1 Introduction
Colorectal cancer (CRC) is the third most common cancer in the world, and the proportion of women is slightly higher than that of men [
1]. As with other types of cancer, mutations in certain genes, such as oncogenes and tumor suppressor genes, may contribute to the development of CRC [
2]. CRC can be divided into sporadic, hereditary and familial based on the different pathways of mutations such as chromosomal instability (CIN), microsatellite instability (MSI) and CpG island methylation phenotype (CIMP) [
3‐
5]. At present, treatment options for CRC patients involve multimodal treatment, such as surgery and radiotherapy [
6]. Considering the limitations of surgery and the side effects of chemotherapy such as low selectivity and systemic toxicity [
7], immunotherapy and targeted therapy have entered clinical application. In addition, there has been significant progress in targeted therapies, such as bevacizumab, aflibercept, regorafenib, cetuximab and panitumumab have been approved for the treatment of metastatic CRC [
8]. Although the treatments are becoming diversified, the results are still not satisfactory. Thus, it is necessary to develop alternative effective target to improve treatment efficiency and reduce side effects for CRC.
In meiosis and mitosis, the separation of chromosomes and the normal division of cells are inseparable from the centromere [
9]. Previously, more than 40 centromere proteins have been identified [
10]. Homo sapiens centromeric protein O (CENPO, NCBI Reference Sequence: NM_024322.3), also known as ICEN36, located on the centromere [
11]. CENP-O, -P, -Q, -R and -50 are all defined as CENPO proteins [
12]. CENPO prevents premature separation of sister chromatids during spindle injury recovery, which is associated with cell death [
12]. Small interference (Si) RNA transfer of CENPO protein will lead to the increase of aneuploidy and aneuploidy chromosomes, which will lead to disease or cancer [
10]. Recent studies have shown that the alteration of CENPO expression can mediate the proliferation of gastric cancer cells [
13]. However, the direct role of CENPO in cancer and the detailed molecular mechanism are currently unclear, especially in CRC. Therefore, the object of this fundamental research will be revealing the role of CENPO in CRC.
This study identified the difference in CENPO expression between cancer and normal tissues in patients with CRC. Subsequently, proliferation, cycle distribution, apoptotic, migration and invasion of CRC cells were detected in loss-of-function assays in vitro. The effects of CENPO knockdown on CRC was evaluated in mice xenograft model. Moreover, the potential mechanism of CENPO in CRC was preliminarily explored.
2 Materials and methods
2.1 Immunohistochemical (IHC) staining
The approval of all experimental procedures related to human CRC samples comes from the ethics committee of Sun Yat-sen Memorial Hospital of Sun Yat-sen University and follows relevant guidelines and regulations. The 100 pairs of cancer tissues and matched non-cancer tissues of CRC patients (Shanghai Outer Biotechnology Company) were used to characterize the expression level of CENPO through IHC experiments. These tissues were fixed with 4% formalin, made into paraffin-embedded sections, dewaxed with xylene, hydrated with alcohol, repaired with sodium citrate, and soaked into 3% H2O2 to remove endogenous catalase. Subsequently, the tissue was eluted with PBS and incubated with the primary antibody against CENPO (1:200, Biorbyt, USA, #orb335144) at 4 °C for 3 h and secondary antibody (1: 400, Abcam, USA, #ab6721) at room temperature for 2 h in sequence. After that, the tissues were treated with DAB, stained with hematoxylin, dehydrated with ethanol gradient, dewaxed with xylene, dried, sealed with neutral gum, and observed under microscope (Nikon C2 + Confocal Microscope, Japan) with magnification of 200 and 400. Notably, high or low expression of CENPO was defined by the median of the scores of each tissue IHC experiment.
2.2 Cell culture condition
The human CRC cell line HCT116 and RKO (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were placed in an incubator (SANYO) at 37 °C with moist air containing 5% CO2, supplemented with Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO) and 10% fetal bovine serum (FBS, GIBCO).
2.3 Establishment of CRC cell lines with CENPO knockdown
CENPO, the mRNA of transcriptional variant 1 was used to design three RNA interference target sequences (Table S1). The fragment was digested by AGE I (5′-ACCGGT-3′, 10 U/µL, NEB) and EcoR I (5′-GAATTC-3′, 10 U/µL, NEB) and inserted into linearized vector BRV-112 (5′-CCGGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTG-3′) (BIOSCIRES, Shanghai, China) using T4 DNA Ligase (Fermentas). Notably, the green fluorescent protein (GFP) tag of the lentiviral vector BRV-112 was used to estimate the transfection efficiency. Subsequently, 5 × 106 HCT116 and RKO cells were co-transfected with 10 μg recombinant BRV-112 plasmids at a multiplicity of infection of 10 using Lipofectamine 3000 (Invitrogen) for 1 h. After transfection with lentivirus, cell transfection efficiency was evaluated under fluorescence microscope (OLYMPUS).
