, we hypothesized the circ_DOCK1/miR-132-3p/ USP11 axis might be an important network for circ_ DOCK1 in colorectal cancer.
In this research, the purposes were to investigate the function of circ_DOCK1 (hsa_circ_0020397) on cell growth, metastasis, and apoptosis in colorectal cancer, and to explore whether it required the circ_DOCK1/ miR-132-3p/USP11 network.

Clinical tissues
Forty-two colorectal cancer patients were listed from Hainan General Hospital. The tumor and normal paracancerous tissues were obtained from same patients by surgical procedures and maintained in liquid nitrogen. All subjects have signed the written informed consents, and this study was permitted via the Ethics Committee of Hainan General Hospital, and performed in line with the Declaration of Helsinki.
Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was isolated with Trizol (Thermo Fisher) according to the protocols. The RNA in nucleus or cytoplasm was prepared with Cytoplasmic & Nuclear RNA Purification kit (Norgen Biotek, Thorold, Canada) according to the manufacturer's instructions. Five hundred nanograms of RNA was used for cDNA synthesis through TaqMan cDNA synthesis kit (Thermo Fisher) following the protocols. The synthesized cDNA was mixed with SYBR (Solarbio, Beijing, China) and primer pairs for qRT-PCR on ABI 7500 FAST Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The primers were synthesized from Sangon (Shanghai, China) and shown in Table 1. U6 (for miRNAs or nucleus) or β-actin (for circRNAs, mRNAs, or cytoplasm) served as endogenous reference. Relative RNA level was analyzed with 2 −ΔΔCt [26].

Circular structure analysis
The circular structure of circ_DOCK1 was analyzed via actinomycin D assay. Briefly, HCT116 and SW480 cells were exposed to 2 μg/mL actinomycin D (Glpbio, Montclair, CA, USA) for 0, 8, 16, or 24 h. Then, cells were harvested for detection of circ_DOCK1 and DOCK1 mRNA levels by qRT-PCR as mentioned above.

Colony formation assay
HCT116 and SW480 cells (600 cells/well) were dispersed in 6-well plates and incubated for 12 days. Then, cells were stained using 0.5% crystal violet (Solarbio) for 10 min. Next, the colonies were imaged and counted.

5-Ethynyl-2′-deoxyuridine (EdU) staining
Cell proliferative ability was investigated using Beyo-Click™ EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime, Shanghai) following the instructions as a previous report [27]. In brief, 4 × 10 4 HCT116 and SW480 cells were seeded in 24-well plates and cultured for 24 h. Then, cells were nurtured with 10 μM EdU for 2 h, followed by fixing with 4% paraformaldehyde (Beyotime) for 15 min and treatment of 0.3% Triton X-100 (Beyotime) for 10 min. Next, cells were incubated with the click reaction solution for 30 min. The nuclei were stained with Hoechst 33342 for 10 min. Cells were observed with a fluorescence microscope (Olympus, Tokyo, Japan). The ratio of EdU-positive cells (EdU-positive cells/Hoechst 33342-stained cells) was calculated to investigate the proliferative ability.

Flow cytometry
For cell cycle distribution analysis, 1 × 10 5 HCT116 and SW480 cells were dispersed in 12-well plates and incubated for 3 days. After fixture using 75% ethanol (Aladdin, Shanghai, China), cells were dyed with propidium iodide (PI) (Beyotime). Cycle process was detected with a flow cytometer (Agilent, Beijing, China). Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Beyotime) was used for detection of cell apoptosis. 1 × 10 5 HCT116 and SW480 cells were added in 12-well plates in triplicate, and then incubated at 37°C for 3 days. Then, cells were resuspended in Annexin V binding buffer (50 mM HEPES, 700 mM sodium chloride, 12.5 mM calcium chloride, and 5% bovine serum albumin), followed by staining with Annexin V-FITC and PI. The apoptotic cells were examined with a flow cytometer.

