Regulation of NCAPG by miR‐99a‐3p (passenger strand) inhibits cancer cell aggressiveness and is involved in CRPC

Abstract Effective treatments for patients with castration‐resistant prostate cancer (CRPC) have not yet been established. Novel approaches for identification of putative therapeutic targets for CRPC are needed. Analyses of RNA sequencing of microRNA (miRNA) expression revealed that miR‐99a‐3p (passenger strand) is significantly downregulated in several types of cancers. Here, we aimed to identify novel miR‐99a‐3p regulatory networks and therapeutic targets for CRPC. Ectopic expression of miR‐99a‐3p significantly inhibited cancer cell proliferation, migration, and invasion in PCa cells. Non‐SMC condensin I complex subunit G (NCAPG) was a direct target of miR‐99a‐3p in PCa cells. Overexpression of NCAPG was detected in CRPC clinical specimens and was significantly associated with shorter disease‐free survival and advanced clinical stage. Knockdown of NCAPG inhibited cancer cell aggressiveness. The passenger strand miR‐99a‐3p acted as an antitumor miRNA in naïve PCa and CRPC. NCAPG was regulated by miR‐99a‐3p, and its overexpression was involved in CRPC pathogenesis. Involvement of passenger strand of miRNA in cancer pathogenesis is novel concept, and identification of antitumor miRNA regulatory networks in CRPC might be provided novel prognostic markers and therapeutic targets for this disease.


Introduction
Analysis of our original miRNA expression signatures of cancers based on RNA sequencing revealed that several passenger strands of miRNAs, for example, miR-145-3p, miR-150-3p, miR-149-3p, miR-199a-3p, and miR-144-5p, are downregulated in several cancer tissues and act as antitumor miRNAs in cancer cells [11][12][13][14][15]. However, this is inconsistent with the paradigm that the guide strand of miRNA is loaded into the miRNA-induced silencing complex (RISC) and represses translation or degradation of target genes [16], whereas the passenger strand of miRNA is thought to be destroyed in the cytoplasm and to have no function [17][18][19].
We have sequentially identified the functional significance of passenger strands of miRNAs in cancer cells based on miRNA signatures [11][12][13][14][15]. In this study, we focused on miR-99a-5p (guide strand) whose expression was significantly downregulated in our miRNA signature of metastatic CRPC [15] and investigated the functional roles including passenger strand miR-99a-3p in naïve PCa and CRPC cells. Previous studies have shown that the guide strand miR-99a-5p has antitumor roles in several cancers [20][21][22][23]. In contrast, no studies have reported the role of the passenger strand miR-99a-3p in cancer cells. Novel strategies based on passenger strands of miRNAs will enhance our understanding of the molecular pathways underlying naïve PCa and CRPC pathogenesis.

Collection of clinical prostate specimens and cell lines
Clinical specimens were collected at Teikyo University Chiba Medical Center and Chiba University Hospital from 2013 to 2016. Patient characteristics and clinical features are summarized in Table 1. The protocol of this study was approved by the Institutional Review Boards of Teikyo University and Chiba University. We have experimented with human PCa cell lines (PC3, DU145, and C4-2). The cells were maintained as previously reported [11,15,24,25].

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)
The procedure of PCR quantification is described in our previous reports [11,15,[24][25][26]. Expression levels of miR-99a-5p and miR-99a-3p normalized to expression of RNU48 were analyzed by TaqMan qRT-PCR. The expression levels of NCAPG and pri-miR-99a were assessed by being normalized with GAPDH or GUSB. Detailed product numbers of reagents used are shown in the  Table S1.

Cell proliferation, migration, and invasion assays
As functional analyses, cell proliferation, migration, and invasion assays were carried out based on our past reports [11,15,[24][25][26]. We confirmed all experiments in triplicate.

Confirmation of miRNAs incorporated into the RNA-induced silencing complex (RISC) by Ago2 immunoprecipitation
To investigate whether exogenous miR-99a-5p and miR-99a-3p were incorporated into the RISC, we carried out immunoprecipitation assays using a microRNA isolation kit for human Ago2 (Wako, Osaka, Japan). The procedure is described in our past reports [11,15].