2.4 Quantitative-PCR (qPCR) analysis
According to the kit instructions (Invitrogen, Carlsbad, CA, USA), RNA extraction from lentivirus-transfected HCT116 and RKO cells. RNA was reverse transcribed into cDNA using the Promega M-MLV kit. Subsequently, template cDNA was used for qPCR analysis with Ace Q qPCR SYBR Green Master Mix (Vazyme, Nanjing, China). The relative mRNA expression of CENPO was quantified with cycle threshold (Ct) values and normalized using the 2−∆∆Cq method. Notably, the primer sequence, and the size of the amplicon synthesized after qPCR were summarized in Table S2. GAPDH as a reference control.
2.5 Western blot
Total protein from lentivirus-transfected HCT116 and RKO cells was extracted using RIPA (Beyotime) and the protein concentration was detected by BCA Protein Assay Kit (Beyotime). Western analysis was separated using SDS-PAGE (10%), transferred to polyvinylidene difluoride (PVDF) membrane, blocked with 5% BSA and 0.5% Tween 20 at 4 °C for 1 h. Next, the protein was co-incubated with the primary antibodies (Table S3) at 4 °C for 3 h and then with the goat anti-rabbit IgG polyclonal antibody (1:3000) labeled with horseradish peroxidase (HRP) at room temperature for 2 h. Finally, protein signal was visualized using chemiluminescence ECL-PLUS kit (Thermo Fisher Scientific).
2.6 MTT assay
After digesting the lentivirus-transfected (shCtrl, shCENPO) HCT116 and RKO cells with trypsin, the cells were resuspended into cell suspension and counted with a counting plate (Cellometer, Cat. #SD-100). The cells were cultured into 6-well plates at a density of 2000 cells/well for 5 days, with 3 replicates in each group. On the second day after plate laying, 20 μL 5 mg/mL MTT and 100 μL DMSO were added in turn for 2–5 min and then placed in the enzyme plate analyzer to detect OD490 nm value.
2.7 Celigo cell counting assay
After digesting the HCT116 and RKO cells [shCtrl, shCENPO, shCENPO + AKT activation (10 μM, SC79, MEC, Cat. #HY-18749)] with trypsin, the cells were resuspended into cell suspension and counted with a counting plate (Cellometer, Cat. #SD-100). The cells were cultured into 6-well plates at a density of 2000 cells/well and the culture system was 100 μL/ well. Celigo (Nexcelom) was monitored once a day for 5 consecutive days. The number of cells was accurately calculated according to the amount of GFP in each scanning orifice. The 5-day data were calculated and the cell proliferation curve was plotted.
2.8 Flow cytometry cell cycle assay
Lentivirus-transfected (shCtrl, shCENPO) HCT116 and RKO cells were inoculated in 6-well plates (2 mL/well) for 5 days. The cells were eluted by PBS, centrifuged for 5 min at 200g, fixed with ethanol, and stained with propidium iodide (PI). The ratio of cells in the G1, S and G2 phases of the CENPO knockdown group and the control group were detected and analyzed by flow cytometry.
2.9 Flow cytometry apoptotic assay
Lentivirus-transfected (shCtrl, shCENPO) HCT116 and RKO cells were inoculated in 6-well plates (2 mL/well) for 5 days. After centrifugation, the cells were successively washed and precipitated by PBS and 1 × binding buffer, stained with 5 μL annexin V-APC in the dark for 15 min. The apoptosis rate was detected by flow cytometry and the results were analyzed by T test.
2.10 Human apoptosis antibody array
Follow the apoptotic antibody array kit (Abcam, USA, #ab134001) instructions, lentivirus-transfected (shCtrl, shCENPO) HCT116 cells protein was diluted with the array diluent buffer kit to 0.5 mg/mL. Subsequently, the array antibody membrane was blocked using blocking buffer at room temperature for 30 min, incubated with HRP linked streptavidin at 4 °C overnight. ChemiDoc XRS chemiluminescence detection and imaging system were used to visually detect proteins.
