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Article

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

by
Reiko Mitsueda
1,
Hiroko Toda
1,
Yoshiaki Shinden
1,
Kosuke Fukuda
1,
Ryutaro Yasudome
1,
Mayuko Kato
2,
Naoko Kikkawa
2,
Takao Ohtsuka
1,
Akihiro Nakajo
1 and
Naohiko Seki
2,*
1
Department of Digestive Surgery, Breast and Thyroid Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8520, Japan
2
Department of Functional Genomics, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan
*
Author to whom correspondence should be addressed.
Cancers 2023, 15(16), 4189; https://doi.org/10.3390/cancers15164189
Submission received: 23 June 2023 / Revised: 26 July 2023 / Accepted: 17 August 2023 / Published: 21 August 2023
(This article belongs to the Special Issue Breast Cancer: Novel Histological and Molecular Markers for Diagnosis, Prognosis, Therapeutic Prediction, and Drug Resistance)
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Abstract

:

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.

1. 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.
BrCa is a heterogeneous cancer and is classified into several subtypes according to histological and molecular classification [3,4,5]. For example, BrCa is classified as luminal, human epidermal growth factor receptor 2-enriched, and triple-negative types depending on the presence or absence of hormone receptors (estrogen and progesterone receptors) and epidermal growth factor receptor 2 in BrCa cells. Furthermore, the luminal type can be classified as luminal-A or luminal-B type according to the expression of Ki-67, a cell cycle marker [6]. Because the malignancy and outcomes of patients vary greatly among subtypes, treatment regimens also differ for each subtype [7,8]. In the future, further classification using molecular markers will facilitate the selection of different therapeutic regimens.
In the post-genome era, researchers have found that various noncoding RNAs are transcribed in cells and are deeply involved in critical functions, such as cell differentiation, proliferation, migration, and immune responses [9,10]. Among these noncoding RNAs, microRNAs (miRNAs) are short, single-stranded RNAs that act as fine controllers of gene expression depending on their sequences. Interestingly, a single miRNA can simultaneously control the expression of many genes [9,11]. Therefore, the presence or absence of miRNAs can disrupt the expression of target genes in cells and contribute to the malignant transformation of normal cells [12,13]. Many studies have revealed that aberrantly expressed miRNAs behave as oncogenes and tumor suppressors through targeting their corresponding genes in cancer cells [14].
Recently, we generated a BrCa miRNA expression signature by RNA sequencing and identified tumor-suppressive miRNAs and their target oncogenes in BrCa cells [15,16]. Our previous study revealed that miR-101-5p was downregulated in BrCa tissues, and its low expression predicted a worse prognosis in patients [16]. Ectopic expression assays showed that miR-101-5p attenuated BrCa malignant phenotypes by controlling several genes (e.g., HMGB3, ESRP1, GINS1, TPD52, SRPK1, VANGL1, and MAGOHB) whose expression levels are closely involved in the molecular pathogenesis of BrCa [16]. Importantly, miR-101-5p is annotated as a passenger strand miRNA derived from pre-miR-101 in miRNA databases.
The general concept of miRNA biogenesis, two types of mature miRNAs are derived from pre-miRNAs. One strand (the guide strand) is selected for loading into the miRNA-Induced Silencing Complex (miRISC). The miRISC (including the guide strand) target mRNAs for silencing based on sequence depending manner. On the contrary, the passenger strand miRNAs (the other strand of pre-miRNA) are thought to be degraded in the cytoplasm and have previously been considered nonfunctional [17]. However, recent studies have shown that some passenger strands of miRNAs act as oncogenes or tumor suppressors in cancer cells [18,19]. Therefore, in cancer-miRNA research, it is essential to consider passenger strands derived from pre-miRNAs as well.
Analysis of our BrCa miRNA signature revealed that miR-30c-1-3p and miR-30c-2-3p (the passenger strands) were downregulated in cancer tissues. A large amount of cohort data obtained from The Cancer Genome Atlas (TCGA) database confirmed that miR-30c-1-3p and miR-30c-2-3p were downregulated in cancer tissues. Moreover, low expression of these targets was closely associated with worse prognosis in patients. Functional assays showed that these miRNAs exerted antitumor functions in BrCa cells by controlling several genes closely involved in BrCa malignant transformation, e.g., TRIP13, CCNB1, RAD51, PSPH, CENPN, KPNA2, and MXRA5.
In this study, we found that miR-30c-3p and its corresponding genes were involved in the malignant transformation of BrCa cells. These miRNA-targeted molecules may be candidates for the early diagnosis and treatment of BrCa.

