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Down-regulation of CIT can inhibit the growth of human bladder cancer cells
Biomedicine & Pharmacotherapy 124 (2020) 109830
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Down-regulation of CIT can inhibit the growth of human bladder cancer cells
T
Zan Liua, Haiyan Yanb, Yang Yanga, Liangjun Weic, Shunyao Xiaa, Youcheng Xiua,d,* a
Department of Urology, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China Department of Anesthesiology, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China c Departments of Urology, Harbin Medical University Cancer Hospital, Harbin, Heilongjiang, China d Heilongjiang Academy of Medical Sciences, Heilongjiang, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Apoptosis Bladder cancer cell Citron rho-interacting Serine/threonine kinase 21 Proliferation
Objective: Our study is to examine the citron rho-interacting, serine/threonine kinase 21 (CIT) in bladder cancer. Methods: We examined CIT level in human bladder cancer tissues by immunohistochemical staining. To explore the impact of CIT on cell proliferation and apoptosis, we down-regulated its expression in two human bladder cancer cell lines, 5367 and T24. We examined cell growth in 5367 and T24. We also performed in vivo analysis using T24 cells. We further used microarray expression profiling to investigate genes differentially expressed in T24 cells with CIT down-regulated. Results: In 100 human samples, CIT was expressed by only 2 of 30 (6.7 %) controls in bladder tissues, whereas by 64 of 70 (91.4 %) cancer patients in tumor tissues (p < 0.001). in vitro analysis demonstrated that CIT knockdown represses cell proliferation by 50 % in both cells and colony formation (77 ± 5 vs. 13 ± 2, p = 0.001 for T24, 58 ± 3 vs. 1 ± 1, p < 0.001 for 5637). We also found CIT knockdown could induce cell cycle arrest, and promote apoptosis in both cells. Tumor-volume monitoring and live in vivo bladder cancer imaging in human xenograft model confirmed that CIT knockdown reduces tumor volume (668.4 ± 333.0 vs. 305.7 ± 170.4 mm3, p = 0.02) and weight (0.27 ± 0.15 vs. 0.57 ± 0.32 g, p = 0.02). Microarray analysis revealed that CIT may regulate cell cycle signalling pathway through various cell cycle regulators. Conclusions: In summary, we provided clinical and experimental evidence that CIT may promote bladder cancer through regulation of cell cycle pathway.
1. Introduction Bladder cancer is the ninth most common cancer worldwide, with an estimated 430,000 new cases diagnosed in 2012 [1]. In China, according to the 2015 National Central Cancer Registry, bladder cancer ranked sixth in male cancers and the most common urologic malignancy [2,3]. In the past few years, the incidence and mortality rates of bladder cancer have increased gradually in China [3]. Despite the development in surgical techniques, perioperative therapies and postoperative management, survival outcomes for bladder cancers patients remained unchanged over the last 30 years [4,5]. To date, clinical management of bladder cancer is largely based on determination of tumor stage and other histopathological parameters. However, studies have suggest that bladder cancer should not be treated exclusively on the basis of pathologic staging, because it does not reflect the overall clinical risk [6]. Therefore, there is an urgent
need for discovery of novel biomarkers and therapeutic targets for the diagnosis and treatment for bladder cancer. Disruption of cell cycle control may lead to genomic instability, neoplastic transformation and tumor progression [7]. Citron rho-interacting, serine/threonine kinase 21 (CIT) is present at the cleavage furrow and the midbody during cytokinesis, a key component for cellular abscission [8,9]. CIT is also found to phosphorylate the regulatory light chain of myosin II, which is the primary motor protein responsible for cytokinesis [10,11]. Although still limited, accumulating evidence is beginning to show the role of CIT in cancer through regulation of cell cycle. Overexpression of CIT has been observed in tumor tissues collected from patients with gastric cancer [12], hepatocellular carcinoma (HCC) [13], colon cancer [14] and multiple myeloma [15]. Experimental studies further demonstrated that CIT knockdown could inhibit cell grown, as well as induce cell cycle arrest and apoptosis in human HCC and colon cancer cell lines [12,13]. Taken together these evidence
⁎ Corresponding author at: Department of Urology, The First Affiliated Hospital of Harbin Medical University, No. 23 Youzheng Street, Harbin, Heilongjiang, 150001, China. E-mail address: [email protected] (Y. Xiu).
https://doi.org/10.1016/j.biopha.2020.109830 Received 15 July 2019; Received in revised form 24 November 2019; Accepted 18 December 2019 0753-3322/ © 2020 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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to a CIT score ≥6.
from human and experimental studies, CIT maybe a novel tumor promoter that can be used as a potential therapeutic target for anti-cancer therapy. However, whether CIT could be a target in bladder cancer is poorly understood. In this study, we demonstrated that the expression level of CIT is higher in human bladder cancer tissues than bladder tissues from controls, and the expression level is not correlated to clinical pathology of bladder cancer. Then, we used lentiviruses expressing CIT-targeting small hairpin RNA (shRNA) approach to knockdown CIT expression in two human bladder cancer cell lines. We found that CIT knockdown represses cell proliferation and colony formation, and induces cell cycle arrest and apoptosis. Tumor-volume monitoring and live in vivo bladder cancer imaging in human xenograft model confirmed that CIT knockdown reduces tumor volume and weight. Microarray analysis revealed that CIT may regulate cell cycle signalling pathway through various cell cycle regulators. In summary, we, for the first time, provided evidence that CIT may promote bladder cancer through regulation of cell cycle pathway.
2.3. Cell lines and Lentiviral infection Human bladder cancer cell lines 5637 and T24 were obtained from Cell Resource Center, Shanghai Institutes for Biological Sciences at the Chinese Academy of Sciences. The cells were cultured in RPMI 1640 medium containing 10 % FBS, streptomycin, and penicillin at 37 °C in an incubator with 5 % CO2. To inhibit CIT expression, small hairpin RNA (shRNA) specifically targeting CIT (target sequence: 5′-GCGTCCTCATACCAGGATAAA-3′) was designed and packed into a lentivirus vector. A scrambled shRNA was used as a negative control using the following sequence: 5′-TTCT CCGAACGTGTCACGT-3′. shRNA of human CIT lentivirus gene transfer vector encoding green Xuorescent protein (GFP) sequence was constructed by Genechem Co., Ltd (Shanghai, China) as described previously [14]. The recombinant lentivirus expressing short hairpin RNA (shRNA) to induce silencing of CIT (sh-CIT) and the control lentivirus (sh-Control) were prepared and titered to 8 × 108 TU/mL (transducing unit). T24 and 5637 cells were plated onto 6-well plates and subsequently infected with sh-CIT or sh-Control as described previously [14].
2. Materials and methods 2.1. Human bladder tissue collection
2.4. Quantitative real-time PCR and Western blotting 100 patients who underwent surgery at Department of Urology, Heilongjiang Nongken General Hospital, China from September 2013 to September 2014 were recruited in our study. Tumor tissue specimens were obtained from 70 bladder cancer patients by transurethral resection. Normal bladder tissues (control) were obtained from 30 patients with renal pelvic carcinoma by laparoscopic nephroureterectomy and confirmed by pathology analysis. All tissues were cut into small pieced (0.8–1.2 cm in diameter), snap-frozen in liquid nitrogen, and then transferred to a low-temperature refrigerator (−80 °C). The study was approved by the Ethics Committee of The First Affiliated Hospital of Harbin Medical University, China. Individual written informed consent was obtained prior to enrolment in the study.
