Molecular Mechanism for Hypertensive Renal Disease: Differential Regulation of Chromogranin A Expression at 3′-Untranslated Region Polymorphism C+87T by MicroRNA-107

Abstract

Chromogranin A (CHGA) is coreleased with catecholamines from secretory vesicles in adrenal medulla and sympathetic axons. Genetic variation in the CHGA 3′-region has been associated with autonomic control of circulation, hypertension, and hypertensive nephropathy, and the CHGA 3′-untranslated region (3′-UTR) variant C+87T (rs7610) displayed peak associations with these traits in humans. Here, we explored the molecular mechanisms underlying these associations. C+87T occurred in a microRNA-107 (miR-107) motif (match: T>C), and CHGA mRNA expression varied inversely with miR-107 abundance. In cells transfected with chimeric luciferase/CHGA 3′-UTR reporters encoding either the T allele or the C allele, changes in miR-107 expression levels had much greater effects on expression of the T allele. Cotransfection experiments with hsa-miR-107 oligonucleotides and eukaryotic CHGA plasmids produced similar results. Notably, an in vitro CHGA transcription/translation experiment revealed that changes in hsa-miR-107 expression altered expression of the T allele variant only. Mice with targeted ablation of Chga exhibited greater eGFR. Using BAC transgenesis, we created a mouse model with a humanized CHGA locus (T/T genotype at C+87T), in which treatment with a hsa-miR-107 inhibitor yielded prolonged falls in SBP/DBP compared with wild-type mice. We conclude that the CHGA 3′-UTR C+87T disrupts an miR-107 motif, with differential effects on CHGA expression, and that a cis:trans (mRNA:miR) interaction regulates the association of CHGA with BP and hypertensive nephropathy. These results indicate new strategies for probing autonomic circulatory control and ultimately, susceptibility to hypertensive renal sequelae.

Hypertension remains the most common and lethal of cardiovascular risk factors,¹ with predisposition to such consequences as renal and cardiac failure. The prevalence of renal failure disease in the United States continues to rise. A common antecedent of this disease, especially in African Americans, is hypertension. We previously showed relationships between renal function, hypertension, and chromogranin A (CHGA). CHGA is the major soluble protein costored and coreleased with catecholamines from secretory vesicles in adrenal medulla and postganglionic sympathetic axons.² CHGA is required for formation of catecholamine secretory vesicles in chromaffin cells,³ and its expression may even be sufficient to induce a regulated secretory system in cells initially without that pathway.⁴ Catecholamine storage vesicles of the adrenal medulla contain remarkably high concentrations of CHGA, catecholamines, ATP, and Ca²⁺. CHGA seems to bind and store both catecholamines and Ca²⁺.⁵ CHGA is also a prohormone that gives rise to biologically active peptides, such as the catecholamine release inhibitor catestatin,⁶ the dysglycemic peptide pancreastatin,^7,8 and the vasodilator vasostatin.⁹

Previously, we found that naturally occurring human genetic variation in the 3′-region of the CHGA gene was associated with autonomic BP regulation and hypertension in several human populations¹⁰ as well as hypertensive renal disease in African Americans,¹¹ and it predicts the rate of chronic GFR decline in such patients.¹² In a case/control study of hypertensive ESRD,¹¹ the 3′-region of CHGA was associated with ESRD, and the effect was replicated in an independent case/control sample.¹¹ A recent report also indicates that CHGA genetic variation predicts renal injury in IgA nephropathy with malignant hypertension in China.¹³

In this study, we explore potential functional motifs in the CHGA 3′-untranslated region (3′-UTR) and identify a partial motif match between microRNA (miR) hsa-miR-107 and the common CHGA 3′-UTR variant C+87T (rs7610), with a superior match for the risk allele +87T. We then investigate whether this motif match regulates CHGA expression using a variety of techniques, proceeding ultimately to in vivo effects on control of BP in the humanized CHGA transgenic mouse. Our results suggest that the interaction of C+87T with hsa-miR-107 may account for alterations in CHGA expression, eventuating in hypertensive nephropathy.

Results

Human CHGA 3′-UTR Variant C+87T: Computational Disruption of an miR Recognition Motif

To explore whether the C+87T variant¹⁰ might be functional, we searched for motifs potentially disrupted by the single-nucleotide polymorphism. A partial match for micro-RNA (miR) hsa-miR-107 was identified that displayed differential binding ability between the C+87 and +87T alleles, with a superior match for the T allele, which was evidenced by a lower minimum free energy and higher alignment score. A sequence alignment of hsa-miR-107 and the CHGA 3′-UTR alleles is shown in Figure 1.