2. Materials and Methods

2.1. Cell Lines and BrCa Clinical Specimens

Two BrCa Cell lines (MDA-MB-157 and MDA-MB-231) were used in this study. Both cell lines were obtained from Public Health England (Salisbury, UK).
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).

2.2. Analysis of miRNAs and miRNA Target Genes in Patients with BrCa

The sequences of miR-30 were confirmed using miRBase ver. 22.1 (https://www.mirbase.org, accessed on 10 July 2020) [20].
We used gene expression profiles of BrCa clinical specimens (GEO accession number: GSE118539) and TargetScanHuman ver.8.0 (https://www.targetscan.org/vert_80/ (accessed on 20 January 2023)) to search for genes regulated by miR-30c-1-3p and miR-30c-2-3p [21].
Expression data of the target genes from BrCa clinical tissues was obtained from GEPIA2 (http://gepia2.cancer-pku.cn/#index (accessed on 10 April 2023)) [22]. The clinical significance of genes in BrCa was obtained from OncoLnc (http://www.oncolnc.org/ (accessed on 10 April 2023)) [23,24,25].

2.3. 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 log2 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].

2.4. 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].

2.5. 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.

2.6. 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].

2.7. 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 listed in Table S2. A list of clinical specimens evaluated by immunohistochemistry is given in Table S3.

2.8. 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.

2.9. 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.

2.10. 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.

3. Results

3.1. Expression and Clinical Significance of miR-30c-1-3p and miR-30c-2-3p in BrCa Clinical Specimens

Analysis of the miRBase database (release 22.1) showed that miR-30c-1-3p and miR-30c-2-3p were annotated as passenger strands of miRNAs and were derived from pre-miR-30c-1 and pre-miR-30c-2, respectively. Seed sequences of these miRNAs were identical (Figure 1A). Human miR-30c-1 was located on chromosome 1p34.2, whereas miR-30c-2 was located on chromosome 6q13.
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.

3.2. Tumor-Suppressive Roles of miR-30c-1-3p and miR-30c-2-3p in BrCa Cells

The tumor-suppressive roles of miR-30c-1-3p and miR-30c-2-3p were evaluated by ectopic expression of miR-30c-1-3p and miR-30c-2-3p in two triple-negative BrCa cell lines, MDA-MB-157 and MDA-MB-231.
Transient transfection of miR-30c-1-3p and miR-30c-2-3p inhibited BrCa cell proliferation (Figure 2A). Cancer cell invasion and migration abilities were markedly inhibited by miR-30c-1-3p and miR-30c-2-3p expression in MDA-MB-157 and MDA-MB-231 cells (Figure 2B,C). Typical images of BrCa cells during invasion and migration assays after miR-30c-1-3p and miR-30c-2-3p transfection are shown in Figure S5.
Based on our current analysis, two miRNAs (miR-30c-1-3p and miR-30c-2-3p) showed tumor-suppressive roles through targeting several oncogenic genes in BrCa cells.

