Research Article KIF20A Predicts Poor Survival of Patients and Promotes Colorectal Cancer Tumor Progression Through the JAK/STAT3 Signaling Pathway

Hindawi Disease Markers Volume 2020, Article ID 2032679, 11 pages https://doi.org/10.1155/2020/2032679

Research Article KIF20A Predicts Poor Survival of Patients and Promotes Colorectal Cancer Tumor Progression through the JAK/STAT3 Signaling Pathway

Qi Zhang,1 Jun Di,1 Zhiyu Ji,2 Aoning Mi,2 Quanying Li,2 Xianghui Du,1 Anpeng Wang,1 Anlian Wang,1 and Changjiang Qin 2,3

1Department of General Surgery, People’s Hospital of Kaifeng, Kaifeng, China 2Department of General Surgery, Huaihe Hospital of Henan University, Kaifeng, China 3Department of Gastroenterology, The First Aﬃliated Hospital of Sun Yat-sen University, Guangzhou, China

Correspondence should be addressed to Changjiang Qin; [email protected]

Qi Zhang, Jun Di, and Zhiyu Ji contributed equally to this work.

Received 4 December 2019; Revised 14 February 2020; Accepted 13 June 2020; Published 9 July 2020

Academic Editor: Ioannis Kosmas

Copyright © 2020 Qi Zhang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Kinesin family member 20A (KIF20A) has been recently reported to be upregulated and associated with increased invasiveness and metastasis in several malignancies. However, the role of KIF20A in colorectal cancer (CRC) is still unclear. This study is aimed at investigating the potential roles of KIF20A in the development of CRC. The results of bioinformatics analysis, immunohistochemical staining, and Western blot analysis showed that KIF20A was overexpressed in CRC tissues compared with adjacent normal tissues. High expression of KIF20A in CRC tissues was associated with depth of invasion, lymphatic node metastasis, distant metastasis, and TNM stage. Moreover, the Kaplan-Meier survival analysis showed that CRC patients with high KIF20A expression had poor prognoses. Cox regression analysis revealed that KIF20A was an independent prognostic factor in patients with CRC. Further studies suggested that knockdown of KIF20A was able to reduce cell proliferation and migration by inhibiting the JAK/STAT3 pathway. Taken together, we propose that KIF20A plays a critical role in the tumorigenesis and tumor progression of colorectal cancer and could represent a potential therapeutic target for CRC.