2.11 Wound-healing assay
Lentivirus-transfected (shCtrl, shCENPO) HCT116 and RKO cells were inoculated in 6-well plates (100 μL/well) at a density of 5 × 104 cells/well for 5 days. In brief, vertical lines for each well were drawn at 0 h, 24 h and 48 h using a pipette. After incubation, the cells were washed with PBS, fixed with 3.7% paraformaldehyde (Corning) for 15 min, and stained with 1% crystal violet (Corning) for 10 min. Finally, cells were placed under a microscope for image acquisition and Image J software (National Institutes of Health) was used to quantify the distance (μm) between the scratches at different time points.
2.12 Transwell invasion assay
Lentivirus-transfected (shCtrl, shCENPO) HCT116 and RKO cells were placed into Transwell chambers (24-well, 8-mm pore) (Corning) at a density of 80,000 cells/well for incubation24 h at 37 °C. The inner compartment contained 100 μL cell suspension and the outer compartment was 500 μL DMEM medium containing 30% FBS. After that, non-invading cells on the upper chamber were removed, the cells attached to the polycarbonate membrane were fixed with 4% precooled paraformaldehyde for 30 min and stained with 0.1% crystal violet at room temperature for 20 min. Afterwards, the cells were placed under a 200 × microscope to capture images from five randomly selected fields.
2.13 Establishment of animal xenograft model
The approval of animal experimental procedures came from the ethics committee of Sun Yat-sen University and followed relevant guidelines and protocols for animal care and protection. Lentivirus-transfected (shCtrl, shCENPO) RKO cells 500 μL were digested with trypsin, resuspended and injected into the right forearm of BALB/c female nude mice (8 × 106 cells/mouse) (Jiesijie Experimental Animals Co., Ltd., Shanghai, China). With 10 mice per group, tumorigenesis rates in the shCtrl group and the shCENPO group were 50% and 20%, respectively. Mice were anesthetized with 0.7% sodium pentobarbital (10 μL/g) and placed in the IVIS spectral fluorescence imaging system (emission wavelength of 510 nm) to assess tumor burden. Tumor size was estimated every other week until 20 days after subcutaneous injection. 26 days later, the mice were treated with cervical dislocation, tumors were taken out, weighed, and photographed. Finally, the expression of CENPO (1:200, Biorbyt, USA, #orb335144), Ki67 (1: 200, Abcam, USA, #ab16667), AKT (1:200, Proteintech, USA, #60203-2-Ig) and p-AKT (1:200, MILLIPORE, USA, #05-1003) was detected by IHC staining in mouse tumor tissues of shCENPO group and shCtrl group as previous described. Images were observed under microscope (Nikon C2 + Confocal Microscope, Japan) with magnification of 200.
2.14 Statistical analysis
The results presented represent experiments repeated at least 3 times and are expressed as mean ± SD. Statistical was analyzed using GraphPad Prism 7.0 software (GraphPad Software Inc., San Diego, CA, USA) and SPSS 21.0 (IBM, Armonk, NY, USA). All tests were analyzed using paired t test and one-way ANOVA followed by Bonferroni’s post hoc test analysis. P < 0.05 were considered statistically significant.
4 Discussion
Centromere is an important component of chromosome separation during meiosis and mitosis of normal cells [
9]. Previous study has reported that abnormal localization or overexpression of the centromere protein CENPO leads to cell division disorders and chromosomal aneuploidy [
11]. The biological process is closely related to the progression of cancers [
17]. For instance, Cao et al. reported that the expression of CENPO was not only related to the prognosis of gastric cancer patients, but also regulated the proliferation of gastric cancer cells [
13]. Moreover, CENPO regulated the cell cycle by mediating mitotic spindle assembly and participated in the BC process [
18]. The present study demonstrated the role of CENPO in promoting CRC. IHC staining used to identify the difference in CENPO expression between cancer and normal tissues in patients with CRC. We found that expression of CENPO was highly expressed in tumor tissues of CRC patients. Expression of CENPO was positively correlated with the deterioration of CRC patients. Additionally, downregulation of CENPO inhibited the malignant progression of CRC cells, such as reduced proliferation, cycle repression in G2 phase, enhanced apoptotic sensitivity and inhibition of migration.
Previous study demonstrated that the migration and metastasis of tumor cells usually required the process of epithelial-mesenchymal transition (EMT) [
15]. N-cadherin, E-cadherin, and Vimentin are regarded as the most common markers in the EMT process [
16]. Choi et al. suggested that the metastasis of CRC cells required the involvement of EMT, such as the abnormal expression of N-cadherin and Vimentin [
19,
20]. Similarly, MicroRNA-1275 regulated Vimentin and E-cadherin to inhibit the migration and invasion of gastric cancer cells [
21]. The study showed that knockdown of CENPO contributed to downregulation of N-cadherin and Vimentin, upregulation of E-cadherin. Thus, CENPO mediated the process of EMT to regulate the migration and invasion of CRC cells.
