© 2015. Published by The Company of Biologists Ltd.

The miR-199/DNM regulatory axis controls receptor-mediated endocytosis

Juan F. Aranda1,2, Alberto Canfrán-Duque1,2, Leigh Goedeke1,2, Yajaira Suárez1,2 and Carlos

Fernández-Hernando1,2, #

1Integrative Cell Signaling and Neurobiology of Metabolism Program, Section of Comparative

Medicine, Yale University School of Medicine, New Haven, Connecticut, USA.

2Vascular Biology and Therapeutics Program, Yale University School of Medicine, New Haven,

Connecticut, USA.

#Corresponding author:

Carlos Fernández-Hernando, PhD

10 Amistad Street, Amistad Research Building, Room 337C, New Haven, CT 06510

Yale University School of Medicine

Tel: (203) 737 4615

Fax: (212) 737 2290

Email: carlos.fernandez@.yale.edu

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JCS Advance Online Article. Posted on 10 July 2015

ABSTRACT

Small non-coding RNAs (microRNAs) are important regulators of gene expression that modulate

many physiological processes, however their role in regulating intracellular transport remains

largely unknown. Intriguingly, we found that the dynamin (DNM) genes, a GTPase family of

proteins responsible for endocytosis in eukaryotic cells, encode the conserved miR-199a/b family

of miRNAs within their intronic sequences. Here, we demonstrate that miR-199a/b regulates

endocytic transport by controlling the expression of important mediators of endocytosis such as

clathrin heavy chain (CLTC), Rab5A, low-density lipoprotein receptor (LDLR) and caveolin-1 (Cav-

1). Importantly, miR-199a/b-5p overexpression markedly inhibits CLTC, Rab5A, LDLR and Cav-1

expression, thus preventing receptor-mediated endocytosis in human cell lines (Huh7 and HeLa).

Of note, miR-199a-5p inhibition increases target gene expression and receptor-mediated

endocytosis. Altogether, our work identifies a novel mechanism by which miRNAs regulate

intracellular trafficking. In particular, we demonstrate that the DNM/miR-199a/b-5p genes act as a

bifunctional locus that regulates endocytosis, thus adding an unexpected layer of complexity in the

regulation of intracellular trafficking.

Keywords: miRNA, endocytosis, LDLR

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ABBREVIATIONS AND ACRONYMS

LDL, Low-density lipoprotein

LDLR, Low-density lipoprotein receptor

CLTC, Clathrin heavy chain

Rab5A, Ras-associated protein 5A

Rab21, Ras-associated protein 21

SREBP, Sterol regulatory element-binding protein

miRNA, microRNA

UTR, Untranslated region

DNM, Dynamin

PTEN, Phosphatase and tensin homologue

Cav-1, Caveolin 1

GTPase, Guanine triphosphate phosphatase

RME, Receptor mediated endocytosis

ER, Endoplasmic reticulum

EE, Early endosomes

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INTRODUCTION

Endocytosis is an essential process in cell physiology by which eukaryotic cells take up

macromolecules and particles from the surrounding medium. Many physiological processes,

including cell migration, angiogenesis, metabolism and development, depend on proper

functioning of endocytosis and thus cells have developed multiple mechanisms to ensure proper

intracellular trafficking and endocytosis (Fernandez-Rojo et al., 2012; Gu et al., 2011; Lee et al.,

2014; Mousley et al., 2012; Parachoniak et al., 2011). There are multiple pathways of endocytosis

into cells; including, clathrin-dependent, caveolin-dependent and clathrin- and caveolin-

independent internalization. In all of them, the material to be internalized is surrounded by an area

of plasma membrane, which then buds off inside the cell to form a vesicle containing the ingested

material that is delivered to intracellular organelles and cytosol (McMahon and Boucrot, 2011).

The best-characterized form of this process is receptor-mediated endocytosis (RME), which

provides a mechanism for the selective uptake of essential nutrients such as LDL via the LDLR

(Brown and Goldstein, 1986) or iron, via TfR (Harding et al., 1983). Thus, factors that are affecting

RME have a direct effect on these receptors, and in the case of LDLR are regulating intracellular

cholesterol levels. In both LDLR and TfR internalization processes, clathrin is playing a key role

during the formation of coated vesicles (Moore et al., 1987). Once vesicles are internalized its

passage through a broad endosomal compartment system is required; first rapidly they are

travelled to early endosomes (EE), where Rab5A is a key regulator (Nielsen et al., 1999), and

subsequently to late endosomes and lysosomes. Whichever route of entry, a crucial step in

endocytosis and intracellular transport is the formation of endocytic vesicles, which specifically

requires the participation of the GTPase dynamin (DNM), to promote its scission and fission from

the plasma membrane (Ferguson and De Camilli, 2012; Jones et al., 1998; Roux et al., 2006;

Takei et al., 1995). DNM is also involved in other membrane remodeling processes, such as

fission of clathrin coated vesicles from the TGN (Cao et al., 2000) and membrane ruffling through

the interaction with actin nucleators (Gu et al., 2010). In mammals the DNM gene family is

encoded by three separate genes: DNM1, 2 and 3. While all three DNM genes share a high

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degree of sequence homology, thus resulting in similar domain-containing proteins, they differ in

their tissue-specific expression (Urrutia et al., 1997).

It is presently accepted, that small non-coding RNAs, microRNAs (miRNAs), are key regulators of

several cellular processes (Bushati and Cohen, 2007). This regulatory control is carried out

through repression of gene expression at the post-transcriptional level by base pairing with

complementary regions mainly within the 3’ untranslated regions (3’UTR) of target mRNAs, thus

promoting mRNA degradation, translational repression, or both (Ambros, 2004; Bartel, 2009;

Filipowicz et al., 2008). Most miRNAs are first transcribed into long transcripts of primary miRNAs

(pri-miRNAs) that are then processed sequentially in the nucleus by Drosha/ DGCR8 to generate

pre-miRNAs, and by Dicer in the cytoplasm to generate the mature ~22 nt miRNA duplex (Lee et

al., 2003) that, after strand selection, mediates the targeting activity when incorporated into the

RISC complex (Chendrimada et al., 2007). There is mounting evidence that suggests that both

strands, the guide or “5p” as well as the miRNA* (also known as the passenger or “3p” strand)

have important regulatory activity (Chamorro-Jorganes et al., 2014; Goedeke et al., 2013).