1. Introduction studying the molecular mechanisms underlying the invasion and metastasis of CRC is of great significance for the preven- Colorectal cancer (CRC) is one of the most common diges- tion and treatment of CRC and for improving the long-term tive system malignancies in the world and is associated with survival rate of patients. poor overall survival due to its high incidence and mortality Kinesins are a superfamily of motor proteins that have rates [1]. In 2016, approximately 134,490 people in the ATP enzyme activity. They are involved in the normal bio- United States were found to have CRC [2]. In the latest statis- logical activities of various cells, including mitosis, meiosis, tics from 2018, CRC ranked third in new cases of malignant and intracellular vesicle transport [6, 7]. Kinesin family tumors in both males and females but second in terms of member 20A (KIF20A, also known as MKLP2) is located mortality [3, 4]. Despite rapid advances in CRC screening, on chromosome 5q31.2 and plays an important role in the the detection rate of early CRC patients has not improved occurrence and development of tumors. Given that KIF20A satisfactorily. Approximately 25% of CRC patients have can directly interact with Rab6 small GTPase-binding pro- metastases at the time of diagnosis. Furthermore, approxi- tein and participate in the dynamics of the Golgi apparatus, mately 50% of CRC patients will develop metastasis, thereby it was first named Rab6-binding kinesin (RAB6KIFL) [8, 9]. contributing to the high mortality of CRC [5]. Therefore, Recently, several studies have demonstrated that KIF20A 2 Disease Markers may play an important role in the development and progres- Table 1: Correlation between KIF20A expression and sion of many different types of cancer, including melanoma clinicopathological variables in 105 CRC patients. [10], breast cancer [11, 12], nasopharyngeal cancer [13], pancreatic cancer [14–16], hepatocellular carcinoma [17], and KIF20A expression c2 P Characteristics Total Low High lung cancer [18, 19]. Therefore, KIF20A is regarded as a value value (N =38) (N =67) novel tumor-associated gene. However, the expression and role of KIF20A in CRC have not yet been examined. The Gender 3.504 0.061 aim of this study was to investigate the roles and underlying Male 65 28 37 mechanism of KIF20A in the progression of CRC. Female 40 10 30 Age 0.070 0.791 2. Materials and Methods <60 years 46 16 30 ≥60 years 59 22 37 2.1. Patients and Tissue Specimens. A total of 105 paraffin Tumor size 0.726 0.394 samples (including CRC tissues and their matched adjacent < tissues) were obtained from CRC patients who underwent 5cm 55 22 33 surgery at the Department of General Surgery, the First Affil- ≥5cm 50 16 34 Differentiation iated Hospital of Sun Yat-sen University (FAHSYSU), in 1.156 0.561 2011. All specimens were pathologically confirmed at the degree Cancer Center of the First Affiliated Hospital of Sun Yat- Well 2 0 2 sen University. The clinical characteristic data of all patients Moderate 84 31 53 are shown in Table 1. None of these patients received preop- Poor 19 7 12 erative chemotherapy or radiotherapy. Histological tumor Tumor location 1.485 0.829 grade and stage were determined using the eighth edition of Ascending the American Joint Committee on Cancer (AJCC) staging 19 8 11 colon system [20]. All patients were followed up. The time of Transverse follow-up was up to December 2017. Fresh tissue specimens 62 4 colon (including CRC tissues and their matched adjacent tissues) Descending were obtained from CRC patients who underwent surgery 20 2 at the Department of General Surgery, Huaihe Hospital of colon Henan University. This study was approved by the Ethics Sigmoid colon 24 9 15 Committees, and written informed consent was obtained Rectum 54 19 35 from each patient. After collection, all tissues were immedi- T stage 12.795 0.005 ° ately snap-frozen in liquid nitrogen and stored at -80 C until 1110 they were used for investigation. 216115 3832657 2.2. Bioinformatics Analysis of TCGA Databases. We applied FireBrowse (http://firebrowse.org/) to retrieve KIF20A 4505 expression in cancer and normal tissues in different cohorts N stage 11.402 0.003 in The Cancer Genome Atlas (TCGA). All available CRC 0592930 mRNA expression data from TCGA-CRC were downloaded 131823 from UCSC Xena (https://xenabrowser.net/). Gene expres- 215114 sion data, including a total of 380 tumor tissue samples com- M stage 5.902 0.015 pared to 50 nontumor samples, were used in this study. Excel 0913754 2016 and GraphPad Prism 6.0 software were used for log2 transformation and data analysis. 114113 TNM stage 18.462 <0.001 2.3. Immunohistochemical (IHC) Staining. Immunohisto- I15114 chemical (IHC) staining was performed as previously II 40 18 22 described, and the method description partly repeats their III 36 8 28 fi ffi wording [21]. Formalin- xed, para n-embedded (FFPE) IV 14 1 13 CRC tissues and their matched adjacent tissues from 105 CRC patients were serially sectioned into 4 μm thick slices. In detail, the sections were dewaxed in xylene and rehydrated nonspecific binding sites. Then, the slides were incubated in a graded alcohol series. The cells were placed in 3% hydro- with a rabbit polyclonal anti-KIF20A antibody (1 : 100, ° gen peroxide for 10 minutes to inhibit endogenous peroxi- Abcam, USA, ab104118) at 4 C overnight and washed three dase activity and then washed with PBS. Next, antigen times with phosphate-buffered saline (PBS) for 5 minutes. retrieval was performed in citrate buffer (pH 6.0) using a After this, the sections were incubated with biotinylated sec- microwave. The samples were immersed in 5% fetal bovine ondary antibodies at room temperature for 1 h and washed serum (FBS) at room temperature for 20 minutes to block three times with PBS for 5 minutes. Finally, the slides were Disease Markers 3 developed with DAB and counterstained with hematoxylin. 2.7. Western Blotting. Western blotting was performed as Protein staining was evaluated via microscopic examination. described previously [23]. Total proteins were extracted with All sections were reviewed and scored independently by radioimmunoprecipitation assay (RIPA) buffer supple- two pathologists of the Cancer Center. To determine IHC mented with 1% phenylmethanesulfonyl fluoride (PMSF). expression of KIF20A in tissues, we applied a scoring system The protein concentrations were quantified using a BCA based on the staining intensity and the percentage of posi- Protein Assay Kit (Solarbio, Beijing, China). Equal concen- tively stained tumor cells. The staining intensity (SI) was trations were separated by 10% SDS-PAGE and electrotrans- graded according to the following criteria: 0, no staining; 1, ferred to PVDF membranes (Millipore, Billerica, MA, USA). weak staining; 2, moderated staining; and 3, strong staining. The membranes were blocked with 5% skim milk in TBS-T The percentage of positive tumor cells among the total for 1 h at room temperature. Then, the blots were incubated observed cells was scored in five randomly selected fields as with primary antibodies against human KIF20A (1 : 500, Pro- follows: 0, no positive tumor cells; 1, 25% positive tumor cells teintech, Wuhan, China) and β-actin (1 : 3000, Sigma- ° or less; 2, 25% to 50% positive tumor cells; 3, 50% to 75% pos- Aldrich, USA) overnight at 4 C and then with rabbit or itive tumor cells; and 4, more than 75% positive tumor cells. mouse secondary antibodies for 1 hour at room temperature In the statistical analysis, the final staining index (SI) was after being washed with TBS-T. After washing, the ECL evaluated by multiplying the percentage of positive tumor Western Blot Kit (CWBIO, Beijing, China) was used to detect cells and staining scores as described by Wang et al. [22]. In the specific bands. view of this method, sections with an SI ≥ 4 were counted as high KIF20A expression, and those with an SI < 4 were 2.8. CCK-8 Array. To further identify the role of KIF20A counted as low KIF20A expression. expression levels on cell proliferation, we used Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Rockville, MD, USA) to measure cell proliferation according to the 2.4. Cell Lines. Human CRC cell lines (HCT116, SW480, manufacturer’s instructions. For cell proliferation, after SW1116, LOVO, and LS174T) used in the current study were transfection for 48 h, cells were harvested and seeded into obtained from the First Affiliated Hospital of Sun Yat-sen 96-well plates at a density of 2000 cells per well. At 0 h, University (FAHSYSU). These cell lines were cultured in 24 h, 48 h, 72 h, and 96 h, 10 μl CCK-8 kit reagent and 90 μl Dulbecco’s modified Eagle’s medium (DMEM, Biological DMEM without 10% FBS were added to each well, and the Industries, USA) supplemented with 10% fetal bovine serum cells were cultured for 4 hours in a humidified atmosphere (FBS, Biological Industries, USA) and 1% antibiotics ° of 5% CO2 at 37 C. The absorbance of each well at a wave- (100 U/ml penicillin and 100 μg/ml streptomycin). All cells length of 450 nm (450 nm OD value) was measured using a were cultured in a humidified atmosphere of 5% CO at ° 2 microplate reader. All data were analyzed with GraphPad 37 C for future use. Prism 6.0 software. 2.9. Transwell Invasion Assay. Transwell chambers (8 μm 2.5. KIF20A Small Interfering RNA Transfection. To further pores, Corning, USA) were used in 24-well plates to detect explore the role of KIF20A expression in tumor proliferation cell invasion. After 48 hours of transfection, first, the cham- and migration, cells were transfected with siRNA purchased bers were placed in 24-well plates containing 500 μl DMEM from Ruibo (Guangzhou, China). The transfections of siRNA without 10% FBS in the lower chamber and 100 μl DMEM ™ were performed using Lipofectamine 3000 reagent (Invi- in the upper for 30 minutes to hydrate chambers; 3×105 cells trogen, Carlsbad, CA, USA) according to the manufacturer’s μ fl were diluted in 100 l serum-free DMEM and seeded into the guidelines. After 48 h, the cells were cultured to 90% con u- upper chamber of the 24-well plates. Next, 500 μl DMEM ence for subsequent analysis. with 10% FBS was added to the lower chamber as a chemoat- ° tractant. After 24 h of incubation in 5% CO2 at 37 C, the cells 2.6. Quantitative Real-Time Polymerase Chain Reaction on the upper surface of the chambers were removed by scrap- (qRT-PCR). To detect the expression levels of KIF20A in ing with a cotton swab, and the cells that migrated to the fi CRC cell lines, quantitative real-time polymerase chain reaction lower membrane were xed with 4% paraformaldehyde for fi analysis was performed. Total RNA from cell lines was 20 min and stained with 0.1% crystal violet. We chose ve fi extracted using TRIzol reagent (Servicebio, Boston, Massachu- random elds to photograph and count. setts, USA), and complementary DNA (cDNA) was extracted 5 2.10. Wound Healing Assay. A total of 3×10 transfected by using the RevertAid First Strand cDNA Synthesis Kit fi cells were seeded into 6-well plates until they reached 70- (Thermo Fisher Scienti c, Waltham, USA) according to the fl fl ’ 80% con uence. The con uent cell layer was wounded by a manufacturer s recommendations for users. The primer μ ′ 10 l pipette tip. Then, the cells were washed with sterile sequences were designed as follows: GAPDH: (forward) 5 PBS, and 2 ml fresh medium without FBS was added to every -ACTTTGGTATCGTGGAAGGACTCAT-3′ and (reverse) 5′ ′ well. The wound healing process was observed at 0 h and 48 h -GTTTTTCTAGACGGCAGGTCAGG-3 ;KIF20A:(forward) to evaluate the migration ability of the tested cells. 5′-ATCAAAATGGCAATCCCTATGTG-3′ and (reverse) 5′ -TCTGGTTCTTACGACCCACTTTT-3′. The expression of 2.11. Statistical Analysis. All data were analyzed using SPSS KIF20A was analyzed using FastStart Universal SYBR Green 19.0 (IBM, USA) software, and all figures were generated Master (Rox) (Roche, Switzerland). using GraphPad Prism 6.0 software. Analysis of the 4 Disease Markers

