Stable duplex-linked antisense targeting miR-148a inhibits breast cancer cell proliferation

MicroRNAs (miRNAs) regulate cancer cell proliferation by binding directly to the untranslated regions of messenger RNA (mRNA). MicroRNA-148a (miR-148a) is expressed at low levels in breast cancer (BC). However, little attention has been paid to the sequestration of miR-148a. Here, we performed a knockdown of miR-148a using anti-miRNA oligonucleotides (AMOs) and investigated the effect on BC cell proliferation. BC cell proliferation was significantly suppressed by AMO flanked by interstrand cross-linked duplexes (CL-AMO), whereas single-stranded and commercially available AMOs had no effect. The suppression was caused by sequestering specifically miR-148a. Indeed, miR-148b, another member of the miR-148 family, was not affected. Importantly, the downregulation of miR-148a induced a greater and longer-lasting inhibition of BC cell proliferation than the targeting of oncogenic microRNA-21 (miR-21) did. We identified thioredoxin-interacting protein (TXNIP), a tumor suppressor gene, as a target of miR-148a and showed that CL-AMO provoked an increase in TXNIP mRNA expression. This study provide evidence that lowly expressed miRNAs such as miR-148a have an oncogenic function and might be a promising target for cancer treatment.

The present study aims to investigate whether the knockdown of miR-148a could affect BC proliferation. We identified direct target mRNAs of miR-148a using mRNA microarray and online database analyses. Our results not only clarify the unknown functions of miR-148a in BC but also demonstrate the advantages of using CL-AMO in miRNA inhibition.
Next, we used a CL-AMO that had CLDs at the 5′-and 3′-ends 17,34 . The CLDs enable CL-AMO to stably hybridize with its target RNA, which allows a long-lasting action in cells 8,17 . The CL-AMO targeting anti-miR-148a (CL-miR148a) was synthesized from MeRNAs to have cross-linked 12-mer duplexes at both the 5′-and 3′-termini of the 22-mer antisense ( Fig. 1 and Supplementary Fig. S3). In addition to CL-miR148a, we constructed a negative control with a scrambled sequence (CL-NC: Supplementary Fig. S4) and a mismatch AMO (CL-miR148aM; Fig. 1), containing two mismatched base pairs in the seed region, which is essential for miRNA-target mRNA binding 35,36 . Surprisingly, CL-miR148a showed a dose-dependent activity (Fig. 2c), and transfection with 20 nM CL-miR148a completely inhibited MCF-7 cell proliferation after 6 days. Transfection of CL-NC and CL-miR148aM had no significant effect (Fig. 2d,e), which confirmed that CL-miR148a antiproliferative action was mediated by its binding to miR-148a. Moreover, we performed the inhibition of miR-148a in ZR-75-1 breast cancer cell line and confirmed the antiproliferative effect of CL-miR148a in another cell line ( Supplementary Fig. S5).
We also investigated the effects of miR-148b in MCF-7 cell. The transfection of the CL-AMO targeting miR-148b (CL-miR148b; Fig. 1) induced a very weak inhibition at 3 and 6 days after transfection, but the effect was lost after 9 days ( Supplementary Fig. S2c). The difference between CL-miR148a and CL-miR148b activities did not result from target amounts as both miRNAs were expressed to similar extent in MCF-7 cells (Supplementary Fig. S2d). It also indicates that the inhibitory effect of CL-miR148a was not mediated by miR-148b downregulation.
