作为图书馆，NLM 提供对科学文献的访问。收录到 NLM 数据库并不意味着 NLM 或美国国立卫生研究院认可或同意这些内容。
Learn more: PMC Disclaimer | PMC Copyright Notice
知道更多： PMC 免责声明 |PMC 版权告示
Onco 瞄准 Ther.2020;13: 2667–2676.
This article has been retracted.
本文已被撤回。
撤回于：Onco Targets Ther.2023 年 5 月 09 日;16：309 Смотритетакже： PMC 撤回政策
KIF4A Promotes Clear Cell Renal Cell Carcinoma (ccRCC) Proliferation in vitro and in vivo
KIF4A 在体外和体内促进透明细胞肾细胞癌 （ccRCC） 增殖
作者信息 文章注释 版权和许可证信息 PMC 免责声明
本文已被撤回。请参阅 Onco Targets Ther.2023 年 5 月 09 日;16: 309.
Abstract 抽象
Purpose 目的
To evaluate the expression in human clear cell renal cell carcinoma (ccRCC) tissues and explore the effects of kinesin family member 4A (KIF4A) on ccRCC progression.
评估人透明细胞肾细胞癌 （ccRCC） 组织中的表达，并探讨驱动蛋白家族成员 4A （KIF4A） 对 ccRCC 进展的影响。
Methods 方法
GEPIA was used to evaluate the mRNA levels of KIF4A in human ccRCC tissues from TCGA database, and Immunohistochemistry (IHC) assays were performed to assess its expression in human ccRCC tissues collected in our hospital. The clinical-pathological analysis was performed to explore the correlation with KIF4A expression. The effects of KIF4A on ccRCC cell proliferation were detected through colony formation and MTT assays. Finally, the effects of KIF4A on tumor growth were measured using a mice model.
采用 GEPIA 评估 TCGA 数据库中人 ccRCC 组织中 KIF4A mRNA 水平，免疫组化 （IHC） 检测其在我院收集的人 ccRCC 组织中的表达。进行临床病理分析以探讨与 KIF4A 表达的相关性。通过集落形成和 MTT 测定检测 KIF4A 对 ccRCC 细胞增殖的影响。最后，使用小鼠模型测量 KIF4A 对肿瘤生长的影响。
Results 结果
Bioinformation results showed the expression of KIF4A mRNA was upregulated in ccRCC tissues and high expression of KIF4A was related with poor prognosis in ccRCC patients. We also found a high expression of KIF4A in human ccRCC tissues collected in our hospital. We also found its expression level was correlated with clinical characteristics, including T stage (P=0.035*) and lymphatic metastasis (P=0.028*). We further confirmed that knockdown of KIF4A suppressed cell proliferation in HTB-47 and CRL-1932 cells. Furthermore, KIF4A contributes to tumor growth of ccRCC cells in mice.
生物信息结果显示 KIF4A mRNA 在 ccRCC 组织中表达上调，KIF4A 高表达与 ccRCC 患者预后不良相关。我们还发现在我们医院收集的人 ccRCC 组织中 KIF4A 的高表达。我们还发现其表达水平与临床特征相关，包括 T 分期 （P=0.035*） 和淋巴转移 （P=0.028*）。我们进一步证实，敲低 KIF4A 抑制了 HTB-47 和 CRL-1932 细胞中的细胞增殖。此外，KIF4A 有助于小鼠 ccRCC 细胞的肿瘤生长。
Conclusion 结论
We found the abnormal high expression of KIF4A in human ccRCC tissues and demonstrated that KIF4A could serve as a tumor induction gene.
我们发现 KIF4A 在人 ccRCC 组织中异常高表达，并证明 KIF4A 可以作为肿瘤诱导基因。
关键词：透明细胞肾细胞癌， ccRCC， KIF4A， 增殖， 预后， 临床病理特征
Introduction 介绍
Renal cell carcinoma is a common urinary disease with a high incidence.1 In the United States, more than 65, 340 newly diagnosed RCC patients and approximately 14, 970 deaths in 2018.2–4 While in 2019, there were 73, 820 new diagnosed RCC patients and approximately 14, 770 deaths.2–4 Clear cell renal cell carcinoma (ccRCC) represents the highly aggressive renal malignant tumor which accounts for nearly 80% of renal cell carcinoma.5 Existing traditional treatments, such as surgical resection, radiation and chemotherapy, seem to be ineffective against this highly aggressive tumor.6 Recently targeted therapy for ccRCC is promising.7 VHL was reportedly considered a potential molecular target for ccRCC, but more mutations were subsequently discovered, such as these mutations in ccRCC leading to further identification of their possible therapeutic role in this cancer.8,9 In order to fight this disease in the future, the development of new molecular tars has potential clinical value.
肾细胞癌是一种常见的泌尿系统疾病，发病率很高。 1 在美国，65， 340 年有超过 2018 名新诊断的 RCC 患者和大约 14， 970 人死亡。 2 – 4 而在 2019 年，有 73， 820 名新诊断的 RCC 患者和大约 14， 770 名死亡患者。 2 – 4 透明细胞肾细胞癌 （ccRCC） 是高度侵袭性的肾恶性肿瘤，占肾细胞癌的近 80%。 5 现有的传统治疗方法，如手术切除、放疗和化疗，似乎对这种高度侵袭性的肿瘤无效。 6 最近 ccRCC 的靶向治疗前景广阔。 7 据报道，VHL 被认为是 ccRCC 的潜在分子靶点，但随后发现了更多突变，例如 ccRCC 中的这些突变，导致进一步确定它们在这种癌症中可能的治疗作用。 8 9 为了将来对抗这种疾病，开发新的分子焦油具有潜在的临床价值。
The kinesin proteins (KIFs) that are mainly involved in cargo transportation belong to the microtubule-based kinesin family.10 More than 45 kinesins have already been identified in human cells.11 Kinesins carry out critical functions in the processes of mitosis and cytokinesis.11 Additionally, previous studies demonstrated that kinesins could promote the separation of sister chromatin.12 Kinesin family member 4A (KIF4A), a motor protein involved in multiple cellular processes such as spindle formation, chromosome segregation, and cytokinesis.13 KIF4A also associates with the regulation of DSB repair-related proteins.14 In recent years, the key role of KIF4A in cancer development has gradually been revealed.15 KIF4A is widely expressed in a variety of human tissues.15 Various studies have reported that KIF4A promoted the progression of several cancers, such as cervical cancer and oral cancer.16 In addition, KIF4A promotes cell proliferation via cell cycle regulation and metastasis in colorectal cancer.15 KIF4A ablation also leads to inhibition of lung cancer cell proliferation.17 However, it is unclear whether KIF4A is involved in the occurrence and development of ccRCC in highly malignant tumors.