This study indicated that decrease of CENPO expression can induce apoptosis of CRC cells. It has been pointed out that the reduction of cell apoptosis is an important factor of tumor genesis and carcinogenesis [
22]. The initiation of apoptosis is an extremely complex process, which is inseparable from the interaction of internal and external pathways [
23,
24]. Generally speaking, the intrinsic pathway is activated by various stimuli, such as DNA damage and loss of cell survival factors, which are controlled by a series of Bcl family members. The binding of the death ligand to the receptor triggers an external pathway [
25]. The present study indicated that knockdown of CENPO contributed to upregulation of Caspase3, Caspase8, HTRA, p53, SMAC, TNF-α, TNF-β and TRAILR-1. On the contrary, Bcl-2, Bcl-w and CLAP-2 were downregulated. The activation of Caspase8 can promote the rupture of Caspase3 and PARP, and activate mitochondrial-mediated endogenous apoptosis [
26]. Cong et al. identified that upregulated expression of Caspase3 induced apoptosis of CRC cells [
26]. In addition, HTRA induces apoptosis by degrading XIAP and activating PI3K/AKT pathway [
26]. Activation of p53 can induce tumor apoptosis and enhance the response of CRC cells to chemotherapy drugs 5-Fluorouracil and Oxaliplatin [
27]. Hehlgans and Grimm et al. pointed that either SMAC or TRAILR-1 can induce apoptosis of CRC cells inhibit DNA damage repair [
28,
29]. Furthermore, Shen et al. and Buhrmann et al. clarified that TNF-
α, TNF-
β are similar [
30], they can induce tumor-related apoptosis to inhibit metastasis of CRC cells [
31,
32]. Huang et al. and Gibson et al. suggested that high expression of anti-apoptotic protein Bcl-2 and Bcl-w are prognostic factors for CRC [
33‐
35]. CLAP-2, also known as CLASP, maintains cell proliferation by regulating the dynamic stability of microtubules [
36,
37]. In this study, we found that knockdown of CENPO contributed to upregulation of pro-apoptosis proteins and downregulation of anti-apoptosis proteins. The alterations of apoptotic signaling pathway were preliminarily explored through the occurrence of apoptosis in tumor cells. Of course, the process of apoptosis signaling pathway required to be further explored, which will provide an important theoretical basis for cancer treatment strategies.
The ability of knockdown CENPO to inhibit tumorigenesis has prompted us to explore downstream pathways that may mediate the carcinogenesis of CENPO. In this study, we demonstrated that CENPO knockdown decreased expression of downstream protein p-AKT, CCND1, PIK3CA, while MAPK9 was increased. Previous study had proved that AKT kinase activation (p-AKT) plays a central role in regulating transcription, cell survival and apoptosis, and is one of the prognostic factors of CRC [
38,
39]. CCND1 regulates DNA repair, and overexpression of this protein may be related to the poor clinical prognosis and distant metastasis of CRC patients, which are considered as the poor prognosis indicator of CRC [
40,
41]. PIK3CA (PI3K) mutation is also a common feature of CRC, which is related to poor prognosis [
42,
43]. Both MAPK and PI3K pathways are involved in CRC cells proliferation and survival. Yaeger et al. illuminated that inhibition of MAPK/PI3K pathway is more effective in the treatment of metastatic CRC [
44]. Consequently, CENPO is implicated in CRC cell progression by regulating downstream pathways p-AKT, CCND1, PIK3CA and MAPK9.
This study demonstrated for the first time the promoting effect of CENPO in CRC. CENPO was not only highly expressed in tumor tissues, but also positively correlated with the deterioration of CRC patients. Additionally, downregulation of CENPO inhibited the malignant progression of CRC cells, such as reduced proliferation, cycle repression in G2 phase, enhanced apoptotic sensitivity and inhibition of migration. CENPO mediated the process of EMT to regulate the migration and invasion of CRC cells. The reduced expression of CENPO attenuated the phosphorylation level of AKT, downregulated CCND1, PIK3CA, and upregulated MAPK9. In vivo experiments further confirmed that CENPO downregulation attenuated tumor growth. In summary, the prominent discovery was the determination of the promoting role of the CENPO in CRC, demonstrating that small molecule inhibitors targeting CENPO were a novel therapeutic strategy for CRC.
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