Unpaired (TCGA cohort) Paired (TCGA cohort) 0.04 0.04 P < 0.001 P < 0.001 0.03 0.03

0.02

0.02 expression mRNA mRNA expression mRNA

0.01 0.01 KIF20A KIF20A in normal and CRC tissues CRC and in normal in normal and CRC tissues CRC and in normal 0.00 0.00 Normal tissues CRC tissues Relative Relative

Relative Relative Normal tissues CRC tissues (N = 50) (N = 380) (N = 32) (N = 32)

(a) (b) Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Case 8

TN TN TN TN TN TN TNTNTN KIF20A

�-Actin

(c)

Figure 1: Expression of KIF20A in CRC tissues. (a) KIF20A mRNA expression was higher in unpaired CRC tissues (n = 380) than in normal tissues (n =50) from TCGA cohort (P <0:001). (b) KIF20A mRNA expression in paired CRC tissues and normal tissues (n =32) in TCGA cohort (P <0:001). (c) KIF20A protein levels were analyzed by Western blotting in 8 paired CRC tissues and adjacent nontumor tissues. relationship between the expression of KIF20A in CRC tis- higher expression in CRC tissues, and 38 patients (36%) sues and clinicopathological features of patient tumor pro- had lower expression or negative expression (P <0:05). gression was carried out using χ2 tests. The survival curves The associations between KIF20A expression level and were evaluated using Kaplan-Meier survival curves and ana- clinical data of CRC patients were analyzed by χ2 tests. lyzed by the log-rank test. Univariate and multivariate analy- Our study results revealed that high expression of KIF20A ses were performed using Cox regression analysis. All was significantly associated with advanced T stage differences were considered to be statistically significant at (P =0:005), N stage (P =0:003), M stage (P =0:015), and P <0:05. TNM stage (P <0:001) (Table 1). Kaplan-Meier survival analysis and log-rank test were carried out to estimate 3. Results the prognostic significance of KIF20A expression level in CRC patients. We found that high KIF20A expression 3.1. Expression of KIF20A in CRC Tissues. To identify the was associated with worse overall survival (OS) and expression level of KIF20A in CRC, we analyzed TCGA disease-free survival (DFS) than low KIF20A expression cohort datasets, and our results showed that the mRNA (P <0:001, Figures 2(c) and 2(d)). expression level of KIF20A was upregulated in unpaired Moreover, univariate and multivariate analyses were used CRC tissues (n = 380) compared to normal tissues (n =50, to compare the impact of KIF20A expression and other prog- Figure 1(a), P <0:001). The mRNA expression level of nostic parameters of CRC patients. Univariate Cox regres- KIF20A in the paired CRC tissues was significantly upregu- sion analysis confirmed that tumor size (HR = 2:086, lated compared with that in the adjacent normal tissues 95%CI = 1:131‐3:848, P =0:019), differentiation degree (n =32, Figure 1(b), P <0:001). As shown in Table 2, the (HR = 5:299, 95%CI = 2:799‐10:030, P <0:001), T stage results showed that the expression level of KIF20A exhibited (HR = 4:469, 95%CI = 2:112‐9:458, P <0:001), N stage a similar trend in other CRC cohorts from the Oncomine (HR = 2:630, 95%CI = 1:817‐3:805, P <0:001), M stage database [24–30]. We found that the protein level of KIF20A (HR = 3:396, 95%CI = 1:661‐6:944, P =0:001), TNM stage was higher in tumor tissues than in normal tissues from our (HR = 2:520, 95%CI = 1:761‐3:608, P <0:001), and KIF20A medical center, as determined by Western blotting (n =8, expression (HR = 4:457, 95%CI = 1:877‐10:579, P =0:001) Figure 1(c)). were significantly associated with worse prognosis in CRC patients. Multivariate Cox regression analysis also demon- 3.2. Overexpression of KIF20A Is Associated with the Clinical strated that differentiation degree (HR = 5:138, 95%CI = Characteristics and Prognosis of CRC Patients. To evaluate 2:374‐11:119, P <0:001) and higher expression of KIF20A the protein expression of KIF20A in human CRC tumor tis- (HR = 2:803, 95%CI = 1:095‐7:175, P =0:032) were nega- sues and adjacent normal tissues, we analyzed KIF20A pro- tively correlated with OS (Table 3). Moreover, KIF20A tein expression in paraffin-embedded human CRC expression was also an independent predictor of DFS specimens by IHC. Figures 2(a) and 2(b) are representative (HR = 2:792, 95%CI = 1:311‐5:946, P =0:008) in CRC KIF20A IHC stainings. As shown, positive staining signals patients (Table 4). Collectively, these results indicated that a of KIF20A were mainly distributed in the cytoplasm of cells. high KIF20A expression level was an independent prognostic The results showed that 67 patients (64%) had significantly factor in CRC patients. Disease Markers 5