T24 cells were used to examine the knockdown efficiency at mRNA and protein levels. Quantitative real-time PCR was preformed to examine the mRNA level of CIT as described previously [14]. The relative expression level was calculated using the 2−ΔΔCT method with GAPDH as the reference gene for normalization. Western blot was used to measure protein level of CIT using primary anti-CIT antibody (1:5000) (ab86782, Abcam, UK). GAPDH was used as control. Protein bands were detected using an ECL-Plus kit (Amersham Biosciences, USA) as described previously [14]. 2.5. In vitro assay of T24 and 5637 cells infected with sh-CIT and shControl
2.2. Immunohistochemistry T24 and 5637 cells were infected with sh-CIT and sh-Control lentivirus and incubated for five days for cell cycle, apoptosis and colony formation assay, or three days for BrDU assay [14]. For cell cycle analysis, cells were trypsinized and fixed in 70 % ethanol at −20 °C for 10 min. The cells were washed with PBS, resuspended in RNase (0.2 mg/mL) and incubated at 37 °C for 10 min. Propidium iodide (PI) was added to obtain a final PI concentration of 300–800 cells/s when analysing on a Guava easyCyte HT flow cytometer (Millipore, USA). For apoptosis assay, cells were collected, washed with PBS and resuspended using staining buffer at a density of 1 × 105 - 1 × 106/mL. Subsequently, 5 μL Annexin V-APC was added to 100 μL of cell suspension and incubated at room temperature for 10−15 min. The signals were detected using a FACS Calibur (Millipore, USA). Cells infected with sh-CIT and sh-Control were seeded in 96-well plates at a density of 2000 cells/well and incubated at 37 °C for 3 days. After the 3-day infection, cells were divided to two groups. One group was used for BrdU incorporation (day 1), and one group was incubated with RPMI 1640 medium for 3 days and then used for BrdU incorporation assay (day 4). Then cells were washed two times with PBS and BrdU reagents (1:100) were subsequently added to each well (10 μL/well). Cell proliferation was analyzed based on the measurement of BrdU incorporation using a BrdU Cell Proliferation ELISA kit (Roche Applied Science, USA) according to the manufacturer’s instructions. We measured BrdU density on days 1 and 4. The BrdU density was 450 nm using Biotek Elx800, and the experiments were performed in triplicate. For colony formation assay, cells were harvested in the logarithmic phase, and plated in triplicate onto 6-well plates at a density of 800 cells/well. After incubation for eight days, cells were washed with PBS
Immunohistochemistry were performed using the SABC kit (suolaibao, China) according to the manufacture’s instruction. The sections were incubated with primary antibody against CIT (1:100) (ab110897, Abcam, UK) at 37 °C for 1 h. Slides containing tissue sections with high expression of CIT were incubated with anti-CIT antibody as positive control, and incubated with PBS as negative control. Then, all sections were incubated with the biotinylated secondary antibody for 20 min at room temperature, followed by a 20 min incubation with streptavidin peroxidase. After rinsing, the results were visualized using Diaminobenzidine and counterstained with hematoxylin. All fields were taken under a light microscope (Olympus Corp., Japan) at 200× magnification. The optical density was quantified by Image-Pro Plus 6.0 in three random high power fields. All immunohistochemistry staining samples were independently estimated by two pathologists in a masked fashion to minimize any possible bias according to the 1997 Union for International Cancer Control (UICC) TNM classification criteria. We assessed the site of staining (cytoplasmic staining was considered a positive expression of CIT), the staining intensity, and extent of CIT expression as follows: Staining intensity was scored based on a range of 0–3, where 0 represents no staining, 1 represents faint yellow, 3 represents brown, and 4 represents dark brown (the strongest staining). The extent was categorized by the percentage of stained cells (staining intensity≥1) as follows: 0–5%=score 0, 6–25%= score 1, 26–50%= score 2, 51–75%= score 3 and 76–100%= score 4. CIT scoring was assigned by multiplying the staining intensity score and the staining extent score. Five fields were randomly selected, and the average score of the five fields was the final IHC staining score. Negative staining refers to a CIT score < 6, and positive staining refers 2
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and subsequently fixed with paraformaldehyde for 30−60 min. The cell colonies were stained with 500 μL of Giemsa for 20 min at room temperature. Images of the cell plates were captured, and the colonies were analyzed.
Table 1 CIT expression and clinicopathological characteristics of bladder cancers (N = 70). Parameter
2.6. In vivo xenograft assay of T24 cells infected with sh-CIT and sh-Control
Age
BALB/c female nude mice at four-week-old were purchased from Lingchang Biological Technology Co., Ltd. (Shanghai, China) and kept in specific pathogen-free conditions. Mice were randomly divided into two groups (n = 10/group), and 1 × 107 T24 cells transfected with shCIT and sh-Control were subcutaneously injected into the right armpits of mice. After initial detection, tumor volumes were evaluated twice a week for 15 days. Tumor sizes were measured with a digital caliper and tumor volume in mm3 was calculated by the formula: volume = (width) 2x length x 3.14/6 [16]. At time of sacrifice, live mouse imaging were conducted using Lumina LT (filters: excitation 672 nm, emission 694 nm; Perkin Elmer, USA) under anaesthesia. On the fluorescence images, freehand regions of interest (ROIs) were drawn carefully along the margins. The mean fluorescence intensities [total radiance efficiency, (p/s)/(μW/cm2)] were generated automatically by Living Image® 4.4 software and recorded. After 1 month of cell inoculation, mice were euthanized by CO2 asphyxiation and only tumors were harvested and weighted. Throughout the experiment, all mice had access to sterilized food and filtered water ad libitum. The vivarium was maintained at 23 °C on a 12 h light/12 h dark cycle. All animal experiments in our study were approved by the Animal Care and Use Committee of the The First Affiliated Hospital of Harbin Medical University hospital, China.
Gender Metastasis Infiltrative Histological grade
T staging
Category
Total (%)
CIT expression Positive (64)
Negative (6)
P-value* 1.000
> 64 yrs ≤64 yrs Male Female Yes No Yes No G1
47 (67.1) 23 (32.9) 49 (70.0) 21 (30.0) 7 (10.0) 63 (90.0) 40 (57.1) 30 (43.9) 17 (24.3)
43 (91.4) 21 (91.3) 46 (93.9) 18 (85.7) 6 (85.7) 58 (92.1) 39 (97.5) 25 (83.3) 13 (76.5)
4 2 3 3 1 5 1 5 4
(8.6) (8.7) (6.1) (14.3) (14.3) (7.9) (2.5) (16.7) (23.5)
G2 G3 Ta-T1 T2 T3-T4
24 29 16 20 34
23 28 13 18 33
1 1 3 2 1
(4.2) (3.4) (18.8) (10.0) (2.9)
(34.3) (41.3) (22.9) (28.6) (45.6)
(95.8) (96.6) (81.3) (90.0) (97.1)
0.355 0.48 0.08 0.90
0.14
* Fisher exact test.
2.8. Statistical analysis A Fisher exact test was used to compare the distributions of categorical variables in study using human samples (Table 1). In experimental studies using human samples, data were expressed as means and standard deviations for continuous variables. T-test was used to compare continuous variables between groups (sh-CIT vs. sh-Control). All statistical analyses were performed using STATA version 14.0 (STATA Corporation, Texas, USA). A two-tailed p value of less than 0.05 was considered as statistically significant.
2.7. Microarray analysis in T24 cells infected with sh-CIT and sh-Control
3. Results
The in vitro and in vivo results demonstrated that CIT was involved in bladder cancer, possibly through regulation of cell growth and survival. We further performed microarray analysis in T24 cells infected with shCIT and sh-Control to better understand the underlying mechanisms of CIT in bladder cancer. Total RNA from T24 cells infected with sh-CIT (n = 3) or sh-Control lentivirus (n = 3) was extracted using Trizol reagent. The quantity of RNA was evaluated using NanoDrop. The integrity was measured by Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., USA). Only samples with high purity (1.7 < A260/ A280 < 2.2) and high integrity (RNA integrity number (RIN) > 8.7), and 28S/18S > 0.7) were subsequently used in microarray experiments. Microarrays were processed to generate gene expression profiles using the Affymetrix human GeneChip PrimeView assay (Affymetrix; Thermo Fisher Scientific, Inc. USA) according to the manufacturer's instructions. Genes that were differentially expressed between shControl and sh-CIT-infected T24 cells were identified based on the following criteria: P < 0.05 and an absolute fold-change > 1.5. Principal component analysis (PCA) was done on the processed data to identify possible outlier samples. We performed functional enrichment analysis of the differentially expressed genes by using Kyoto Encyclopedia of Genes and Genomes (KEGG) and BioCarta pathway. Five genes, including cyclin D1 (CCND1), cyclin dependent kinase 1 (CDK1), minichromosome maintenance complex component 3 (MCM3), MCM7, and proliferating cell nuclear antigen (PCNA), were selected for validation using western blot as described above. The primary antibodies used included anti-CCND1 (1:150, ab16663, Abcam, UK), antiCDK1 (1:3000, ab32384, Abcam, UK), anti-MCM3 (1:1000, #4003 s, Cell Signaling Technology, USA), anti-MCM7 (1:1000, #4018, Cell Signaling Technology, USA), and anti-PCNA (1:2000, #2586s, Cell Signaling Technology, USA).