Figure 1.

Human CHGA 3′-UTR variant C+87T (rs7610): T allele has a superior match to micro-RNA hsa-miR-107. Alignment of human CHGA 3′-UTR C+87T variant alleles T versus C with miR hsa-miR-107. The T allele exhibits a superior match to hsa-miR-107 by both minimum free energy and alignment score. The scores are explained at the bottom of the panel.

Relative Abundances of Endogenous CHGA and miR-107

The endogenous CHGA mRNA and miR-107 were investigated by quantitative RT-PCR (qRT-PCR) in cell lines and tissues from rat and human, including rat pheochromocytoma/chromaffin (PC12) cells, normal (WKY) rat brain stem, normal (WKY) rat adrenal glands, human neuroblastoma cells (SH-SY5Y), and human embryonic kidney (HEK293T) cells. We observed a roughly inverse relationship between the expressions of CHGA mRNA and miR-107 (Figure 2), with kidney cells displaying the highest miR-107 but lowest CHGA mRNA abundance.

Figure 2.

CHGA mRNA abundance: Inverse dependence on hsa-miR-107 across cell lines and tissues. Results were obtained by qRT-PCR in replicate samples and plotted±SEMs for each group. mRNA results were normalized to GAPDH mRNA in the same sample, whereas the miR-107 results are normalized to the small RNA SNORD61. CHGA mRNA abundance is inversely proportional to miR-107 abundance (R=0.96, P=0.005). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Differential Activity of CHGA 3′-UTR Variant C+87T Alleles: 3′-UTR/Luciferase Reporter Studies

To test whether the CHGA 3′-UTR C+87T variant is itself functional, we inserted the 409-bp 3′-UTR into a reporter plasmid just downstream from the luciferase open reading frame and derived the minor allele version (+87T) by site-directed mutagenesis (Supplemental Figure 1). Luciferase activities of C+87 or +87T alleles were measured after cotransfection of the reporter plasmid with hsa-miR-107 or its antagomir into human HEK293T, PC12, or SH-SY5Y cells.

hsa-miR-107 Effects in Human HEK293T Cells

Endogenous hsa-miR-107

Human HEK293T cells express a large amount of endogenous hsa-miR-107 but small amount of CHGA (Figure 2), making it a useful line to study the effect of endogenous miR on CHGA gene expression. Because of inhibition of hsa-miR-107, reporter expression was greater by 18.1% for the +87T allele (P=0.004) but not for the C+87 allele (P>0.05) (Figure 3A).

Figure 3.

Hsa-miR-107 regulates human CHGA 3′-UTR expression in human HEK293T cells. Luciferase reporter activity of CHGA 3′-UTR C+87T in human HEK293T cells: effects of hsa-miR-107. Results are analyzed by two-way ANOVA factoring for miR (present versus mock) and allele (T versus C). The values are expressed as ±SEMs. (A) Effect of hsa-miR-107 inhibitor. (B) Effect of hsa-miR-107 mimic.

Exogenous hsa-miR-107

The cotransfected miR-107 mimic decreased reporter expression for both C+87 and +87T alleles (Figure 3B), with a significant difference between the two allele responses (6.7% for the C version [P=0.005] versus 18.5% for the T version [P<0.001]).

In an additional experiment controlling for transfection efficiency by cotransfection of the LacZ reporter plasmid pSV–β-galactosidase, the ratio of luciferase/β-galactosidase enzymatic activities was greater for the C>T alleles (P=0.02) (Supplemental Figure 2), paralleling the C>T difference seen when luciferase activity was simply normalized to protein concentration in the sample (Figure 3).

hsa-miR-107 Effects in Rat PC12 and Human SH-SY5Y Cells

PC12 Pheochromocytoma Cells

PC12 cells express relatively large amounts of endogenous CHGA but little miR-107 (Figure 2). Exogenous miR-107 decreased reporter expression on both alleles, but the difference between the two alleles (C+87T) was appreciable (18.2% on C versus 34.9% on T; P=0.05) (Supplemental Figure 3A).