We performed immunoprecipitation (IP) using anti-Ago2 antibodies after CL-miR148a transfection to investigate whether CL-miR148a binds to the miRNA-induced silencing complex (miRISC). CL-miR148a was recovered in the IP samples, confirming that CL-miR148a binds to miRISC ( Fig. 2f and Supplementary Fig. S6). www.nature.com/scientificreports/ miR-21 is one of the most well-defined oncomiRs, and the downregulation of miR-21 suppresses cell proliferation of BC cells 6,7 . We prepared a CL-AMO-targeting miR-21 (CL-miR21; Supplementary Fig. S4) and compared the inhibition activities of CL-miR21 and CL-miR148a. As previously reported, CL-miR21 inhibited cell proliferation at 10 and 20 nM 6 days after transfection (Fig. 3). Interestingly, the antiproliferative activity of CL-miR148a was greater than the CL-miR21 effect at all concentrations tested. The biggest significant difference was observed for the 20 nM samples. It is notable that the inhibitory activity of CL-miR148a was maintained even after 9 days whereas CL-miR21 did not display a suppressive effect of this duration (data not shown).
We performed quantitative polymerase chain reaction (qPCR) to measure miRNA expression level after CL-AMO transfection but did not obtain reproducible results (data not shown). This is consistent with previous results reporting that high-affinity AMOs do not mediate the degradation of miRNAs 17,37 and sometimes directly prevent qPCR reaction 38 .

Investigation of target genes regulated by CL-miR148a.
We analyzed the gene expression profile by mRNA microarrays to identify the genes controlled by miR-148a. Genes were considered differentially expressed when more than 1.5-fold changes were measured upon CL-miR148a addition. We found 461 genes that were upregulated (Fig. 4a) and could constitute direct targets of miR-148a, and 462 genes were downregulated (Fig. 4b). An online database search using TargetScan 7.2 identified 802 genes predicted to contain binding sequences to miR-148a in the 3′-UTR of their mRNAs. Using microarray and online database analysis, we selected five candidates as direct miR-148a target: solute carrier family 7 member 11 (SLC7A11), thioredoxininteracting protein (TXNIP), cytoplasmic polyadenylation element-binding protein 4 (CPEB4), solute carrier family 7 member 5 (SLC7A5), and laminin subunit alpha 4 (LAMA4) ( Fig. 4c and Supplementary Fig. S7). MCF-7 cells were transfected with CL-miR148a, and qPCR analysis of SLC7A11, TXNIP, CPEB4, SLC7A5, and LAMA4 was performed. The mRNA expression levels of TXNIP, CPEB4, and SLC7A5 significantly increased, whereas SLC7A11 and LAMA4 mRNA were only slightly increased (Fig. 5). These results indicate that TXNIP, CPEB4, and SLC7A5 were plausible candidate genes involved in the inhibition of MCF-7 cell proliferation. CL-AMOs that have no complementary sequences to miR-148a (CL-miR148aM, CL-miR148b, and CL-NC) did not affect the mRNA expression level of these genes (Fig. 5). We considered that TXNIP was the most promising miR-148a target gene in MCF-7 cells as it was shown to contribute to a better prognosis of BC 39 and inhibits BC cell proliferation 40 . Recently, the direct binding between miR-148a and the 3′-UTR of TXNIP was reported in noncancerous cells. Moreover, an anti-miR-148a oligonucleotide was shown to upregulate TXNIP protein in hepatocytes 41 and cardiomyocytes 42 . We also investigated whether ssAMO or TuD-AMO-targeting miR-148a upregulated TXNIP expression. As shown in Supplementary Fig. S8, ssAMO had no effect, whereas TuD elicited  www.nature.com/scientificreports/ only a slight effect on TXNIP expression. Therefore, TXNIP expression might be closely associated with BC cell proliferation regulated by CL-miR148a, as discussed below.

Discussion
Aberrant expression of miRNAs can cause various cancers, which attracted attention on miRNAs as potential therapeutic targets and biomarkers of cancer 43,44 . Inhibition of miR-21, one of the earliest identified oncomiRs 45 , represents a potential therapeutic target 33 . In BC, expression of miR-148a is low, and its overexpression inhibits BC cell proliferation and migration [21][22][23]25 . As miR-148a has an antioncogenic function, knockdown experiments of miR-148a in BC were not considered.