3.3. 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.
The TargetScan Human database (release 8.0) revealed that a total of 3,154 genes had miR-30c-1-3p and miR-30c-2-3p binding sites in their 3′ untranslated regions (UTRs). Using BrCa clinical specimens, a gene expression profile was obtained (Gene Expression Omnibus accession number: GSE118539); 525 genes were identified as upregulated (log2 fold ratio > 2.0) in cancer tissues compared with normal tissues. By merging the two datasets, 62 genes were selected as miR-30c-1-3p and miR-30c-2-3p targets in BrCa cells (Table 1).
Cancers 15 04189 g003
Figure 3. Strategy for identification of miR-30c-1-3p and miR-30c-2-3p targets in BrCa cells. To identify target genes, we used our original mRNA profile (GEO accession number: GSE118539) and the TargetScan Human database (release 8.0). By merging the two sets of data, we identified 62 genes as miR-30c-1-3p and miR-30c-2-3p targets. Among 62 targets, we narrowed down the clinically significant genes in BrCa using two databases, GEPIA2 (http://gepia2.cancer-pku.cn/#analysis (accessed on 10 April 2023) and OncoLnc (http://www.oncolnc.org/ (accessed on 10 April 2023). Finally, seven oncogenic genes were selected as miR-30c-1-3p and miR-30c-2-3p targets in BrCa cells.
Figure 3. Strategy for identification of miR-30c-1-3p and miR-30c-2-3p targets in BrCa cells. To identify target genes, we used our original mRNA profile (GEO accession number: GSE118539) and the TargetScan Human database (release 8.0). By merging the two sets of data, we identified 62 genes as miR-30c-1-3p and miR-30c-2-3p targets. Among 62 targets, we narrowed down the clinically significant genes in BrCa using two databases, GEPIA2 (http://gepia2.cancer-pku.cn/#analysis (accessed on 10 April 2023) and OncoLnc (http://www.oncolnc.org/ (accessed on 10 April 2023). Finally, seven oncogenic genes were selected as miR-30c-1-3p and miR-30c-2-3p targets in BrCa cells.
Cancers 15 04189 g003

3.4. 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).
We finally selected seven genes (TRIP13, CCNB1, RAD51, PSPH, CENPN, KPNA2, and MXRA5) as targets of miR-30c-1-3p and miR-30c-2-3p in BrCa cells. Our results showed that these genes were upregulated in BrCa tissues (Figure 4), and their high expression significantly predicted poor prognosis (10-year overall survival) in patients with BrCa (Figure 5).

3.5. 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 well-known clinical prognostic factors (age, T stage, N stage, and M stage; Figure 7A).

3.6. 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).

3.7. Direct Regulation of TRIP13 by miR-30c-1-3p and miR-30c-2-3p in BrCa Cells

First, we investigated whether the expression of TRIP13 was controlled by miR-30c-1-3p and miR-30c-2-3p in BrCa cells. The expression levels of TRIP13 mRNA and protein were markedly suppressed by ectopic expression of miR-30c-1-3p and miR-30c-2-3p in BrCa cells (Figure 8A,B). The full western blotting image is shown in Figure S6.
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.
To investigate whether miR-30c-1-3p and miR-30c-2-3p bound directly to the 3′ UTR of TRIP13 in BrCa cells, dual-luciferase reporter assays were conducted (Figure 9A). TargetScan database analysis revealed that miR-30c-1-3p and miR-30c-2-3p shared a binding site. The luminescence intensity was significantly decreased following co-transfection with 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 (Figure 9B). Conversely, co-transfection of a construct without the miR-30c-3p binding site (deleted miR-30c-3p binding site) showed no decrease in the luminescence intensity (Figure 9B). These data indicated that miR-30c-1-3p and miR-30c-2-3p bound directly to TRIP13 and regulated TRIP13 expression in BrCa cells.

3.8. 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.
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).

4. 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.
Based on the BrCa miRNA signature, we focused on miR-30c-1-3p and miR-30c-2-3p and demonstrated that these miRNAs behaved as tumor-suppressive miRNAs by controlling several oncogenic genes in BrCa cells. The miR-30 family consists of six miRNAs (miR-30a, miR-30b, miR-30c-1, miR-30c-2, miR-30d, and miR-30e), each of which generates guide and passenger strands from their respective precursors. Interestingly, the six miRNAs are clustered in pairs on three chromosomes (miR-30e/miR-30c-1 on chromosome 1p34.2, miR-30c-2/miR-30a on chromosome 6q13, and miR-30b/miR-30d on chromosome 8q24.22). The seed sequences of miR-30c-1-3p and miR-30c-2-3p that control the target genes are identical (UGGGAG).
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.

5. 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 miRNAs 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.

