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Regulation of spindle and kinetochore‐associated protein 1 by antitumor miR‐10a‐5p in renal cell carcinoma

Analysis of our original microRNA (miRNA) expression signature of patients with advanced renal cell carcinoma (RCC) showed that microRNA‐10a‐5p (miR‐10a‐5p) was significantly downregulated in RCC specimens. The aims of the present study were to investigate the antitumor roles of miR‐10a‐5p and the novel cancer networks regulated by this miRNA in RCC cells. Downregulation of miR‐10a‐5p was confirmed in RCC tissues and RCC tissues from patients treated with tyrosine kinase inhibitors (TKI). Ectopic expression of miR‐10a‐5p in RCC cell lines (786‐O and A498 cells) inhibited cancer cell migration and invasion. Spindle and kinetochore‐associated protein 1 (SKA1) was identified as an antitumor miR‐10a‐5p target by genome‐based approaches, and direct regulation was validated by luciferase reporter assays. Knockdown of SKA1 inhibited cancer cell migration and invasion in RCC cells. Overexpression of SKA1 was observed in RCC tissues and TKI‐treated RCC tissues. Moreover, analysis of The Cancer Genome Atlas database demonstrated that low expression of miR‐10a‐5p and high expression of SKA1 were significantly associated with overall survival in patients with RCC. These findings showed that downregulation of miR‐10a‐5p and overexpression of the SKA1 axis were highly involved in RCC pathogenesis and resistance to TKI treatment in RCC.

Analysis of our original microRNA (miRNA) expression signature of patients with advanced renal cell carcinoma (RCC) showed that microRNA-10a-5p (miR-10a-5p) was significantly downregulated in RCC specimens. The aims of the present study were to investigate the antitumor roles of miR-10a-5p and the novel cancer networks regulated by this miRNA in RCC cells. Downregulation of miR-10a-5p was confirmed in RCC tissues and RCC tissues from patients treated with tyrosine kinase inhibitors (TKI). Ectopic expression of miR-10a-5p in RCC cell lines (786-O and A498 cells) inhibited cancer cell migration and invasion. Spindle and kinetochore-associated protein 1 (SKA1) was identified as an antitumor miR-10a-5p target by genome-based approaches, and direct regulation was validated by luciferase reporter assays. Knockdown of SKA1 inhibited cancer cell migration and invasion in RCC cells. Overexpression of SKA1 was observed in RCC tissues and TKI-treated RCC tissues. Moreover, analysis of The Cancer Genome Atlas database demonstrated that low expression of miR-10a-5p and high expression of SKA1 were significantly associated with overall survival in patients with RCC. These findings showed that downregulation of miR-10a-5p and overexpression of the SKA1 axis were highly involved in RCC pathogenesis and resistance to TKI treatment in RCC.
R enal cell carcinoma accounts for approximately 2-5% of adult malignancies worldwide, and approximately 350 000 new cases are diagnosed, with over 140 000 deaths, each year. (1,2) Most cases of RCC (approximately 80%) are classified as ccRCC. (3) In the majority of patients, ccRCC is associated with dysfunction of the VHL gene. (4,5) Lack of VHL function causes activation of HIF and VEGF pathways in ccRCC cells. (6,7) The mTOR pathway is also activated by dysregulation of HIF and VEGF pathways in patients with ccRCC. (8,9) Based on this information regarding the molecular pathogenesis of ccRCC, molecular targeted therapies for patients with advanced and metastatic RCC have been developed during the past decade. (10) The molecular-targeted agents sorafenib, sunitinib, pazopanib, axitinib, bevacizumab, and cabozantinib inhibit VEGF and VEGF receptor pathways, and temsirolimus and everolimus inhibit the mTOR pathway; treatment with these agents has resulted in significant benefits to patients with advanced RCC. (10,11) However, the curative effects of these treatments are limited because cancer cells exhibit activation of several alternative signal cascades and acquire resistance to these treatments during therapeutic processes. (11,12) Treatment strategies for drug-resistant cancer cells are limited, and the prognosis of these patients is extremely poor. However, the molecular mechanisms of resistance to molecular-targeted therapies in RCC cells are still unclear. miRNAs act as pivotal players that regulate the expression control of protein-coding/protein-noncoding RNAs in a sequence-dependent manner. (13,14) Notably, a single miRNA can directly control