3.1. CIT level in bladder cancers and normal bladder tissues in human using immunohistochemistry The expression of CIT in bladder cancer tissues from cancer patients and normal bladder tissues from cancer-free patients was assessed by immunohistochemistry. Fig. 1 shows that CIT was predominantly localized to the cytoplasm. Overall, the intensity and extent of CIT level increased across control (normal bladder tissues, IHC score = 1.4 ± 1.6), grade 1 (least aggressive, IHC score = 7.6 ± 3.1), grade 2 (moderately aggressive, IHC score = 8.3 ± 1.8) and grade 3 tumor (most aggressive and most likely to spread, IHC score = 9.0 ± 1.8). The positive expression of CIT was detected in only 2 of 30 (6.7 %) controls in bladder tissues, whereas in 64 of 70 (91.4 %) cancer patients in tumor tissues (IHC score = 8.4 ± 2.2) (p < 0.001), indicating that CIT is unregulated in bladder cancers. We then analyse the relationship between CIT expression with basic and clinicopathological characteristics among the 70 bladder cancer patients. Table 1 shows that the percentage of patients with CIT-positive cancer is not different by age (> 64 vs. ≤64), gender, and clinicopathological of bladder cancers. These results suggest CIT is up-regulated in bladder cancer tissues independent of metastatic status or cancer T staging. 3.2. CIT promotes bladder cancer cell growth and inhibits apoptosis in vitro We evaluated CIT expression using real-time PCR in 5637 and T24 cells, finding expression of CIT in both cell lines (Fig. 2A). Therefore, we knocked down CIT with lentivirus-medicated sh-CIT and sh-Control in both cells to investigate the role of CIT in cell proliferation and apoptosis. T24 cells infected with lentivirus expressing CIT-shRNA and control-shRNA was used to examine the knockdown efficiency of CIT. Fig. 2B and C show significantly reduced CIT expression at mRNA and 3
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Fig. 1. Immunohistochemistry against CIT in (A) bladder tissues from controls and (B–D) bladder tumor samples from bladder cancer patients. Original magnification: 200×.
3.4. CIT inhibits cell cycle pathways
protein level in T24 and 5637 cells, suggesting an effective inhibition of CIT using the lentiviral-based shRNA strategy. Fig. 2D–I compared cell proliferation, colony formation, cell cycle, and apoptosis in T24 and 5637 cells infected with sh-CIT and sh-Control. BrDU incorporation assay shows that sh-CIT significantly inhibited cell proliferation by approximately 50 % in T24 (Fig. 2D) and 5637 cells (Fig. 2E). We performed a colony formation assay to determine the colonogenic ability. The results show that sh-CIT significantly reduced colony formation in T24 (77 ± 5 vs. 13 ± 2, p = 0.001) (Fig. 2F) and 5637 cells (58 ± 3 vs. 1 ± 1, p < 0.001) (Fig. 2G). These results demonstrated that CIT is involved in bladder cancer cell proliferation and colony formation. The PI staining revealed that sh-CIT results in significant decrease of cells in the S phase and decrease of cells in the G2/M phase in T24 (Fig. 2H) and 5637 cells (Fig. 2I). An annexin VAPC apoptosis assay showed that sh-CIT significantly promoted cell apoptosis in both cells (p < 0.05, Fig. 2J and K). Taken together, these results demonstrated that suppression of CIT in human bladder cancer cells could inhibit cell proliferation and colony formation, induce cell cycle arrest, and promote apoptosis.
To understand the underlying mechanisms of CIT in bladder cancer, we used microarray to investigate gene expression profiles in T24 cell infected with sh-CIT (KD) and sh-Control (NC). The distribution and similarities of six samples (three KD and three NC cells) were shown in Supplementary Fig. 1. Fig. S1A shows an overview of the sample relations based on PCA. Pearson's correlation of the signal value (Pearson's correlation coefficient > 0.95; Fig. S1B) indicated a good overall correlation of individual gene expression measurements across chips. Signal value distribution (Fig. S1C) and relative signal box plot graphs (Fig. S1D) demonstrated the expression values of all microarray probe distribution statistics and all samples were reproducible. Scatterplot graphs (Fig. S1E) demonstrated differences in gene expression of all microarray probe distribution statistics between KD and NC. Heatmap shows significant differential expression patterns between KD and NC cells, including 364 up-regulated genes and 471 downregulated genes by CIT knockout (Fig. 4A). KEGG and BioCarta pathway analysis demonstrated that the differentially expressed genes were enriched in ten pathways (Fig. 4B). Functional analysis by GO terms showed that some of these genes are implicated in cancer development or cancer-related biological pathways, such as cell cycle and p53 pathway (the major apoptosis signalling pathway). Therefore, we analysed the functional interaction network between CIT and cell cycle pathway (the one with significantly enriched pathway in cells with CIT knockdown), finding 17 cell cycle regulators in cell cycle pathway were down-regulated significantly by CIT knockdown with a fold-change > 1.5 (p < 0.05) (Fig. 4C). Five genes were selected for validation based on statistical significance and cancerrelated biological functions, including cyclin D1 (CCND1), cyclin dependent kinase 1 (CDK1), minichromosome maintenance complex component 3 (MCM3), MCM7, and proliferating cell nuclear antigen (PCNA). Western blots demonstrated that the protein level of CCND1, MCM3, and MCM7 were downregulated by 62 %, 88 %, 76 %, respectively. The expression of CKD1 was decreased to undetectable level. We did not observe significant down-regulation of PCNA (Fig. 4D).
3.3. CIT promotes bladder cancer cell growth in vivo We evaluated xenograft formation of T24 cells infected with sh-CIT (KD) and sh-Control (NC). After inoculation of cells for 21 days, tumors in KD group were significantly smaller than those in NC group, and the difference remained at sacrifice (668.4 ± 333.0 vs. 305.7 ± 170.4 mm2, p = 0.02) (Fig. 3B). Tumor weight is also significantly lower in KD group than those in NC group (0.27 ± 0.15 vs. 0.57 ± 0.32 g, p = 0.02) (Fig. 3C). Fluorescence intensities of the tumors in KD and NC groups were shown in Fig. 3D. In the KD mice, we could observe lower fluorescence intensities than those in NC mice. The average of total radiant efficiency is significantly lower in KD group than those in NC group (2.0e+10 ± 5.1e+09 vs.4.0e+10 ± 1.6e+9 [p/s]/[μW/cm2]) (p = 0.003), suggesting the lower intensities of tumor with knockdown of CIT.