主要参与货物运输的驱动蛋白 （KIF） 属于基于微管的驱动蛋白家族。 10 已在人类细胞中鉴定出超过 45 种驱动蛋白。 11 驱动蛋白在有丝分裂和胞质分裂过程中执行关键功能。 11 此外，先前的研究表明，驱动蛋白可以促进姐妹染色质的分离。 12 驱动蛋白家族成员 4A （KIF4A），一种参与纺锤体形成、染色体分离和胞质分裂等多种细胞过程的运动蛋白。 13 KIF4A 还与 DSB 修复相关蛋白的调节有关。 14 近年来，KIF4A 在癌症发展中的关键作用逐渐显现。 15 KIF4A 在多种人体组织中广泛表达。 15 各种研究报告称，KIF4A 促进了多种癌症的进展，例如宫颈癌和口腔癌。 16 此外，KIF4A 通过结直肠癌的细胞周期调节和转移促进细胞增殖。 15 KIF4A 消融还导致肺癌细胞增殖的抑制。 17 然而，目前尚不清楚 KIF4A 是否参与高度恶性肿瘤中 ccRCC 的发生和发展。
Here, we declared that KIF4A is involved in the development of ccRCC and found that KIF4A is highly expressed in ccRCC tissue samples. Our data further confirmed that KIF4A is correlated with the clinical pathology of ccRCC patients such as tumor stage and tumor size. Furthermore, our results indicated that KIF4A depletion dramatically inhibited ccRCC cell proliferation and inhibited tumor growth in mice. Therefore, KIF4A may become a promising therapy for ccRCC.
在这里，我们宣布 KIF4A 参与 ccRCC 的发育，并发现 KIF4A 在 ccRCC 组织样本中高表达。我们的数据进一步证实 KIF4A 与 ccRCC 患者的临床病理相关，例如肿瘤分期和肿瘤大小。此外，我们的结果表明，KIF4A 耗竭显着抑制小鼠 ccRCC 细胞增殖并抑制肿瘤生长。因此，KIF4A 可能成为 ccRCC 的一种有前途的疗法。
Materials and Methods 材料和方法
Bioinformatical Analyze 生物信息学分析
GEPIA (http://gepia.cancer-pku.cn/detail.php?gene=KIF4A) was used to analyze data from TCGA (The Cancer Genome Atlas) for differential expressed genes, and the median was utilized as the threshold to separate the patients into two groups for Kaplan-Meier survival analysis.
GEPIA （ http://gepia.cancer-pku.cn/detail.php?gene=KIF4A） 用于分析来自 TCGA （The Cancer Genome Atlas） 的差异表达基因数据，以中位数为阈值，将患者分为两组进行 Kaplan-Meier 生存分析。
Antibodies, Primers and shRNA Plasmids
抗体、引物和 shRNA 质粒
Anti-KIF4A (for IHC assays, 1:400 dilution, for immunoblot assays, 1:1000 dilution, ab12227, Abcam, Cambridge, UK), Anti-β-actin (1:1000 dilution, ab8226, Abcam), Anti-Ki67 (1:1000 dilution, ab16667, Abcam), Anti-proliferating cell nuclear antigen (PCNA) (1:500 dilution, ab92552, Abcam).
KIF4A 抗体（用于 IHC 检测，1：400 稀释，用于免疫印迹检测，1：1000 稀释，ab12227，Abcam，Cambridge，UK）、β-肌动蛋白抗体（1：1000 稀释，ab8226，Abcam）、Ki67 抗体（1：1000 稀释，ab16667，Abcam）、抗增殖细胞核抗原 （PCNA）（1：500 稀释，ab92552，Abcam）。
The quantitative RT-PCR primer sequences of KIF4A were as follows: Forward, 5ʹ -TCTGTTTCAGGCTGCTTTCA-3ʹ and Reverse, 5ʹ-GGATGACCTTGCCCACAGCCT-3ʹ; The quantitative RT-PCR primer sequences of GAPDH were as follows: Forward, 5ʹ-CATCTCTGCCCCCTCTGCTGA-3ʹ and Reverse, 5ʹ-GGATGACCTTGCCCACAGCCT-3ʹ.
KIF4A 的定量 RT-PCR 引物序列如下：正向 5ʹ -TCTGTTTCAGGCTCTTTCA-3 和反向 5ʹ-GGATGACCTTGCCCACAGCCT-3ʹ;GAPDH 的定量 RT-PCR 引物序列如下：正向 5ʹ-CATCTCTGCCCCCTGCTGA-3 和反向 5ʹ-GGATGACCTTGCCCAGCCT-3。
KIF4A shRNA clone was conducted in our laboratory, and the targeted sequences were as follows: 5ʹ-AACAGGAAGAAGTCTTCAATACA-3ʹ.