Table 2: Oncomine analysis of KIF20A expression in colorectal cancer (total 9 colorectal cancer cohorts).

Cohort no. Cohort Sample (N) t-test Fold change P value 1 Hong colorectal [23] Colorectal carcinoma (70) vs. normal (12) 10.005 2.891 7:36E − 09 Colon carcinoma (5) vs. normal (10) 9.362 5.046 1:49E − 06 Colon adenoma (5) vs. normal (10) 7.856 4.145 3:09E − 06 2 Skrzypczak colorectal 2 [24] Colon carcinoma epithelia (5) vs. normal (10) 5.083 2.268 2:54E − 04 Colon adenoma epithelia (5) vs. normal (10) 3.671 1.828 0.002 3 Ki colon [25] Colon adenocarcinoma (50) vs. normal (28) 2.233 1.177 0.014 Colorectal carcinoma (36) vs. normal (24) 5.842 2.198 2:14E − 07 4 Skrzypczak colorectal [24] Colorectal adenocarcinoma (45) vs. normal (24) 5.596 2.058 6:10E − 07 5 Graudens colon [26] Colorectal carcinoma (18) vs. normal (12) 3.397 1.308 0.001 Rectal adenoma (7) vs. normal (32) 7.728 3.537 2:17E − 05 6 Sabates-Bellver colon [27] Colon adenoma (25) vs. normal (32) 11.436 3.033 4:11E − 16 Rectal mucinous adenocarcinoma (4) vs. normal (5) 2.892 1.588 0.014 Cecum adenocarcinoma (17) vs. normal (5) 4.901 2.127 0.002 Rectosigmoid adenocarcinoma (10) vs. normal (5) 2.989 1.727 0.007 7 Kaiser colon [28] Colon adenocarcinoma (41) vs. normal (5) 4.544 1.976 0.003 Colon mucinous adenocarcinoma (13) vs. normal (5) 3.27 1.893 0.004 Rectal adenocarcinoma (8) vs. normal (5) 1.685 1.458 0.06 Cecum adenocarcinoma (22) vs. normal (22) 8.124 2.891 3:81E − 10 Colon mucinous adenocarcinoma (22) vs. normal (22) 8.048 2.77 7:14E − 10 Rectosigmoid adenocarcinoma (3) vs. normal (22) 2.789 1.767 0.031 8 TCGA colorectal Colon adenocarcinoma (101) vs. normal (22) 8.765 2.678 1:34E − 09 Rectal adenocarcinoma (60) vs. normal (22) 8.145 2.563 2:62E − 09 Rectal mucinous adenocarcinoma (6) vs. normal (22) 6.021 3.112 5:97E − 05 9 Gaspar colon [29] Colorectal adenoma epithelia (56) vs. normal (22) 2.908 1.213 0.003