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Fig. 2. Cell-based assay in T24 and 5637 cells infected with sh-CIT and sh-Control. (A) mRNA level of CIT in wild T24 and 5637 cells. (B) mRNA and (C) protein level of CIT was reduced significantly by using real-time PCR and Western Blotting following CIT knockdown using lentiviral-based shRNA in T24 cells and 5637 cells. ShCIT significantly inhibited cell proliferation using BrDU incorporation assay and colony formation in T24 (D, F) and 5637 cells (E,G). CIT-CIT significantly inhibited colony formation in T24 (F) and 5637 cells (G). sh-CIT increased the percentage of cells with S and G2/M phases, and decrease in the percentage of cells with G1 phase in T24 (H) and 5637 cells (J). sh-CIT significantly promoted cell apoptosis in both cells in T24 (J) and 5637 cells (K). **P < 0.05 vs. sh-Ctrl. 5
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it remains unclear whether there is increased CIT level in adjacent normal tissues, which may increase tumor susceptibility. Another limitation is that, we didn’t compare clinical outcome (i.e., overall survival) between CIT-positive and CIT-negative cancer patients as in a previous studies of colon cancer and multiple myeloma [14,15]. Such analysis using survival data by follow-up records is needed in the future to understand the prognostic significance of CIT in bladder cancer. The other limitations include small sample size and a single-center design; therefore, our results require further verification in a larger sample and multi-centric design with long-term follow-up. We then performed loss-of-function experiments in vitro and in vivo to elucidate the underlying mechanism of CIT. We found that CIT knockdown represses cell proliferation and colony formation, which is consistent with previous findings in experimental studies in HCC [13], colon cancer cells [14] and multiple myeloma cells [15]. We also found that CIT regulates G2/M transition, suggesting that CIT knockdown cells underwent complete DNA replication but were blocked at cytokinesis. Our finding agrees with previous reports demonstrating that CIT play a role in the G2/M transition of hepatocytes in both cell [13] and animal models [20]. However, some studies show that CIT inhibits the G1/S transition in colon cancer cells and multiple myeloma cells [14,15]. The different repair machinery systems in different cancer cell lines might partially explain the difference. Tumor-volume monitoring and live in vivo bladder cancer imaging in human xenograft model confirmed that CIT knockdown reduces the growth rate of bladder cancer cells and the final tumor volume and weight. More in vivo experiments using 5637 cells as well as using an inducible knockout system are needed to confirm the conclusion. Taken together, CIT might regulate cell cycle and survival to promote the development of colon cancer, thus serving as a potential therapeutic target for bladder cancer therapy. To further understand the intracellular signalling pathway induced by CIT, we examined gene expression profile in CIT knock-down T24 cells. GO functional analysis showed that differentiated genes are enriched in cells cycle pathway, which further confirms that CIT is a cell cycle‑related regulatory factor in bladder cancer. Functional interaction network and validation assay revealed that CIT interacts with several cell cycle and apoptosis regulators with known cancer-related biological functions, such as CDK1, MCM7 and CCND1 (detected in both cell cycle and p53 pathways). Experimental studies demonstrated that inhibition of CDK, a key regulator of cell cycle in many tumorigenic events [21], could result in growth inhibition and apoptosis in bladder cancer cells in vitro and in vivo [22]. MCM proteins participate in the initiation and elongation steps of DNA replication [23]. MCM7 is found to be overexpressed in various types of cancers, including human bladder cancer tissues [24]. Overexpression of CCND1 has been found in various cancers. In a recent review, Ren et al. has reported significant association between increased CCND1 expression and prognosis of bladder cancer [25]. Therefore, microarray analysis further suggested that CIT is involved in deregulation of cell cycle and apoptosis, thus contributing to tumorigenesis in bladder cancer. More protein levels of biomarkers are needed to better understand the role of CIT in apoptosis and cell death, such as cleaved caspases, cleaved PARP and caspase inhibitor (i.e., Z-VAD). In summary, we provide clinical evidence of overexpression of CIT bladder cancer tissues. We also provide experimental evidence that CIT may promote bladder cancer through regulation of cell cycle pathway.
Fig. 3. CIT knockdown reduces tumor volume and weight. (A) Subcutaneous tumors in nude mice and isolated tumors 1-month after inoculation of T24 cells infected with NC or KD (n = 10/group). (B) Tumor growth curves of xenografts in nude mice. (C) Tumor weights of the xenografts. (D). in vivo bladder cancer imaging. In the KD mice, we observed lower fluorescence intensities than those in NC mice. The color scale bar shows the range of strongest (red) to weakest signal (blue).
4. Discussion We demonstrated that the expression level of CIT is higher in human bladder cancer tissues compared with bladder tissues from normal subject. In vitro study showed that CIT knockdown represses cell proliferation and colony formation, and induces cell cycle arrest and apoptosis in human bladder cancer cells. We also demonstrated that CIT knockdown could reduce bladder cancer cell growth in vivo. Microarray analysis further revealed that CIT may regulate cell cycle signalling pathway. In summary, we, for the first time, provided clinical and experimental evidence that CIT may promote bladder cancer possibly through regulation of cell cycle pathway. Biomarkers have been suggested to incorporate into standard practice to improve the current diagnostic practice for bladder cancer [17]. Recent studies demonstrated that the development and progression of bladder cancer involves alterations in several molecular pathways [5], and cell-cycle regulation is one of the most extensively characterized cellular process [18]. CIT, a downstream effector of Rho family GTPases involved in cell cycle regulation, have been studied in several cancers [19]. Three studies using human samples demonstrated that CIT is up-regulated in gastric cancer [12], HCC [13] and colon cancer [14] and multiple myeloma [15] compared with adjacent nontumor tissues or matched normal control tissues. In this study, compared with control tissues, CIT expression at protein level is up-regulated in human bladder cancer tissues. Our findings not only agreed well with previous studies, but also demonstrated that CIT maybe a potential biomarker for bladder cancer. However, since we did not measure CIT level in adjacent normal tissues in bladder cancer patients,
Ethics approval and consent to participate Ethical approval was given by the Ethics Committee of The First Affiliated Hospital of HarbinMedical University. All patients gave their written information consent.
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Fig. 4. Cell cycle pathway is affected by down-regulation of CIT. (A) Heatmap of 835 genes (364 up-regulated and 471 down-regulated genes) in KD cell. The heatmap color corresponds to gene expression level (red, up-regulated; green, down-regulated) (P < 0.05 and absolute fold change > 1.5). (B) Functional pathway enrichment of differential genes analyzed based on the KEGG and BIOCARTA. (C) Interaction network between CIT and the genes involved in the cell cycle pathway. (red, up-regulated; green, down-regulated; gray, no change). (D) Validation of five top differentiated genes (CCND1, MCM3, MCM7, PCNA, and CDK1) using western blot.
Consent for publication
data and performed the data analysis. All authors prepared the manuscript. YX, ZL, YS and HY amended the manuscript critically.
Not applicable. Declaration of Competing Interest Availability of data and material All the authors declare that they have no conflict of interest. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Acknowledgements None.
Funding
Appendix A. Supplementary data
This work was supported by Natural Science Foundation of Heilongjiang Province of China, grant number ZD201516, QC2014C112; China Postdoctoral Science Foundation, grant number 2016M601450; Heilongjiang Province Postdoctoral Science Foundation, grant number LBH-Z16139; 1st Affiliate Hospital of Harbin Medical University Research Foundation, grant number 2014L01.