Author Contributions

Conceptualization, N.S.; methodology, N.S.; formal analysis, R.M., K.F., R.Y., and N.S.; investigation, R.M., K.F., and R.Y.; data curation, R.M., K.F., and R.Y.; writing—original draft, R.M. and N.S.; writing—review and editing, R.M., H.T., Y.S., K.F., R.Y., T.O., A.N., and N.S.; visualization, R.M. and N.S.; supervision, N.S.; project administration, N.S.; funding acquisition, H.T., Y.S., M.K., N.K., and N.S. All authors have read and agreed to the published version of the manuscript.

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.

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Figure 1. Expression and clinical significance of miR-30c-1-3p and miR-30c-2-3p in BrCa clinical specimens. (A) 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.
Figure 1. Expression and clinical significance of miR-30c-1-3p and miR-30c-2-3p in BrCa clinical specimens. (A) 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.
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Figure 2. Functional assays of miR-30c-1-3p and miR-30c-2-3p in BrCa cell lines (MDA-MB-157 and MDA-MB-231). (A) Cell proliferation was assessed using XTT assays 72 h after miRNA transfection. (B) Cell invasion was assessed using Matrigel invasion assays at 48 h after seeding miRNA-transfected cells into the chambers. (C) Cell migration was assessed using a membrane culture system at 48 h after seeding miRNA-transfected cells into the chambers.
Figure 2. Functional assays of miR-30c-1-3p and miR-30c-2-3p in BrCa cell lines (MDA-MB-157 and MDA-MB-231). (A) Cell proliferation was assessed using XTT assays 72 h after miRNA transfection. (B) Cell invasion was assessed using Matrigel invasion assays at 48 h after seeding miRNA-transfected cells into the chambers. (C) Cell migration was assessed using a membrane culture system at 48 h after seeding miRNA-transfected cells into the chambers.
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Figure 4. Expression levels of seven targets in BrCa specimens. Analysis of the expression levels of seven genes (TRIP13, CCNB1, RAD51, PSPH, CENPN, KPNA2, and MXRA5) in BrCa clinical specimens using TCGA-BRCA datasets. All these genes were upregulated in BrCa tissues (n = 1085) compared with normal tissues (n = 291) (* p < 0.01).
Figure 4. Expression levels of seven targets in BrCa specimens. Analysis of the expression levels of seven genes (TRIP13, CCNB1, RAD51, PSPH, CENPN, KPNA2, and MXRA5) in BrCa clinical specimens using TCGA-BRCA datasets. All these genes were upregulated in BrCa tissues (n = 1085) compared with normal tissues (n = 291) (* p < 0.01).
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Figure 5. Clinical significance of seven targets in BrCa specimens. Kaplan–Meier curves of 10-year overall survival rates according to the expression levels of the seven target genes (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.
Figure 5. Clinical significance of seven targets in BrCa specimens. Kaplan–Meier curves of 10-year overall survival rates according to the expression levels of the seven target genes (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.
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Figure 6. Expression of TRIP13 in BrCa tissues. Immunohistochemical staining of TRIP13 was confined to cancer tissues, whereas weak staining was observed in the noncancerous area. Magnification: 40× (upper) and 200× (lower).
Figure 6. Expression of TRIP13 in BrCa tissues. Immunohistochemical staining of TRIP13 was confined to cancer tissues, whereas weak staining was observed in the noncancerous area. Magnification: 40× (upper) and 200× (lower).