The Cancer Genome Atlas (TCGA) database analyses of PCa
To identify the clinical significance of NCAPG, we applied to TCGA database. The gene expression and clinical data were analyzed using cBioportal (http://www.cbioportal. org/) [27]. The data were obtained on 30 May 2017.

Statistical analysis
The relationship between the two groups was analyzed using the Mann-Whitney U test. The relationship of three variables or more was analyzed using Bonferroniadjusted Mann-Whitney U tests. The correlation between

Effects of restoring miR-99a-5p/3p on cell proliferation, migration, and invasion activities in PCa cell lines
To confirm the tumor-suppressive roles of miR-99a-5p and miR-99a-3p, we carried out ectopic expression assays by miRNA transfection into PC3, DU145, and C4-2 cells. According to the results of functional assays, cancer cell proliferation, migration activity, and invasion activity were all remarkably inhibited by transfection with miR-99a-3p compared with those of mock-or miR-control-transfected PC3, DU145 C4-2 cells (P < 0.0001, P < 0.0001, and P < 0.0001, respectively; Fig. 1D-F, S4A and B). Cell proliferation assay was also performed in LNCaP cells, and its ability was suppressed by transfection with miR-99a-3p (data not shown). In contrast, miR-99a-5p showed no significant antitumor effects (Fig. 1D-F).

Search for putative oncogenes regulated by miR-99a-3p in PCa cells
We focused on miR-99a-3p, which showed marked antitumor effects. The selection strategy of miR-99a-3p target genes is shown in Figure 2A. Initially, we used the TargetScan Human 7.1 database and found that 1591 genes had theoretical target sites for miR-99a-3p in their 3′-UTRs. Next, we extracted genes whose expression levels were decreased by transfection with miR-99a-3p by gene expression analysis (GEO accession number: GSE85614). Genes that were markedly decreased by transfection into PC3 cells with miR-99a-3p are shown in Table 2 (foldchange log 2 < −2.0). In this study, a total of 30 putative oncogenic targets of miR-99a-3p regulation were identified in PC cells. We investigated further whether it has related to the pathogenesis of PCa and these targets using TCGA database. Among these targets, 17 genes (NCAPG, SGOL1,  RRM2, ESCO2, ZNF695, CDK1, NEK2, FANCI, FAM64A,  ZWINT, PIGL, KIF11, MCM4, BRCA1, CDKN3, GRIA2, and MKI67) were involved in PCa pathogenesis, high expression of these genes were significantly associated with disease-free survival rate (Figs 2B, 3).
Finally, we focused on NCAPG, which showed the greatest reduction in expression following transfection with miR-99a-3p.

Clinical significance of NCAPG in PCa
According to TCGA database, NCAPG expression levels were closely related to prognosis and clinical stage in patients with PCa. High NCAPG expression group had remarkably shorter disease-free survival (DFS) than that of the low expression group in patients with PCa (P = 0.0009, Fig. 2B). Moreover, the expression levels of NCAPG were markedly increased in cases with advanced T stage, advanced N stage, and high Gleason Score (Fig. 2C). These results indicated that NCAPG may affect disease progression and malignancy in PCa.

NCAPG was directly regulated by miR-99a-3p in PCa cells
The expression of NCAPG mRNA was significantly decreased by miR-99a-3p transfection compared to that of mock-or miR-control-transfected cells (Fig. 4A). Consistent with this, NCAPG protein expression was reduced by miR-99a-3p transfection (Fig. 4B).
To validate direct binding of miR-99a-3p in NCAPG mRNA, we performed luciferase reporter assays. The TargetScan database predicted that miR-99a-3p joined at position 462-468 in the 3′-UTR of NCAPG. The luminescence intensity was remarkably reduced by cotransfection with miR-99a-3p and wild-type vector of 3′-UTR of NCAPG. In contrast, using the vector in which the target site of miR-99a-3p was deleted, the luminescence intensity did not change (Fig. 4C).
Furthermore, to analyze NCAPG protein expression, immunohistochemistry was performed with PCa clinical Regulation of NCAPG by miR-99a-3p in CRPC T. Arai et al. specimens (Table 1). In CRPC specimens, NCAPG protein was strongly expressed in metastatic tissues from patients with CRPC, compared with non-PCa or HSPC specimens ( Fig. 5C and D).