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2020.109830. References [1] S. Antoni, J. Ferlay, I. Soerjomataram, A. Znaor, A. Jemal, F. Bray, Bladder cancer incidence and mortality: a global overview and recent trends, Eur. Urol. 71 (1) (2017) 96–108. [2] K. Li, T. Lin, C. Chinese Bladder Cancer, W. Xue, X. Mu, E. Xu, et al., Current status
Author’s contributions YX and ZL contributed to the study design; all authors collected the 7
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[15] I. Sahin, Y. Kawano, R. Sklavenitis-Pistofidis, M. Moschetta, Y. Mishima, S. Manier, et al., Citron Rho-interacting kinase silencing causes cytokinesis failure and reduces tumor growth in multiple myeloma, Blood Adv. 3 (7) (2019) 995–1002. [16] M. Tomayko, P. Reynolds, Determination of subcutaneous tumor size in athymic (nude) mice, Cancer Chemother. Pharmacol. 24 (3) (1989) 148–154. [17] F. Darwiche, D.J. Parekh, M.L. Gonzalgo, Biomarkers for non-muscle invasive bladder cancer: current tests and future promise, Indian J. Urol. 31 (4) (2015) 273–282. [18] A.P. Mitra, D.E. Hansel, R.J. Cote, Prognostic value of cell-cycle regulation biomarkers in bladder cancer, Semin. Oncol. 39 (5) (2012) 524–533. [19] N. Wettschureck, S. Offermanns, Rho/Rho-kinase mediated signaling in physiology and pathophysiology, J. Mol. Med. (Berl.) 80 (10) (2002) 629–638. [20] H. Liu, F. Di Cunto, S. Imarisio, L.M. Reid, Citron kinase is a cell cycle-dependent, nuclear protein required for G2/M transition of hepatocytes, J. Biol. Chem. 278 (4) (2003) 2541–2548. [21] U. Asghar, A.K. Witkiewicz, N.C. Turner, E.S. Knudsen, The history and future of targeting cyclin-dependent kinases in cancer therapy, Nat. Rev. Drug Discov. 14 (2) (2015) 130–146. [22] A. Wirger, F.G. Perabo, S. Burgemeister, L. Haase, D.H. Schmidt, C. Doehn, et al., Flavopiridol, an inhibitor of cyclin-dependent kinases, induces growth inhibition and apoptosis in bladder cancer cells in vitro and in vivo, Anticancer Res. 25 (6B) (2005) 4341–4347. [23] M. Lei, B.K. Tye, Initiating DNA synthesis: from recruiting to activating the MCM complex, J. Cell. Sci. 114 (Pt 8) (2001) 1447–1454. [24] G. Toyokawa, K. Masuda, Y. Daigo, H.S. Cho, M. Yoshimatsu, M. Takawa, et al., Minichromosome Maintenance Protein 7 is a potential therapeutic target in human cancer and a novel prognostic marker of non-small cell lung cancer, Mol. Cancer 10 (2011) 65. [25] B. Ren, W. Li, Y. Yang, S. Wu, The impact of cyclin D1 overexpression on the prognosis of bladder cancer: a meta-analysis, World J Surg Onco. 12 (2014) 55.
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Down-regulation of CIT can inhibit the growth of human bladder cancer cells
T
Zan Liua, Haiyan Yanb, Yang Yanga, Liangjun Weic, Shunyao Xiaa, Youcheng Xiua,d,* a
Department of Urology, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China Department of Anesthesiology, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China c Departments of Urology, Harbin Medical University Cancer Hospital, Harbin, Heilongjiang, China d Heilongjiang Academy of Medical Sciences, Heilongjiang, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Apoptosis Bladder cancer cell Citron rho-interacting Serine/threonine kinase 21 Proliferation
Objective: Our study is to examine the citron rho-interacting, serine/threonine kinase 21 (CIT) in bladder cancer. Methods: We examined CIT level in human bladder cancer tissues by immunohistochemical staining. To explore the impact of CIT on cell proliferation and apoptosis, we down-regulated its expression in two human bladder cancer cell lines, 5367 and T24. We examined cell growth in 5367 and T24. We also performed in vivo analysis using T24 cells. We further used microarray expression profiling to investigate genes differentially expressed in T24 cells with CIT down-regulated. Results: In 100 human samples, CIT was expressed by only 2 of 30 (6.7 %) controls in bladder tissues, whereas by 64 of 70 (91.4 %) cancer patients in tumor tissues (p < 0.001). in vitro analysis demonstrated that CIT knockdown represses cell proliferation by 50 % in both cells and colony formation (77 ± 5 vs. 13 ± 2, p = 0.001 for T24, 58 ± 3 vs. 1 ± 1, p < 0.001 for 5637). We also found CIT knockdown could induce cell cycle arrest, and promote apoptosis in both cells. Tumor-volume monitoring and live in vivo bladder cancer imaging in human xenograft model confirmed that CIT knockdown reduces tumor volume (668.4 ± 333.0 vs. 305.7 ± 170.4 mm3, p = 0.02) and weight (0.27 ± 0.15 vs. 0.57 ± 0.32 g, p = 0.02). Microarray analysis revealed that CIT may regulate cell cycle signalling pathway through various cell cycle regulators. Conclusions: In summary, we provided clinical and experimental evidence that CIT may promote bladder cancer through regulation of cell cycle pathway.
1. Introduction Bladder cancer is the ninth most common cancer worldwide, with an estimated 430,000 new cases diagnosed in 2012 [1]. In China, according to the 2015 National Central Cancer Registry, bladder cancer ranked sixth in male cancers and the most common urologic malignancy [2,3]. In the past few years, the incidence and mortality rates of bladder cancer have increased gradually in China [3]. Despite the development in surgical techniques, perioperative therapies and postoperative management, survival outcomes for bladder cancers patients remained unchanged over the last 30 years [4,5]. To date, clinical management of bladder cancer is largely based on determination of tumor stage and other histopathological parameters. However, studies have suggest that bladder cancer should not be treated exclusively on the basis of pathologic staging, because it does not reflect the overall clinical risk [6]. Therefore, there is an urgent
need for discovery of novel biomarkers and therapeutic targets for the diagnosis and treatment for bladder cancer. Disruption of cell cycle control may lead to genomic instability, neoplastic transformation and tumor progression [7]. Citron rho-interacting, serine/threonine kinase 21 (CIT) is present at the cleavage furrow and the midbody during cytokinesis, a key component for cellular abscission [8,9]. CIT is also found to phosphorylate the regulatory light chain of myosin II, which is the primary motor protein responsible for cytokinesis [10,11]. Although still limited, accumulating evidence is beginning to show the role of CIT in cancer through regulation of cell cycle. Overexpression of CIT has been observed in tumor tissues collected from patients with gastric cancer [12], hepatocellular carcinoma (HCC) [13], colon cancer [14] and multiple myeloma [15]. Experimental studies further demonstrated that CIT knockdown could inhibit cell grown, as well as induce cell cycle arrest and apoptosis in human HCC and colon cancer cell lines [12,13]. Taken together these evidence
⁎ Corresponding author at: Department of Urology, The First Affiliated Hospital of Harbin Medical University, No. 23 Youzheng Street, Harbin, Heilongjiang, 150001, China. E-mail address: [email protected] (Y. Xiu).
https://doi.org/10.1016/j.biopha.2020.109830 Received 15 July 2019; Received in revised form 24 November 2019; Accepted 18 December 2019 0753-3322/ © 2020 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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to a CIT score ≥6.
from human and experimental studies, CIT maybe a novel tumor promoter that can be used as a potential therapeutic target for anti-cancer therapy. However, whether CIT could be a target in bladder cancer is poorly understood. In this study, we demonstrated that the expression level of CIT is higher in human bladder cancer tissues than bladder tissues from controls, and the expression level is not correlated to clinical pathology of bladder cancer. Then, we used lentiviruses expressing CIT-targeting small hairpin RNA (shRNA) approach to knockdown CIT expression in two human bladder cancer cell lines. We found that CIT knockdown represses cell proliferation and colony formation, and induces cell cycle arrest and apoptosis. Tumor-volume monitoring and live in vivo bladder cancer imaging in human xenograft model confirmed that CIT knockdown reduces tumor volume and weight. Microarray analysis revealed that CIT may regulate cell cycle signalling pathway through various cell cycle regulators. In summary, we, for the first time, provided evidence that CIT may promote bladder cancer through regulation of cell cycle pathway.
2.3. Cell lines and Lentiviral infection Human bladder cancer cell lines 5637 and T24 were obtained from Cell Resource Center, Shanghai Institutes for Biological Sciences at the Chinese Academy of Sciences. The cells were cultured in RPMI 1640 medium containing 10 % FBS, streptomycin, and penicillin at 37 °C in an incubator with 5 % CO2. To inhibit CIT expression, small hairpin RNA (shRNA) specifically targeting CIT (target sequence: 5′-GCGTCCTCATACCAGGATAAA-3′) was designed and packed into a lentivirus vector. A scrambled shRNA was used as a negative control using the following sequence: 5′-TTCT CCGAACGTGTCACGT-3′. shRNA of human CIT lentivirus gene transfer vector encoding green Xuorescent protein (GFP) sequence was constructed by Genechem Co., Ltd (Shanghai, China) as described previously [14]. The recombinant lentivirus expressing short hairpin RNA (shRNA) to induce silencing of CIT (sh-CIT) and the control lentivirus (sh-Control) were prepared and titered to 8 × 108 TU/mL (transducing unit). T24 and 5637 cells were plated onto 6-well plates and subsequently infected with sh-CIT or sh-Control as described previously [14].
