Oncogenic Targets Regulated by Tumor-Suppressive miR-30c-1-3p and miR-30c-2-3p: TRIP13 Facilitates Cancer Cell Aggressiveness in Breast Cancer

Simple Summary Two passenger strand microRNAs (miRNAs), miR-30c-1-3p and miR-30c-2-3p, were identified as tumor-suppressive miRNAs in breast cancer (BrCa) cells. Seven genes (TRIP13, CCNB1, RAD51, PSPH, CENPN, KPNA2, and MXRA5) were putative targets of these miRNAs, and their expression was closely involved in BrCa molecular pathogenesis. Among these targets, inhibition of TRIP13 significantly suppressed aggressive phenotypes of BrCa cells. Abstract Accumulating evidence suggests that the miR-30 family act as critical players (tumor-suppressor or oncogenic) in a wide range of human cancers. Analysis of microRNA (miRNA) expression signatures and The Cancer Genome Atlas (TCGA) database revealed that that two passenger strand miRNAs, miR-30c-1-3p and miR-30c-2-3p, were downregulated in cancer tissues, and their low expression was closely associated with worse prognosis in patients with BrCa. Functional assays showed that miR-30c-1-3p and miR-30c-2-3p overexpression significantly inhibited cancer cell aggressiveness, suggesting these two miRNAs acted as tumor-suppressors in BrCa cells. Notably, involvement of passenger strands of miRNAs is a new concept of cancer research. Further analyses showed that seven genes (TRIP13, CCNB1, RAD51, PSPH, CENPN, KPNA2, and MXRA5) were putative targets of miR-30c-1-3p and miR-30c-2-3p in BrCa cells. Expression of seven genes were upregulated in BrCa tissues and predicted a worse prognosis of the patients. Among these genes, we focused on TRIP13 and investigated the functional significance of this gene in BrCa cells. Luciferase reporter assays showed that TRIP13 was directly regulated by these two miRNAs. TRIP13 knockdown using siRNA attenuated BrCa cell aggressiveness. Inactivation of TRIP13 using a specific inhibitor prevented the malignant transformation of BrCa cells. Exploring the molecular networks controlled by miRNAs, including passenger strands, will facilitate the identification of diagnostic markers and therapeutic target molecules in BrCa.


Introduction
According to a report by the World Health Organization, breast cancer (BrCa) is the most common cancer among women worldwide; approximately 2.3 million women are diagnosed each year, with 700,000 BrCa-related deaths [1]. It is estimated that one in five women will develop BrCa during their lifetime [2]. The development of methods for the early diagnosis of BrCa and the discovery of new treatment regimens are important issues in BrCa research.
The current study was conducted in accordance with the guidelines of the Declaration of Helsinki and was approved by the Ethics Committee of Kagoshima University (approval number 160038 28-65; date of approval: 19 March 2021).

Analysis of Molecular Pathways Using Gene Set Enrichment Analysis (GSEA) Software
We explored TRIP13-mediated molecular pathways by GSEA 4.3.2. From the TCGA-BRCA data, TRIP13 expression levels were divided into high and low expression groups according to Z-score for BrCa patients. A ranked list of genes was created based on the log 2 ratio comparing the expression level of each gene between the two groups. Gene ranking was performed by comparing the expression level of each gene between the two groups. Further analysis was performed by applying the Hallmark gene set from the Molecular Signatures Database [26][27][28].

RNA Extraction and Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR)
Total RNA from BrCa cell lines was isolated using Isogen II (NIPPON GENE Co., Ltd., Tokyo, Japan). cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (catalog no.: 4368814, ThermoFisher Scientific Inc., Waltham, MA, USA). Gene expression was analyzed by real time PCR using SYBR green assay (ThermoFisher Scientific) on StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). An internal control in gene expression assays was β-Glucuronidases (GUSB). The sequences of primers for SYBR Green assays are summarized in Table S1. The reagents used in this study were listed in Table S2. The procedures for RNA extraction and qRT-PCR were described in our previous studies [29,30].

Transfection with Small Interfering RNA (siRNAs) and miRNAs
Opti-MEM (Gibco, Carlsbad, CA, USA) and LipofectamineTM RNAiMax Transfection Reagent (Invitrogen, Carlsbad, CA, USA) were used for transfection of small-sized RNA (siRNA and miRNA) into BrCa cell lines. The experimental protocol conforms to our previous studies [29,30]. The siRNAs and miRNAs used in this study are listed in Table S2.

