miR-25 Downregulates MCU and Protects from Ca2+-Dependent Apoptosis
In the last two decades, mitochondrial Ca2+
homeostasis has been shown to participate in the control of the intrinsic pathway of apoptosis and to be influenced by oncogenes [3–6
], thus suggesting that it is a signaling checkpoint in tumorigenesis. However, direct evidence and mechanistic insight were still lacking. The recent identification of the mitochondrial Ca2+
channel (mitochondrial calcium uniporter, MCU) [1, 2
] and of the associated regulator MICU1 (also known as CBARA1) [7
] now allow molecular investigation of the process, including the regulation of their expression by microRNAs (miRNAs). miRNAs are a class of small (19–25 nt), noncoding regulatory RNAs that regulate gene expression, causing target mRNA degradation or suppressing mRNA translation [8
]. In human cancers, specific miRNAs are up- or downregulated, with consequent alteration in the expression of target proteins [9, 10
By filtering the output of four target prediction algorithms (TargetScan [11
], MicroT [12
], MicroCosm [13
], and miRanda [14
]; see Table S1
available online), we identified five cancer-related miRNA families (miR-15, miR-17, miR-21, miR-25, and miR-137) that could be predicted to target MCU and/or MICU1. We thus tested their effect on mitochondrial Ca2+
homeostasis by expressing them in HeLa cells and measuring mitochondrial [Ca2+
] with a targeted aequorin-based Ca2+
probe (mtAEQ) [15
]. The data (A) showed that only miR-25 caused a marked reduction in the [Ca2+
rise evoked by cell stimulation with 100 μM histamine, an agonist coupled to the generation of inositol 1,4,5-trisphosphate (InsP3
) and the release of Ca2+
from the endoplasmic reticulum (ER). Accordingly, overexpression of an anti-miR-25 increases the mitochondrial Ca2+
uptake to agonist stimulation (Figure S1
A), with a slight decrease in cytosolic [Ca2+
), probably due to increased Ca2+
clearance by mitochondria (Figure S1
miR-25 Reduces [Ca2+]m and Protects from Apoptosis by Downregulation of MCU mRNA and Protein Levels
The effects were predicted to depend on MCU downregulation. Indeed, the bioinformatics analysis of the 1,896 nt 3′ UTR of MCU revealed a 100% match target seed sequence for miR-25 at nt 1075–1081, highly conserved across seven species (B), and insertion of the 759 nt 3′ UTR of MCU (but not of the 569 nt 3′ UTR of MICU1) downstream of the luciferase gene in a reporter plasmid led to significant miR-25-dependent decrease of reporter activity (Figures S1
C and S1D). We thus tested MCU expression by immunoblotting and detected a marked reduction in the protein level upon miR-25 overexpression (C) and an increase in anti-miR-25-expressing cells (Figure S1
E). As expected, MCU mRNA abundance was significantly decreased by miR-25 (D), whereas anti-miR-25 increased it (Figure S1
F). MCU downregulation was also evident using an immunofluorescence technique: Figure S1
G shows that miR-25 expression drastically decreased MCU antibody reactivity.
The effect of miR-25 is shared by the other members of the miRNA family: miR-92a and miR-363 target MCU mRNA and reduce MCU protein levels and, accordingly, inhibit mitochondrial Ca2+ uptake, without affecting [Ca2+]c and [Ca2+]er (data not shown).
We investigated whether miR-25-dependent reduction in mitochondrial Ca2+
uptake correlates with increased resistance to apoptotic challenges. Microscopy counts of cell viability after treatment with H2
, C2-ceramide, or staurosporine (STS) revealed that miR-25-expressing HeLa cells were strongly protected from death caused by C2-ceramide and H2
(E), apoptotic challenges for which mitochondrial Ca2+
loading acts as a sensitizing factor [16–18
], whereas the sensitivity to STS was unaffected. Accordingly, PARP and caspase-3 cleavage upon C2-ceramide treatment were markedly reduced in miR-overexpressing cells (F). These results were also confirmed by cellular positivity to the apoptotic marker annexin V (Figure S1
miR-25 Induces Reduction of Mitochondrial Ca2+ Uptake Exclusively through MCU
We then proceeded to rule out that the effect on [Ca2+]m was secondary to alterations of global Ca2+ signaling patterns or to morphological or functional dysregulation of mitochondria. On the former aspect, we investigated the cytosolic [Ca2+] changes and the state of filling and release kinetics of the ER. The results showed that miR-25, when expressed in HeLa cells, caused no difference in the amplitude of the [Ca2+]c rise evoked by histamine (A), nor in the steady state [Ca2+]er or in the release caused by the agonist (B). Thus, the effect of miR-25 on Ca2+ homeostasis is exclusively mitochondrial.
miR-25 Inhibits Mitochondrial Ca2+ Uptake without Causing Morphological Rearrangement or Changes in the Electrochemical Gradient
We then investigated the mitochondrial membrane potential (ΔΨm), the driving force for Ca2+
accumulation, and the morphology of mitochondria, i.e., both the contacts with the ER (which were shown to be a critical determinant of rapid Ca2+
transfer between the two organelle [19–21
]) and the formation of largely interconnected tubules, which favors Ca2+
diffusion within mitochondria. On the former aspect, measurements with the ΔΨm-sensitive fluorescent dye tetramethylrhodamine methyl ester (TMRM) revealed no difference between miR-overexpressing and control HeLa cells (C). As to morphology, mitochondrial labeling with the fluorescent probe mtDsRed showed that miR-25 overexpression causes no significant difference in mitochondrial volume or number (D). Similarly, cotransfection with mtDsRed and an ER-targeted GFP showed no difference in the number of contact sites (D, contact sites in white).