SH-SY5Y Neuroblastoma Cells

SH-SY5Y cells express intermediate amounts of endogenous CHGA and miR-107. Exogenous hsa-miR-107 inhibitor augmented CHGA expression on both alleles, but the two alleles (C+87T) increased differentially (+43% on the C version [P=0.001] versus +121% on the T version [P=0.004]) (Supplemental Figure 3B).

The 3′-UTR in Natural Context: miR-107 Effect on Eukaryotic CHGA Expression by the Full-Length cDNA/mRNA

The hsa-miR-107 oligonucleotides were cotransfected with the pCMV-CHGA plasmid into human HEK293T cells, and CHGA protein expression was monitored by immunoblot. Because of hsa-miR-107 inhibition, the CHGA level was substantially greater for the T allele (P=0.01) (Figure 4A) but had no effect on the C allele (P>0.05). Inhibition of CHGA expression by hsa-miR-107 mimic was greater on the T allele (P=0.02) (Figure 4B), although there was no effect on the C allele (P>0.05).

Figure 4.

Hsa-miR-107 effect on CHGA protein expression in human HEK293T cells. CHGA 3′-UTR C+87T in the full-length cDNA: effects of hsa-miR-107. HEK293T cells were transfected with pCMV-hCHGA, in which the strong pCMV promoter drives expression of the full-length human CHGA cDNA. The electrophoresed/membrane-transferred proteins are detected by antibodies (anti-CHGA or anti-GAPDH) with intensity scored by ImageJ (http://rsbweb.nih.gov/ij/). Numbers show the ratio of CHGA to GAPDH. In a parallel experiment, when HEK293T cells were transfected with empty expression vector (pCMV plasmid) rather than the expression plasmid containing the human CHGA cDNA insert (pCMV→hCHGA), there was no expression of CHGA detected on immunoblots. The data are expressed as ±SEMs. (A) Effect of hsa-miR-107 inhibitor in human HEK293T cells. (B) Effect of hsa-miR-107 mimic in human HEK293T cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

The 3′-UTR in Natural Context: Coupled In Vitro Transcription/Translation of the Full-Length CHGA cDNA/mRNA

Wild-type (C+87) or variant (+87T) CHGA cDNAs were expressed by the T7 promoter and then translated by rabbit reticulocytes. The wild-type (C allele) cDNA exhibited greater expression than the variant (T allele) when visualized by the anti-CHGA immunoblotting (Supplemental Figure 4). Expression of the +87T version was less after the hsa-miR-107 mimic and more with hsa-miR-107 inhibitor, whereas the C+87 version was unchanged by mimic or inhibitor treatments.

Chga and Renal Function in the Mouse In Vivo

In the absence of Chga expression (by targeted homozygous deletion in the Chga[−/−] mouse) (Figure 5), plasma creatinine concentration was reduced by 10% (P=0.03), whereas eGFR was reciprocally greater, indicating glomerular hyperfiltration in the face of hypertension.

Figure 5.

Loss of CHGA results in higher eGFR and hyperfiltration. eGFR in the Chga(−/−) mouse. Plasma creatinine was determined by HPLC (to avoid the influence of noncreatinine chromogens in plasma), and eGFR was estimated from creatinine data. Nine Chga(+/+) (control) and 10 Chga(−/−) mice were evaluated. Because data were not normally distributed, results were analyzed by nonparametric (Mann–Whitney) statistical tests. The eGFR values are expressed as ±SEMs.

Effect of miR-107 on BP In Vivo

The availability of our humanized CHGA mouse strain^14,15 allowed us to test whether hsa-miR-107 influenced CHGA expression in vivo, because the human CHGA +87T variant displays a match with hsa-miR-107 that is markedly superior to the complementary mouse Chga alignment (Figure 6A).

Figure 6.

Inhibition of hsa-miR-107 in vivo results in drop of BP. (A) Alignment of hsa-miR-107 with human and mouse versions of the CHGA 3′-UTR in the homologous region of C+87T. At C+87T in both human and mouse versions of CHGA in these models, the diploid genotype is T/T. (B) Differential effects of an miR-107 inhibitor/antagomir on mouse BP and HR in humanized (CHGA+/+;Chga−/−) versus wild-type (CHGA−/−;Chga+/+) mice. Male humanized (CHGA+/+;Chga−/−; n=7) and wild-type (CHGA−/−;Chga+/+; n=8) mice were studied at 16–18 weeks of age. The single-stranded oligonucleotide was delivered by intraperitoneal injection (at 3.3 µg/g body wt) at time 0 hours (10:00 hours) into mice with telemetric continuous recording of BP and HR. Values are expressed as ±SEMs. (C) Statistics were done by repeated measures ANOVA using linear mixed model, and data are presented.