在我们的实验室进行了 KIF4A shRNA 克隆，靶向序列如下：5ʹ-AACAGGAAGAAGTCTTCAATACA-3ʹ。
Human Tissues Collection and Analysis
人体组织采集和分析
The ccRCC tissue samples and paired adjacent non-tumor tissues were obtained from 78 ccRCC patients at Shanxi Bethune hospital. The clinical-pathological characteristics, including ages, genders, tumor stages, tumor grades, and tumor size were collected and briefly listed in Table 1. The use of human samples in this study was approved by the Ethics Committee of Shanxi Bethune Hospital. The research involving human participants experiments had been approved by our hospital and our equivalent committee. The participants provided their written informed consents to participate in this study.
ccRCC 组织样本和配对的癌旁非肿瘤组织取自山西白求恩医院的 78 例 ccRCC 患者。收集临床病理特征，包括年龄、性别、肿瘤分期、肿瘤分级和肿瘤大小，并在表 1 中简要列出。本研究中人体样本的使用得到了山西白求恩医院伦理委员会的批准。涉及人类参与者实验的研究已获得我们医院和我们同等委员会的批准。参与者提供了参与本研究的书面知情同意书。
Table 1 表 1
Relationships of KIF4A and Clinicopathological Characteristics in 78 Patients with Clear Cell Renal Cell Carcinoma
KIF4A 与 78 例透明细胞肾细胞癌患者临床病理特征的关系
| Feature 特征 | All n=78 全部 n=78 | KIF4A Expression KIF4A 表达 | χ2 2 χ | P | |
|---|---|---|---|---|---|
| Low 低 | High 高 | ||||
| n=40 | n=38 | ||||
| Age (year) 年龄 （岁） | 2.834 | 0.092 | |||
| < 55 | 48 | 21 | 27 | ||
| ≥ 55 | 30 | 19 | 11 | ||
| Gender 性 | 1.238 | 0.266 | |||
| Male 雄 | 44 | 25 | 19 | ||
| Female 女性 | 34 | 15 | 19 | ||
| T stage T 期 | 4.468 | 0.035* | |||
| T1–2 吨 1–2 | 32 | 21 | 11 | ||
| T3–4 吨 3–4 | 46 | 19 | 27 | ||
| Tumor grade 肿瘤分级 | 2.651 | 0.103 | |||
| Low 低 | 34 | 21 | 13 | ||
| High 高 | 44 | 19 | 25 | ||
| Tumor size 肿瘤大小 | 4.803 | 0.028* | |||
| < 4 cm < 4 厘米 | 28 | 19 | 9 | ||
| ≥ 4 cm ≥ 4 厘米 | 50 | 21 | 29 | ||
Note: *P < 0.05. 注：*P < 0.05。
To investigate the possible relationship between KIF4A levels and ccRCC, immunohistochemistry (IHC) assays were subsequently performed. Briefly, the samples were fixed with 4% PFA, then cut into 5-um slices and blocked with 2% BSA in PBS for 30 mins. The Slides were then incubated with an antibody targeting KIF4A at 4°C overnight. Subsequently, after rinsing in PBS several times, the slides were maintained in a biotinylated secondary antibody conjugated to horseradish peroxidase (HRP). Subsequently, a staining reaction was performed by using a DAB kit.
为了研究 KIF4A 水平与 ccRCC 之间的可能关系，随后进行了免疫组化 （IHC） 测定。简而言之，用 4% PFA 固定样品，然后切成 5 um 切片，并用 2% BSA 的 PBS 溶液封闭 30 分钟。然后将玻片与靶向 KIF4A 的抗体在 4°C 下孵育过夜。随后，在 PBS 中冲洗数次后，将玻片保存在与辣根过氧化物酶 （HRP） 偶联的生物素化二抗中。随后，使用 DAB 试剂盒进行染色反应。
KIF4A is mainly located in the nucleus of ccRCC cells. The percentage of positively stained cells was scored as follows: 0, negative staining; 1, 10–50% positive tumor cells and 2, >50% positive-stained cells. The intensity of KIF4A staining was evaluated as 0 (no staining), 1 (moderate staining) and 2 (strong staining). The expression level of KIF4A was analysed based on the staining index: the staining intensity score was multiplied by the positive tumor cell staining score. KIF4A staining scored 0, 1 and 2 were considered low-expression, other scores of 3 and 4 were evaluated as high-expression.
KIF4A 主要位于 ccRCC 细胞的细胞核中。阳性染色细胞的百分比评分如下：0，阴性染色;1， 10–50% 阳性肿瘤细胞和 2， >50% 阳性染色细胞。KIF4A 染色强度评价为 0 （无染色） 、 1 （中度染色） 和 2 （强染色）。根据染色指数分析 KIF4A 的表达水平：染色强度评分乘以阳性肿瘤细胞染色评分。KIF4A 染色评分为 0 、 1 和 2 分为低表达，其他 3 分和 4 分评价为高表达。
Cell Culture and Transfection
细胞培养和转染
Human ccRCC cell lines, HTB-47 and CRL-1932, were obtained from ATCC. Both HTB-47 and CRL-1932 cells were maintained in RPMI1640 medium with 10% fetal bovine serum addition (Gibico, CA, USA) in a 5% CO 2 incubator.
人 ccRCC 细胞系 HTB-47 和 CRL-1932 购自 ATCC。HTB-47 和 CRL-1932 细胞均维持在添加 10% 胎牛血清RPMI1640培养基（Gibico，CA，USA）中，在 5% CO 2 培养箱中。
KIF4A shRNA plasmids were transfected into HTB-47 and CRL-1932 cells were conducted using lipofectamine 2000 (11668019, Invitrogen, Carlsbad, CA, USA). Stable knockdown of HTB-47 cells was obtained by its shRNA lentivirus infection and the selection was performed by puromycin supplementation and used in animal experiments.
将 KIF4A shRNA 质粒转染到 HTB-47 中，并使用 lipofectamine 2000（11668019，Invitrogen，Carlsbad，CA，USA）进行 CRL-1932 细胞的检测。通过其 shRNA 慢病毒感染获得 HTB-47 细胞的稳定敲低，并通过补充嘌呤霉素进行筛选并用于动物实验。
Quantitative PCR Assay 定量 PCR 检测
Total RNA from human ccRCC cells were extracted via Trizol reagent (15596026, Invitrogen, Carlsbad, CA, USA). Then, the RNA was reverse-transcribed into cDNA by M-MLV reverse transcriptase (M1701, Promega, Madison, Wisconsin, USA).
通过 Trizol 试剂 （15596026， Invitrogen， Carlsbad， CA， USA） 从人 ccRCC 细胞中提取总 RNA。然后，通过 M-MLV 逆转录酶 （M1701， Promega， Madison， Wisconsin， USA） 将 RNA 逆转录成 cDNA。
SYBR Ex Taq kit (638319, Takara, Japan) was utilized to conduct Quantitative RT-PCR and the relative KIF4A expression level was normalized to the level of GAPDH.