Cell Proliferation, Invasion and Migration Assays in BrCa Cells
Cell proliferation, invasion, and migration assays were performed in BrCa cells. Briefly, cell proliferation was assessed using XTT assays (Sigma-Aldrich, St. Louis, MO, USA); invasion was evaluated using Matrigel chamber assays with Corning BioCoat Matrigel (Corning, New York, NY, USA); and migration was examined using chamber assays with Corning BioCoat cell culture chambers (Corning). Details of the procedures are included in our previous studies [29,30].

Western Blotting and Immunohistochemistry
Western blotting and immunohistochemical analysis were performed according to our previous studies [29,30]. Anti-thyroid hormone receptor interactor 13 (TRIP13) human rabbit polyclonal IgG was used as a primary antibody. The antibodies used in the study are  Table S2. A list of clinical specimens evaluated by immunohistochemistry is given  in Table S3.

RNA Immunoprecipitation (RIP) Assays
RIP assays were performed using a MagCapture microRNA Isolation Kit, Human Ago2 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) according to the manufacturer's protocol. The expression level of TRIP13 bound to Ago2-conjugated miRNAs was assessed using qRT-PCR.

Plasmid Construction and Dual-Luciferase Reporter Assays
Vector construction and dual-luciferase reporter assays were performed as described in our previous studies [19,31]. The vector insertion sequences are shown in Figure S1, and the reagents used are listed in Table S2.

Statistical Analyses
Statistical analyses were performed using JMP Pro 16 (SAS Institute Inc., Cary, NC, USA). Differences between two groups were analyzed using Welch's t-test, and those between multiple groups were analyzed using Dunnett's test. Survival rates were analyzed by Kaplan-Meier survival curves and log-rank test.
Accordingly, we next validated the expression levels of the miR-30 family using TCGA datasets. The expression levels of miR-30c-1-3p and miR-30c-2-3p were significantly reduced in BrCa tissues ( Figure 1B). Moreover, low expression of these miRNAs was associated with The mature sequences of miR-30c-1-3p and miR-30c-2-3p are given. Seed sequences of these miRNAs are shown in red. (B) Expression levels of miR-30c-1-3p and miR-30c-2-3p were evaluated by TCGA-BRCA database analysis. In total, 643 BrCa tissues and 81 normal epithelial tissues were analyzed. (C) Kaplan-Meier survival curve analyses of patients with BrCa using data from TCGA-BRCA dataset. The patients were divided into high and low expression groups according to their miRNA expression (based on median expression). The red line shows the high expression group, and the blue line shows the low expression group.
Analysis of our original miRNA expression signature created by RNA sequencing showed that some members of the miR-30 family (miR-30a, miR-30b, miR-30c, miR-30d, and miR-30e) exhibited low expression in cancer tissues compared with normal tissues.
Accordingly, we next validated the expression levels of the miR-30 family using TCGA datasets. The expression levels of miR-30c-1-3p and miR-30c-2-3p were significantly reduced in BrCa tissues ( Figure 1B). Moreover, low expression of these miRNAs was associated with a significantly poor prognosis (based on the 10-year survival rate) compared with high expression of these miRNAs ( Figure 1C). The expression levels of the two miRNAs were compared across patient subtypes. The expression level of miR-30c-1-3p was higher in TNBC than in luminal. miR-30c-2-3p showed lower expression in TNBC compared to luminal ( Figure S2). Examination of prognosis by patient subtype showed that in luminal patients, patients with low miR-30c-1-3p expression had a poor prognosis ( Figure S3). The miR-30c-5p (the guide strand) was also analyzed in the same way. Expression of miR-30c-5p was reduced in BrCa tissues compared with normal tissues. However, miR-30c-5p expression did not affect the prognosis of BrCa patients ( Figure S4).
Our recent studies revealed that some passenger strands of miRNAs were closely involved in the molecular pathogenesis of human cancers. In this study, we focused on miR-30c-1-3p and miR-30c-2-3p and investigated the functional significance of these miRNAs with the aim of identifying their target genes in BrCa cells.

Identification of Genes Controlled by miR-30c-1-3p and miR-30c-2-3p in BrCa Cells
To detect genes that were controlled by miR-30c-1-3p and miR-30c-2-3p in BrCa cells, we carried out in silico database analysis and combined these findings with our gene expression data. Our strategy for miRNA target searching is shown in Figure 3.

Clinical Significance of Putative Target
Genes of miR-30c-1-3p and miR-30c-2-3p in BrCa Moreover, the 62 selected genes were subjected to clinicopathological analysis using the TCGA-BRCA dataset. Among these genes, 37 genes were significantly overexpressed in BrCa tissues (n = 1085) compared with normal tissues (n = 291; p < 0.01). In addition, 12 genes showed statistically significant correlations with poor overall survival (based on the 10-year survival rate, p < 0.05).