In this study, we examined the effects of miR-148a knockdown. As the expression of miR-148a is low, we took advantage of CL-AMOs, which have high binding affinity for target miRNAs and a durable action in cells, to sequester miR-148a. CL-miR148a clearly inhibited BC cell proliferation in a dose-dependent manner (Fig. 2c and Supplementary Fig. S5), and the effect lasted for 9 days after transfection. Conversely, ssAMO-148a or TuD AMOs had no significant effect even at a concentration tenfold higher than that of CL-miR-148a (Fig. 2a, b, and Supplementary Fig. S2a, b). The mismatched CL-AMO (CL-miR148aM) did not inhibit BC cell proliferation (Fig. 2e). CL-miR148a was found to be bound to the Ago2-miRNA complex by IP (Fig. 2f), which confirmed that CL-miR148a functioned by binding between CL-AMO and miR-148a.
BC cell proliferation was not affected by CL-miR148b, which was specific for miR-148b ( Supplementary  Fig. S2c). This excluded a possible cross-reaction between CL-miR148a and miR-148b. Thus, miR-148b, unlike miR-148a, is irrelevant for suppressing BC cell growth.
Notably, even though miR-148a had a much lower expression level than miR-21 in MCF-7 cells (Supplementary Fig. S1) 46 , the downregulation of miR-148a inhibited cell growth more efficiently than the decrease of miR-21 did (Fig. 3). This result suggests that miRNAs with low expression levels are also involved in cancer cell proliferation.
The CLD structure is able to stabilize the hybridization of adjacent single-stranded antisense. We previously reported that CL-AMOs had a much higher melting temperature when bound to target RNAs than standard single-stranded antisense RNAs had 17 . Thus, we speculate that CL-AMOs tightly bind to their targets irreversibly, resulting in the complete sequestration or degradation of the target miRNA (Fig. 6, top).
To identify the CL-miR148a target genes associated with the inhibition of MCF-7 cell proliferation, we conducted mRNA microarray analysis after CL-miR148a transfection. We considered upregulated genes as direct targets of miR-148a (Fig. 4a) and narrowed them down to five genes by referring to an online database for miR-148a target prediction ( Fig. 4c and Supplementary Fig. S7). Importantly, three of the five genes were upregulated upon the addition of CL-miR148a (Fig. 5). We specifically focused on TXNIP, which was first identified as a 1,25-dihydroxyvitamin D 3 -inducible gene 47 , because recent studies have reported the direct binding of miR-148a to the 3′-UTR of TXNIP in hepatocytes 41 and cardiomyocytes 42 . In contrast to CL-miR148a, ssAMO-148a did not affect TXNIP, whereas TuD-148a induced a slight dose-dependent upregulation of TXNIP, consistent with its effect on cell proliferation (Supplementary Fig. S8). These results suggest that inhibition of BC cell proliferation is caused by changes in the TXNIP expression level.
TXNIP directly binds to the reduced form of thioredoxin (TRX) (Fig. 6, middle) 48 . TRX is an antioxidant protein that reduces oxidized proteins and protects cells from oxidative stress-induced apoptosis. It was also shown to contribute to cancer progression (TRX system; Fig. 6, bottom) 49,50 . TRX is overexpressed in BC 51,52 , and TRX-transfected MCF-7 cells display a higher proliferation rate 53 . Therefore, the TRX system is closely related to MCF-7 cell growth 54 . The binding of TXNIP to TRX decreases the amounts of the TRX reduced form, resulting in the inhibition of BC tumorigenesis 39,40 . CL-miR148a transfection upregulated TXNIP mRNA expression ( Fig. 5) but caused no change in TRX expression level ( Supplementary Fig. S9). TXNIP inhibits TRX activity by protein-protein interaction 48 but does not regulate TRX mRNA expression 55 . Therefore, it is plausible that CL-miR148a caused the inhibition of BC cell proliferation through the TXNIP and TRX system. This is the first study reporting that miR-148a downregulation inhibits BC cell proliferation. We showed that TXNIP gene expression is under the control of miR-148a in MCF-7 cells (Fig. 5). CL-miR148a could reduce miR-148a to even a lower concentration in cells, resulting in the inhibition of BC cell proliferation via TXNIP upregulation (Fig. 6, top). These results indicate that complete inhibition of miRNA unveils unknown cellular responses, which might lead to the discovery of unknown functions of miRNAs.