使用 SYBR Ex Taq 试剂盒 （638319， Takara， Japan） 进行定量 RT-PCR，并将相对 KIF4A 表达水平标准化为 GAPDH 水平。
Immunoblot Assays 免疫印迹检测
Extract ccRCC cells or tissue proteins with RIPA buffer (9800, Cell Signaling, Danvers, MA). Then, the protein was separated by SDS-PAGE, and subsequently transferred protein onto polyvinylidene fluoride (PVDF) membranes and blocked with 5% BSA. Membranes were incubated with primary antibodies targeting KIF4A, Ki67, PCNA, and β-actin, respectively, and then incubated with HRP-conjugated secondary antibodies for 1 hr. Visualize signals with ECL kit. To analyze the intensity of each band, ImageJ software was used.
用 RIPA 缓冲液（9800，Cell Signaling，Danvers，MA）提取 ccRCC 细胞或组织蛋白。然后，通过 SDS-PAGE 分离蛋白质，随后将蛋白质转移到聚偏二氟乙烯 （PVDF） 膜上，并用 5% BSA 封闭。将膜与分别靶向 KIF4A、Ki67、PCNA 和 β-肌动蛋白的一抗一起孵育，然后与 HRP 偶联的二抗孵育 1 小时。使用 ECL 试剂盒可视化信号。为了分析每个波段的强度，使用了 ImageJ 软件。
Cell Proliferation Assays
细胞增殖检测
For colony formation assay, about 500 ccRCC cells transfected with control or KIF4A shRNA plasmids were seeded into 6-well culture plates. After culturing for 2 weeks, the cells were fixed with PFA and dyed with 0.2% crystal violet for 20 mins at room temperature, and then manually counted the number of colonies.
对于集落形成测定，将约 500 个用对照或 KIF4A shRNA 质粒转染的 ccRCC 细胞接种到 6 孔培养板中。培养 2 周后，用 PFA 固定细胞，并在室温下用 0.2% 结晶紫染色 20 分钟，然后手动计数菌落数。
For MTT assays, cells were seeded in 96-well culture dishes at a density of about 1000 cells. After adherence, incubated cells with MTT. After washing with PBS, dimethyl sulfoxide (DMSO) was added to each well, and the absorbance was measured at 570 nm using a microplate reader.
对于 MTT 测定，将细胞以约 1000 个细胞的密度接种在 96 孔培养皿中。贴壁后，用 MTT 孵育细胞。用 PBS 洗涤后，向每个孔中加入二甲基亚砜 （DMSO），并使用酶标仪在 570 nm 处测量吸光度。
Tumor Growth Assays 肿瘤生长检测
Nude BalB/c mice (6–8 weeks, 18–22 g, female) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). For tumor growth assay, HTB-47 cells transfected with control or KIF4A shRNA lentivirus were injected subcutaneously into athymic Nude BalB/c mice. After 2 weeks, the tumor of each mouse was isolated and photographed. And the tumor volume was monitored every 3 days and marked the growth curve of tumor growth. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The animal study was carried out in accordance with the guidelines approved by the Animal Experimentation Ethics Committee of Shanxi Bethune Hospital. The protocol was approved by the Committee, and all surgery and all efforts were made to minimize suffering.
裸体 BalB/c 小鼠 （6-8 周，18-22 g，雌性） 购自北京维达河实验动物科技有限公司 （中国北京）。对于肿瘤生长测定，将转染对照或 KIF4A shRNA 慢病毒的 HTB-47 细胞皮下注射到无胸腺裸 BalB/c 小鼠中。2 周后，分离每只小鼠的肿瘤并拍照。每 3 d 监测一次肿瘤体积，并标记肿瘤生长的生长曲线。遵循了所有适用的国际、国家和/或机构关于动物护理和使用指南。动物研究是根据山西白求恩医院动物实验伦理委员会批准的指南进行的。该方案得到了委员会的批准，所有手术和所有努力都已降至最低。
Semi-Quantitative Analysis Assay for the Results of Immunohistochemistry in vivo
体内免疫组织化学结果的半定量分析测定
The expression of KIF4A or Ki67 was determined with anti-KIF4A or anti-Ki67 antibody in paraffin tumor using an immunohistochemistry kit (ZSGB-BIO, pv6000, Chian) according to the manufacture’s protocols. Briefly, isolated tumor tissues were fixed into 10% neutral-buffered formalin and then embedded in paraffin blocks. Then, the embedded tissues were cut into 4 μm sections. After antigens retrieval in a microwave oven for 15 mins and endogenous peroxidase blockage, sections were added with indicated antibodies overnight at 4°C. And then, goat anti-rabbit antibody was added at 37°C for 1 hr. Added DAB to visualize signals. Images were taken under a microscope (Olympus BX43).
根据制造商的方案，使用免疫组织化学试剂盒 （ZSGB-BIO， pv6000， Chian） 用抗 KIF4A 或抗 Ki67 抗体在石蜡瘤中测定 KIF4A 或 Ki67 的表达。简而言之，将分离的肿瘤组织固定到 10% 中性缓冲福尔马林中，然后包埋在石蜡块中。然后，将包埋的组织切成 4 μm 的切片。在微波炉中修复抗原 15 分钟并封闭内源性过氧化物酶后，在 4°C 下加入指定抗体切片过夜。 然后，在 37°C 下加入山羊抗兔抗体 1 小时。添加了 DAB 以可视化信号。在显微镜下拍摄图像 （Olympus BX43）。
Statistics 统计学
GraphPad 6 was used for data analysis in this study. All results were represented as mean ± SD. For statistical significance, Student’s t-test was used. The Kaplan–Meier curve method was performed for survival analysis. Additionally, P<0.05 was considered significantly different.