3.3. Expression of KIF20A in CRC Cell Lines. Given the signif- assays indicated that the migration of the si-KIF20A group icant correlation between high expression of KIF20A and was significantly reduced compared with that of the si-NC poor prognosis, we further analyzed the mRNA and protein group (Figure 4(c)). expression of KIF20A among five CRC cell lines by RT- PCR and Western blot analysis, respectively (Figures 3(a) 3.5. KIF20A Regulates the JAK/STAT3 Signaling Pathway. and 3(b)), and found that HCT116 and SW1116 had high Previous studies have reported the important role of JAK/- expression of KIF20A, so they were chosen for subsequent STAT3 signaling in CRC tumorigenesis [31, 32]. Hence, we study. To further identify the effect of KIF20A in CRC examined whether there was a connection between KIF20A in vitro, siRNA transfection technology was used to silence and JAK/STAT3 signaling. In this study, KIF20A knock- its expression in the cell lines with high expression of down reduced MMP2, phosphorylated JAK2, and phosphor- KIF20A, and the transfection efficiency was detected by ylated STAT3 expression at the protein level (Figure 4(d)). Western blot assays and RT-PCR (Figures 3(c) and 3(d)). These data imply that KIF20A could activate the JAK/- STAT3 signaling pathway, which consequently promotes 3.4. Knockdown of KIF20A Inhibits the Proliferation and CRC carcinogenesis. Migration Ability of CRC Cells In Vitro. To explore the biological function of KIF20A in CRC progression, siRNA was 4. Discussion transfected into CRC cell lines, and cell proliferation and migration ability were measured. The CCK-8 assay showed Many investigators have found that overexpression of that cell proliferative activity was significantly decreased with KIF20A plays a crucial role in tumorigenesis and cancer pro- KIF20A knockdown compared with that in control cells gression in various human cancers. KIF20A, a member of the (Figure 4(a)). We also examined the effect of KIF20A on cell kinesin superfamily, is characterized by a motor domain that migration ability by wound healing and Transwell assays. is highly conserved in the evolution of biological species [33, Our data from the wound healing assay showed that the cell 34]. KIF20A was first reported to localize to the Golgi appa- wound closure ability decreased after silencing KIF20A com- ratus, where it plays an important role in organelle dynamics pared with that of the control group (Figure 4(b)). Transwell with the GDP-bound form of Rab6 [8]. Moreover, KIF20A 6 Disease Markers

Figure 2: Increased KIF20A protein expression predicts poor prognosis in patients with CRC. (a, b) IHC staining analysis of KIF20A protein levels in CRC patient tissues. (c, d) Kaplan-Meier analysis revealed that patients with higher KIF20A expression had worse OS and DFS than those with lower KIF20A expression (P <0:001). Disease Markers 7

Table 3: Cox proportional hazard regression analysis for overall survival.

Univariate analysis Multivariate analysis Characteristics 95% CI 95% CI HR P value HR P value Lower Upper Lower Upper Gender 1.357 0.743 2.477 0.320 Age 0.692 0.380 1.258 0.227 Tumor size 2.086 1.131 3.848 0.019 Diﬀerentiation degree 5.299 2.799 10.030 <0.001 5.138 2.374 11.119 <0.001 Tumor location 1.218 0.970 1.530 0.090 T stage 4.469 2.112 9.458 <0.001 N stage 2.630 1.817 3.805 <0.001 M stage 3.396 1.661 6.944 0.001 TNM stage 2.520 1.761 3.608 <0.001 KIF20A expression 4.457 1.877 10.579 0.001 2.803 1.095 7.175 0.032 Abbreviations: HR: hazard ratio; CI: conﬁdence interval.

Table 4: Cox proportional hazard regression analysis for disease-free survival.

Univariate analysis Multivariate analysis Characteristics 95% CI 95% CI HR P value HR P value Lower Upper Lower Upper Gender 1.393 0.817 2.374 0.224 Age 0.798 0.469 1.356 0.404 Tumor size 2.037 1.186 3.498 0.010 Diﬀerentiation degree 3.788 2.077 6.912 <0.001 3.357 1.676 6.772 0.001 Tumor location 1.180 0.974 1.430 0.091 T stage 3.591 1.876 6.876 <0.001 N stage 2.069 1.481 2.891 <0.001 M stage 2.630 1.319 5.245 0.006 TNM stage 1.974 1.455 2.680 <0.001 KIF20A expression 3.599 1.809 7.161 <0.001 2.792 1.311 5.946 0.008 Abbreviations: HR: hazard ratio; CI: conﬁdence interval.

1.5 10

8 1.0 6 LOVO HCT116 SW1116 LS174T SW480

KIF20A 4 0.5 2 �-Actin 0.0 0 KIF20A mRNA expression level KIF20A protein expression level LOVO LOVO SW480 SW480 LS174T LS174T SW1116 HCT116 SW1116 HCT116 (a) (b) 1.5 1.5 ⁎⁎ ⁎⁎ 1.0 1.0 Si-SW1116 NC Si-HCT116 NC

KIF20A 0.5 0.5

�-Actin Expression level of KIF20A 0.0 Expression level of KIF20A 0.0 SW1116 HCT116 Si-SW1116 NC Si-HCT116 NC (c) (d)

Figure 3: Expression of KIF20A in CRC cell lines. KIF20A expression was examined at the (a) mRNA and (b) protein levels in ﬁve CRC cell lines. The siRNA transfection eﬃciency was detected by (c) Western blot assays and (d) RT-PCR. 8 Disease Markers

SW1116 HCT116 1.5 1.0 ⁎⁎ ⁎⁎ 0.8 ⁎⁎ 1.0 ⁎ 0.6

0.4 0.5 0.2 OD value (450 nm) OD value (450 nm) 0.0 0.0 02448 72 96 0 24487296 Time (h) Time (h)

Si-SW1116 Si-HCT116 NC NC (a) SW1116 HCT116

Si-SW1116 NC Si-HCT116 NC 1.0 0.6 ⁎ ⁎⁎ 0.8 0 h 0 h 0.4 0.6

0.4 0.2 48 h Percent wound closure 0.2 48 h Percent wound closure

0.0 Si-SW1116 NC (b) 500 400 SW1116 HCT116 ⁎⁎ ⁎⁎ Si-SW1116 NC 400 Si-HCT116 NC 300

300 200 200 100 100 Number of cells per feld Number of cells per feld 0 0 Si-SW1116 NC Si-HCT116 NC (c) SW1116 HCT116

NC Si-SW1116 NC Si-HCT116

MMP2

p-JAK2

p-STAT3

�-Actin

(d)