2. Materials and methods 2.1. Human bladder tissue collection
2.4. Quantitative real-time PCR and Western blotting 100 patients who underwent surgery at Department of Urology, Heilongjiang Nongken General Hospital, China from September 2013 to September 2014 were recruited in our study. Tumor tissue specimens were obtained from 70 bladder cancer patients by transurethral resection. Normal bladder tissues (control) were obtained from 30 patients with renal pelvic carcinoma by laparoscopic nephroureterectomy and confirmed by pathology analysis. All tissues were cut into small pieced (0.8–1.2 cm in diameter), snap-frozen in liquid nitrogen, and then transferred to a low-temperature refrigerator (−80 °C). The study was approved by the Ethics Committee of The First Affiliated Hospital of Harbin Medical University, China. Individual written informed consent was obtained prior to enrolment in the study.
T24 cells were used to examine the knockdown efficiency at mRNA and protein levels. Quantitative real-time PCR was preformed to examine the mRNA level of CIT as described previously [14]. The relative expression level was calculated using the 2−ΔΔCT method with GAPDH as the reference gene for normalization. Western blot was used to measure protein level of CIT using primary anti-CIT antibody (1:5000) (ab86782, Abcam, UK). GAPDH was used as control. Protein bands were detected using an ECL-Plus kit (Amersham Biosciences, USA) as described previously [14]. 2.5. In vitro assay of T24 and 5637 cells infected with sh-CIT and shControl
2.2. Immunohistochemistry T24 and 5637 cells were infected with sh-CIT and sh-Control lentivirus and incubated for five days for cell cycle, apoptosis and colony formation assay, or three days for BrDU assay [14]. For cell cycle analysis, cells were trypsinized and fixed in 70 % ethanol at −20 °C for 10 min. The cells were washed with PBS, resuspended in RNase (0.2 mg/mL) and incubated at 37 °C for 10 min. Propidium iodide (PI) was added to obtain a final PI concentration of 300–800 cells/s when analysing on a Guava easyCyte HT flow cytometer (Millipore, USA). For apoptosis assay, cells were collected, washed with PBS and resuspended using staining buffer at a density of 1 × 105 - 1 × 106/mL. Subsequently, 5 μL Annexin V-APC was added to 100 μL of cell suspension and incubated at room temperature for 10−15 min. The signals were detected using a FACS Calibur (Millipore, USA). Cells infected with sh-CIT and sh-Control were seeded in 96-well plates at a density of 2000 cells/well and incubated at 37 °C for 3 days. After the 3-day infection, cells were divided to two groups. One group was used for BrdU incorporation (day 1), and one group was incubated with RPMI 1640 medium for 3 days and then used for BrdU incorporation assay (day 4). Then cells were washed two times with PBS and BrdU reagents (1:100) were subsequently added to each well (10 μL/well). Cell proliferation was analyzed based on the measurement of BrdU incorporation using a BrdU Cell Proliferation ELISA kit (Roche Applied Science, USA) according to the manufacturer’s instructions. We measured BrdU density on days 1 and 4. The BrdU density was 450 nm using Biotek Elx800, and the experiments were performed in triplicate. For colony formation assay, cells were harvested in the logarithmic phase, and plated in triplicate onto 6-well plates at a density of 800 cells/well. After incubation for eight days, cells were washed with PBS
Immunohistochemistry were performed using the SABC kit (suolaibao, China) according to the manufacture’s instruction. The sections were incubated with primary antibody against CIT (1:100) (ab110897, Abcam, UK) at 37 °C for 1 h. Slides containing tissue sections with high expression of CIT were incubated with anti-CIT antibody as positive control, and incubated with PBS as negative control. Then, all sections were incubated with the biotinylated secondary antibody for 20 min at room temperature, followed by a 20 min incubation with streptavidin peroxidase. After rinsing, the results were visualized using Diaminobenzidine and counterstained with hematoxylin. All fields were taken under a light microscope (Olympus Corp., Japan) at 200× magnification. The optical density was quantified by Image-Pro Plus 6.0 in three random high power fields. All immunohistochemistry staining samples were independently estimated by two pathologists in a masked fashion to minimize any possible bias according to the 1997 Union for International Cancer Control (UICC) TNM classification criteria. We assessed the site of staining (cytoplasmic staining was considered a positive expression of CIT), the staining intensity, and extent of CIT expression as follows: Staining intensity was scored based on a range of 0–3, where 0 represents no staining, 1 represents faint yellow, 3 represents brown, and 4 represents dark brown (the strongest staining). The extent was categorized by the percentage of stained cells (staining intensity≥1) as follows: 0–5%=score 0, 6–25%= score 1, 26–50%= score 2, 51–75%= score 3 and 76–100%= score 4. CIT scoring was assigned by multiplying the staining intensity score and the staining extent score. Five fields were randomly selected, and the average score of the five fields was the final IHC staining score. Negative staining refers to a CIT score < 6, and positive staining refers 2
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and subsequently fixed with paraformaldehyde for 30−60 min. The cell colonies were stained with 500 μL of Giemsa for 20 min at room temperature. Images of the cell plates were captured, and the colonies were analyzed.
Table 1 CIT expression and clinicopathological characteristics of bladder cancers (N = 70). Parameter
2.6. In vivo xenograft assay of T24 cells infected with sh-CIT and sh-Control
Age
BALB/c female nude mice at four-week-old were purchased from Lingchang Biological Technology Co., Ltd. (Shanghai, China) and kept in specific pathogen-free conditions. Mice were randomly divided into two groups (n = 10/group), and 1 × 107 T24 cells transfected with shCIT and sh-Control were subcutaneously injected into the right armpits of mice. After initial detection, tumor volumes were evaluated twice a week for 15 days. Tumor sizes were measured with a digital caliper and tumor volume in mm3 was calculated by the formula: volume = (width) 2x length x 3.14/6 [16]. At time of sacrifice, live mouse imaging were conducted using Lumina LT (filters: excitation 672 nm, emission 694 nm; Perkin Elmer, USA) under anaesthesia. On the fluorescence images, freehand regions of interest (ROIs) were drawn carefully along the margins. The mean fluorescence intensities [total radiance efficiency, (p/s)/(μW/cm2)] were generated automatically by Living Image® 4.4 software and recorded. After 1 month of cell inoculation, mice were euthanized by CO2 asphyxiation and only tumors were harvested and weighted. Throughout the experiment, all mice had access to sterilized food and filtered water ad libitum. The vivarium was maintained at 23 °C on a 12 h light/12 h dark cycle. All animal experiments in our study were approved by the Animal Care and Use Committee of the The First Affiliated Hospital of Harbin Medical University hospital, China.
Gender Metastasis Infiltrative Histological grade
T staging
Category
Total (%)
CIT expression Positive (64)
Negative (6)
P-value* 1.000
> 64 yrs ≤64 yrs Male Female Yes No Yes No G1
47 (67.1) 23 (32.9) 49 (70.0) 21 (30.0) 7 (10.0) 63 (90.0) 40 (57.1) 30 (43.9) 17 (24.3)
43 (91.4) 21 (91.3) 46 (93.9) 18 (85.7) 6 (85.7) 58 (92.1) 39 (97.5) 25 (83.3) 13 (76.5)
4 2 3 3 1 5 1 5 4
(8.6) (8.7) (6.1) (14.3) (14.3) (7.9) (2.5) (16.7) (23.5)
G2 G3 Ta-T1 T2 T3-T4
24 29 16 20 34
23 28 13 18 33
1 1 3 2 1
(4.2) (3.4) (18.8) (10.0) (2.9)
(34.3) (41.3) (22.9) (28.6) (45.6)
(95.8) (96.6) (81.3) (90.0) (97.1)
0.355 0.48 0.08 0.90
0.14
* Fisher exact test.