Transwell assay
Cell migration and invasion were examined in 24-well plates with transwell inserts (Corning, Corning, NY, USA). For migration analysis, HCT116 and SW480 cells (3 × 10 4 cells per well) in serum-free DMEM were placed in the upper chambers. For invasion analysis, the inserts were pre-coated with Matrigel, and 1 × 10 5 cells in serum-free medium were added in the upper chambers. The lower chambers were filled with DMEM plus 10% fetal bovine serum. After the incubation for 24 h, cells in the lower chambers were stained using 0.1% crystal violet, and imaged with a × 100 magnification microscope (Olympus).
Dual-luciferase reporter, RNA pull-down, and immunoprecipitation (RIP) assays The wild-type (WT) sequence of circ_DOCK1 or USP11 with miR-132-3p binding sites was cloned in pGL3 vector (Promega, Madison, WI, USA), forming the circ_ DOCK1-WT and USP11-WT luciferase reporter vectors. The mutant (MUT) luciferase reporter vectors (circ_ DOCK1-WT and USP11-MUT) were constructed using the mutated sequences. The constructed vectors and miR-NC or miR-132-3p mimic were transfected in HCT116 and SW480 cells for 24 h. Luciferase activity was examined with a dual-luciferase assay kit (Promega).
A Pierce™ Magnetic RNA-Protein Pull-Down kit (Thermo Fisher) was used to analyze the binding of circ_DOCK1 and miR-132-3p following the instructions. In brief, the biotin-labeled circ_DOCK1-WT (Bio-circ_ DOCK1-WT), Bio-circ_DOCK1-MUT, or negative control (Bio-NC) was generated through the RNA 3' end desthiobiotinylation kit (Thermo Fisher) and incubated with the streptavidin magnetic beads and lysates of HCT116 and SW480 cells overnight. The enriched miR-132 level was measured via qRT-PCR.

Xenograft model
The animal research was approved via the ethics committee of Hainan General Hospital and was processed following the experimental animal use guidelines of the National Institutes of Health. The lentivirus vectors of shRNA against circ_DOCK1 (sh-circ_DOCK1) or negative control (sh-NC) were formed via GeneChem (Shanghai, China), and transfected in HCT116 cells. Five-week-old male BALB/c nude mice were obtained from Charles River (Beijing, China) and subcutaneously injected with 2 × 10 6 sh-circ_DOCK1-or sh-NCtransfected HCT116 cells (n=5). The tumor volume was examined weekly after cell injection for 10 days and calculated via 0.5 × length × width 2 . After 30 days, the mice were killed via inhalation anesthesia of 5% isoflurane (Sigma), and tumor weight was measured. The levels of circ_DOCK1, miR-132-3p, USP11, and Ki67 in tumor tissues were measured via qRT-PCR, Western blotting, or immunohistochemistry.

Statistical analysis
The graphic and statistical analysis were performed with GraphPad Prism 6 (GraphPad Inc., La Jolla, CA, USA). The experiments were repeated three times with 3 technical replicates, unless otherwise indicated. The results were expressed as mean ± standard deviation (SD). The linear correlation was investigated by Pearson test. The comparison was conducted using Student's t-test or ANOVA, and it was significant at P<0.05.

Circ_DOCK1 is upregulated in colorectal cancer
To probe the role of circ_DOCK1 in colorectal cancer development, its expression was examined. By detecting circ_DOCK1 expression in 42 paired tumor and normal tissues, results showed circ_DOCK1 (hsa_circ_0020397) level was significantly increased in colorectal cancer tissues compared with normal samples (Fig. 1a), while hsa_circ_0020394 level was not obviously changed in tumor and normal samples (Supplementary Figure 1). Furthermore, higher level of circ_DOCK1 was displayed in colorectal cancer cell lines (HCT116 and SW480) than human normal colonic epithelial cell line FHC cells (Fig. 1b). Moreover, the circular structure of circ_DOCK1 was confirmed by actinomycin D (a transcriptional inhibitor) assay, which showed circ_DOCK1 was more resistant to actinomycin D treatment than DOCK1 mRNA ( Fig. 1 c and d). Additionally, circ_DOCK1 abundance was detected in nucleus and cytoplasm of HCT116 and SW480 cells. Results showed circ_ DOCK1 was mostly expressed in the cytoplasm ( Fig.  1 e and f). These results suggested increased circ_ DOCK1 might be associated with colorectal cancer development.