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Figure 7. Clinical significance of TRIP13 in BrCa specimens. (A) Forest plot showing multivariate Cox proportional hazards regression analysis of 10-year overall survival. Patients with high TRIP13 expression showed significantly low overall survival. The data were obtained from TCGA-BRCA datasets. (B) TRIP13-mediated pathways identified by gene set enrichment analysis. The top three enrichment plots (E2F targets, G2M checkpoint, and MYC targets) are presented in the high TRIP13 expression group for patients with BrCa.
Figure 7. Clinical significance of TRIP13 in BrCa specimens. (A) Forest plot showing multivariate Cox proportional hazards regression analysis of 10-year overall survival. Patients with high TRIP13 expression showed significantly low overall survival. The data were obtained from TCGA-BRCA datasets. (B) TRIP13-mediated pathways identified by gene set enrichment analysis. The top three enrichment plots (E2F targets, G2M checkpoint, and MYC targets) are presented in the high TRIP13 expression group for patients with BrCa.
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Figure 8. Control of TRIP13 expression by miR-30c-1-3p and miR-30c-2-3p in BrCa cells. (A) qRT-PCR analyses demonstrating the downregulation of TRIP13 mRNA expression at 72 h after miR-30c-1-3p and miR-30c-2-3p transfection into BrCa cells. GUSB was used as an internal control. (B) Western blot analyses showing significantly reduced expression of TRIP13 protein 72 h after miR-30c-1-3p and miR-30c-2-3p transfection into BrCa cells. β-actin was used as an internal loading control. (C) Isolation of RISC-incorporated TRIP13 mRNA by Ago2 immunoprecipitation. Direct TRIP13 expression by miR-30c-1-3p and miR-30c-2-3p in BrCa cells is demonstrated. Schematic illustration of the RIP assay is shown in the right. qRT-PCR suggested that TRIP13 mRNA was significantly incorporated into the RISC.
Figure 8. Control of TRIP13 expression by miR-30c-1-3p and miR-30c-2-3p in BrCa cells. (A) qRT-PCR analyses demonstrating the downregulation of TRIP13 mRNA expression at 72 h after miR-30c-1-3p and miR-30c-2-3p transfection into BrCa cells. GUSB was used as an internal control. (B) Western blot analyses showing significantly reduced expression of TRIP13 protein 72 h after miR-30c-1-3p and miR-30c-2-3p transfection into BrCa cells. β-actin was used as an internal loading control. (C) Isolation of RISC-incorporated TRIP13 mRNA by Ago2 immunoprecipitation. Direct TRIP13 expression by miR-30c-1-3p and miR-30c-2-3p in BrCa cells is demonstrated. Schematic illustration of the RIP assay is shown in the right. qRT-PCR suggested that TRIP13 mRNA was significantly incorporated into the RISC.
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Figure 9. miR-30c-1-3p and miR-30c-2-3p bound directly to the 3′ UTR of TRIP13 in BrCa cells. (A) TargetScan database analysis predicting putative miR-30c-3p-binding sites in the 3′ UTR of TRIP13. The sequence of the binding sites is highlighted in red. (B) In dual luciferase reporter assays, co-transfection 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).
Figure 9. miR-30c-1-3p and miR-30c-2-3p bound directly to the 3′ UTR of TRIP13 in BrCa cells. (A) TargetScan database analysis predicting putative miR-30c-3p-binding sites in the 3′ UTR of TRIP13. The sequence of the binding sites is highlighted in red. (B) In dual luciferase reporter assays, co-transfection 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).