Figure 4: Knockdown of KIF20A inhibits CRC cell proliferation and migration ability through the JAK/STAT3 signaling pathway. (a) Cell proliferation activity after knockdown of KIF20A was assessed by CCK-8 assay. (b) Cell wound closure ability was analyzed by wound healing assay. (c) Transwell analysis was used to determine cell migration ability with or without KIF20A knockdown. Statistical significance was analyzed according to the number of migrated cells. (d) MMP2, phosphorylation of JAK, and STAT3 expression after KIF20A knockdown were determined by Western blot analysis. Data are presented as the mean ± SEM. ∗P <0:05; ∗∗P <0:01. binds to microtubules and couples adenosine triphosphate revealed that aberrant expression of KIF20A is closely asso- hydrolysis to generate mechanical force [6], which is ciated with tumorigenesis. believed to play a central role in mitosis and cytokinesis A number of studies indicate that KIF20A is upregulated [35]. KIF20A has been shown to accumulate in mitotic cells, in many human malignant tumors. In the present study, we which is involved in the formation of the mitotic spindle, found that KIF20A was overexpressed in CRC tumors and and plays an important role in cytokinesis [9]. These results associated with clinicopathological features and prognosis Disease Markers 9 in CRC. TCGA cohort analysis revealed that the KIF20A STAT3 signaling pathway promotes the development of mRNA expression level was significantly higher in CRC tis- colon carcinoma cells [42]. Moreover, a previous report has sues than in normal tissues. Consistent with the KIF20A level illustrated that STAT3 targets MMP2 in cell migration and in TCGA cohort, the data in the Oncomine database and invasion of esophageal squamous carcinoma cells [43]. In from our center also further validated a higher level of the present study, KIF20A knockdown inhibited CRC cell KIF20A. In addition, our results showed that 64% of patients proliferation and migration, which are related to reduced had high KIF20A expression in CRC tissues, and KIF20A MMP2, phosphorylated JAK2, and phosphorylated STAT3 expression was significantly associated with advanced T protein expression in CRC cells. Therefore, these observa- stage, N stage, M stage, and TNM stage. Moreover, overex- tions indicate that KIF20A may promote CRC cell growth pression of KIF20A was closely correlated with worse and migration through JAK/STAT3-dependent mechanisms prognosis. This observation is similar to the recent finding and suggest that KIF20A can be further evaluated as a poten- in other tumors; it has been reported that KIF20A is an inde- tial therapeutic target for CRC. pendent factor for overall survival (OS) and progression-free survival (PFS) in patients with nasopharyngeal carcinoma 5. Conclusion [13]. Zhang et al. revealed that overexpression of KIF20A was closely associated with many clinicopathological factors, In summary, our data demonstrated that KIF20A was over- including HPV infection, stage, recurrence, lymphovascular expressed in CRC tissues and was significantly associated space involvement, nodal status, and poor prognosis in with poor prognosis in patients with CRC. Silencing of patients; moreover, upregulation of KIF20A also predicted KIF20A expression inhibited the proliferation and migration poor OS and disease-free survival (DFS) in early cervical ability of CRC cell lines via the JAK/STAT3 signaling path- squamous cell carcinoma patients [36]. Based on the above way. These data suggested that KIF20A is an important reg- findings, we found that KIF20A might play a role in cancer ulator in CRC progression, which may provide a new progression and serve as a novel biomarker of prognosis for therapeutic target or clinical biomarker for treating patients CRC patients. with CRC. Invasion and metastasis are important biological characteristics of malignant tumors and are the pathological bases Data Availability leading to tumor recurrence, disease deterioration, and death fi [37]. Zhao et al. confirmed that KIF20A is highly expressed in The data used to support the ndings of this study are lung adenocarcinoma and may serve as an independent included within the article. prognostic factor for OS. Moreover, knockdown of KIF20A could inhibit cell proliferation through G1 phase arrest and Conflicts of Interest induce apoptosis [18]. In ovarian clear-cell carcinoma, high fl KIF20A expression may contribute to disease progression, The authors declare that they have no con ict of interest. which is principally attributable to its asymptomatic intra- ’ peritoneal dissemination with or without distant metastases Authors Contributions to parenchymal organs [38]. A previous study showed that Qi Zhang and Changjiang Qin conceived and designed the high expression of KIF20A can promote the proliferation, experiments. Qi Zhang, Jun Di, and Zhiyu Ji carried out the invasion, and migration of pancreatic cancer cells; in con- experiment of molecular biology and drafted the manuscript. trast, targeted silencing of KIF20A can reduce the prolifera- Aoning Mi and Quanying Li carried out the cell experiment. tion, migration, and invasion [16]. Yamashita et al. found Xianghui Du, Anpeng Wang, and Anlian Wang analyzed the that KIF20A is a novel melanoma-associated antigen that data. All authors read and approved the final manuscript. Qi may be considered to be a diagnostic and prognostic marker Zhang, Jun Di, and Zhiyu Ji contributed equally to this work. for melanoma [10]. Zou et al. demonstrated that KIF20A could be a potential new prognostic factor and target for endocrine therapy-resistant patients with breast cancer Acknowledgments [12]. Consistent with the research described above, the This study was supported by grants from the National Natu- fi results of this study con rmed that silencing KIF20A could ral Science Foundation of China (NSFC-U1504818), the Sci- inhibit the proliferation capacity of cells. Transwell and ence and Technology Foundation of Henan Province wound healing assays also showed that the migration ability (172102310152), the Natural Science Foundation of Henan of CRC cell lines was inhibited after knockdown of KIF20A. Province of China (182300410359), the Medical Science These data strongly suggest that KIF20A may act as an onco- and Technique Foundation of Henan Province gene in the tumorigenesis and progression of CRC; however, (201601029), and the Education Foundation of Henan Prov- its molecular mechanism in CRC is not yet clear. ince (19A320020). JAK/STAT3 signaling is a well-identified oncogenic pathway that plays a pivotal role in various cellular biological References activities important for carcinogenesis, including cell survival, cell cycle, and angiogenesis in multiple cancers [39], [1] R. L. Siegel, K. D. Miller, S. A. Fedewa et al., “Colorectal cancer and the activation of the JAK/STAT3 pathway in CRC has statistics, 2017,” CA: a Cancer Journal for Clinicians, vol. 67, been largely indicated [32, 40, 41]. Activating the JAK/- no. 3, pp. 177–193, 2017. 10 Disease Markers