Our findings provided new knowledge regarding miR-148a function in BC, and they may contribute to the development of promising strategies for BC treatment. Additionally, it showed the importance of miRNAs with low expression levels. Finally, AMO-mediated knockdown of miRNAs can provide important insights into the functions of these miRNAs.

Methods
AMOs. The anti-miR-148a tough decoy RNA was purchased from GeneDesign Inc. (Osaka, Japan). 22-mer single-stranded anti-hsa-miR-148a-3p and all CL-AMOs (CL-miR148a, CL-miR148aM, and CL-miR148b) were chemically synthesized from 2′-O-methyl RNA (MeRNA) (GeneDesign Inc.). CL-AMOs were prepared according to previous reports 17 with slight modifications (see scheme in Supplementary Fig. S3). The molecular weights of CL-miR148a, CL-miR148b, and CL-miR148aM were confirmed by liquid chromatography-mass spectrometry (LC-MS). The ESI-MS data of all CL-AMOs are shown below. www.nature.com/scientificreports/ Switzerland). The cell number was expressed relatively to the value for no AMO control (single-point analysis at 6 days post-transfection) or for the day of transfection (day0) (time-course analysis for 9 days).
Quantitative analysis of miRNA expression. Endogenous miR-148a and miR-148b expression in MCF-7 cells was analyzed as previously described 8 using the TaqMan MicroRNA Assay (Thermo Fisher Scientific) according to the manufacturer's instructions. U6 RNA was used as an endogenous control to normalize the relative quantitation of target miRNAs.
Immunoprecipitation of Ago2 complex and detection of CL-miR148a. Briefly, we seeded 2.8 × 10 6 MCF-7 cells separately in three 75 cm 2 flasks and transfected them with CL-miR148a at a final concentration of 2 nM. We harvested the cells 24 h post-transfection and performed IP of the Ago2 complex using the micro-RNA Isolation Kit, Human Ago2 (Wako, Osaka, Japan), according to the manufacturer's instructions. After ethanol precipitation, we merged the IP samples from the three flasks into one sample. Northern blot analysis of CL-miR148a was performed as previously described 17 . For CL-miR148a detection, we prepared a digoxigenin (DIG)-tailed deoxyoligonucleotide probe for the miR-148a sequence using the DIG Oligonucleotide Tailing Kit, 2nd Generation (Roche, Mannheim, Germany), and subjected it to hybridization with CL-miR148a on the nylon membrane overnight. mRNA microarray. MCF-7 cells mock-transfected or transfected with CL-miR148a at a final concentration of 20 nM were harvested using TRI Reagent 3 days post-transfection. Purification of mRNA and mRNA microarray analysis were performed at Macrogen Japan (Tokyo, Japan) using the human Clariom S Assay (Thermo Fisher Scientific). The data were analyzed using Affymetrix GeneChip Command Console software (Thermo Fisher Scientific), and we compared differences in gene expression between mock-transfected and CL-miR148atransfected samples.
Quantitative analysis of mRNA expression. MCF-7 cells were seeded onto a 48-well plate at a density of 4 × 10 3 cells/well and transfected with AMOs, as described above, at a final concentration of 20 or 50 nM. After 3 days, the cells were harvested, merging three wells into one sample, using the RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany). We purified total RNAs and synthesized cDNA as previously described 8 . Then, we performed qPCR according to a previous report 8