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Figure 10. Oncogenic function of TRIP13 in BrCa cells. (A) The inhibitory effects of two different siRNAs targeting TRIP13 (siTRIP13-1 and siTRIP13-2) expression were examined. TRIP13 mRNA levels were effectively inhibited by each siRNA. (B) TRIP13 protein levels were effectively inhibited by each siRNA (siTRIP13-1 and siTRIP13-2). (C) Cell proliferation was assessed using XTT assays 72 h after siRNA transfection into MDA-MB-231 cells. Cell proliferation ability was significantly reduced after knockdown of TRIP13. (D) MDA-MB-231 cells were treated with DCZ0415, a TRIP13 inhibitor. Cell proliferation ability was significantly blocked by DCZ0415 in a concentration-dependent manner.
Figure 10. Oncogenic function of TRIP13 in BrCa cells. (A) The inhibitory effects of two different siRNAs targeting TRIP13 (siTRIP13-1 and siTRIP13-2) expression were examined. TRIP13 mRNA levels were effectively inhibited by each siRNA. (B) TRIP13 protein levels were effectively inhibited by each siRNA (siTRIP13-1 and siTRIP13-2). (C) Cell proliferation was assessed using XTT assays 72 h after siRNA transfection into MDA-MB-231 cells. Cell proliferation ability was significantly reduced after knockdown of TRIP13. (D) MDA-MB-231 cells were treated with DCZ0415, a TRIP13 inhibitor. Cell proliferation ability was significantly blocked by DCZ0415 in a concentration-dependent manner.
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Table 1. Candidate gene targets of miR-30c-1-3p/miR-30c-2-3p significantly upregulated in our breast cancer mRNA profile.
Table 1. Candidate gene targets of miR-30c-1-3p/miR-30c-2-3p significantly upregulated in our breast cancer mRNA profile.
Entrez Gene IDGene SymbolGene NamemiR-30c-1-3p/
miR-30c-2-3p
Total Binding Sites		mRNA
Profile
log2 Fold Change		Gene
Expression
p Value		10-Year Overall
Survival
p Value
10024TROAPTrophinin associated protein14.59<0.010.256
9319TRIP13Thyroid hormone receptor interactor 1313.72<0.010.032
83461CDCA3Cell division cycle associated 313.62<0.01NA 1
8968HIST1H3FHistone cluster 1, H3f13.50>0.010.733
9123SLC16A3Solute carrier family 16 (monocarboxylate transporter), member 323.38<0.010.300
113730KLHDC7BKelch domain containing 7B13.35>0.010.300
952CD38CD38 molecule13.31>0.010.068
891CCNB1Cyclin B133.24<0.010.004
147841SPC24SPC24, NDC80 kinetochore complex component23.23<0.010.532
1281COL3A1Collagen, type III, alpha 113.20<0.010.310
92359CRB3Crumbs family member 313.19<0.010.531
5888RAD51RAD51 recombinase33.07<0.010.006
8638OASL2’-5’-oligoadenylate synthetase-like13.05<0.010.596
6664SOX11SRY (sex determining region Y)-box 1113.03>0.010.027
4085MAD2L1MAD2 mitotic arrest deficient-like 1 (yeast)12.99<0.010.058
5723PSPHPhosphoserine phosphatase12.98<0.010.015
84900RNFT2Ring finger protein, transmembrane 222.95<0.010.623
3017HIST1H2BDHistone cluster 1, H2bd32.89<0.010.244
6772STAT1Signal transducer and activator of transcription 1, 91kDa12.88<0.010.810
2537IFI6Interferon, alpha-inducible protein 612.83<0.010.813
1462VCANVersican12.80<0.010.095
317754POTEDPOTE ankyrin domain family, member D62.78>0.01NA
2065ERBB3erb-b2 receptor tyrosine kinase 322.66<0.010.131
57156TMEM63CTransmembrane protein 63C12.64<0.010.725
10051SMC4Structural maintenance of chromosomes 412.59<0.010.271
79814AGMATAgmatine ureohydrolase (agmatinase)12.58>0.010.050
55423SIRPGSignal-regulatory protein gamma22.57>0.010.021
4261CIITASlass II, major histocompatibility complex, transactivator72.56>0.010.017
2151F2RL2Coagulation factor II (thrombin) receptor-like 242.51<0.010.970
8534CHST1Carbohydrate (keratan sulfate Gal-6) sulfotransferase 122.50>0.010.052
154467CCDC167Coiled-coil domain containing 16712.48<0.010.479