Approximately half of the miRNA genes can be found in intergenic regions, whereas the intragenic

miRNAs are predominantly located inside introns and usually oriented on the same DNA strand of

the host gene (Saini et al., 2007). Intergenic miRNA genes have their own promoter region and

their expression is regulated by the same molecular mechanisms that control the expression of

protein-coding genes. Alternatively, same-strand intronic miRNAs are co-transcribed with their

host gene (Rodriguez et al., 2004), and then processed to finally become mature functional

miRNAs. A number of studies have shown that intronic miRNAs localized in the same orientation

as their host genes usually cooperate with them to regulate similar cellular functions (Rayner et

al., 2010; van Rooij et al., 2009). However, exceptions to this common scheme of co-transcription

have been reported. In fact, about 26% of intronic miRNAs are antisense orientated and

transcribed independently of their host genes (Rodriguez et al., 2004; Siegel et al., 2009). Despite

of this, intronic miRNA can support the function of its host gene by silencing genes that are

functionally antagonistic to the host, or act synergistically with the host by coordinating the

expression of genes with related functions.

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Interestingly, miR-199a/b family members are encoded within introns of the DNM genes in the

opposite orientation of their host gene. MiR-199a/b family is composed of three members, miR-

199a1, miR-199a2 and miR-199b located within the DNM2, DNM3 and DNM1 genes respectively.

MiR-199a/b gene sequences exhibit high conservation across species and share the same seed

sequence, thereby potentially targeting the same group of genes. Interestingly, predicted target

genes for miR-199a/b-5p (guide) strands are broadly conserved among species compared to the

miR-199a/b-3p (passenger) strand. Therefore, here we investigated miR-199a/b-5p potential

target genes using several miRNA target bioinformatic algorithms. Importantly, we identified

putative binding sites for miR-199a/b-5p in the 3’UTR of genes involved in vesicle mediated

transport and endocytosis. Of note, our present findings indicate that miR-199a/b-5p regulates the

expression of multiple genes participating clathrin-dependent (Cltc, Rab5A, Rab21 and Ldlr) and

independent endocytosis (Cav-1). Furthermore, we demonstrated that miR-199a/b-5p inhibited the

clathrin-mediated endocytosis through the regulation of CLTC, Rab5A and Rab21 expression,

affecting the normal function of receptors located in the plasma membrane such as LDLR and

transferrin receptor (TfR). Altogether, our work showed a novel mechanism by which miRNAs

regulate intracellular trafficking. In particular, we describe that the DNM genes along with miR-199

act as a bifunctional locus encoding DNM, a GTPase that is a critical mediator of endocytosis, and

miR-199a/b, which regulates intracellular trafficking as well, thus adding an unexpected layer of

complexity in the regulation of endocytosis.

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RESULTS

MiR-199a/b-5p are potential regulators of transport and vesicle-mediated trafficking

processes

While investigating the genomic location of miRNAs encoded in the human genome, we noted the

intriguing presence of a highly conserved miRNA family, miR-199a/b, embedded within the

intronic sequences of the DNM genes (Fig. 1A). The miR-199a/b family consists of three

members, miR-199a-1, miR-199a-2 and miR-199b, which are transcribed from conserved

antisense intronic transcripts of the DNM2 locus (human chromosome 19), DNM3 locus (human

chromosome 1) and DNM1 locus (human chromosome 9), respectively (Fig. 1A). Human miR-

199a1-5p and miR-199a2-5p have identical mature sequences, while the miR-199b-5p mature

sequence differs in two nucleotides outside of the seed sequence (Fig. 1B). The miR-199a-5p

mature sequences show higher conservation among vertebrate species than miR-199b-5p

(Supplementary material Fig. S1A), indicating that miR-199a1 and miR-199a2 are evolutionary

conserved. Since this seed sequence conservation is retained, we focused our study on miR-

199a-5p.

Mammalian miRNAs are present in the genome as independent transcriptional units or embedded

within the introns of protein-coding genes. To determine whether the expression of the miR-

199a/b family members and DNM genes are co-regulated, we measured their expression in

different human tissues. As seen in Fig. 1C and supplementary material Fig. S1B, we observed

that the expression of mature miR-199a-5p (miR-199a1-5p and miR-199a2-5p), miR-199b-5p and

their respective precursors (pre-miR-199a-1, pre-miR-199a-2 and pre-miR-199b) (Supplementary

material Fig. S1C) were widely expressed in most tissues. Remarkably, compared with other

tissues, mature miR-199a-5p is expressed at very low levels in the brain that expresses high

levels of DNM3 (Fig. 1C). Similarly, the expression of miR-199b-5p in the brain is markedly

reduced compared with other tissues (supplementary material Fig. S1B). Interestingly, miR-199b-

5p levels correlate inversely with DNM1 expression (Fig. S1B), suggesting that miR-199b-5p is

regulated independently of its host gene.

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We next sought to ascertain the potential function of miR-199a/b-5p. To this end, we employed a

combination of bioinformatic algorithms (Targetscan, [http://www.targetscan.org] and miRanda

[http://www.microrna.org]) that predict miRNA targets largely based on the ability of the miRNA

sequence to undergo specific base-pairing within the putative 3’UTR target. The predicted miR-

199a/b-5p target genes were assigned to several functional annotation clusters and networks as

shown in Fig. 1D. Interestingly, using gene ontology software analysis (Panther

[http://www.pantherdb.org/]) (Thomas et al., 2003), and the protein–protein interaction database,

String, (http://string-db.org/) (Szklarczyk et al., 2011), we observed that the most represented

cluster was associated with genes involved in cellular transport (Fig. 1D). Among them

specifically, miR-199a/b-5p were predicted to target a vast network of genes associated with

endocytic functions, including CLTC, Cav-1, Rab5A, LDLR and Rab21 (Fig. 1D) (Bucci et al.,

1994; Dorsey et al., 2007; Doyon et al., 2011; Pellinen et al., 2008; Simpson et al., 2004; Singh et

al., 2003). This intriguing observation led us to investigate the biological role of miR-199a/b-5p in

controlling receptor-mediated endocytosis.

MiR-199a-5p regulates the expression of endocytosis mediators

To evaluate the effect of miR-199a/b-5p on CLTC, LDLR, Rab5A and Rab21 expression, we

transfected human hepatic Huh7 cells with synthetic miR-199a-5p mimics. As seen in Fig. 2A,

CLTC, LDLR, Rab5A and Rab21 mRNA expression was inhibited in cells overexpressing miR-

199a-5p compared to cells transfected with a non-targeting control miRNA mimic (CM).