Overall, the data reveal that the [Ca2+]m reduction caused by miR-25 should be ascribed to reduction of mitochondrial Ca2+ uptake through MCU. To further confirm this notion, we measured mitochondrial Ca2+ accumulation in permeabilized cells. For this purpose, HeLa cells were perfused with a solution mimicking the intracellular milieu (IB), supplemented with 2 mM EGTA, and permeabilized with digitonin for 1 min. The perfusion buffer was then changed to IB with an EGTA-buffered [Ca2+] of 4 μM (E) or 1 μM (F), eliciting a gradual rise in [Ca2+]m that reached a plateau value of ~80 and ~20, respectively. At both buffered [Ca2+], miR-25 overexpression causes a marked reduction in the rate of Ca2+ accumulation into mitochondria.
alterations induced by miR-25 could be reverted by MCU re-expression in miR-25-expressing cells (Figure S2
A) and, accordingly, this rescued Ca2+
affinity was mirrored in enhanced susceptibility to Ca2+
-dependent apoptosis (Figure S2
B). Moreover, 22Rv1 prostatic cells, which possess very high levels of miR-25 (see ), were strongly sensitized to apoptosis after MCU overexpression (Figure S2
C). The increased ability of mitochondria to accumulate Ca2+
is a fundamental aspect in MCU-related promotion of cell death: indeed, apoptosis induction observed in MCU-overexpressing HeLa cells was almost abolished in the presence of intracellular Ca2+
buffer BAPTA (Figure S2
Inhibition of MCU Levels by miR-25 Is a Key Aspect in Human Colon Cancer Progression
Finally, although miR-25 has also been reported to exert antiapoptotic effects via interference with the expression of proapoptotic proteins, such as Bim [22
], TRAIL [23
], and PTEN [24
], these results show how MCU can be considered a fundamental target of miR-25-dependent apoptosis inhibition.
Inhibition of MCU Levels by miR-25 Is a Key Aspect in Human Colon Cancer Progression
We then extended the analysis to cancer cells and tissues. We first evaluated cell lines derived from human carcinomas, in which miR-25 was reported to be highly expressed [24–26
]. Both in PC3, LnCaP, and 22Rv1 (derived from prostate cancer) and in HCT116, RKO, SW80, and WiDr (derived from colon cancer) cell lines, we detected an inverse correlation between miR-25 levels and MCU mRNA expression, with high miR-25 levels and low MCU expression levels in cancer lines, compared to primary nonneoplastic cells (A). We then directly investigated human poorly differentiated colonic adenocarcinoma samples by immunohistochemistry and microarray. Also in this case, a significant difference in miR-25 expression levels was detected (B), which correlates with a downregulation of MCU expression. Indeed, in colonic adenocarcinoma samples with high miR-25 expression levels, MCU was virtually undetectable by immunohistochemistry in cancerous tissues, compared to relatively high protein abundance in the normal mucosa (C).
To validate that miR-25 exerts its biological activity through its effect on MCU, we transfected HeLa cells with short hairpin RNA (shRNA) targeting MCU: as for miR-25, shRNA-MCU decreases MCU abundance and increases proliferation (Figure S3
A), indicating that MCU targeting is important for the growth-promoting activity of miR-25. We also tested the ability of MCU to inhibit the proliferation. We generated PC3 cells that stably expressed a MCU-FLAG-tagged construct (MCU-FLAG), in which MCU level and activity was increased relative to that in empty vector (pcDNA3) stable clones (Figures S3
B and S3C), and found that they formed lower numbers of colonies in soft agar compared to control pcDNA3 stable clones (Figure S3
We then investigated whether miR-25-dependent inhibition of mitochondrial Ca2+
uptake, and the ensuing resistance to apoptosis, could be specifically reversed in cancer cells. For this purpose, we overexpressed anti-miR-25 in the PC3 and HCT116 cells lines investigated in . In both cell types, anti-miR-25 expression caused an ~40% increase in the [Ca2+
rise evoked by 100 μM ATP (A and 4B). Accordingly, sensitivity to C2-ceramide and H2
were enhanced, as revealed by the lower viability (C and 4D) and increased PARP and caspase-3 cleavage (E and 4F) detected in anti-miR-25-expressing cells. These data were also confirmed measuring cellular positivity to annexin V (Figures S4
A and S4B).
Regulation of miR-25 Levels Strongly Sensitizes Cells to Ca2+-Dependent Apoptotic Stimuli
Overall, the data identify a microRNA (miR-25), highly expressed in cancer cells, that by targeting the newly discovered calcium channel of mitochondria reduces the sensitivity of cancer cells to apoptotic agents. This not only represents conclusive evidence of the key role of organelle Ca2+ accumulation in the mitochondria-dependent apoptotic routes but also highlights a novel, unexpected target in cancer therapy. Now, the exciting task of unveiling the structural and functional properties of this long-awaited component of the calcium signaling machinery of the cell finds an immediate translational application in a disease area of paramount importance.