many mRNAs in human cells. (15) Therefore, aberrantly expressed miRNAs can disrupt the tight control of RNA expression in cancer cells. Moreover, dysregulation of miRNAs is deeply involved in cancer cell progression, metastasis, and drug resistance. (16)(17)(18)(19) In RCC, miRNAs are closely related to the development of cancer, and previous studies have reported the relationships among many miRNAs and RCC. For example, the miR-200 family, containing miR-141, miR200a/b/c, and miR-429, forms two clusters, miR-200a/200b/429 and miR-141/200c, and expression of the miR-200 family is markedly downregulated in RCC tissues. (20) Additionally, the miR-200 family has been reported to be involved in the EMT in several cancers, and miR-141 and miR-200c function as tumor suppressors in RCC by inhibiting the EMT through targeting of ZFHX1B, a transcriptional repressor for CDH1/E-cadherin. (20,21) In this way, investigation of molecular networks based on miRNAs may help to elucidate the molecular mechanisms mediating the progression of RCC. , and schematic representation of the chromosomal location of human miR-10a. (a) Kaplan-Meier survival curve for overall survival rate based on miR-10a-5p expression in patients with ccRCC from The Cancer Genome Atlas (TCGA) database. (b) miR-10a is located on human chromosome 17q21.32. Mature microRNAs (miRNAs), miR-10a-5p (guide strand) and miR-10a-3p (passenger strand), are derived from pre-miR-10a. Analysis of our original miRNA expression signature of TKI failure in patients with RCC showed that antitumor miR-101 directly regulated ubiquitin-like with PHD and ring finger domains 1 (UHRF1), which acted as an oncogene in RCC cells. (22) Based on this signature, we focused on miR-10a-5p because miR-10a-5p is significantly downregulated in TKItreated ccRCC compared with primary ccRCC. (22) Moreover, The Cancer Genome Atlas (23) database showed that the overall survival of patients in the low miR-10a-5p expression group was significantly shorter than that of patients in the high expression group in ccRCC (P = 0.00991, Fig. 1a).
The aims of the present study were to investigate the functional significance of miR-10a-5p and to identify the molecular targets regulated by miR-10a-5p in ccRCC cells. Our data showed that restoration of mature miR-10a-5p inhibited ccRCC cell proliferation, migration, and invasion. Furthermore, our data demonstrated that the SKA1 gene was overexpressed in primary RCC and advanced RCC specimens and was directly regulated by miR-10a-5p in RCC cells. These results demonstrated that SKA1 was involved in RCC pathogenesis, implying that SKA1 could be a novel diagnostic and therapeutic target for patients with advanced RCC.

Materials and Methods
ccRCC clinical specimens and cell culture. A total of 15 pairs of ccRCC specimens and adjacent noncancerous specimens were collected from patients who had undergone radical nephrectomy at Chiba University Hospital (Chiba, Japan) from 2012 to 2015. Clinicopathological characteristics of the patients are summarized in Table 1. Four patients who died of ccRCC after TKI treatment underwent autopsy at Teikyo University Chiba Medical Center Hospital from 2012 to 2016, as summarized in Table 2. These specimens were staged according to the General Rule for Clinical and Pathological Studies on Renal Cell Carcinoma based on the American Joint Committee on Cancer (AJCC)-UICC TNM classification. (24) Written consent for tissue donation for research purposes was obtained from each patient before tissue collection. We used two human ccRCC cell lines (786-O and A498) obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) as previously described. (22,(25)(26)(27)(28) RNA extraction. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol, as described previously. (22,(25)(26)(27)(28) RNA quality was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Statistical analysis. Relationships between two groups and the numerical values obtained by qRT-PCR were analyzed using Mann-Whitney U-tests and paired t-tests. Spearman's rank test was used to evaluate the correlation between the expression levels of miR-10a-5p and SKA1. Relationships among more than three variables and numerical values were analyzed using Bonferroni-adjusted Mann-Whitney U-tests. Survival analysis was carried out using the Kaplan-Meier method and log-rank tests with JMP software (version 13; SAS Institute Inc., Cary, NC, USA); all other analyses were carried out using Expert StatView (version 5; SAS Institute Inc.).