4939OAS22’-5’-oligoadenylate synthetase 2, 69/71kDa22.48<0.010.506
55839CENPNCentromere protein N12.48<0.010.025
22797TFECTranscription factor EC12.46>0.010.731
8477GPR65G protein-coupled receptor 6522.41>0.010.315
921CD5CD5 molecule12.37>0.010.001
554313HIST2H4BHistone cluster 2, H4b12.37<0.01NA
1951CELSR3Cadherin, EGF LAG seven-pass G-type receptor 322.37>0.010.719
4582MUC1Mucin 1, cell surface associated12.36<0.010.905
4860PNPPurine nucleoside phosphorylase12.32>0.010.110
55824PAG1Phosphoprotein membrane anchor with glycosphingolipid microdomains 132.32>0.010.197
3838KPNA2Karyopherin alpha 2 (RAG cohort 1, importin alpha 1)12.32<0.010.013
1122CHMLChoroideremia-like (Rab escort protein 2)22.31>0.010.170
7371UCK2Uridine-cytidine kinase 212.28>0.010.062
1734DIO2Deiodinase, iodothyronine, type II32.28<0.010.411
653269POTEIPOTE ankyrin domain family, member I32.26>0.01NA
22996TTC39ATetratricopeptide repeat domain 39A22.24<0.010.189
9735KNTC1Kinetochore associated 112.22>0.010.173
9603NFE2L3Nuclear factor, erythroid 2-like 332.22>0.010.972
3070HELLSHelicase, lymphoid-specific12.21<0.010.327
8038ADAM12ADAM metallopeptidase domain 1212.17<0.010.344
25878MXRA5Matrix-remodelling associated 512.17<0.010.041
27338UBE2SUbiquitin-conjugating enzyme E2S12.16<0.010.800
55248TMEM206Transmembrane protein 20622.15<0.010.214
11006LILRB4Leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 422.12<0.010.246
150372NFAM1NFAT activating protein with ITAM motif 132.07>0.010.304
83481EPPK1Epiplakin 122.06<0.010.341
201254STRA13Stimulated by retinoic acid 1312.04>0.010.487
2187FANCBFanconi anemia, complementation group B12.02>0.010.017
4495MT1GMetallothionein 1G12.01>0.010.442
8270LAGE3L antigen family, member 322.01<0.010.862
10962MLLT11Myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila); translocated to, 1112.01>0.010.602
1 Not Available.
Table 2. TRIP13-mediated pathways identified by gene set enrichment analysis (GSEA).
Table 2. TRIP13-mediated pathways identified by gene set enrichment analysis (GSEA).
NameNormalized Enrichment ScoreFDR q-Value
HALLMARK_E2F_TARGETS3.561q < 0.001
HALLMARK_G2M_CHECKPOINT3.395q < 0.001
HALLMARK_MYC_TARGETS_V13.038q < 0.001
HALLMARK_MYC_TARGETS_V22.602q < 0.001
HALLMARK_MTORC1_SIGNALING2.477q < 0.001
HALLMARK_MITOTIC_SPINDLE2.473q < 0.001
HALLMARK_UNFOLDED_PROTEIN_RESPONSE1.975q < 0.001
HALLMARK_SPERMATOGENESIS1.906q < 0.001
HALLMARK_DNA_REPAIR1.6910.003
HALLMARK_INTERFERON_ALPHA_RESPONSE1.6550.003
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Mitsueda, R.; Toda, H.; Shinden, Y.; Fukuda, K.; Yasudome, R.; Kato, M.; Kikkawa, N.; Ohtsuka, T.; Nakajo, A.; Seki, N. Oncogenic Targets Regulated by Tumor-Suppressive miR-30c-1-3p and miR-30c-2-3p: TRIP13 Facilitates Cancer Cell Aggressiveness in Breast Cancer. Cancers 2023, 15, 4189. https://doi.org/10.3390/cancers15164189

AMA Style

Mitsueda R, Toda H, Shinden Y, Fukuda K, Yasudome R, Kato M, Kikkawa N, Ohtsuka T, Nakajo A, Seki N. Oncogenic Targets Regulated by Tumor-Suppressive miR-30c-1-3p and miR-30c-2-3p: TRIP13 Facilitates Cancer Cell Aggressiveness in Breast Cancer. Cancers. 2023; 15(16):4189. https://doi.org/10.3390/cancers15164189

Chicago/Turabian Style

Mitsueda, Reiko, Hiroko Toda, Yoshiaki Shinden, Kosuke Fukuda, Ryutaro Yasudome, Mayuko Kato, Naoko Kikkawa, Takao Ohtsuka, Akihiro Nakajo, and Naohiko Seki. 2023. "Oncogenic Targets Regulated by Tumor-Suppressive miR-30c-1-3p and miR-30c-2-3p: TRIP13 Facilitates Cancer Cell Aggressiveness in Breast Cancer" Cancers 15, no. 16: 4189. https://doi.org/10.3390/cancers15164189

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