Effects of silencing NCAPG in PCa cell lines
We examined the effects of NCAPG knockdown in PC3, DU145, and C4-2 cells using two types of si-NCAPG (si-NCAPG-1 and si-NCAPG-2). Two siRNAs effectively downregulated NCAPG mRNA and NCAPG protein expression in PC3, DU145, and C4-2 cells (Fig. 6A and B). Additionally, functional assays indicated that cell proliferation, migration, and invasion were significantly inhibited by knockdown of NCAPG in comparison with mock-or si-control-transfected cells (Fig. 6C-E, S5A and B). Even in LNCaP cells, cell proliferation assay was performed, and its ability was markedly suppressed by knockdown of NCAPG (data not shown).

Effects of cotransfection with NCAPG/ miR-99a-3p in PC3 cells
We performed NCAPG rescue experiments by cotransfection with NCAPG and miR-99a-3p into PC3 cells. Western blot analysis of NCAPG protein expression is shown in Figure 7A and B. According to Western blotting, NCAPG protein levels were recovered by cotransfection with NCAPG and miR-99a-3p in PC3 cells. Moreover, the proliferation, migration, and invasion capacities of PC3 cells were recovered by cotransfection with NCAPG and miR-99a-3p compared with cells transfected with miR-99a-3p only ( Fig. 7C-E, S6A and B). These results indicated that NCAPG affected the aggressiveness of PC3 cells.