Effects of restoring miR-10a-5p on cell proliferation, migration, and invasion activities in ccRCC cell lines. To investigate the functional efficacy of miR-10a-5p, we carried out gain-of-function studies using miRNA transfection into 786-O and A498 cells. XTT assays showed that cell proliferation was significantly inhibited in miR-10a-5p transfectants compared with that in mock or miR-control transfectants (Figs 2b; S1a). Migration assays showed that cell migration activity was significantly inhibited in miR-10a-5p transfectants in comparison with those in mock or miR-control transfectants (Figs 2c;  S2a). Similarly, Matrigel invasion assays showed that cell invasion activity was significantly inhibited in miR-10a-5p transfectants in comparison with those in mock or miR-control transfectants (Figs 2d; S2b).
Identification of candidate genes regulated by miR-10a-5p in ccRCC cells. To further elucidate the molecular mechanisms and pathways regulated by antitumor miR-10a-5p in ccRCC cells, we carried out a combination of in silico analyses and oligo microarray analyses using miR-10a-5p transfectants. The strategy for selection of miR-10a-5p target genes is shown in Figure 3. First, we used TargetScanHuman 7.1 database and identified that 3661 genes had putative target sites for miR-10a-5p in their 3 0 -UTR. Next, we paired down the 3661 genes based on gene expression data (GEO database accession number: GSE93290), and 31 genes were markedly downregulated after transfection of 786-O and A498 cells with miR-10a-5p (fold-change log 2 < À2.0; Table 3). Finally, we checked the expression status of these 31 genes and the clinical significance of ccRCC using the OncoLnc database (http://www.onc olnc.org/). Kaplan-Meier survival curves showed that high expression of eight genes was associated with poor prognosis in ccRCC (Table 3; Figs 4a, 5). Among these genes, we focused on SKA1, which was most downregulated by transfection of miR-10a-5p, and showed the most dramatic difference in OncoLnc prognostic analysis (P = 2.72EÀ08, Fig. 4a).
Expression of SKA1 in ccRCC clinical specimens. A total of 15 pairs of primary ccRCC specimens, adjacent noncancerous specimens, and TKI-treatment failure autopsy specimens from 15 sites were used for the expression analysis of SKA1 by qRT-PCR. Expression of SKA1 was significantly upregulated in primary cancer tissues compared with that in normal tissues (P = 0.0011; Fig. 4b) and was significantly higher in autopsy specimens than in primary cancer tissues (P = 0.0011; Fig. 4b). Additionally, Spearman's rank test indicated a negative correlation between the expression levels of miR-10a-5p and SKA1 (Fig. 4c).
Furthermore, to analyze SKA1 protein expression, immunohistochemistry was carried out with a ccRCC tissue   TKI treatment compared with that in normal kidney (Fig. 6).
SKA1 was directly regulated by miR-10a-5p transfection in ccRCC cells. We carried out qRT-PCR and western blotting to validate whether restoration of miR-10a-5p in 786-O and A498 cells reduced the expression of SKA1. Expression of SKA1 mRNA was significantly suppressed by miR-10a-5p transfection compared with that in mock-or miR-control-transfected cells (Fig. 7a). Similarly, SKA1 protein expression was repressed in the miR-10a-5p transfectants (Fig. 7b).
Next, we carried out luciferase reporter assays to determine whether SKA1 mRNA had a functional target site. The Tar-getScan database predicted that miR-10a-5p bound at position 28-35 in the 3 0 -UTR of SKA1. We used vectors encoding a partial wild-type sequence of the 3 0 -UTR of SKA1 mRNA, including the predicted miR-10a-5p target site, or a vector lacking the miR-10a-5p target site. Luminescence intensity was significantly reduced by cotransfection with miR-10a-5p and the vector carrying the wild-type 3 0 -UTR of SKA1. However, luminescence intensity was not suppressed when the target site of miR-10a-5p was deleted from the vectors (Fig. 7c).