Circ_DOCK1 knockdown inhibits cell growth and metastasis and triggers apoptosis in colorectal cancer
To evaluate the influence of circ_DOCK1 on colorectal cancer development, HCT116 and SW480 cells were transfected with si-circ_DOCK1 or si-NC. The addition of si-circ_DOCK1 effectively reduced circ_DOCK1 expression but showed little influence on DOCK1 mRNA expression (Fig. 2 a and b). Moreover, cell growth was evaluated via colony formation, Edu staining, MTT, cycle distribution, and related protein expression. By detecting these events, results showed that circ_DOCK1 knockdown evidently reduced growth of HCT116 and SW480 cells by decreasing colony-formation ability, EdU-positive ratio, cell proliferation, and CyclinD1 expression, and inducing cycle arrest at G0/G1 phase (Fig. 2c-i). Additionally, metastasis was assessed by cell migration and invasion. Results displayed that circ_DOCK1 silence obviously inhibited the migratory and invasive abilities (Fig. 2 j and  k). Furthermore, circ_DOCK1 interference led to clearly higher apoptotic production in HCT116 and SW480 cells (Fig. 2l). These data suggested silence of circ_DOCK1 could suppress colorectal cancer development.

MiR-132-3p is targeted by circ_DOCK1 and downregulated in colorectal cancer
To analyze the mechanism addressed via circ_DOCK1 in colorectal cancer, the targets of circ_DOCK1 were predicted via circBank. The target sites of circ_DOCK1 on miR-132-3p are shown in Fig. 3a. The circ_DOCK1-WT and circ_ DOCK1-MUT vectors were constructed, and miR-132-3p mimic markedly inhibited luciferase activity of circ_DOCK1-WT but did not change the activity of circ_DOCK1-MUT (Fig. 3 b and c). Furthermore, RNA pull-down assay showed miR-132-3p could be bound to circ_DOCK1 in bio-circ_  DOCK1-WT group, while the enrichment was abolished in bio-circ_DOCK1-MUT group (Fig. 3d). Additionally, miR-132-3p abundance was increased via circ_DOCK1 silence (Fig. 3e). Moreover, miR-132-3p abundance was evidently reduced in colorectal cancer cells and tumor tissues (Fig. 3 f  and g). And it was inversely associated with circ_DOCK1 level in colorectal cancer (Fig. 3h). These results suggested miR-132-3p was sponged via circ_DOCK1.
MiR-132-3p constrains cell growth and metastasis and increases apoptosis by decreasing USP11 in colorectal cancer cells To study the function of miR-132-3p/USP11 axis on colorectal cancer progression, HCT116 and SW480 cells were transfected with miR-NC, miR-132-3p mimic, miR- Fig. 7 Circ_DOCK1 knockdown reduces cell growth in xenograft model. The sh-NC or sh-circ_DOCK1-transfetced HCT116 cells were subcutaneously injected in the nude mice, and mice were classified as sh-NC or sh-circ_DOCK1 group. n=5. a, b Tumor volume and weight were detected. c-f Circ_DOCK1, miR-132-3p, and USP11 levels were examined via qRT-PCR and Western blotting. g USP11 and Ki67 levels were measured via immunohistochemistry in tumor tissues. *P<0.05 Fig. 8 The schematic diagram of this study. Circ_DOCK1 sponges miR-132-3p to regulate USP11, thus increasing cell proliferation, migration, and invasion and suppressing apoptosis in colorectal cancer 132-3p mimic + pcDNA, or USP11 overexpression vector. USP11 abundance was significantly decreased by miR-132-3p overexpression, and it was upregulated via addition of USP11 overexpression vector (Fig. 6 a and  b). Moreover, miR-132-3p overexpression evidently decreased cell growth by repressing the colony-formation ability, EdU-positive ratio, cell proliferation, and CyclinD1 expression, and increasing cycle arrest at G0/ G1 phase, which were attenuated via USP11 upregulation (Fig. 6c-i). Additionally, miR-132-3p mimic obviously repressed cell migration and invasion in HCT116 and SW480 cells, and this effect was mitigated via addition of USP11 overexpression vector (Fig. 6 j and k). Furthermore, miR-132-3p mimic resulted in obvious apoptotic production in HCT116 and SW480 cells, and it was weakened via restoration of USP11 (Fig. 6l). These results suggested miR-132-3p overexpression could repress colorectal cancer development by targeting USP11.