Conversely, Huh7 cells transfected with antisense oligonucleotides directed against miR-199a-5p

(inh-199a-5p) had significantly increased CLTC, LDLR and Rab5A mRNA expression compared to

cells transfected with a non-targeting control inhibitor (CI) (Fig. 2B). Consistent with this,

overexpression of miR-199a-5p markedly reduced CLTC, LDLR, Rab5A and Rab21 protein

expression (Fig. 2C, quantified in right panels). Moreover, inhibition of endogenous miR-199a-5p

increased LDLR, Rab5A and Rab21 protein in Huh7 cells, suggesting that miR-199a/b-5p plays a

physiological role in regulating cellular endocytosis (Fig. 2D, quantified in right panels).

CLTC, LDLR, Rab5A and Rab21 have one or more miR-199a/b-5p predicted binding sites that are

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conserved across mammals (Fig. 2E and supplementary material Fig. S1D). To determine

whether miR-199a-5p specifically targets the 3’UTR of CLTC, LDLR, Rab5A and Rab21, we

cloned the entire 3’UTR of the aforementioned genes into a luciferase reporter vector and

assessed whether miR-199a-5p overexpression could reduce luciferase reporter activity. As seen

in Fig. 2F, miR-199a-5p markedly repressed CLTC, LDLR and Rab21 3’UTR activity. Surprisingly,

Rab5A 3’UTR activity was not influenced by miR-199a-5p overexpression (Fig. 2F), despite its

inhibitory effect on Rab5A mRNA and protein expression (Fig. 2A, C). As expected, mutation of

the miR-199a-5p target sites relieved miR-199a-5p repression of CLTC, LDLR and Rab21 3’UTR

activity, consistent with a direct interaction of miR-199a-5p with these sites (Fig. 2F). In addition to

these genes, we also identified Cav-1 as a predicted miR-199a-5p target gene. As Cav-1 is

expressed at very low levels in hepatic cell lines, we analyzed the role of miR-199a-5p in

regulating Cav-1 expression in HeLa cells, which express significantly higher levels of this protein.

The results show that miR-199a-5p overexpression markedly reduces Cav-1 mRNA and protein

expression compared with cells transfected with CM (supplementary material Fig. S2A, C, left

panels). A similar effect was observed when we analyzed the expression of Cav-1 by

immunofluorescence (supplementary material Fig. S2D). Importantly, inhibition of endogenous

miR-199a-5p led to an increase in Cav-1 expression (supplementary material Fig. S2A, C, right

panels and supplementary Fig. S2D). We finally confirmed that miR-199a-5p directly targets Cav-

1 by assessing the Cav-1 3’UTR luciferase activity in cells transfected with miR-199a-5p mimics

(supplementary Fig. S2B). Taken together these results identify CLTC, LDLR, Rab21 and Cav-1

as direct targets of miR-199a-5p.

MiR-199a-5p inhibits receptor-mediated endocytosis

CLTC, Rab5A and Rab21 are essential components of receptor-mediated endocytosis (RME), a

process by which cells internalize molecules (de Hoop et al., 1994; Doyon et al., 2011; Simpson et

al., 2004). RME is widely used for the specific uptake of substances required by the cell, including

LDL via the LDLR (Kang and Folsch, 2011; Mettlen et al., 2010), and iron, through the TfR (Gan et

al., 2002; Tosoni et al., 2005). The LDLR binds to LDL particles and mediates their endocytosis

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along with CLTC, necessary for coated vesicle formation and the Rab5A and Rab21 GTPases,

which are involved in regulating vesicle trafficking in the early endosomal compartment

(Semerdjieva et al., 2008; Simpson et al., 2004). Our previous results suggested that miR-199a-

5p may control LDLR activity by directly targeting the LDLR (Fig. 2) but also by regulating its

endocytosis through repression of Rab5A, Rab21 and CLTC (Fig. 2). Therefore, to functionally

assess the role of miR-199a-5p in regulating LDLR activity in human hepatic cells, we

overexpressed or inhibited miR-199a-5p and examined fluorescence-labeled LDL (DiI-LDL)

binding (4C during 2 h) and uptake (37C during 5, 15 and 30 min) by flow cytometry (Fig. 3A).

Transfection of Huh7 cells with miR-199a-5p mimics markedly reduced DiI-LDL specific uptake at

different time points. Importantly, the reduction in DiI-LDL internalization at 30 min was

significantly greater (34%) than the DiI-LDL binding before incubating cells at 37C (22%) (Fig.

3B). These results suggest that both DiI-LDL binding and uptake are regulated by miR-199a-5p.

As expected, DiI-LDL uptake was significantly reduced in Huh7 cells transfected with miR-199a-5p

at longer time points (2 and 4 h) and treated with Dynasore, a widely used DNM inhibitor (Macia et

al., 2006) (Fig. 3C and supplementary material Fig. S3A, C). Conversely, cells treated with a miR-

199a-5p inhibitor (inh-199a-5p) had increased DiI-LDL uptake during 2 and 4 h (supplementary

material Fig. S3B, C). Treatment of Huh7 with U18666A, a compound that blocks cholesterol

trafficking from late endosomes and lysosomes to ER resulting in enhanced processing of the

sterol regulatory binding protein 2 (SREBP2) and increased expression of LDLR (Liscum and

Faust, 1989) was used as a positive control (supplementary material Fig. S3B right panel and

S3D). To further confirm the effect of miR-199a-5p in regulating LDLR expression and activity, we

next assessed LDLR-antibody internalization by immunofluorescence. As seen in supplementary

material Fig. S4, we found a marked reduction of LDLR internalization as well as a concomitant

decrease in CLTC staining in cells transfected with miR-199a-5p mimics compared to CM.

Upon internalization, LDL is delivered first to early endosomes and then to lysosomes where LDL-

derived cholesteryl esters are hydrolyzed to unesterified cholesterol (Brown and Goldstein, 1986).