Effects of silencing SKA1 in ccRCC cell lines. To examine the functional significance of SKA1, we carried out loss-of-function studies using si-SKA1 transfectants. First, we evaluated the knockdown efficiency of si-SKA1 transfection in 786-O and A498 cells. In this study, we used two types of si-SKA1 (si-SKA1-1 and si-SKA1-2). qRT-PCR and western blotting analyses showed that transfection with both siRNAs effectively downregulated SKA1 mRNA and SKA1 protein expression in 786-O and A498 cells (Fig. 8a,b). Furthermore, functional assays indicated that si-SKA1 transfection markedly inhibited cell proliferation, migration, and invasion in comparison with mock-or si-control-transfected cells (Figs 8c; S1b, S3a, S4a).
Effects of cotransfection of SKA1/miR-10a-5p in 786-O cells. To validate whether the molecular pathway of SKA1/miR-10a-5p was critical for the progression of ccRCC, we carried out SKA1 rescue experiments by cotransfection with SKA1 and miR-10a-5p in 786-O cells. SKA1 protein expression by Western blotting analysis is shown in Figure 9a. Functional assays showed that the migration and invasion abilities of ccRCC cells were recovered by SKA1 and miR-10a-5p transfection compared with cells with restored miR-10a-5p only (Figs 9b-Fig. 6. Expression of SKA1 in clinical clear cell renal cell carcinoma (ccRCC) specimens using a tissue microarray and autopsy tissues. Representative immunohistochemical staining for SKA1 in a ccRCC tissue microarray (cat. no. KD806; US Biomax, Inc., Rockville, MD, USA) and autopsy specimens after tyrosine kinase inhibitor (TKI) treatment (Patient D, Table 2 d; S1c, S5a,b). These results supported that SKA1 affected the aggressiveness of ccRCC cells.
Clinical significance of SKA1 in ccRCC. To explore the clinical significance of SKA1 in ccRCC, we analyzed Kaplan-Meier curves of DFS rates according to the expression level of SKA1, and the relationships among SKA1 expression and cancer stage, tumor stage, and histological grade in ccRCC were evaluated using the TCGA-KIRC database. Kaplan-Meier curves for DFS rates showed that the DFS of the high SKA1 expression group was significantly shorter than that of the low expression group in ccRCC (P < 0.0001, Fig. 10a). Additionally, expression levels of SKA1 were significantly increased in cases of advanced disease stage, advanced T stage, and advanced histological grade (Fig. 10b-d). These analyses suggested that SKA1 affected disease progression and malignancy in ccRCC. Similarly, TCGA data analysis results of clinical significance for the other seven genes are shown in Figures S6-S9.

Discussion
Based on the underlying molecular oncogenic mechanisms of RCC, several molecular targeted agents have been developed to improve the prognosis of patients with advanced RCC. (10,11) However, although almost all patients with RCC respond to initial treatment with molecular targeted therapies, cancer cells ultimately become resistant to these treatments. (12,32,33) Several molecular mechanisms of drug resistance have been reported in RCC; however, all of these mechanisms are not sufficient to explain the observed changes in cancer cells. Understanding these molecular mechanisms using current genomic approaches is the first step to overcoming drug resistance in RCC cells.
To investigate the molecular mechanisms of drug resistance in RCC cells, we constructed a miRNA expression signature using autopsy specimens from patients with ccRCC who exhibited TKI-treatment failure. (22) Our present data demonstrated that miR-10a-5p acted as an antitumor miRNA in RCC cells. TCGA database analyses indicated that downregulation of miR-10a-5p was associated with poor prognosis in patients with RCC. miR-10a belongs to the miR-10 family together with miR-10b, and miR-10a-5p is a guide strand of miR-10a. Previous studies have indicated that miR-10a is deregulated in several types of cancers. (34)(35)(36)(37) In different types of cancer, miR-10a has dual functions as either a cancer-promoting or cancer-suppressing miRNA. In RCC, a recent study showed that miR-10a-5p is a predictor of progression and survival for RCC; (38) however, the functional significance and relevant molecular mechanisms of miR-10a-5p in cancer progression are still unknown.