2.8. Statistical analysis A Fisher exact test was used to compare the distributions of categorical variables in study using human samples (Table 1). In experimental studies using human samples, data were expressed as means and standard deviations for continuous variables. T-test was used to compare continuous variables between groups (sh-CIT vs. sh-Control). All statistical analyses were performed using STATA version 14.0 (STATA Corporation, Texas, USA). A two-tailed p value of less than 0.05 was considered as statistically significant.
2.7. Microarray analysis in T24 cells infected with sh-CIT and sh-Control
3. Results
The in vitro and in vivo results demonstrated that CIT was involved in bladder cancer, possibly through regulation of cell growth and survival. We further performed microarray analysis in T24 cells infected with shCIT and sh-Control to better understand the underlying mechanisms of CIT in bladder cancer. Total RNA from T24 cells infected with sh-CIT (n = 3) or sh-Control lentivirus (n = 3) was extracted using Trizol reagent. The quantity of RNA was evaluated using NanoDrop. The integrity was measured by Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., USA). Only samples with high purity (1.7 < A260/ A280 < 2.2) and high integrity (RNA integrity number (RIN) > 8.7), and 28S/18S > 0.7) were subsequently used in microarray experiments. Microarrays were processed to generate gene expression profiles using the Affymetrix human GeneChip PrimeView assay (Affymetrix; Thermo Fisher Scientific, Inc. USA) according to the manufacturer's instructions. Genes that were differentially expressed between shControl and sh-CIT-infected T24 cells were identified based on the following criteria: P < 0.05 and an absolute fold-change > 1.5. Principal component analysis (PCA) was done on the processed data to identify possible outlier samples. We performed functional enrichment analysis of the differentially expressed genes by using Kyoto Encyclopedia of Genes and Genomes (KEGG) and BioCarta pathway. Five genes, including cyclin D1 (CCND1), cyclin dependent kinase 1 (CDK1), minichromosome maintenance complex component 3 (MCM3), MCM7, and proliferating cell nuclear antigen (PCNA), were selected for validation using western blot as described above. The primary antibodies used included anti-CCND1 (1:150, ab16663, Abcam, UK), antiCDK1 (1:3000, ab32384, Abcam, UK), anti-MCM3 (1:1000, #4003 s, Cell Signaling Technology, USA), anti-MCM7 (1:1000, #4018, Cell Signaling Technology, USA), and anti-PCNA (1:2000, #2586s, Cell Signaling Technology, USA).
3.1. CIT level in bladder cancers and normal bladder tissues in human using immunohistochemistry The expression of CIT in bladder cancer tissues from cancer patients and normal bladder tissues from cancer-free patients was assessed by immunohistochemistry. Fig. 1 shows that CIT was predominantly localized to the cytoplasm. Overall, the intensity and extent of CIT level increased across control (normal bladder tissues, IHC score = 1.4 ± 1.6), grade 1 (least aggressive, IHC score = 7.6 ± 3.1), grade 2 (moderately aggressive, IHC score = 8.3 ± 1.8) and grade 3 tumor (most aggressive and most likely to spread, IHC score = 9.0 ± 1.8). The positive expression of CIT was detected in only 2 of 30 (6.7 %) controls in bladder tissues, whereas in 64 of 70 (91.4 %) cancer patients in tumor tissues (IHC score = 8.4 ± 2.2) (p < 0.001), indicating that CIT is unregulated in bladder cancers. We then analyse the relationship between CIT expression with basic and clinicopathological characteristics among the 70 bladder cancer patients. Table 1 shows that the percentage of patients with CIT-positive cancer is not different by age (> 64 vs. ≤64), gender, and clinicopathological of bladder cancers. These results suggest CIT is up-regulated in bladder cancer tissues independent of metastatic status or cancer T staging. 3.2. CIT promotes bladder cancer cell growth and inhibits apoptosis in vitro We evaluated CIT expression using real-time PCR in 5637 and T24 cells, finding expression of CIT in both cell lines (Fig. 2A). Therefore, we knocked down CIT with lentivirus-medicated sh-CIT and sh-Control in both cells to investigate the role of CIT in cell proliferation and apoptosis. T24 cells infected with lentivirus expressing CIT-shRNA and control-shRNA was used to examine the knockdown efficiency of CIT. Fig. 2B and C show significantly reduced CIT expression at mRNA and 3
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Fig. 1. Immunohistochemistry against CIT in (A) bladder tissues from controls and (B–D) bladder tumor samples from bladder cancer patients. Original magnification: 200×.
3.4. CIT inhibits cell cycle pathways
protein level in T24 and 5637 cells, suggesting an effective inhibition of CIT using the lentiviral-based shRNA strategy. Fig. 2D–I compared cell proliferation, colony formation, cell cycle, and apoptosis in T24 and 5637 cells infected with sh-CIT and sh-Control. BrDU incorporation assay shows that sh-CIT significantly inhibited cell proliferation by approximately 50 % in T24 (Fig. 2D) and 5637 cells (Fig. 2E). We performed a colony formation assay to determine the colonogenic ability. The results show that sh-CIT significantly reduced colony formation in T24 (77 ± 5 vs. 13 ± 2, p = 0.001) (Fig. 2F) and 5637 cells (58 ± 3 vs. 1 ± 1, p < 0.001) (Fig. 2G). These results demonstrated that CIT is involved in bladder cancer cell proliferation and colony formation. The PI staining revealed that sh-CIT results in significant decrease of cells in the S phase and decrease of cells in the G2/M phase in T24 (Fig. 2H) and 5637 cells (Fig. 2I). An annexin VAPC apoptosis assay showed that sh-CIT significantly promoted cell apoptosis in both cells (p < 0.05, Fig. 2J and K). Taken together, these results demonstrated that suppression of CIT in human bladder cancer cells could inhibit cell proliferation and colony formation, induce cell cycle arrest, and promote apoptosis.
To understand the underlying mechanisms of CIT in bladder cancer, we used microarray to investigate gene expression profiles in T24 cell infected with sh-CIT (KD) and sh-Control (NC). The distribution and similarities of six samples (three KD and three NC cells) were shown in Supplementary Fig. 1. Fig. S1A shows an overview of the sample relations based on PCA. Pearson's correlation of the signal value (Pearson's correlation coefficient > 0.95; Fig. S1B) indicated a good overall correlation of individual gene expression measurements across chips. Signal value distribution (Fig. S1C) and relative signal box plot graphs (Fig. S1D) demonstrated the expression values of all microarray probe distribution statistics and all samples were reproducible. Scatterplot graphs (Fig. S1E) demonstrated differences in gene expression of all microarray probe distribution statistics between KD and NC. Heatmap shows significant differential expression patterns between KD and NC cells, including 364 up-regulated genes and 471 downregulated genes by CIT knockout (Fig. 4A). KEGG and BioCarta pathway analysis demonstrated that the differentially expressed genes were enriched in ten pathways (Fig. 4B). Functional analysis by GO terms showed that some of these genes are implicated in cancer development or cancer-related biological pathways, such as cell cycle and p53 pathway (the major apoptosis signalling pathway). Therefore, we analysed the functional interaction network between CIT and cell cycle pathway (the one with significantly enriched pathway in cells with CIT knockdown), finding 17 cell cycle regulators in cell cycle pathway were down-regulated significantly by CIT knockdown with a fold-change > 1.5 (p < 0.05) (Fig. 4C). Five genes were selected for validation based on statistical significance and cancerrelated biological functions, including cyclin D1 (CCND1), cyclin dependent kinase 1 (CDK1), minichromosome maintenance complex component 3 (MCM3), MCM7, and proliferating cell nuclear antigen (PCNA). Western blots demonstrated that the protein level of CCND1, MCM3, and MCM7 were downregulated by 62 %, 88 %, 76 %, respectively. The expression of CKD1 was decreased to undetectable level. We did not observe significant down-regulation of PCNA (Fig. 4D).