本研究使用 GraphPad 6 进行数据分析。所有结果均表示为 SD ±平均值。对于统计学意义，使用 Student t 检验。采用 Kaplan-Meier 曲线法进行生存分析。此外，P<0.05 被认为存在显著差异。
Results 结果
KIF4A Expression Correlates with ccRCC
KIF4A 表达与 ccRCC 相关
KIF4A has been reported to be important in the promotion of cancer development. Bioinformatic analysis was conducted to analyze the expression level of KIF4A in ccRCC tissues and normal kidney tissues. As shown in Figure 1, KIF4A expression in ccRCC tissues (n = 523) was increased at mRNA levels compared to normal tissues (n = 100) (Figure 1A). According to the expression pattern KIF4A in patients, all ccRCC patients can be divided into high group (n=254, from TCGA dataset) and low group (n=257, from TCGA data set), as shown in Figure 1B. We noticed patients with high KIF4A expression had poor overall survival (OS) and disease-free survival (DFS). These data suggest that KIF4A may be an adverse prognosis in ccRCC patients. To further verify the potential role of KIF4A in ccRCC, we detected its expression level in ccRCC tissue samples by IHC assays. We found KIF4A mainly located in the nucleus of ccRCC cells (Figure 2A). Consistently, adjacent tissues exhibited low or no KIF4A expression compared with ccRCC tissues (Figure 2A and andBB).
据报道，KIF4A 在促进癌症发展方面很重要。进行生物信息学分析，分析 kif4A 在 ccRCC 组织和正常肾组织中的表达水平。如图 1 所示，与正常组织 （n = 100） 相比，ccRCC 组织 （n = 523） 中 KIF4A 在 mRNA 水平上的表达增加（图 1A）。根据 KIF4A 患者表达模式，所有 ccRCC 患者可分为高组（n=254，来自 TCGA 数据集）和低组（n=257，来自 TCGA 数据集），如图 1B 所示。我们注意到 KIF4A 高表达的患者总生存期 （OS） 和无病生存期 （DFS） 较差。这些数据表明 KIF4A 可能是 ccRCC 患者的不良预后。为了进一步验证 KIF4A 在 ccRCC 中的潜在作用，我们通过 IHC 测定检测了其在 ccRCC 组织样本中的表达水平。我们发现 KIF4A 主要位于 ccRCC 细胞的细胞核中 （图 2A）。与 ccRCC 组织相比，癌旁组织始终表现出低或无 KIF4A 表达（图 2A 和 andBB）。
KIF23 expression at mRNA level on pancreatic tumor tissue and normal pancreatic tissues and is associated with poor prognosis. (A) KIF4A expression at mRNA level was high in 523 tumor tissues and low in 100 normal tissues. (B) Pancreatic cancer patients with high KIF4A expression had poor overall survival (OS) and poor disease-free survival (DFS).
KIF23 在胰腺肿瘤组织和正常胰腺组织上的 mRNA 水平表达，与不良预后相关。（A） 523 个肿瘤组织中 mRNA 水平的 KIF4A 表达高，100 个正常组织中低。（B） KIF4A 高表达的胰腺癌患者总生存期 （OS） 和无病生存期 （DFS） 较差。
KIF4A was highly expressed in human ccRCC tissues. (A) Immunohistochemical assays were performed, and the representative photographs of KIF4A expression in ccRCC tissues were shown (×100 and ×200 magnification, respectively). (B) IHC staining revealed the expression level of KIF4A in the adjacent liver tissues (×100 and ×200 magnification, respectively).
KIF4A 在人 ccRCC 组织中高表达。（A） 进行免疫组织化学测定，并显示 KIF4A 在 ccRCC 组织中表达的代表性照片 （分别为 ×100 和 ×200 放大倍数）。（B） IHC 染色显示 KIF4A 在癌旁肝组织中的表达水平 （分别为 ×100 和 ×200 放大）。
Then, 78 patient samples were divided into KIF4A low and high-expression groups based on the staining scored listed in Materials and methods (Figure 2A and Table 1). Forty patients were evaluated as low expression of KIF4A, whereas 38 of them exhibited high-expression levels (Table 1).
然后，根据材料和方法中列出的染色评分，将 78 例患者样本分为 KIF4A 低表达组和高表达组（图 2A 和表 1）。40 例患者被评估为 KIF4A 低表达，而其中 38 例表现出高表达水平（表 1）。
We then analysed the clinical-pathological characteristics, such as age, gender, tumor stage, tumor grade and size in ccRCC patients. It is worth noting that no significant correlations were found between patient age, gender and tumor grade between KIF4A low and high-expression groups (Table 1). Interestingly, the expression level of KIF4A was correlated with tumor stage (P=0.034) and tumor size (P=0.028) in ccRCC patients (Table 1).
然后，我们分析了 ccRCC 患者的临床病理特征，例如年龄、性别、肿瘤分期、肿瘤分级和大小。值得注意的是，在 KIF4A 低表达组和高表达组之间未发现患者年龄、性别和肿瘤分级之间存在显着相关性（表 1）。有趣的是，KIF4A 的表达水平与 ccRCC 患者的肿瘤分期 （P=0.034） 和肿瘤大小 （P=0.028） 相关 （表 1）。
Taken together, our results demonstrated that KIF4A correlates with ccRCC.
综上所述，我们的结果表明 KIF4A 与 ccRCC 相关。
Knockdown of KIF4A Weakened ccRCC Cell Proliferation in vitro
敲低 KIF4A 减弱 ccRCC 细胞体外增殖
To explore the potential function of KIF4A in ccRCC, the KIF4A shRNA plasmids were transfected into t HTB-47 and CRL-1932 cells, to knockdown KIF4A expression. The reduction of KIF4A in HTB-47 and CRL-1932 cells confirmed the knockdown efficiency by quantitative RT-PCR assays (Figure 3A). The reduction of KIF4A expression was further proved via Immunoblot assays in HTB-47 and CRL-1932 cells (Figure 3B).
为探究 KIF4A 在 ccRCC 中的潜在功能，将 KIF4A shRNA 质粒转染到 t HTB-47 和 CRL-1932 细胞中，以敲低 KIF4A 表达。HTB-47 和 CRL-1932 细胞中 KIF4A 的减少证实了定量 RT-PCR 测定的敲低效率（图 3A）。通过在 HTB-47 和 CRL-1932 细胞中进行免疫印迹测定进一步证明 KIF4A 表达的降低（图 3B）。
KIF4A expression was effectively inhibited in both HTB-47 and CRL-1932 human ccRCC cells caused by its shRNA plasmids. (A) Quantitative RT-PCR assays revealed the dramatically reduced expression level of KIF4A caused by its shRNA in HTB-47 and CRL-1932 cells, respectively. (B) Immunoblot assays confirmed the efficiently silencing of KIF4A caused by its shRNA in HTB-47 and CRL-1932 cells. Results are presented as mean ± SD, *P < 0.05.