When administered in vivo, the hsa-miR-107 antagomir (Figure 6B) resulted in prolonged declines in both systolic BP (SBP) and diastolic BP (DBP) in the CHGA humanized but not wild-type mice, and all mice survived during the entire recording period. The SBP decline was maximal by approximately 3 hours, whereas that of DBP was maximal by approximately 4 hours. Statistically, there were significant effects for time (after antagomir), strain (CHGA[+/+] versus Chga[+/+]), and time-by-strain interaction (each P<0.05). By contrast, heart rate (HR) was not affected by the antagomir, and HR did not differ by strain; thus, the fall in SBP/DBP after the antagomir was not accompanied by reflex tachycardia. Control intraperitoneal injections (saline) changed BP/HR transiently in response to stress for both strains. The elevated BP returned to normal after approximately 60 minutes in wild-type mice and approximately 30 minutes for humanized CHGA mice (data not shown). The antagomir-treated humanized CHGA mice tissues were also analyzed for CHGA mRNA expression. Both the adrenal and brain stem tissues showed elevation of CHGA mRNA 5 hours post-treatment (Supplemental Figure 5).

Discussion

Overview: CHGA in Hypertension and Nephropathy

The human CHGA 3′-UTR C+87T (rs7610) common (approximately 10% allele frequency) polymorphism is associated with autonomic BP regulation and hypertension in several human populations¹⁰ as well as hypertensive renal disease in African Americans.¹¹

Is there a role for CHGA in control of hypertension and renal function across different human biogeographic ancestries? Previously, we¹⁶ discovered that the C+87T 3′-UTR variant occurs in a variety of population samples, including African Americans (MAF of approximately 9.6%), white/European ancestry (MAF of approximately 27.4%), Hispanic/Mexican Americans (MAF of approximately 34%), and east Asians (MAF of approximately 7.0%); these allele frequencies are borne out in more recent National Center for Biotechnology Information data deposits, including the Hap-Map. We found that human CHGA genetic variation associates with the heritable risk trait of renal albumin excretion in healthy white twin pairs.¹⁷ In a biogeographically broader arena, genetic variation at the CHGA locus was associated with renal injury in Chinese patients with IgA nephropathy and malignant hypertension.¹³ In the glomerulus, CHGA itself triggers profibrotic responses in cultured glomerular capillary endothelial and mesangial cells.¹²

Here, we provide evidence of the functionality of the CHGA 3′-UTR variant and its differential interaction with a specific miR. miRs are a class of noncoding small RNAs that regulate gene expression by complementary base pairing to target motifs, typically in 3′-UTR regions of mRNAs, with consequent transcript cleavage (in the case of a perfect match) or translational repression (in the case of a partial match). Single-nucleotide polymorphisms that reside in miR target motifs¹⁸ may either abolish existing binding motifs or create novel sites. Such variants are potentially implicated in a broad range of human traits,¹⁹ despite stronger negative selection on miR sites than other motifs of the 3′-UTR.²⁰ We found that the T allele at C+87T in the CHGA 3′-UTR creates a superior motif for hsa-miR-107 binding (Figure 1), with consequences for CHGA gene expression in cultured cells and finally, BP in vivo.

In the mouse with targeted Chga ablation (and hence, severe hypertension in the face of catestatin deficiency,³ plasma creatinine was diminished (Figure 5) with a coordinate greater eGFR, indicating hyperfiltration in the setting of systemic hypertension. Of potential pertinence, glomerular hyperfiltration is a risk factor for accelerated loss of function in the context of progressive renal decline.^21,22 Indeed, increases in GFR correspond to elevated renal albumin excretion, even within the physiologic range in healthy individuals¹⁷ It is intriguing to note that hypertensive African Americans,²³ the group at highest risk of hypertensive nephropathy,¹¹ also exhibit evidence of glomerular hyperfiltration (as elevated creatinine clearance) in the salt-loaded state.²³