To ascertain whether miR-199a-5p influences intracellular trafficking of LDL-derived cholesterol,

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we transfected Huh7 cells with a CM, miR-199a-5p mimic and assessed DiI-LDL uptake and

subcellular localization by immunofluorescence. Interestingly, we observed that overexpression of

miR-199a-5p resulted in accumulation of DiI-LDL particles in early endosomal compartment as

seen by co-staining with EEA1 marker at early time points (Fig. 3D,E). We also analyzed the

intracellular co-localization of DiI-LDL with Rab11, a recycling endosomal and the late

endosome/lysosomal compartment CD63 during the time course experiment. While miR-199a-5p

levels did not influence the co-localization of Rab11 and DiI-LDL after culturing the cells in

presence of DiI-LDL for 20 min, we found a little co-localization of DiI-LDL with CD63 in cells

transfected with miR-199-5p after 30 min of incubation with DiI-LDL at 37C (Fig. 3D,E). We also

observed a marked reduction in DiI-LDL/CD63 co-localization in cells transfected with miR-199a-

5p 4h after DiI-LDL incubation (supplementary material Fig. S3C). As expected, cells treated with

U18666A accumulated DiI-LDL in the lysosome compartments, while Dynasore significantly

reduced LDL internalization and colocalization with lysosomal CD63 protein (supplementary

material Fig. S3D). Cells transfected with Inh-199a-5p had no significant effect on the DiI-

LDL/CD63 colocalization (supplementary material Fig. S3C, right panels). Since the cholesterol

uptake in the cells is mediated mainly by the internalization of LDL via LDLR, we next assessed

the intracellular location of cholesterol using Filipin, a dye that stains unesterified cholesterol.

Consistent with the diminished LDL uptake observed in miR-199a-5p transfected cells, we found a

striking accumulation of free cholesterol at the plasma membrane compared with CM treated cells

(Fig. 4 middle panel and intensity plot). Inhibition of endogenous miR-199a-5p expression showed

similar free cholesterol distribution than Huh7 cells transfected with CM (Fig. 4). Taken together,

these results suggest that cells overexpressing miR-199a-5p have a trafficking defect that causes

missorting of LDL particles after LDLR internalization.

Because miR-199a-5p inhibits numerous components of endocytic pathway as well as the LDLR,

the study of the contribution of miR-199a-5p in regulating RME by assessing DiI-LDL uptake could

be influenced by the significant reduction in LDLR expression observed in miR-199a-5p

overexpressing cells. To avoid this caveat, we transfected Huh7 cells with a LDLR-GFP cDNA

construct that lacked the 3’UTR, thereby making it resistant to miR-199a-5p’s inhibitory action,

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and assessed DiI-LDL cellular localization in cells transfected with miR-199a-5p mimics. The

results shown that miR-199a-5p overexpression caused a marked retention of LDLR-GFP in the

plasma membrane as shown by actin co-staining, compared to cells transfected with a CM (Fig.

5A, intensity plots in right panels). We further analyze the LDLR internalization by flow cytometry.

To this end, Huh7 cells were transfected with CM or miR-199a-5p and LDLR-GFP construct and

incubated with a LDLR Ab (labeled with PE) during 2 h at 4ºC. Then, cells were incubated at 37ºC

at different time points to allow LDLR/LDLR-Ab internalization. Importantly, we found that miR-

199a-5p overexpression in LDLR-GFP positive cells impairs surface internalization of LDLR upon

engagement with antibodies and 37ºC incubation (Fig. 5B). These results suggest that the

reduction of DiI-LDL uptake is mediated by two mechanisms; direct inhibition of LDLR expression

and the repression of numerous components of the endocytic machinery.

Given the direct effect of miR-199a-5p on LDLR expression and activity, we wondered whether or

not other receptor-mediated processes were affected by miR-199a-5p. Therefore we next

assessed the effect of miR-199a-5p on transferrin receptor (TfR) endocytosis. TfR regulates the

import of the transferrin-iron complex via clathrin-mediated endocytosis (Killisch et al., 1992). To

this end, we transfected human epithelial HeLa cells with miR-199a-5p and incubated them with

FITC-conjugated transferrin at various time points. As shown in Fig. 6A, miR-199a-5p significantly

inhibited transferrin internalization as assessed on a single cell-basis by flow cytometry. As

expected, Dynasore treatment inhibited also transferrin internalization (Fig 6A). This effect was

independent of total and surface TfR expression levels (Fig. 6B,D), suggesting that the observed

internalization defect is due to other endocytic proteins rather than the absence of TfR. To rule out

the possibility that miR-199a-5p may be also affecting exocytosis, we transfected HeLa cells with

miR-199a-5p mimics for 48h and then treated with the endocytosis inhibitor, Dynasore. As seen in

Fig. 6A, Tf-FITC internalization under these conditions did not change compare to cells treated

with Dynasore only, suggesting that exocytosis was not affected by miR-199a-5p. We next

analyzed the intracellular localization of Tf-FITC after miR-199a-5p transfection using confocal

microscopy. As expected, miR-199a-5p overexpressing cells showed a reduced Rab5 staining

compared with CM transfected cells (Fig. 6C). Tf-FITC internalization in CM transfected cells

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showed a partial co-localization with EEA1 10 min after incubating cells with Tf-FITC (Fig. 6C).

Most importantly, miR-199a-5p overexpression increased the co-localization of Tf-FITC with EEA1

suggesting a defect in the endocytic process (Fig. 6C). This effect was not due to changes in

EEA1 protein levels as measured in cells transfected with miR-199a-5p mimics (Fig. 6B),

suggesting that miR-199a-5p overexpression in cells triggers malfunctioning of the endosome

compartment. Taken together, these results suggest that miR-199a-5p influences endocytic

compartment functioning.

DISCUSSION

Although a large number of studies have shown the participation of several miRNAs in cancer and

diseases development (Cuk et al., 2013; Pignot et al., 2013; Srivastava et al., 2013), little is known

about the role of miRNAs related to a central process in cell biology -intracellular trafficking- on

which other cellular functions depend for proper operation. The most salient finding of this study is

the identification of miR-199a-5p as an important regulator of endocytosis. In particular, our study

expands the current understanding of how miRNAs, specifically miR-199a/b-5p, contribute to

intracellular trafficking control. Remarkably, the miR-199a/b family is encoded within the DNM

genes, critical components of the endocytic machinery, suggesting that DNM and miR-199a/b

form a genomic locus that control intracellular transport pathways (Fig. 7). Similar to other intronic

miRNAs, such as miR-33a/b and miR-208 (Callis et al., 2009; Rayner et al., 2010), we found that

miR-199a/b-5p regulates related physiological processes to those controlled by the host genes in

which they are encoded. This finding prompted us to the identification and further characterization

of target genes associated with cellular trafficking. Interestingly, we found that a significant

number of predicted target genes for miR-199a/b-5p were associated with cellular transport.

Our results also demonstrate that miR-199a-5p plays an opposite role to DNM in controlling

endocytosis. DNM1 is selectively expressed at very high levels in neurons, where it is crucial for

synapses to efficiently recycle synaptic vesicles during intense activity (Bauerfeind et al., 1995).