[2] R. L. Siegel, K. D. Miller, and A. Jemal, “Cancer statistics, [18] X. Zhao, L. L. Zhou, X. Li et al., “Overexpression of KIF20A 2016,” CA: a Cancer Journal for Clinicians, vol. 66, no. 1, confers malignant phenotype of lung adenocarcinoma by pro- pp. 7–30, 2016. moting cell proliferation and inhibiting apoptosis,” Cancer [3] R. L. Siegel, K. D. Miller, and A. Jemal, “Cancer statistics, Medicine, vol. 7, no. 9, pp. 4678–4689, 2018. 2018,” CA: a Cancer Journal for Clinicians, vol. 68, no. 1, [19] G. Xiu, X. Sui, Y. Wang, and Z. Zhang, “FOXM1 regulates pp. 7–30, 2018. radiosensitivity of lung cancer cell partly by upregulating [4] F. Bray, J. Ferlay, I. Soerjomataram, R. L. Siegel, L. A. Torre, KIF20A,” European Journal of Pharmacology, vol. 833, and A. Jemal, “Global cancer statistics 2018: GLOBOCAN esti- pp. 79–85, 2018. mates of incidence and mortality worldwide for 36 cancers in [20] M. B. Amin, S. B. Edge, F. L. Greene et al., American Joint 185 countries,” CA: a Cancer Journal for Clinicians, vol. 68, Committee on Cancer. AJCC Cancer Staging Manual, no. 6, pp. 394–424, 2018. pp. 113–123, Springer, New York, NY, 8th edition, 2017. [5] E. Van Cutsem, A. Cervantes, B. Nordlinger, and D. Arnold, [21] C. J. Qin, X. M. Song, Z. H. Chen et al., “XRCC2 as a predictive “Metastatic colorectal cancer: ESMO Clinical Practice Guide- biomarker for radioresistance in locally advanced rectal cancer lines for diagnosis, treatment and follow-up†,” Annals of patients undergoing preoperative radiotherapy,” Oncotarget, Oncology, vol. 25, pp. iii1–iii9, 2014. vol. 6, no. 31, pp. 32193–32204, 2015. [6] N. Hirokawa, Y. Noda, and Y. Okada, “Kinesin and dynein [22] D. Wang, S. Zhang, Y. Chen, B. Hu, and C. Lu, “Low expres- superfamily proteins in organelle transport and cell division,” sion of NKD2 is associated with enhanced cell proliferation Current Opinion in Cell Biology, vol. 10, no. 1, pp. 60–73, 1998. and poor prognosis in human hepatocellular carcinoma,” [7] N. Hirokawa, Y. Noda, Y. Tanaka, and S. Niwa, “Kinesin Human Pathology, vol. 72, pp. 80–90, 2018. superfamily motor proteins and intracellular transport,” [23] Q. Li, H. Tang, F. Hu, and C. Qin, “Knockdown of A-kinase Nature Reviews. Molecular Cell Biology, vol. 10, no. 10, anchor protein 4 inhibits hypoxia-induced epithelial-to- pp. 682–696, 2009. mesenchymal transition via suppression of the Wnt/β- [8] A. Echard, F. Jollivet, O. Martinez et al., “Interaction of a catenin pathway in human gastric cancer cells,” Journal of Golgi-associated kinesin-like protein with Rab6,” Science, Cellular Biochemistry, vol. 119, no. 12, pp. 10013–10020, vol. 279, no. 5350, pp. 580–585, 1998. 2018. [9] E. Hill, M. Clarke, and F. A. Barr, “The Rab6-binding kinesin, [24] Y. Hong, T. Downey, K. W. Eu, P. K. Koh, and P. Y. Cheah, “A Rab6-KIFL, is required for cytokinesis,” The EMBO Journal, ‘metastasis-prone’ signature for early-stage mismatch-repair vol. 19, no. 21, pp. 5711–5719, 2000. proficient sporadic colorectal cancer patients and its implica- ” [10] J. Yamashita, S. Fukushima, M. Jinnin et al., “Kinesin family tions for possible therapeutics, Clinical & Experimental Metas- – member 20A is a novel melanoma-associated antigen,” Acta tasis,vol.27,no.2,pp.83 90, 2010. Dermato-Venereologica, vol. 92, no. 6, pp. 593–597, 2012. [25] M. Skrzypczak, K. Goryca, T. Rubel et al., “Correction: Model- [11] P. Khongkow, A. R. Gomes, C. Gong et al., “Paclitaxel targets ing oncogenic signaling in colon tumors by multidirectional FOXM1 to regulate KIF20A in mitotic catastrophe and breast analyses of microarray data directed for maximization of ana- ” cancer paclitaxel resistance,” Oncogene, vol. 35, no. 8, pp. 990– lytical reliability, PLoS One, vol. 5, no. 12, 2010. 1002, 2016. [26] D. H. Ki, H. C. Jeung, C. H. Park et al., “Whole genome anal- ” [12] J. X. Zou, Z. Duan, J. Wang et al., “Kinesin family deregulation ysis for liver metastasis gene signatures in colorectal cancer, – coordinated by bromodomain protein ANCCA and histone International Journal of Cancer, vol. 121, no. 9, pp. 2005 methyltransferase MLL for breast cancer cell growth, survival, 2012, 2007. and tamoxifen resistance,” Molecular Cancer Research, vol. 12, [27] E. Graudens, V. Boulanger, C. Mollard et al., “Deciphering cel- no. 4, pp. 539–549, 2014. lular states of innate tumor drug responses,” Genome Biology, [13] S. L. Liu, H. X. Lin, F. Qiu et al., “Overexpression of kinesin vol. 7, no. 3, p. R19, 2006. family member 20A correlates with disease progression and [28] J. Sabates-Bellver, L. G. van der Flier, M. de Palo et al., “Tran- poor prognosis in human nasopharyngeal cancer: a retrospec- scriptome profile of human colorectal adenomas,” Molecular tive analysis of 105 patients,” PLoS One, vol. 12, no. 1, Cancer Research, vol. 5, no. 12, pp. 1263–1275, 2007. p. e0169280, 2017. [29] S. Kaiser, Y. K. Park, J. L. Franklin et al., “Transcriptional reca- [14] K. Taniuchi, M. Furihata, and T. Saibara, “KIF20A-mediated pitulation and subversion of embryonic colon development by RNA granule transport system promotes the invasiveness of mouse colon tumor models and human colon cancer,” pancreatic cancer cells,” Neoplasia, vol. 16, no. 12, pp. 1082– Genome Biology, vol. 8, no. 7, p. R131, 2007. 1093, 2014. [30] C. Gaspar, J. Cardoso, P. Franken et al., “Cross-species com- [15] K. Imai, S. Hirata, A. Irie et al., “Identification of HLA-A2- parison of human and mouse intestinal polyps reveals con- restricted CTL epitopes of a novel tumour-associated antigen, served mechanisms in adenomatous polyposis coli (APC)- KIF20A, overexpressed in pancreatic cancer,” British Journal driven tumorigenesis,” The American Journal of Pathology, of Cancer, vol. 104, no. 2, pp. 300–307, 2011. vol. 172, no. 5, pp. 1363–1380, 2008. [16] D. Stangel, M. Erkan, M. Buchholz et al., “Kif20a inhibition [31] M. L. Slattery, A. Lundgreen, S. A. Kadlubar, K. L. Bondurant, reduces migration and invasion of pancreatic cancer cells,” and R. K. Wolff, “JAK/STAT/SOCS-signaling pathway and The Journal of Surgical Research, vol. 197, no. 1, pp. 91–100, colon and rectal cancer,” Molecular Carcinogenesis, vol. 52, 2015. no. 2, pp. 155–166, 2013. [17] I. Gasnereau, M. Boissan, G. Margall-Ducos et al., “KIF20A [32] H. Xiong, Z. G. Zhang, X. Q. Tian et al., “Inhibition of JAK1, mRNA and its product MKlp2 are increased during hepato- 2/STAT3 signaling induces apoptosis, cell cycle arrest, and cyte proliferation and hepatocarcinogenesis,” The American reduces tumor cell invasion in colorectal cancer cells,” Neopla- Journal of Pathology, vol. 180, no. 1, pp. 131–140, 2012. sia, vol. 10, no. 3, pp. 287–297, 2008. Disease Markers 11