Results in Context with the Literature

Of note, human miR-107, the gene encoding hsa-miR-107 located on human chromosome 10q23, is within a QTL region for both BP^24–26 and body weight.²⁷ miR-107 is reportedly upregulated in obese mice and seems to have a key role in insulin sensitivity.²⁸ Here, we show that miR-107 binds to the CHGA 3′-UTR (especially on the T allele) and decreases gene expression, perhaps leading to derangement of catecholamine uptake, storage, and release²; we speculate that such changes may contribute to alterations of insulin sensitivity. Therefore, regulation of CHGA expression by miR-107 possibly links both BP and insulin associations, because decrease in CHGA also predicts decreased CHGA-derived peptides catestatin and pancreastatin. Catestatin is known for its hypotensive effect by inhibiting catecholamine secretion,^3,29 and pancreastatin regulates insulin sensitivity.³⁰ A recent study reported that miR-107 is upregulated in hypoxia to prevent endothelial progenitor cell differentiation through its target hypoxia-inducible factor-1β.³¹ We have recently reported a polymorphism in the Chga 3′-UTR that alters the miR-22 motif in the hypertensive SHR rat strain, leading to dysregulation of CHGA expression in the brain stem and contributing to pathogenesis of hypertension.³²

Patients with hypertension display elevated plasma concentrations of CHGA,³³ whereas CHGA knockout mice exhibit substantially higher BP³; indeed, the effect of CHGA expression on catecholamine release and BP seems to be biphasic, with elevations of BP noted at very low and very high CHGA expressions achieved by manipulating CHGA gene copy number.¹⁵ Here, we treated mice with an inhibitor/antagomir of miR-107, leading to a fall in SBP/DBP but not HR in humanized CHGA(+/+) mice; this effect was not seen in wild-type Chga(+/+) mice (Figure 6B). Because the only difference between the two strains is the human versus mouse version of CHGA, we argue that this effect proceeds through augmented expression of CHGA and hence, catestatin, the CHGA-encoded peptide that inhibits release of catecholamine and is, thus, associated with lower BP.³⁴

Advantages and Limitations of This Study

We undertook functional studies at a positional candidate genetic locus on chromosome 14q, CHGA, to document biologic mechanisms underlying the genetic associations with cardiorenal traits. The use of luciferase reporter genes allowed us to quantify expression and therefore, document effects of the variant as well as the transacting factor miR-107, thereby permitting quantitative verification to augment the plausibility of our conclusions. CHGA 3′-UTR C+87T (rs7610) is a human variant, and therefore, we performed many of our experiments on the HEK cell line HEK293T that expresses both CHGA and hsa-miR-107 (Figure 2). Finally, the mouse studies indicated an effect of the miR in vivo (Figure 6B). However, the bulk of our studies was conducted in cell lines; future studies with additional animal models (or even human subjects) should assist in elaborating hsa-miR-107 mechanisms in vivo.

Although we showed a clear effect of miR-107 on the human CHGA gene to influence BP (Figure 6B), an effect of the cis-/trans-CHGA/miR-107 interaction on progressive renal disease has not been specifically investigated by these rodent studies; because chronic renal disease develops only slowly, additional rodent experiments over a time course of months to years may be required to understand the mechanism of the renal disease association.

Although we documented the effect of miR-107 on the CHGA 3′-UTR, closely related miR-103, with a binding motif that resembles that of miR-107,³⁵ was not investigated here.

Finally, recent allelic association studies of hypertensive ESRD in African Americans have uncovered disease predisposition alleles in another locus: APOL1 on chromosome 22q12.³⁶ The precise mechanism whereby APOL1 variants cause renal damage is not completely clear, but because CHGA is located on a different chromosome (14q32), the phenotypic effects of the two genes would be expected to segregate independently.

Perspectives

Previously, we determined that human CHGA 3′-UTR rs7610 +87T allele conferred elevated risk for developing hypertension¹⁰ and hypertensive renal disease.¹¹ Now, we find that, both computationally and experimentally, the +87T allele seems to exhibit enhanced inhibition by the miR hsa-miR-107, decreasing CHGA as well as catestatin expression and perhaps leading to higher BP and ultimately, ESRD. Our in vivo results with an antagomir of miR-107 (Figure 6B) seem to bear out this hypothesis. Although in this study, we focused only on the miR/3′-UTR step in the causal sequence of events, the overall pathophysiologic chain of events that we propose is outlined sequentially in Figure 7. The results point to new molecular strategies for probing autonomic control of the circulation and ultimately, the susceptibility to and pathogenesis of cardiovascular disease states, such as hypertension and nephropathy.

Figure 7.