Discussion
One of the main challenges in the treatment of CRPC is the control of aggressive and lethal metastatic PCa cells. According to TCGA database, the expression levels of NCAPG were significantly increased in cases of advanced T stage, advanced N stage, and high Gleason score. *P < 0.01, **P < 0.001, and ***P < 0.0001.
In general miRNA biogenesis, guide strand of miRNA is incorporated into RISC (RNA-induced silencing complex) and acts as a fine-tuner of expression control. In contrast, passenger strand of miRNA is disassembled and has no function [17][18][19]. In miRNA biology, miRNA strand selection process is still obscure that which strand become the guide strand or the passenger strand from a miRNA duplex. Recent studies suggested that the thermodynamic character of the duplex seems to play an important role [35]. An important feature of the miRNA guide strand is the U-bias at the 5′end and excess purine, and the passenger strand has a C-bias at the 5′ end and an excess of pyrimidine [35]. The molecular dynamics of miRNA (guide strand and passenger strand) degradation and stabilization in normal and disease cells remain largely unknown.
In this study, we focused on miR-99a-5p whose expression was significantly downregulated in our miRNA signature of metastatic CRPC and investigated the functional roles including passenger strand miR-99a-3p in PCa cells.
As the results, we indicated that miR-99a-3p has potent antitumor effects in PCa cells. The expression levels of the two miRNAs, miR-99a-5p and miR-99a-3p, were obviously different in clinical specimens and cancer cell lines. We do not see any clear answer as to why this kind of difference will arise. This challenge is an important issue for miRNA research. In addition, a more detailed study on the concentration of miRNAs to be transfected into cancer cells and antitumor effects will be necessary.
In contrast, the passenger strand miR-99a-3p has been reported as a diagnostic marker of the chemotherapy response in patients with advanced colorectal cancer [47]; however, there are no reports examining the functional significance of miR-99a-3p in cancer cells. Our previous studies of miRNA signatures showed that miR-99a-3p was significantly downregulated in bladder cancer, renal cell carcinoma, and head and neck squamous cell carcinoma, suggesting miR-99a-3p has antitumor roles in these cancers [48][49][50]. Moreover, TCGA database revealed that low expression of miR-99a-3p was significantly associated with poor prognosis in head and neck cancer and lung adenocarcinoma (Fig. S7). This is the first report demonstrating that miR-99-3p may function as an antitumor miRNA in naïve PCa and CRPC cells.
Unique nature of miRNA, single miRNA controls vast number of genes in normal and cancer cells. We performed gene expression analyses and in silico database search to identify miR-99a-3p regulated oncogenic genes in PCa cells. Interestingly, a large number of cohort analyses by TCGA database showed several targets were deeply involved PCa pathogenesis. These genes might be important tools for elucidating the molecular pathogenesis of PCa and CRPC.
In this study, by focusing on miR-99a-3p, which had not been well studied in previous reports, we found that NCAPG was directly regulated by miR-99a-3p in PCa cells. Overexpression of NCAPG was observed in CRPC clinical specimens, and its expression was found to be essential for PCa pathogenesis, as demonstrated by analysis of TCGA database. Interestingly, our previous study indicated that NCAPG was regulated by miR-145-3p in PCa cells [15]. Thus, NCAPG is a candidate gene controlled by multiple antitumor miRNAs in CRPC, and its function in the pathogenesis of PCa may be important. However, the cancer-promoting functions of this molecule are still not well known.
NCAPG is involved in mitotic chromosome condensation and is related to the cell cycle. Mitotic chromosome condensation is an essential cellular property of all proliferating cells and results in reconstitution of chromosomes into rod-like mitotic chromosomes, ensuring separation of sister chromatids during cell division. In vertebrates, there are two types of condensin complexes, type I and II complexes, both of which contain nonstructural maintenance of chromosomes (non-SMC) regulatory subunits. Defects in one of the subunits cause incomplete chromosome condensation [51,52]. NCAPG exists in the condensin I complex and is associated with proper segregation of sister chromatids in the condensation and fission of mitotic chromosomes [53]. Previous studies showed that NCAPG was involved in the cell cycle and had cancerpromoting functions in several types of cancers [54,55]. A recent study showed that knockdown of NCAPG induced apoptosis, reduced cancer cell survival, and suppressed the epithelial-mesenchymal transition (EMT) in cancer cells via upregulation of Bax, cleaved caspase-3, and E-cadherin and downregulation of cyclin A1, CDK2, Bcl-2, N-cadherin, and HOXB9 in hepatocellular carcinoma [55]. Our present data showed that aberrant expression of NCAPG enhanced PCa cell aggressiveness. Thus, these data suggested that NCAPG had clinical significance in PCa pathogenesis and could have applications as a therapeutic target in CRPC.
In conclusion, both strands of pre-miR-99a, that is, miR-99a-5p and miR-99a-3p, were significantly reduced in naïve PCa and CRPC clinical specimens. The passenger strand, miR-99a-3p, had potent antitumor effects via targeting of the oncogene NCAPG in PCa cells. NCAPG was markedly elevated in CRPC and was involved in CRPC pathogenesis, suggesting that NCAPG could have applications as a therapeutic target in CRPC. The involvement of passenger strand miRNAs in cancer cells is novel concept of naïve PCa and CRPC pathogenesis.
T. Arai et al. Regulation of NCAPG by miR-99a-3p in CRPC Figure S3. Both strands of miR-99a-5p and miR-99a-3p incorporated into the RISC. Figure S4. Phase micrographs of wound healing and invasion assays following transfection with miR-99a-5p/3p in PCa cell lines. Figure S5. Phase micrographs of wound healing and invasion assays following transfection with si-NCAPG in PCa cell lines. Figure S6. Phase micrographs of wound healing and invasion assays following cotransfection with NCAPG/ miR-99a-3p in PC3 cells. Figure S7. Kaplan-Meier survival curves based on miR-99a-3p expression in patients with Head and Neck squamous cell carcinoma and Lung adenocarcinoma. Table S1. Product numbers of reagents.