[33] R. D. Vale, T. S. Reese, and M. P. Sheetz, “Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility,” Cell, vol. 42, no. 1, pp. 39–50, 1985. [34] N. Hirokawa and Y. Noda, “Intracellular transport and kinesin superfamily proteins, KIFs: structure, function, and dynamics,” Physiological Reviews, vol. 88, no. 3, pp. 1089– 1118, 2008. [35] P. A. Nguyen, A. C. Groen, M. Loose et al., “Spatial organiza- tion of cytokinesis signaling reconstituted in a cell-free system,” Science, vol. 346, no. 6206, pp. 244–247, 2014. [36] W. Zhang, W. He, Y. Shi et al., “High expression of KIF20A is associated with poor overall survival and tumor progression in early-stage cervical squamous cell carcinoma,” PLoS One, vol. 11, no. 12, p. e0167449, 2016. [37] G. P. Gupta and J. Massague, “Cancer metastasis: building a framework,” Cell, vol. 127, no. 4, pp. 679–695, 2006. [38] Y. Kawai, K. Shibata, J. Sakata et al., “KIF20A expression as a prognostic indicator and its possible involvement in the proliferation of ovarian clear-cell carcinoma cells,” Oncology Reports, vol. 40, no. 1, pp. 195–205, 2018. [39] H. Yu, H. Lee, A. Herrmann, R. Buettner, and R. Jove, “Revisit- ing STAT3 signalling in cancer: new and unexpected biological functions,” Nature Reviews. Cancer, vol. 14, no. 11, pp. 736– 746, 2014. [40] Q. Liang, D. Ma, X. Zhu et al., “RING-finger protein 6 ampli- fication activates JAK/STAT3 pathway by modifying SHP-1 ubiquitylation and associates with poor outcome in colorectal cancer,” Clinical Cancer Research, vol. 24, no. 6, pp. 1473– 1485, 2018. [41] X. Xue, S. K. Ramakrishnan, K. Weisz et al., “Iron uptake via DMT1 integrates cell cycle with JAK-STAT3 signaling to promote colorectal tumorigenesis,” Cell Meta, vol. 24, no. 3, pp. 447–461, 2016. [42] X. Xue, K. Jungles, G. onder, J. Samhoun, B. Győrffy, and K. M. Hardiman, “HIF-3α1 promotes colorectal tumor cell growth by activation of JAK-STAT3 signaling,” Oncotarget, vol. 7, no. 10, pp. 11567–11579, 2016. [43] Y. Ou, L. Liu, L. Xue et al., “TRAP1 shows clinical significance and promotes cellular migration and invasion through STAT3/MMP2 pathway in human esophageal squamous cell cancer,” Journal of Genetics and Genomics, vol. 41, no. 10, pp. 529–537, 2014.