DNM2 is ubiquitously expressed and DNM3 is found most prominently in the brain and in the

testis (Cao et al., 1998). For instance, while DNM2 is required for receptor-mediated endocytosis,

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miR-199a-5p inhibits the expression of numerous genes associated with this process including

CLTC and Rab GTPases. Our expression profile analysis is in agreement with the results

obtained in other studies, which indicate that miR-199a/b-5p strands are moderately expressed in

several tissues (Gu and Chan, 2012; Sakurai et al., 2011). As expected by their opposing roles,

the expression of miR-199 members and their respective DNM host genes were inversely

expressed in most human tissues. This is particularly remarkable in the case of the brain, where

the expression of DNM1 is markedly high (Cao et al., 1998) compared with its intronic miRNA,

miR-199b-5p, which is expressed at significantly low levels (Fig. S2A). The opposite is also

observed in the heart, where the expression of miR-199a-5p and miR-199b-5p (da Costa Martins

et al., 2010) is high in contrast with the moderate expression of DNM2 and almost null expression

of DNM1 and DNM3 (Fig. 1C). This observation suggests that organs that require active

trafficking, such as the brain (Faire et al., 1992) express significant amounts of DNM and very low

levels of miR-199a/b.

So far, very few studies have shown previously the participation of miRNAs in receptor mediated

internalization or trafficking (Lin et al., 2014; Serva et al., 2012; Yang et al., 2013). In the case of

miR-199a/b family, both strands of miR-199a-1 and miR-199a-2 namely miR-199a-5p and miR-

199a-3p are expressed in human tissues (Shatseva et al., 2011; Shen et al., 2010) and their

expression is originated from the DNM2 and DNM3 introns. Interestingly, miR-199a-3p that is

highly expressed in some tumor cells was reported to target caveolin-2 (Shatseva et al., 2011), a

key structural protein regulating endocytosis. In addition to miR-199a, the same DNM3 intron also

contains miR-214 that is expressed as a cluster together with miR-199a and subsequently

processed to render mature forms. Of note, miR-214 is known to target PTEN (phosphatase and

tensin homolog), which interacts with DNM and regulate receptor recycling (Yang et al., 2008).

More interesting, the mirror miRNA miR-3120, that is fully complementary of miR-214 in the DNM3

intron, is co-expressed with its host gene mRNA and regulates uncoating of clathrin-coated

vesicles by targeting Hsc70 and auxilin (Scott et al., 2012). These studies are in agreement with

our hypothesis that DNM intronic miRNAs regulate important aspects of cellular functions that are

similar to that of its host gene.

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Aside from using bioinformatic prediction tools, we performed several experimental approaches to

dissect the role of miR-199a-5p in regulating endocytosis including; the identification of putative

regulators by mRNA expression profiling and analysis of protein level changes upon expression

and/or inhibition of miR-199a-5p levels together with fluorescence confocal microscopy-based

assay and rescue of function analysis. In this study we have characterized a prominent role of

miR-199a-5p in regulating endocytosis that mechanistically is facilitated by the direct

downregulation of multiple genes involved in the endocytic pathway (Fig. 7). As a result,

overexpression of miR-199a-5p inhibited clathrin-mediated endocytosis as shown by our in vitro

LDL and transferrin internalization assays. This observation suggests the ability of miR-199a-5p in

a comprehensive manner to control the internalization at different levels (LDLR and CLTC) of

essential nutrients, such as cholesterol, present in LDL, and iron. Interestingly, antagonism of

miR-199a-5p enhances LDLR, Rab21 and Rab5A protein expression levels significantly. This

observation suggest that miR-199a-5p plays a role in regulating constitutive levels of these

proteins, increasing LDL-uptake and binding in Huh7 cells. In accordance with this, we observed

an increase in the intracellular pool of LDL when inhibiting endogenous expression of miR-199a-

5p, suggesting a physiological role in vivo for miR-199a-5p in controlling internalization pathways

in the cell. These results also suggest that antagonism of endogenous miR-199a/b-5p might have

a potential therapeutic effect for increasing levels of LDLR expression. It is also interesting to note

that miR-199a/b-5p host genes, DNM, associate with CTLC and mediate the formation of vesicles

after endocytosis (Takei et al., 1999). Disruption of DNM function resulted in reduced

internalization of receptors (Girard et al., 2011; Gray et al., 2003; Shajahan et al., 2004), such as

LDLR. Given that we have hypothesized a major role of miR-199a-5p in endocytosis, our rescue

of function experiment with ectopic LDLR-GFP shows the inability to completely restore

intracellular levels of DiI-LDL as expected, and consistently with the finding that many other

endocytic components are affected when miR-199a-5p levels are elevated. In this regard, a recent

study has reported that miR-199a-5p targets a well-known endocytosis regulator, such Cav-1

(Lino Cardenas et al., 2013). We also confirmed that miR-199a-5p overexpression inhibits Cav-1

expression but further studies will assess the contribution of this miRNA to Cav-1 functions.

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One important question to address further is whether or not changes in miR-199a/b-5p

endogenous levels influence endocytosis. The documented observation that in most of cancers

miR-199a-5p expression is downregulated supports the idea that cancer cells can exploit this fact

to ensure the intracellular trafficking necessary for growth therefore enabling cancer progression

(Ramsay et al., 2007; Xu et al., 2012). Intriguingly, miR-199a-5p overexpression inhibits tumor

cells migration without affecting cellular proliferation and viability (Cheung et al., 2011; Duan et al.,

2011). To do that, we speculate that miR-199a-5p might regulate key endocytic intermediates as

described here to fine-tune intracellular trafficking routes that are implicated in cancer progression.

As discussed previously miRNAs is known to regulate many aspects of cancer cell biology (Iorio

and Croce, 2012) and individual miRNAs could be differentially expressed under different stimuli.

Because of their different genomic location, future experiments will further characterize the

specific roles of miR-199a1-5p, miR-199a2-5p and miR-199b-5p in regulating endocytosis in this

pathological context. In light of our findings it is plausible to assume that miR-199a/b-5p may

function in the opposite way of DNM host genes, but additional work is needed to clarify whether

or not miR-199/DNM locus coordinates a unique biological functional response.

In summary, our study uncovers an elegant mechanism by which DNM/miR-199 genomic locus

coordinately regulates cellular endocytosis.