Identification of Genes Controlled by miR-30c-1-3p and miR-30c-2-3p in BrCa Cells
To detect genes that were controlled by miR-30c-1-3p and miR-30c-2-3p in BrCa cells, we carried out in silico database analysis and combined these findings with our gene expression data. Our strategy for miRNA target searching is shown in Figure 3.

Clinical Significance of TRIP13 in BrCa
Among these targets, we focused on TRIP13 and performed further analyses of its function in BrCa cells. Recent studies have shown that TRIP13 is a key regulator of meiotic recombination and the spindle assembly checkpoint [32,33]. Exploitation of the checkpoint inhibition process may have applications in the treatment of BrCa.
Immunohistochemistry was performed to analyze TRIP13 expression in BrCa clinical specimens. TRIP13 protein was highly expressed in cancer lesions but weakly expressed

Clinical Significance of TRIP13 in BrCa
Among these targets, we focused on TRIP13 and performed further analyses of its function in BrCa cells. Recent studies have shown that TRIP13 is a key regulator of meiotic recombination and the spindle assembly checkpoint [32,33]. Exploitation of the checkpoint inhibition process may have applications in the treatment of BrCa.
Immunohistochemistry was performed to analyze TRIP13 expression in BrCa clinical specimens. TRIP13 protein was highly expressed in cancer lesions but weakly expressed in noncancerous areas ( Figure 6). Tissue information is shown in Table S3.  (TRIP13, CCNB1,  RAD51, PSPH, CENPN, KPNA2, and MXRA5). The patients (n = 1006) were divided into high and low expression groups according to the median gene expression level. The red lines represent the high expression group, and the blue lines represent the low expression group. High expression levels of these genes were significantly correlated with poor prognosis in patients with BrCa.

Clinical Significance of TRIP13 in BrCa
Among these targets, we focused on TRIP13 and performed further analyses of its function in BrCa cells. Recent studies have shown that TRIP13 is a key regulator of meiotic recombination and the spindle assembly checkpoint [32,33]. Exploitation of the checkpoint inhibition process may have applications in the treatment of BrCa.
Immunohistochemistry was performed to analyze TRIP13 expression in BrCa clinical specimens. TRIP13 protein was highly expressed in cancer lesions but weakly expressed in noncancerous areas ( Figure 6). Tissue information is shown in Table S3. A multivariate Cox proportional hazards model showed that high expression of TRIP13 was an independent prognostic factor for overall survival after adjusting for wellknown clinical prognostic factors (age, T stage, N stage, and M stage; Figure 7A).

TRIP13-Mediated Molecular Pathways in BrCa Cells
To investigate TRIP13-mediated molecular pathways in BrCa cells, we performed GSEA using TCGA-BRCA RNA-sequencing data. "E2F targets", "G 2 M checkpoint", and "MYC target" pathways were enriched in patients showing high TRIP13 expression compared with those in patients showing low expression (Table 2, Figure 7B).
A multivariate Cox proportional hazards model showed that high expression of TRIP13 was an independent prognostic factor for overall survival after adjusting for well-known clinical prognostic factors (age, T stage, N stage, and M stage; Figure 7A).

TRIP13-Mediated Molecular Pathways in BrCa Cells
To investigate TRIP13-mediated molecular pathways in BrCa cells, we performed GSEA using TCGA-BRCA RNA-sequencing data. "E2F targets", "G2M checkpoint", and "MYC target" pathways were enriched in patients showing high TRIP13 expression compared with those in patients showing low expression (Table 2, Figure 7B).   To confirm the incorporation of TRIP13 mRNA into the RNA-induced silencing complex (RISC) in BrCa cells, RIP assays were conducted ( Figure 8C). Ago2-bound miRNAs and mRNAs were isolated via immunoprecipitation of Ago2, which plays a central role in the RISC. qRT-PCR using immunoprecipitation-isolated samples demonstrated significantly higher levels of TRIP13 mRNA in miR-30c-1-3p and miR-30c-2-3p transfected cells compared with control cells. These findings provided evidence of the significant incorporation of TRIP13 into the RISC.