KIF4A 在其 shRNA 质粒引起的 HTB-47 和 CRL-1932 人 ccRCC 细胞中表达均被有效抑制。（A） 定量 RT-PCR 测定显示 KIF4A 的 shRNA 在 HTB-47 和 CRL-1932 细胞中的表达水平分别显著降低。（B） 免疫印迹测定证实了 KIF4A 在 HTB-47 和 CRL-1932 细胞中由 shRNA 引起的有效沉默。结果以 SD ±平均值表示，*P < 0.05。
To evaluate the role of KIF4A in cell proliferation, colony formation assays were performed. The knockdown of KIF4A dramatically inhibited the proliferation of ccRCC cells, assessed by the significant reduction in colony numbers (Figure 4A). Similarly, through MTT assays, we observed an obvious decreased absorbance value in both HTB-47 and CRL-1932 cells with KIF4A ablation (Figure 4B). We also assessed the expression levels of cell proliferation markers, Ki67 and PCNA. Consistently, KIF4A ablation resulted in an obvious reduction in Ki67 and PpCNA expression levels in HTB-47 and CRL-1932 cells (Figure 4C and andDD).
为了评估 KIF4A 在细胞增殖中的作用，进行了集落形成测定。KIF4A 的敲除显着抑制了 ccRCC 细胞的增殖，通过菌落数量的显着减少来评估（图 4A）。同样，通过 MTT 测定，我们观察到 KIF4A 消融的 HTB-47 和 CRL-1932 细胞的吸光度值明显降低（图 4B）。我们还评估了细胞增殖标志物 Ki67 和 PCNA 的表达水平。一致地，KIF4A 消融导致 HTB-47 和 CRL-1932 细胞中 Ki67 和 PpCNA 表达水平明显降低（图 4C 和 andDD）。
KIF4A promotes ccRCC cell proliferation in vitro. (A) HTB-47 and CRL-1932 cells transfected with control or KIF4A shRNA, and the proliferation capacity was quantified by colony formation assays. (B) The results of MTT assays showed the inhibition of cell proliferation caused by KIF4A depletion. (C) Immunoblot assays showed Ki67 expression level in control or KIF4A knockdown HTB-47 and CRL-1932 cells. (D) Immunoblot assays revealed the expression level of PCNA in control or KIF4A ablation ccRCC cells. Results are presented as mean ± SD, *P < 0.05.
KIF4A 在体外促进 ccRCC 细胞增殖。（A） 用对照或 KIF4A shRNA 转染的 HTB-47 和 CRL-1932 细胞，并通过集落形成测定对增殖能力进行定量。（B） MTT 测定结果显示 KIF4A 耗竭引起的细胞增殖受到抑制。（C） 免疫印迹试验显示 Ki67 在对照或 KIF4A 敲低 HTB-47 和 CRL-1932 细胞中的表达水平。（D） 免疫印迹法显示 PCNA 在对照或 KIF4A 消融 ccRCC 细胞中的表达水平。结果以 SD ±平均值表示，*P < 0.05。
In conclusion, our data suggested that KIF4A promotes ccRCC cell proliferation in vitro.
总之，我们的数据表明 KIF4A 在体外促进 ccRCC 细胞增殖。
KIF4A Promotes ccRCC Growth in Mice
KIF4A 促进小鼠 ccRCC 生长
Similar to our previous results, KIF4A ablation led to the inhibition of ccRCC cell proliferation in vitro, and then we speculated that KIF4A stimulated the development of ccRCC in mice.
与我们之前的结果类似，KIF4A 消融导致体外 ccRCC 细胞增殖受到抑制，然后我们推测 KIF4A 刺激了小鼠 ccRCC 的发育。
To confirm our hypothesis, HTB-47 cells with control or KIF4A shRNA lentivirus infection were subcutaneously injected into nude mice. After 2 weeks, the tumor was isolated, photographed, and measured. According to the tumor volume measured every 3 days, we plotted the tumor growth curve in each group. Representative photographs of tumors are displayed in Figure 5A. Interestingly, the volume of tumors in KIF4A-depleted groups was significantly smaller than the control groups (Figure 5A).
为了证实我们的假设，将感染对照或 KIF4A shRNA 慢病毒的 HTB-47 细胞皮下注射到裸鼠体内。2 周后，分离肿瘤，拍照并测量。根据每 3 天测量一次的肿瘤体积，我们绘制了每组的肿瘤生长曲线。肿瘤的代表性照片如图 5A 所示。有趣的是，KIF4A 耗竭组的肿瘤体积显着小于对照组（图 5A）。
KIF4A facilitated ccRCC growth in mice. (A) Hep3B cells infected with control or KIF4A shRNA lentivirus were subcutaneously implanted into nude mice. After 2 weeks, tumors were isolated, and volume was examined every 3 days (n=5 in each group). Tumor growth curve was calculated and analyzed according to the average volume of six tumors in each group. (B) IHC assays indicated the expression level of KIF4A in control or KIF4A depletion tumor tissues isolated from mice. (C) IHC assays revealed the expression level of Ki67 in control or KIF4A depletion tumor tissues taken from mice. Results are presented as mean ± SD, *P < 0.05.
KIF4A 促进了小鼠 ccRCC 的生长。（A） 将感染对照或 KIF4A shRNA 慢病毒的 Hep3B 细胞皮下植入裸鼠体内。2 周后，分离肿瘤，每 3 天检查一次体积 （每组 n=5）。根据每组 6 个肿瘤的平均体积计算分析肿瘤生长曲线。（B） IHC 测定显示 KIF4A 在对照或从小鼠分离的 KIF4A 耗竭肿瘤组织中的表达水平。（C） IHC 测定揭示了 Ki67 在对照或小鼠 KIF4A 耗竭肿瘤组织中的表达水平。结果以 SD ±平均值表示，*P < 0.05。
Additionally, silencing of KIF4A was confirmed by IHC assays (Figure 5B). We also examined the expression level of Ki67 in control and KIF4A knockdown groups by IHC assays. As expected, Ki67 reduction was observed in tumors in the KIF4A knockdown groups, which indicates that KIF4A knockdown impaired proliferation capacity (Figure 5C). Therefore, we showed that the involvement of KIF4A in the regulation of ccRCC development in mice.