MATERIALS AND METHODS

Materials

Chemicals were obtained from Sigma-Aldrich unless otherwise noted. 1,1’-Dioctadecyl-3,3,3,3’-

tetramethylindocarbocyanineperclorate (DiI) was purchased from Molecular Probes (Invitrogen). A

rabbit polyclonal antibody against LDLR was obtained from Cayman Chemical and mouse

monoclonal antibodies against HSP90, Rab5A, Rab21 and EEA1 were purchased from BD

Bioscience. The mouse monoclonal antibody against LDLR and the goat antibody for TfR were

obtained from Santa Cruz Biotechnology. The rabbit polyclonal antibodies to CLTC and Rab11

were purchased from Cell Signaling Technology. Tf-FITC and secondary fluorescently labeled

antibodies were from Molecular Probes (Invitrogen). MiRNA mimics and inhibitors were obtained

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from Dharmacon. The LDLR-GFP plasmid was kindly provided by Prof. Peter Tontonoz (UCLA,

Los Angeles, CA).

Cell culture

Human (Huh7) hepatic, cervix carcinoma (HeLa) and monkey kidney fibroblast (COS7) cells were

obtained from American Type Tissue Collection. Huh7, HeLa and COS7 cells were maintained in

Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 2%

penicillin-streptomycin in 10 cm2 dishes at 37°C and 5% CO2. For DiI-LDL uptake and binding

experiments, Huh7 cells were incubated in DMEM containing 10% lipoprotein-deficient serum

(LPDS) supplemented with 30 μg/ml DiI-LDL cholesterol.

Bioinformatic analysis of miRNA target genes.

Target genes for hsa-miR-199a/b were identified and compared using the online target prediction

algorithm, miRWalk (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/), which provides target

interaction information from eight different prediction algorithms. Specifically, the programs

miRanda, miRWalk and TargetScan were used. The putative targets produced by all three of

these algorithms for miR-199a were uploaded into the gene classification system, PANTHER v8.0

(http://www.pantherdb.org) to identify gene targets that were mapped to the transport process

(GO:0006810). The functional interactions of these predicted targets for miR-199a/b-5p described

in STRING v9.05 (http://string-db.org) were then combined with the functional annotation groups

described in DAVID. Matlab and Cytoscape v2.8.3 were used to create the visualization networks,

as previously described (Huang da et al., 2009). STRING interactions with a confidence score of

0.4 or higher were added and highlighted in bold. Smaller annotation clusters and unconnected

genes were left out of the visualization due to space constraints.

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miRNA mimic/inhibitor transfections

For mimic and inhibitor transfections, Huh7 and HeLa cells were transfected with 40 nM miRIDIAN

miRNA mimics (miR-199a-5p) or with 60 nM miRIDIAN miRNA inhibitors (Inh-199a-5p)

(Dharmacon) using RNAimax (Invitrogen) or Lipofectamine 2000 (Invitrogen) for co-transfection

experiments with the LDLR-GFP plasmid. All experimental control samples were treated with an

equal concentration of a non-targeting control mimic sequence (CM) or inhibitor negative control

sequence (CI) for use as controls for non-sequence-specific effects in miRNA experiments.

Verification of miR-199a-5p over-expression and inhibition was determined using qRT-PCR, as

described below.

RNA isolation and quantitative real-Time PCR

Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s

protocol. For mRNA quantification, cDNA was synthesized using iScript RT Supermix (Bio-Rad),

following the manufacturer’s protocol. Quantitative real-time PCR (qRT-PCR) analysis was

performed in triplicate using iQ SYBR green Supermix (BioRad) on an iCycler Real-Time

Detection System (Eppendorf). The mRNA level was normalized to GAPDH (glyceraldehyde-3-

phosphate dehydrogenase) as a house keeping gene. The human primer sequences used were:

GAPDH, 5’-TTGATTTTGGAGGGATCTCG-3’ and 5’-CAATGACCCCTTCATTGACC-3’; LDLR, 5’-

TGATGGGTTCATCTGACCAGT-3’ and 5’-AGTTGGCTGCGTTAATGTGAC-3’; CLTC 5’-

TGAGGCGACTGGGCGGAGTT-3’ and 5’-CCGGGGACGCAGGAAACTGG-3’; cav-1 5’-

AGTGCATCAGCCGTGTCTATTCCA-3’ and 5’-TCTGCAAGTTGATGCGGACATTGC-3’; Rab5A

5’-GGGGCTGCTTTTCTAACCCA-3’ and 5’-TTTGCTAGGTCGGCCTTGTT-3’; Rab21 5’-

CCTCCGGTGCCTGACGTGGT-3’ and 5’-CAGCCTTCCCCCAGCAGCAC-3’, DNM1 5’-

CACCGTTAGACAGTGCACCA-3’ and 5’-CCCTTGCGGATGACCAGAAT-3’, DNM2 5’-

CACAGCCCCACTCCACAGCG-3’ and 5’- CCTGGGGGAATCCCTGGGGG-3’, DNM3 5’-

CCCCCACTCTGGGGCTCCTC-3’ and 5’- GATGGGGGTGGTCTCCGGCT-3’ TfR 5’-

GAACTACACCGACCCTCGTG-3’ and 5’-TGCCACACAGAAGAACCTGC-3’. For miRNA

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quantification, total RNA was reverse transcribed using the miScript II RT Kit (Qiagen). Primers

specific for human pre-miR-199a1, pre-miR-199a2, pre-miR-199b, miR-199a-5p and miR-199a-3p

(Qiagen) were used and values normalized to SNORD68 (Qiagen) as a housekeeping gene.

Western blot analysis

Cells were lysed in ice-cold buffer containing 50 mM Tris-HCl, pH 7.5, 125 mM NaCl, 1% NP-40,

5.3 mM NaF, 1.5 mM NaP, 1 mM orthovanadate and 1 mg/ml of protease inhibitor cocktail

(Roche) and 0.25 mg/ml AEBSF (Roche). Cell lysates were rotated at 4ºC for 1h before the

insoluble material was removed by centrifugation at 12000 x g for 10 min. After normalizing for

equal protein concentration, cell lysates were resuspended in SDS sample buffer before

separation by SDS-PAGE. Following overnight transfer of the proteins onto nitrocellulose

membranes, the membranes were probed with the following antibodies: LDLR (1:500), Rab5

(1:1000), CLTC (1:1000), Rab21 (1:500), EEA-1 (1:1000), TfR (1:1000) and HSP90 (1:1000).

Protein bands were visualized using the Odyssey Infrared Imaging System (LI-COR

Biotechnology). Densitometry analysis of the gels was carried out using ImageJ software from the

NIH (http://rsbweb.nih.gov/ij/).