Effect of TRIP13 siRNA and the TRIP13 Inhibitor DCZ0415 on TRIP13 Function in BrCa Cell
Next, to analyze the oncogenic roles of TRIP13 in MDA-MB-231 BrCa cells, we performed knockdown assays using siRNAs targeting TRIP13. Two types of siRNAs (siTRIP13-1 and siTRIP13-2) markedly suppressed TRIP13 expression at both the mRNA and protein levels in BrCa cells ( Figure 10A,B). Full western blotting images are shown in Figure S7.
In an analysis using a TRIP13 inhibitor (DCZ0415), cell proliferation was suppressed in a concentration-dependent manner ( Figure 10D). The sequence of the binding sites is highlighted in red. (B) In dual luciferase reporter assays, cotransfection of miR-30c-1-3p or miR-30c-2-3p, and a vector containing the miR-30c-3p binding site in the 3 UTR of TRIP13 showed decreased luminescence activity in BrCa cells (N.S.: not significant compared with the mock group).

Effect of TRIP13 siRNA and the TRIP13 Inhibitor DCZ0415 on TRIP13 Function in BrCa Cell
Next, to analyze the oncogenic roles of TRIP13 in MDA-MB-231 BrCa cells, we performed knockdown assays using siRNAs targeting TRIP13. Two types of siRNAs (siTRIP13-1 and siTRIP13-2) markedly suppressed TRIP13 expression at both the mRNA and protein levels in BrCa cells ( Figure 10A,B). Full western blotting images are shown in Figure S7.

Discussion
Our previous studies demonstrated that miR-99a-3p (the passenger strand of pre-miR-99a) and miR-101-5p (the passenger strand of pre-miR-101) functioned as tumor-suppressive miRNAs in BrCa cells. Their target genes FAM64A and GINS1 were shown to be aberrantly expressed in BrCa tissues, and their expression levels were closely associated with the molec- Cell proliferation assays showed that siTRIP13-transfected MDA-MB-231 cells show significantly reduced cell growth ( Figure 10C).
In an analysis using a TRIP13 inhibitor (DCZ0415), cell proliferation was suppressed in a concentration-dependent manner ( Figure 10D).

Discussion
Our previous studies demonstrated that miR-99a-3p (the passenger strand of pre-miR-99a) and miR-101-5p (the passenger strand of pre-miR-101) functioned as tumor-suppressive miRNAs in BrCa cells. Their target genes FAM64A and GINS1 were shown to be aberrantly expressed in BrCa tissues, and their expression levels were closely associated with the molecular pathogenesis of BrCa [16,30]. Such new findings demonstrating that the passenger strands of miRNAs derived from pre-miRNAs are involved in cancer pathogenesis indicate that passenger strands of miRNAs should be analyzed alongside guide strands.
Previous studies have shown the downregulation of miR-30c-5p in several types of cancers, including breast cancer, and the oncogenes it regulates have been implicated in various cancer pathways, such as cell proliferation, metastasis, and drug resistance. On the contrary, miR-30c-3p (the passenger strand) has not been reported in detail [34][35][36][37]. A large number of cohort analysis by TCGA database revealed that low expression levels of miR-30c-5p did not affect the prognosis of BrCa patients. In contrast, BrCa patients with low miRNAs (miR-30c-1-3p and miR-30c-2-3p) expressions had clear impact on prognosis. Therefore, this study focused on two types of miRNAs, miR-30c-1-3p and miR-30c-2-3p. In the estrogen receptor-negative BrCa subtype, nuclear factor kappa B (NF-κB) signaling is frequently activated. Additionally, miR-30c-2-3p has been shown to act as a negative regulator of NF-κB signaling, and ectopic expression of miR-30c-2-3p attenuates cell proliferation by targeting TRADD and CCNE1 in BrCa cells [38]. In another study, overexpression of the circular RNA circ0072995 was shown to promote the invasion and migration of cancer cells by adsorbing miR-30c-2-3p in MDA-MB-231 cells [39]. These reports are consistent with our current findings and strongly indicated that miR-30c-2-3p acted as a tumor-suppressive miRNA in BrCa cells.
Some reports have described the roles of miR-30c-1-3p and miR-30c-2-3p in other cancer types. For example, in lung adenocarcinoma, the long noncoding RNA LINC00346 was shown to adsorb miR-30c-2-3p and abolish its tumor-suppressive function. Overexpression of LINC00346 promotes the development of lung adenocarcinoma through regulation of the miR-30c-2-3p/cell cycle signaling pathway [40]. N6-methyladenosine (m6A) is the most common modification in the mammalian RNA transcriptome and is broadly present in mRNAs and certain noncoding RNAs [41]. Recent studies have suggested that alterations in m6A modification patterns are deeply involved in tumorigenesis [42]. Methyltransferase-like 14 (METTL14) is a key RNA methyltransferase involved in m6A modification. A recent study showed that METTL14 enhances the maturation of miR-30c-1-3p and that miR-30c-1-3p expression inhibits lung cancer malignant transformation [43]. Moreover, METTL14-mediated m6A modification has also been reported to be involved in miR-30c-2-3p regulation in gastric cancer [44]. Aberrant expression of genes involved in m6A modification and regulation of miRNAs in BrCa cells will be important research topics in the future.
A feature of miRNAs is that the target genes they control differ depending on the type of cancer. In this study, we attempted to search for genes regulated by tumor-suppressive miR-30c-1-3p and miR-30c-2-3p in BrCa cells. In total, seven genes (TRIP13, CCNB1, RAD51, PSPH, CENPN, KPNA2, and MXRA5) were identified as putative targets of miR-30c-1-3p and miR-30c-2-3p, and their expression levels were found to be closely associated with poor prognosis in patients. As a future study, it will be necessary to analyze miRNAs and target molecules controlled by miRNAs by subtype of BrCa patients.
Based on these findings, we focused on TRIP13, a member of the large superfamily of AAA+ ATPase proteins [45]. The AAA+ ATPase family is involved in a wide range of biological processes, including protein folding and DNA recombination, replication, and repair [46,47]. Our data showed that aberrant expression of TRIP13 was deeply involved in the malignant transformation of BrCa cells. Notably, TRIP13 has been shown to be overexpressed in several types of cancers, and its aberrant expression is involved in the malignant transformation of various types of cancer cells, including BrCa cells [48,49]. In lung cancer cells, TRIP13 knockdown inhibited malignant phenotypes, e.g., increased apoptosis, induced cell cycle arrest, and inhibited the proliferation, invasion, and migration abilities. Furthermore, overexpression of TRIP13 was associated with tumor metastasis through activation epithelial-mesenchymal transformation pathways [48]. TRIP13 is a novel mitotic checkpoint-silencing protein. Overexpression of TRIP13 is a hallmark of cancer cells exhibiting chromosomal instability, especially in certain BrCa with poor prognosis [49]. In head and neck cancer, overexpressed TRIP13 interacts with the DNA-protein kinase C complex and activates the DNA repair process, thereby affecting drug resistance [50]. A recent study demonstrated that the TRIP13 inhibitor DCZ0415 impairs nonhomologous end-joining repair and attenuates cancer cell growth in hepatocellular carcinoma [51]. Furthermore, combining DCZ0415 and olaparib (a poly [ADP-ribose] polymerase [PARP1] inhibitor) has synergistic anticancer effects against hepatocellular carcinoma cells [51]. PARP1 inhibitors and CDK4/6 inhibitors have also been used in the treatment of BrCa. Combining these drugs with TRIP13 inhibitors may lead to synergistic anticancer effects, thereby facilitating the development of new treatment regimens.