One of the main challenges in miRNA studies is identification of miRNA-target genes and RNA networks mediated by antitumor miRNAs in cancer cells. A total of 31 genes were identified as putative targets of miR-10a-5p regulation in ccRCC cells in this study. Among them, eight genes (SKA1, P4HB, ELOVL2, KCTD13, RTN4R, APAF1, ANXA7, and IMPAD1) were associated with poor prognosis in patients with RCC by TCGA analyses. We focused on the SKA1 gene because overexpression of this gene showed the most significant association with poor prognosis in RCC. Another seven genes may also be involved in the pathology of RCC. Analyses of these genes are important for elucidating the molecular mechanisms of RCC oncogenesis, metastasis and drug resistance.
It is well known that one mRNA was regulated by a number of miRNAs. TargetScan database searching showed that SKA1 was a putative target for miR-10b-5p, miR-24-3p and miR-23b-3p which were significantly downregulated in our miRNA signature of TKI treatment of RCC specimens. (22) Our previous studies demonstrated that miR-24-3p and miR-23b-3p acted as antitumor miRNAs targeting several oncogenic genes. (39,40) Also, miR-10b-5p functioned as an antitumor miRNA in several types of cancers including RCC. (41)(42)(43) We made the following hypothesis from these findings, SKA1 is regulated by several antitumor miRNAs which contribute to RCC pathogenesis and drug resistance. From the analyses of these miRNAs, it can be expected to lead to elucidation of the mechanism of drug resistance of RCC.
Several studies have indicated that SKA1 is involved in the growth and proliferation of various types of cancer, including oral adenosquamous and hepatocellular carcinoma, bladder cancer, gastric cancer, prostate cancer, thyroid cancer, non-small cell lung cancer, and glioblastoma. (44)(45)(46)(47)(48)(49)(50)(51) In the human genome, SKA1, SKA2, and SKA3 form the SKA complex. During mitosis, the SKA complex is localized between the outer kinetochore interface and the spindle microtubules. This complex is indispensable for stabilizing adhesion of spindle microtubules to kinetochores and maintaining the metaphase plate; therefore, this complex is critical for appropriate chromosome segregation during mitosis. (52)(53)(54) Interestingly, the expression of SKA1 contributes to cisplatin resistance in lung cancer cells by protecting the cells from cisplatin-induced apoptosis. (50) Knockdown of SKA1 decreases the activation of extracellular signal-regulated kinase (ERK1/2) and AKT-mediated signaling pathways in lung cancer cells. (50) Another study also demonstrated that knockdown of SKA1 alleviated the activation of ERK1/2 and AKT in bladder cancer cells. (46) In adenoid cystic carcinoma, knockdown of SKA1 inhibited cell proliferation, invasion, and migration, and cell cycle arrest by regulating cell cycle-promoting genes and the matrix metalloproteinase-9 gene. (55) These findings suggested that the expression of SKA1 may be induced by cancer-promoting genes and could contribute to cancer cell aggressiveness and drug resistance. Many reports have demonstrated that acquired resistance of RCC cells to molecular targeted therapies induces cancer-promoting genes and activates several alternative pathways. (12,56,57) A previous study showed that sunitinib treatment significantly suppressed phosphorylation of ERK1/2 and AKT in TKI-sensitive RCC cells, whereas inhibition of phosphorylation was not observed in TKI-resistant RCC cells. (58) In this study, phosphorylation of ERK1/2 and AKT was suppressed by knockdown of SKA1 and restoration of miR-10a-5p. This suggests that downregulation of miR-10a-5p and overexpression of the SKA1 axis may be involved in resistance to VEGF-and mTOR-targeted treatments in RCC. In conclusion, downregulation of miR-10a-5p was detected in the miRNA signature of TKI-failure RCC and acted as an antitumor miRNA in RCC cells. To the best of our knowledge, this is the first study showing that antitumor miR-10a-5p directly regulated SKA1 in RCC cells. Overexpression of SKA1 was observed in primary and TKI-failure RCC specimens. Moreover, downregulation of miR-10a-5p and overexpression of SKA1 were associated with poor prognosis in patients with RCC. Elucidation of miR-10a-5p/SKA1-mediated molecular networks may improve our understanding of the pathogenesis of primary RCC and molecular targeted treatment failure in RCC and facilitate the development of new treatment strategies.

Disclosure Statement
Authors declare no conflicts of interest.

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