此外，通过 IHC 测定证实了 KIF4A 的沉默（图 5B）。我们还通过 IHC 测定检测了对照组和 KIF4A 敲低组中 Ki67 的表达水平。正如预期的那样，在 KIF4A 敲低组的肿瘤中观察到 Ki67 减少，这表明 KIF4A 敲低损害了增殖能力（图 5C）。因此，我们表明 KIF4A 参与小鼠 ccRCC 发育的调节。
Discussion 讨论
There are four types of renal carcinoma: clear cell renal carcinoma, granulosa cell renal carcinoma, mixed cell renal carcinoma, and undifferentiated cell renal carcinoma.18 Among them, the vast majority are renal clear cell carcinoma, accounting for 70%~80% of renal carcinoma.19,20 Renal clear cell carcinoma is often asymptomatic in the early stage, or only fever, fatigue and other systemic symptoms; tumor volume was found to increase. However, in the advanced stage, existing treatments have little effect on ccRCC.21,22 New treatments are urgently needed to combat this disease. Through the bioinformation analysis, we noticed the high mRNA levels of KIF4A in human ccRCC tissues, and KIF4A expression was correlated with the prognosis of ccRCC patients, indicating that KIF4A can be used as a prognostic indicator of ccRCC.
肾癌有四种类型：透明细胞肾癌、颗粒细胞肾癌、混合细胞肾癌和未分化细胞肾癌。 18 其中，绝大多数为肾透明细胞癌，占肾癌的 70%~80%。 19 20 肾透明细胞癌早期常无症状，或仅出现发热、乏力等全身症状;发现肿瘤体积增加。然而，在晚期，现有治疗方法对 ccRCC 影响不大。 21 22 迫切需要新的治疗方法来对抗这种疾病。通过生物信息分析，我们注意到人 ccRCC 组织中 KIF4A 的 mRNA 水平很高，KIF4A 表达与 ccRCC 患者的预后相关，表明 KIF4A 可以作为 ccRCC 的预后指标。
Performing IHC assays, we noticed 78 human ccRCC tissue samples and their corresponding non-tumor adjacent tissue samples, we found a relatively high-expression level of KIF4A in tumor tissues, and the expression level of KIF4A was remarkably associated with clinical features including tumor size and the number of tumor nodes. Similarly, another study indicated that high expression of KIF4A is correlated with lymph node metastasis in patients with colorectal cancer.16 These clinical data testified the possible role of KIF4A in the histopathology and progression of tumorigenesis. Further investigations are consistent with the hypothesis that KIF4A serves as a trigger of ccRCC progression. Performing IHC assays, we also found the decrease expression of Ki67 in tumor tissues isolated from mice of KIF4A depletion groups, which further confirmed the critical role of KIF4A on the regulation of ccRCC cell proliferation.
进行 IHC 分析，我们注意到 78 例人 ccRCC 组织样本及其相应的非肿瘤邻近组织样本，我们发现 KIF4A 在肿瘤组织中的表达水平相对较高，并且 KIF4A 的表达水平与肿瘤大小和肿瘤淋巴结数量等临床特征显著相关。同样，另一项研究表明，KIF4A 的高表达与结直肠癌患者的淋巴结转移相关。 16 这些临床数据证明了 KIF4A 在组织病理学和肿瘤发生进展中的可能作用。进一步的研究与 KIF4A 是 ccRCC 进展的触发因素的假设一致。进行 IHC 测定，我们还发现从 KIF4A 耗竭组小鼠分离的肿瘤组织中 Ki67 的表达降低，这进一步证实了 KIF4A 在调节 ccRCC 细胞增殖中的关键作用。
KIF4A is a member of KIF4 subfamily, which consists of KIF4A, KIF4B, KIF21A and KIF21B.23,24 Various studies have reported that the KIF4 subfamily is critical in tumorigenesis and progression.25,26 It is reported that KIF4A was a new component of the chromosome segregation machinery and acted critical roles in mediating spindle organization and cytokinesis.23 All these functions suggest that KIF4A may have an impact on tumorigenesis and development. In fact, KIF4A plays as an oncogene and participates in the progression of several malignancies, including breast cancer, oral cancer, and cervical cancer.16,27 KIF4A stimulates cell migration and invasion in lung cancer,17 and it was found to regulate cell proliferation by the activation of spindle assembly checkpoint (SAC).28 Additionally, KIF4A promotes the proliferation of colorectal cancer by the regulation of p21-mediated cell cycle progression, and they found KIF4A knockdown obviously decreased the expression level of pAKT, and the expression level of MEK and ERK was comparable, suggesting that KIF4A promotes CRC proliferation through PI3K/AKT signaling pathway.15 Interestingly, we found that KIF4A contributed to cell proliferation of ccRCC in vitro and in mice. Whether KIF4A promotes ccRCC through this signaling pathway also requires further study.
KIF4A 是 KIF4 亚家族的成员，该亚家族由 KIF4A、KIF4B、KIF21A 和 KIF21B 组成。 23 24 各种研究报道，KIF4 亚家族在肿瘤发生和进展中起关键作用。 25 26 据报道，KIF4A 是染色体分离机制的新组成部分，在介导纺锤体组织和胞质分裂中起关键作用。 23 所有这些功能表明 KIF4A 可能对肿瘤发生和发展产生影响。事实上，KIF4A 作为癌基因发挥作用，参与多种恶性肿瘤的进展，包括乳腺癌、口腔癌和宫颈癌。 16 27 KIF4A 刺激肺癌中的细胞迁移和侵袭， 17 并发现它通过激活纺锤体组装检查点 （SAC） 来调节细胞增殖。 28 此外，KIF4A 通过调节 p21 介导的细胞周期进程促进结直肠癌的增殖，他们发现 KIF4A 敲低显著降低了 pAKT 的表达水平，MEK 和 ERK 的表达水平相当，表明 KIF4A 通过 PI3K/AKT 信号通路促进 CRC 增殖。 15 有趣的是，我们发现 KIF4A 在体外和小鼠中促进了 ccRCC 的细胞增殖。KIF4A 是否通过该信号通路促进 ccRCC 也需要进一步研究。
KIF4A was known to be involved in the regulation of mitosis.29 Previous studies have indicated KIF4A could be phosphorylated by CDK1 at S1186, triggered chromosome compaction.23,30 Meanwhile, KIF4A affected the spindle formation and chromosome segregation in oocytes.31,32 These studies suggest KIF4A has the potential to affect the cell cycle and proliferation of cancer cells. Interestingly, here we noticed that KIF4A promoted cell proliferation in ccRCC, and therefore thought KIF4A affected the proliferation via mediating mitosis.