LDL receptor and TfR activity assays

Human LDL was isolated and labeled with the fluorescent probe DiI as previously reported (Calvo

et al., 1998). Huh7 cells were transfected in 6- or 12-well plates with miRNA mimics and inhibitors

in DMEM containing 10% LPDS for 48h. Then, cells were washed once in 1x PBS and incubated

in fresh media containing DiI-LDL (30 g cholesterol/ml). Non-specific uptake was determined in

extra wells containing a 50-fold excess of unlabeled native LDL (nLDL). Cells were incubated for 5

min-4h at 37°C to allow for DiI-LDL uptake. In other instances, cells were incubated for 120 min at

4°C to assess LDLR antibody binding. At the end of the incubation period, cells were washed,

resuspended in 1 ml of PBS and analyzed by flow cytometry (FACScalibur, Becton Dickinson), as

previously described (Suarez et al., 2004). The results are expressed in terms of % of DiILDL

specific uptake after subtracting autofluorescence of cells incubated in the absence of DiI-LDL

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calculated from median intensity of fluorescence (M.I.F.) relative to 2h time point. For time course

experiments, DiI-LDL uptake is represented as M.I.F. in arbitrary units.

For analysis of transferrin internalization cells were incubated for 45 min at 37°C in serum-free

DMEM. Cells were first incubated on ice for 20 min followed by addition of 50 g/mL FITC-

Transferrin (Sigma) in serum-free media. After 30 min on ice cells were washed with iced cold

PBS and transferred to a 37°C/5% CO2 incubator in the presence of unlabeled Transferrin for the

indicated times. Cells were acid washed to remove surface-bound transferrin and analyzed by

FACS or fixed in 4% PFA 15 min for fluorescence microscopy analysis.

Fluorescence microscopy

For LDLR-Ab internalization and DiI-LDL uptake assays, Huh7 cells were grown on coverslips and

transfected with a miR-199a-5p mimic and a negative control mimic (CM) in DMEM containing

10% LPDS. 48h post transfection, cells were cooled to 4ºC for 20 min to stop membrane

internalization. Cells were then incubated with LDLR mAb (C7) (Santa Cruz) and 30 g/ml DiI-LDL

for 40 min at 4ºC. Following incubation, cells were gently washed twice with cold medium and

shifted to 37ºC to allow for internalization of both LDLR-Ab complexes and DiI-LDL for the

indicated times, acid stripped and fixed with 4% PFA. After 5 min of Triton X-100 0.2%

permeabilization and 15 min of blocking (PBS-BSA 3%), cells were stained with anti-mouse Alexa

488 (Molecular Probes) and TO-PRO 3 (Life Technologies) for 1h at room temperature. After this,

cells were washed twice with 1x PBS and mounted on glass slides with Prolong-Gold (Life

Technologies).

For LDLR-GFP rescue experiments, Huh7 cells were grown on coverslips and co-transfected with

1 g LDLR-GFP and 40 nM of a control mimic CM or miR-199a-5p mimic. 48h post transfection

cells were incubated with 30 g/ml DiI-LDL for 1h at 37ºC (uptake). Then, cells were washed

twice with 1x PBS, fixed with 4% PFA, and blocked (3% BSA in 1x PBS) for 15 min. Following

this, cells were washed twice, stained with Phalloidin to visualized F-actin and mounted on glass

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slides with Prolong-Gold (Life Technologies). All images were analyzed using confocal microscopy

(Leica SP5 II) equipped with a 63X Plan Apo Lenses. All gains for the acquisition of comparable

images were maintained constant. Analysis of different images was performed using ImageJ (NIH)

and Adobe Photoshop CS5.

3’UTR luciferase reporter assays

cDNA fragments corresponding to the entire 3’UTR of human LDLR, Cav-1, Rab5A, Rab21 and

CLTC were amplified by RT-PCR from total RNA extracted from Huh7 cells with XhoI and NotI

linkers. The PCR product was directionally cloned downstream of the Renilla luciferase open

reading frame of the psiCHECK2TM vector (Promega) that also contains a constitutively expressed

firefly luciferase gene, which is used to normalize transfections. Point mutations in the seed region

of the predicted miR-199a binding sites within all the above 3’UTR were generated using the

Multisite-Quickchange Kit (Stratagene), according to the manufacturer’s protocol. All constructs

were confirmed by sequencing. COS7 cells were plated into 12-well plates (Costar) and co-

transfected with 1 μg of the indicated 3’UTR luciferase reporter vectors and miR-199a-5p mimics,

or control mimics (CM) (Dharmacon) utilizing Lipofectamine 2000 (Invitrogen). Luciferase activity

was measured using the Dual-Glo Luciferase Assay System (Promega). Renilla luciferase activity

was normalized to the corresponding firefly luciferase activity and plotted as a percentage of the

control (cells co-transfected with the corresponding concentration of control mimic). Experiments

were performed in triplicate wells of a 12-well plate and repeated at least three times.

Statistics

All data are expressed as mean ±SEM. Statistical differences were measured using an unpaired

Student’s t test. A value of P≤0.05 was considered statistically significant. Data analysis was

performed using GraphPad Prism Software Version 6.03 (GraphPad, San Diego, CA). *P≤0.05.

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ACKNOWLEDGMENTS

We thank Dr. Peter Tontonoz for generously providing the LDLR-GFP plasmid. This work was

supported by grants from the National Institutes of Health, R01HL107953 and R01HL106063 (to

CF-H) and 1F31AG043318 (to LG).

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Figures

Figure 1. MiR-199/DNM loci genomic location, human tissue expression and bioinformatic

analysis of miR-199a/b predicted target genes. (A) Schematic representation of genomic

location of DNM genes and their miR-199a/b intronic family members. Intronic miR-199a2-5p is

co-transcribed in a cluster with miR-214. Note that the three members of miR-199a/b are encoded

in the opposite strand of DNM host genes. (B) Sequence alignment between human miR-199

family members. Seed sequences are indicated in boxes. Red color in miR-199b sequence

indicates those nucleotides that have diverged with respect to miR-199a. Stem loop, mature 5p

and 3p forms are indicated. (C) Gene expression analyses (qRT-PCR) of miR-199a-5p, DNM2

and DNM3 in different human tissues normalized to snoRD68 for miR-199a-5p and GAPDH, for

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DNM2 and DNM3 genes. Data in B and C were relatives to miR-199a-5p transcripts expressed in

adipose tissue. (D) Gene ontology analysis of the miR-199a/b predicted target genes using

Panther software (upper right and bottom panels). Protein–protein interaction analysis scheme of

selected miR-199a-5p–predicted target genes using String 9.1 software and Navigator 2.2. is

shown in the upper left panel.