3.3. CIT promotes bladder cancer cell growth in vivo We evaluated xenograft formation of T24 cells infected with sh-CIT (KD) and sh-Control (NC). After inoculation of cells for 21 days, tumors in KD group were significantly smaller than those in NC group, and the difference remained at sacrifice (668.4 ± 333.0 vs. 305.7 ± 170.4 mm2, p = 0.02) (Fig. 3B). Tumor weight is also significantly lower in KD group than those in NC group (0.27 ± 0.15 vs. 0.57 ± 0.32 g, p = 0.02) (Fig. 3C). Fluorescence intensities of the tumors in KD and NC groups were shown in Fig. 3D. In the KD mice, we could observe lower fluorescence intensities than those in NC mice. The average of total radiant efficiency is significantly lower in KD group than those in NC group (2.0e+10 ± 5.1e+09 vs.4.0e+10 ± 1.6e+9 [p/s]/[μW/cm2]) (p = 0.003), suggesting the lower intensities of tumor with knockdown of CIT.
4
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Fig. 2. Cell-based assay in T24 and 5637 cells infected with sh-CIT and sh-Control. (A) mRNA level of CIT in wild T24 and 5637 cells. (B) mRNA and (C) protein level of CIT was reduced significantly by using real-time PCR and Western Blotting following CIT knockdown using lentiviral-based shRNA in T24 cells and 5637 cells. ShCIT significantly inhibited cell proliferation using BrDU incorporation assay and colony formation in T24 (D, F) and 5637 cells (E,G). CIT-CIT significantly inhibited colony formation in T24 (F) and 5637 cells (G). sh-CIT increased the percentage of cells with S and G2/M phases, and decrease in the percentage of cells with G1 phase in T24 (H) and 5637 cells (J). sh-CIT significantly promoted cell apoptosis in both cells in T24 (J) and 5637 cells (K). **P < 0.05 vs. sh-Ctrl. 5
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it remains unclear whether there is increased CIT level in adjacent normal tissues, which may increase tumor susceptibility. Another limitation is that, we didn’t compare clinical outcome (i.e., overall survival) between CIT-positive and CIT-negative cancer patients as in a previous studies of colon cancer and multiple myeloma [14,15]. Such analysis using survival data by follow-up records is needed in the future to understand the prognostic significance of CIT in bladder cancer. The other limitations include small sample size and a single-center design; therefore, our results require further verification in a larger sample and multi-centric design with long-term follow-up. We then performed loss-of-function experiments in vitro and in vivo to elucidate the underlying mechanism of CIT. We found that CIT knockdown represses cell proliferation and colony formation, which is consistent with previous findings in experimental studies in HCC [13], colon cancer cells [14] and multiple myeloma cells [15]. We also found that CIT regulates G2/M transition, suggesting that CIT knockdown cells underwent complete DNA replication but were blocked at cytokinesis. Our finding agrees with previous reports demonstrating that CIT play a role in the G2/M transition of hepatocytes in both cell [13] and animal models [20]. However, some studies show that CIT inhibits the G1/S transition in colon cancer cells and multiple myeloma cells [14,15]. The different repair machinery systems in different cancer cell lines might partially explain the difference. Tumor-volume monitoring and live in vivo bladder cancer imaging in human xenograft model confirmed that CIT knockdown reduces the growth rate of bladder cancer cells and the final tumor volume and weight. More in vivo experiments using 5637 cells as well as using an inducible knockout system are needed to confirm the conclusion. Taken together, CIT might regulate cell cycle and survival to promote the development of colon cancer, thus serving as a potential therapeutic target for bladder cancer therapy. To further understand the intracellular signalling pathway induced by CIT, we examined gene expression profile in CIT knock-down T24 cells. GO functional analysis showed that differentiated genes are enriched in cells cycle pathway, which further confirms that CIT is a cell cycle‑related regulatory factor in bladder cancer. Functional interaction network and validation assay revealed that CIT interacts with several cell cycle and apoptosis regulators with known cancer-related biological functions, such as CDK1, MCM7 and CCND1 (detected in both cell cycle and p53 pathways). Experimental studies demonstrated that inhibition of CDK, a key regulator of cell cycle in many tumorigenic events [21], could result in growth inhibition and apoptosis in bladder cancer cells in vitro and in vivo [22]. MCM proteins participate in the initiation and elongation steps of DNA replication [23]. MCM7 is found to be overexpressed in various types of cancers, including human bladder cancer tissues [24]. Overexpression of CCND1 has been found in various cancers. In a recent review, Ren et al. has reported significant association between increased CCND1 expression and prognosis of bladder cancer [25]. Therefore, microarray analysis further suggested that CIT is involved in deregulation of cell cycle and apoptosis, thus contributing to tumorigenesis in bladder cancer. More protein levels of biomarkers are needed to better understand the role of CIT in apoptosis and cell death, such as cleaved caspases, cleaved PARP and caspase inhibitor (i.e., Z-VAD). In summary, we provide clinical evidence of overexpression of CIT bladder cancer tissues. We also provide experimental evidence that CIT may promote bladder cancer through regulation of cell cycle pathway.
Fig. 3. CIT knockdown reduces tumor volume and weight. (A) Subcutaneous tumors in nude mice and isolated tumors 1-month after inoculation of T24 cells infected with NC or KD (n = 10/group). (B) Tumor growth curves of xenografts in nude mice. (C) Tumor weights of the xenografts. (D). in vivo bladder cancer imaging. In the KD mice, we observed lower fluorescence intensities than those in NC mice. The color scale bar shows the range of strongest (red) to weakest signal (blue).
4. Discussion We demonstrated that the expression level of CIT is higher in human bladder cancer tissues compared with bladder tissues from normal subject. In vitro study showed that CIT knockdown represses cell proliferation and colony formation, and induces cell cycle arrest and apoptosis in human bladder cancer cells. We also demonstrated that CIT knockdown could reduce bladder cancer cell growth in vivo. Microarray analysis further revealed that CIT may regulate cell cycle signalling pathway. In summary, we, for the first time, provided clinical and experimental evidence that CIT may promote bladder cancer possibly through regulation of cell cycle pathway. Biomarkers have been suggested to incorporate into standard practice to improve the current diagnostic practice for bladder cancer [17]. Recent studies demonstrated that the development and progression of bladder cancer involves alterations in several molecular pathways [5], and cell-cycle regulation is one of the most extensively characterized cellular process [18]. CIT, a downstream effector of Rho family GTPases involved in cell cycle regulation, have been studied in several cancers [19]. Three studies using human samples demonstrated that CIT is up-regulated in gastric cancer [12], HCC [13] and colon cancer [14] and multiple myeloma [15] compared with adjacent nontumor tissues or matched normal control tissues. In this study, compared with control tissues, CIT expression at protein level is up-regulated in human bladder cancer tissues. Our findings not only agreed well with previous studies, but also demonstrated that CIT maybe a potential biomarker for bladder cancer. However, since we did not measure CIT level in adjacent normal tissues in bladder cancer patients,
Ethics approval and consent to participate Ethical approval was given by the Ethics Committee of The First Affiliated Hospital of HarbinMedical University. All patients gave their written information consent.
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Fig. 4. Cell cycle pathway is affected by down-regulation of CIT. (A) Heatmap of 835 genes (364 up-regulated and 471 down-regulated genes) in KD cell. The heatmap color corresponds to gene expression level (red, up-regulated; green, down-regulated) (P < 0.05 and absolute fold change > 1.5). (B) Functional pathway enrichment of differential genes analyzed based on the KEGG and BIOCARTA. (C) Interaction network between CIT and the genes involved in the cell cycle pathway. (red, up-regulated; green, down-regulated; gray, no change). (D) Validation of five top differentiated genes (CCND1, MCM3, MCM7, PCNA, and CDK1) using western blot.
Consent for publication
data and performed the data analysis. All authors prepared the manuscript. YX, ZL, YS and HY amended the manuscript critically.
Not applicable. Declaration of Competing Interest Availability of data and material All the authors declare that they have no conflict of interest. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Acknowledgements None.
Funding
Appendix A. Supplementary data
This work was supported by Natural Science Foundation of Heilongjiang Province of China, grant number ZD201516, QC2014C112; China Postdoctoral Science Foundation, grant number 2016M601450; Heilongjiang Province Postdoctoral Science Foundation, grant number LBH-Z16139; 1st Affiliate Hospital of Harbin Medical University Research Foundation, grant number 2014L01.
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