Circ_DOCK1 silence reduces tumor growth
To further probe the function of circ_DOCK1 in colorectal cancer, sh-circ_DOCK1-or sh-NC-transfected HCT116 cells were applied to establish the xenograft model. The mice were classified as sh-circ_DOCK1 or sh-NC group (n=5). Tumor volume and weight were markedly declined in the sh-circ_DOCK1 group in comparison to the sh-NC group (Fig. 7 a and b). In addition, circ_DOCK1 and USP11 abundances were clearly reduced, but miR-132-3p level was increased in the sh-circ_DOCK1 group compared with the sh-NC group (Fig. 7c-g). Additionally, the proliferationrelated marker Ki67 expression was detected in tumor tissues. The data of immunohistochemistry showed Ki67 level was clearly lowered in the sh-circ_DOCK1 group (Fig. 7g). The schematic diagram of circ_ DOCK1-drived mechanism is exhibited in Fig. 8, which showed that circ_DOCK1 regulated USP11 by sponging miR-132-3p, thus contributing to cell proliferation, migration, and invasion and repressing apoptosis in colorectal cancer.

Discussion
Colorectal cancer is a common malignancy with high incidence and mortality [28]. Exploring new mechanism is helpful for finding novel strategy for colorectal cancer treatment. The noncoding RNAs like circRNAs have potential for the detection and therapy of colorectal cancer [29]. Our study evaluated the oncogenic function of circ_ DOCK1 on colorectal cancer development, and we firstly confirmed the circ_DOCK1/miR-132-3p/USP11 axis.
Multiple isoforms of circ_DOCK1 have been reported to play important roles in colorectal cancer. For example, circ_DOCK1 (hsa_circ_0007142) facilitated cell proliferation, migration, and invasion by modulating miR-122-5p/cell division cycle 25A (CDC25A) in colorectal cancer [11]. Similarly, another isoform of circ_ DOCK1 (hsa_circ_0020397) also modulated cell viability, apoptosis, and invasion through miR-138/telomerase reverse transcriptase (TERT)/programmed death-ligand 1 (PD-L1) axis in colorectal cancer [12]. These all suggested the oncogenic role of circ_DOCK1 in cancers. Here, we focused on the circ_DOCK1 (hsa_circ_ 0020397) isoform, and confirmed circ_DOCK1 knockdown could repress cell growth and metastasis, and promoted apoptosis in colorectal cancer, which suggested circ_DOCK1 might function as an important target for colorectal cancer treatment.
Then, we analyzed the downstream of miR-132-3p and firstly confirmed miR-132-3p could target USP11. USP11 acted as a common deubiquitinase that can promote cell growth via modulating cell cycle processes and DNA repair [22,34]. Moreover, previous evidences suggested USP11 could promote epithelial-to-mesenchymal transition to increase cell metastasis in ovarian cancer and breast cancer [35,36]. Additionally, USP11 could promote cell proliferation and metastasis via regulating nuclear factor 90 (NF90) in hepatocellular carcinoma [37]. More importantly, USP11 could promote cell growth and metastasis in colorectal cancer [23,38]. These reports indicated the oncogenic role of USP11 in human tumors, including colorectal cancer. Consistent with these reports, our study found USP11 reversed the anti-cancer function of miR-132-3p, indicating miR-132-3p could modulate colorectal cancer development by targeting USP11. Furthermore, we found circ_DOCK1 regulated USP11 indirectly via miR-132-3p. In this way, the circ_DOCK1/miR-132-3p/USP11 axis participated in colorectal cancer development. However, the current work did not explore the downstream and specific function of USP11 in colorectal cancer, which would be explored in future.
In conclusion, our study displayed that circ_DOCK1 was upregulated in colorectal cancer, and circ_DOCK1 knockdown repressed growth and metastasis, and promoted apoptosis of colorectal cancer cells, possibly via increasing miR-132-3p and decreasing USP11. This study provided a new insight for understanding the mechanism of colorectal cancer and provided a target for colorectal cancer treatment.