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Figure 2. MiR-199a/b regulates LDLR, CLTC, Rab5A and Rab21 expression in Huh7 cells. (A

and B) qRT-PCR analysis of LDLR, CLTC, Rab5A and Rab21 expression in Huh7 cells

transfected with non-targeting control mimic (CM), miR-199a-5p mimic, or control inhibitor (CI) and

miR-199a-5p inhibitor. (C and D) Western blot analysis of LDLR, CLTC, Rab5A and Rab21 in

Huh7 cells transfected with CM or miR-199a-5p mimic (C) and CI or inh-199a-5p (D).

Densitometry analysis is shown in the corresponding histograms in the right panels. (E) Human

LDLR, CLTC, Rab21 and Rab5a 3’UTR containing the indicated point mutations (PM) in the miR-

199a/b-5p target sites. (F) Luciferase reporter activity in COS7 cells transfected with CM or miR-

199a-5p mimic and the indicated human 3’UTR containing or not the indicated PM in the miR-

199a-5p target binding sites. In panels A and B, data are expressed as mean ± SEM and

representative of ≥ 3 experiments in triplicate. *, p≤ 0.05. In panel E, data are expressed as mean

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percentage of 3’UTR activity of CM ± SEM and are representative of ≥ 3 experiments in triplicate.

*, p≤ 0.05. In panels C, Hsp90 was used as a loading control. Quantification of the band

densitometry analysis is shown in the bottom of the blot images.

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Figure 3. MiR-199a/b-5p regulates the LDLR activity. (A) Schematic diagram showing time

course experiment followed in B. (B) Flow cytometry analysis of DiI-LDL uptake in Huh7 cells

transfected with a control mimic (CM) or miR-199a-5p mimic and incubated with 30 μg/ml DiI-LDL

for the indicated times at 37ºC. Data correspond to the MIF S.E.M. of three experiments. MIF,

median intensity of fluorescence; a.u.f., arbitrary units of fluorescence. (C) DiI-LDL uptake in Huh7

cells treated or not with Dynasore for 2 h. Data are expressed as percentage of the control (in

absence of Dynasore). Data correspond to the meanS.E.M. of three experiments. (D)

Representative confocal immunofluorescence images of Huh7 cells transfected as indicated and

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subjected to 10-30 min of DiI-LDL uptake at 37ºC, fixed and stained for the Early Endosome

Antigen-1, (EEA1), Rab11, lysosomal marker CD63, and TOPRO for the nuclei. Scale bar, 10 μm

(E) Histograms showing co-localization analysis of indicated markers and DiI-LDL in Huh7 cells

transfected as indicated by measuring mander’s coefficient. Data are representative of ≥ 3

experiments. *, p≤ 0.05 and #, p> 0.05.

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Figure 4. MiR-199a-5p regulates free cholesterol intracellular localization. Representative

immunofluorescence analysis of free cholesterol (Filipin), F-actin and DiI-LDL in Huh7 cells

transfected with non-targeting control miRNA (CM-CI), miR-199a-5p mimics or inh-199a-5p and

incubated with 30 μg/ml DiI-LDL for 1 h at 37ºC. Fluorescence intensity plots are shown in the

right panels. White arrows indicate the region of cell subjected to image analysis. Scale bar, 10

μm.

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Figure 5. MiR-199a-5p regulates internalization of LDLR. (A) Representative confocal images

of DiI-LDL, LDLR-GFP and F-actin expression in Huh7 cells co-transfected with LDLR-GFP and

CM or miR-199a-5p mimics and incubated with 30 μg/ml DiI-LDL for 1 h at 37ºC. Scale bar, 10μm.

Magnification insets are shown; scale bar, 2.5m. Fluorescence Intensity plots for LDLR-GFP

(green) F-actin (blue) and DiI-LDL (red) signals along the arrow in the image are shown in the right

panels. (B) Schematic representation showing the time course experiment followed in left panel.

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Huh7 cells transfected with LDLR-GFP and CM or miR-199a-5p was incubated with anti-LDLR

antibodies at 4ºC for 2 h, washed and then switched to 37ºC to allow internalization at 5 and 10

min. Cells were then stained with anti-phycoerythrin (PE) secondary antibodies, washed, fixed

with PFA and analyzed by FACS. Histogram shows level of LDLR internalization in gated LDLR-

GFP positive cells as % PE fluorescence G mean. Data are expressed as geometrical mean

percentage of CM ± SEM and are representative of ≥ 3 experiments in triplicate. *, p≤ 0.05.

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Figure 6. MiR-199a-5p regulates transferrin uptake. (A) Flow cytometry analysis of transferrin-

FITC internalization assay as described in methods in HeLa cells transfected with negative control

miRNA (CM) or miR-199a-5p mimics and treated as indicated. Data are expressed as the

difference of fluorescence geometrical mean of each time point minus fluorescence G mean of

time 0 min ± SEM and are representative of ≥ 3 experiments. (B) Western blot analysis of TfR and

EEA1 in HeLa cells transfected with CM or miR-199a-5p mimics. Hsp90 was used as loading

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control. Quantification of protein fold change is shown in the histogram. (C) Representative

confocal images of Tf, Rab5 and EEA1 expression in HeLa cells transfected with CM or miR-

199a-5p mimics, incubated at different times with FITC labelled transferrin (Tf-FITC). Histograms

in the bottom right show EEA1/Tf-FITC colocalization analysis. Data are representative of ≥ 3

experiments. *, p≤0.05. (D, left panel) Representative confocal images of membrane Tf-FITC

localization in HeLa cells treated as (C). (D, right panel) Flow cytometry analysis of surface Tf-

FITC binding in HeLa cells transfected with CM and miR-199a-5p

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Figure 7. Proposed model of DNM/miR-199a/b target gene regulation. Sense strands of the

DNM genes are transcribed and translated to synthetize DNM proteins that are involved in

endosome trafficking. MiR-199a-5p is transcribed in the nucleus from the antisense strand of

introns in the DNM2 and DNM3 gene and regulates receptor mediated endocytosis and

intracellular cholesterol levels by balancing the posttranscriptional repression of genes involved in

endocytosis such as LDLR, CLTC, Cav-1, Rab5A and Rab21.

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