已知 KIF4A 参与有丝分裂的调节。 29 既往研究表明 KIF4A 可以在 S1186 位点被 CDK1 磷酸化，触发染色体压缩。 23 30 同时，KIF4A 影响卵母细胞的纺锤体形成和染色体分离。 31 32 这些研究表明 KIF4A 有可能影响癌细胞的细胞周期和增殖。有趣的是，我们注意到 KIF4A 促进了 ccRCC 中的细胞增殖，因此认为 KIF4A 通过介导有丝分裂影响增殖。
In addition to KIF4A, other members of KIF family also play various roles in the tumor development and metastasis.33 KIF7 could promote the growth of prostate tumor through LKB1-mediated AKT inhibition.34 KIFC1 was associated with the progression of HCC.35 Additionally, KIF3A participated in the development of breast cancer.36 These studies, together with our results, indicate the possible roles of KIFs as potential therapeutic targets.
除了 KIF4A，KIF 家族的其他成员也在肿瘤发生和转移中发挥各种作用。 33 KIF7 可通过 LKB1 介导的 AKT 抑制促进前列腺肿瘤的生长。 34 KIFC1 与 HCC 的进展相关。 35 此外，KIF3A 参与了乳腺癌的发展。 36 这些研究以及我们的结果表明 KIF 作为潜在治疗靶点的可能作用。
Collectively, our results demonstrated that KIF4A was highly expressed in human HCC tissues. We also found a correlation between KIF4A expression level and clinical characteristics in ccRCC patients. Furthermore, KIF4A promoted the proliferation of ccRCC cells in vitro and in mice. Therefore, we have a preliminary mechanism study of KIF4A in ccRCC development and provide a novel therapeutic target for the treatment of ccRCC.
总的来说，我们的结果表明 KIF4A 在人 HCC 组织中高表达。我们还发现 ccRCC 患者 KIF4A 表达水平与临床特征之间存在相关性。此外，KIF4A 在体外和小鼠中促进 ccRCC 细胞的增殖。因此，我们对 KIF4A 在 ccRCC 开发中的机制进行了初步研究，并为 ccRCC 的治疗提供了新的治疗靶点。
Data Sharing Statement 数据共享声明
The dataset supporting the conclusions of this article is included in the article.
支持本文结论的数据集包含在文章中。
Disclosure 披露
The authors declare that they have no competing interests in this work.
作者声明他们在这项工作中没有利益冲突。
References 引用
Bie L， Zhao G， Cheng P， et al.通过测量有丝分裂纺锤体检查点基因表达，可以提高胶质瘤患者生存时间预测的准确性。公共科学图书馆一号。2011;6（10）：e25631。doi： 10.1371/journal.pone.0025631 [ PMC 免费文章] [ PubMed] [ CrossRef] [ Google Scholar]
墨菲 KA、詹姆斯 BR、威尔伯 A、格里菲斯 TS。转移性肾细胞癌的同基因小鼠模型，用于临床前治疗的定量和纵向评估。J Vis Exp. 2017;122：e55080 的。[ PMC 免费文章][ 公共医学][ 谷歌学术搜索]
Lane BR，Kattan MW。预测肾细胞癌的预后。Curr Opin Urol. 2005;15(5):289–297.doi： 10.1097/01.mou.0000178336.94991.17 [ PubMed] [ CrossRef] [ 谷歌学术]
检查点抑制剂是肾细胞癌治疗的新时代。Med Oncol.2018;35(6):85.doi： 10.1007/s12032-018-1147-y [ PubMed] [ CrossRef] [ Google 学术搜索]
Wang X， Wang T， Chen C， et al.血清外泌体 miR-210 作为透明细胞肾细胞癌的潜在生物标志物。J 细胞生物化学。2019;120(2):1492–502.[ 公共医学][ 谷歌学术搜索]
转移性透明细胞肾细胞癌治疗的不断发展前景。Clin Adv Hematol Oncol.2018;16(10):677–686.[ 公共医学][ 谷歌学术搜索]
晚期透明细胞肾细胞癌患者潜在预测标志物和对靶向治疗的反应分析。中国医学杂志 （Engl）.2015;128(15):2026–2033.doi： 10.4103/0366-6999.161353 [ PMC 免费文章] [ PubMed] [ CrossRef] [ Google Scholar]
Ricketts CJ， Linehan WM. 对 VHL 缺陷性透明细胞肾细胞癌表观遗传重塑的见解。癌症 Discov。2017;7(11):1221–1223.doi： 10.1158/2159-8290.CD-17-0971 [ PubMed] [ CrossRef] [ 谷歌学术]
Hogner A， Krause H， Jandrig B， et al. PBRM1 和 VHL 表达在人透明细胞肾细胞癌中与患者总生存期的差异相关性相关。乌罗尔·昂科尔。2018;36（3）：94e91–94 e14。doi： 10.1016/j.urolonc.2017.10.027 [ PubMed] [ CrossRef] [ Google 学术搜索]
Hirokawa N， Tanaka Y. 驱动蛋白超家族蛋白 （KIF）：各种功能及其与生活和疾病中重要现象的相关性。Exp Cell Res. 2015 年;334(1):16–25.doi： 10.1016/j.yexcr.2015.02.016 [ PubMed] [ CrossRef] [ Google Scholar]
新泽西州卡姆林，麦克劳克林 EA，霍尔特 JE。开车通过：驱动蛋白超家族蛋白在女性减数分裂中的作用。Hum Reprod 更新。2017;23(4):409–420.doi： 10.1093/humupd/dmx010 [ PubMed] [ CrossRef] [ 谷歌学术]