Conclusions
In this study, TCGA analysis revealed that low expression levels of miR-30c-1-3p and miR-30c-2-3p adversely affected the prognosis of patients with BrCa. Ectopic expression of these miRNAs attenuated the malignant phenotypes of BrCa cells, suggesting that these miRNAs acted as tumor-suppressive miRNAs in BrCa cells. In total, seven genes (TRIP13, CCNB1, RAD51, PSPH, CENPN, KPNA2, and MXRA5) were putative targets of miR-30c-1-3p and miR-30c-2-3p, and their high expression levels were associated with a worse prognosis in patients. TRIP13 was directly regulated by miR-30c-1-3p and miR-30c-2-3p, and its overexpression facilitated BrCa cell aggressiveness. Based on the tumor-suppressive miR-NAs analysis, it was possible to identify genes that were closely related to the molecular pathogenesis of BrCa.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cancers15164189/s1, Figure S1: The vector insertion sequences by luciferase reporter assay; Figure S2: The expression levels of the two miRNAs compared across patient subtypes. Figure S3: Clinical significance of miR-30c-1-3p and miR-30c-2-3p compared between the patient subtypes. Figure S4: Clinical significance of miR-30a-5p in BrCa patients; Figure S5: Typical images of BrCa cells during invasion and migration assays by miR-30c-1-3p and miR-30c-2-3p expression; Figure S6: Full size images of WB in Figure 8B; Figure S7: Full size images of WB in Figure 10B. Table S1: The sequences of primers used for SYBR Green assays; Table S2: Reagents used in this study; Table S3: Clinical characteristics of patients with breast cancer who provided specimens for immunohistochemical staining of TRIP13. Funding: The present study was supported by KAKENHI grant numbers 21K09367, 21K09577, 22K08705, 22K09679, and 23K08094.

Institutional Review Board Statement:
The study was conducted in accordance with the guidelines of the Declaration of Helsinki and was approved by the Ethics Committee of Kagoshima University (approval number 160038 28-65; date of approval: 19 March 2021).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.