Hypothetical schema. Hypertension, hypertensive renal disease, and the CHGA pathway from 3′-UTR variation to disease. A sequence of proposed pathophysiologic events on the basis of our results and previous¹¹ experimental results is shown.

Concise Methods

Plasmid Preparations

Human CHGA 3′-UTR/Luciferase Reporter

To test whether the CHGA 3′-UTR C+87T variant is itself functional, we inserted the entire 409-bp 3′-UTR into a reporter plasmid just downstream from the luciferase open reading frame and derived the minor allele version (+87T) by site-directed mutagenesis (Supplemental Figure 1) followed by sequence verification.

Prokaryotic and Eukaryotic Expression of Full-Length Human CHGA cDNA

The wild-type pCMV-CHGA (C+87 allele in 3′-UTR) eukaryotic expression plasmid, which also has a T7 promoter flanking the insert, was purchased from Origene (SC119356; Origene, Rockville, MD). The +87T allele was generated by site-directed mutagenesis (200519; Agilent Technologies, Santa Clara, CA).

Cell Biology

HEK293T cells were cultured in high-glucose DMEM (11995; Invitrogen, Carlsbad, CA), 10% FBS, and 1× penicillin/streptomycin/glutamine. Human neuroblastoma cells (SH-SY5Y) were cultured in MEM with high glucose (12491; Invitrogen), Ham’s F12 (11765; Invitrogen), 10% FBS, and 1× penicillin/streptomycin/glutamine. Rat pheochromacytoma cells (PC12) were cultured in a medium containing DMEM high glucose (11965; Invitrogen), 10% horse serum, 5% FBS, and 1× penicillin/streptomycin/glutamine. These cell lines were cultured in an incubator with 6% CO₂ at 37°C, and cell passage number (since initiation of the line) was between 10 and 25 in these experiments.

Endogenous RNA Expression

The relative abundance of CHGA mRNAs and miR-107 in the cell lines and tissues was measured by qRT-PCR and normalized by housekeeping RNAs—either GAPDH mRNA (for CHGA mRNA) or SNORD61 small RNA (for miR-107). Total RNA was extracted from each cell or tissue, and real-time PCR was done with fluorescent reporter-tagged oligonucleotide primers on an ABI-7700 TaqMan platform (Life Technologies). Threshold cycle (C_t) is determined for both the specific target RNA as well as the housekeeping RNA, and the difference in C_t (target RNA versus housekeeper RNA) is normalized to the average for that state (e.g., cell or tissue type) by the comparative C_t (i.e., 2^−∆∆Ct) method.³⁷ Nonetheless, expression comparisons across cells, tissues, and species are only approximate, because different efficiencies of the amplification kinetics of the target and reference gene assays may generate uncertainties of estimation.

The relative amounts of miR-107 as well as most common mature miRs across cells, tissues, and species have been quantified by RNA-Seq³⁸ and archived for download as normalized counts in Excel workbooks at http://www.microrna.org. In human cells and tissues, miR-107 is widely distributed in brain (frontal cortex, hippocampus, and midbrain), lymphocytes (B and T), breast, cervix, epididymis, hepatocytes, liver, ovary, and pituitary.

Human CHGA 3′-UTR/Luciferase Reporter Activity Assay

After transfection (transfectin lipid reagent; 170–3350; Bio-Rad) and cell growth over 24 hours, cells were harvested with passive lysis buffer (E194A; Promega) for sequential measurement of luciferase enzymatic activity and protein concentration assay. Luciferase enzymatic activity was measured using d-luciferin (L6882; Promega) as the substrate on a Luminometer Autolumat 953 (EG&G Berthold, Bad Wildbad, Germany). Protein concentration was measured using a dye-binding protein assay (500–0006; Bio-Rad) on a SmartSpec Plus spectrophotometer (Bio-Rad). Promoter activity results are expressed as the ratio of luciferase activity to protein concentration. In some experiments, we also evaluated the role of transfection efficiency between the 3′-UTR alleles (T versus C plasmids) by cotransfecting a different plasmid (pSV–β-galactosidase), wherein the SV40 early promoter drives expression of the β-galactosidase (LacZ) gene (E1081; Promega); cotransfected cell lysates were then assayed for β-galactosidase enzymatic activity (E2000; Promega) by incubation for 30 minutes with an equal volume of the substrate o-nitrophenyl-β-d-galactopyranoside in assay buffer followed by termination of the reaction by adding 1 M Na₂CO₃. Then, absorbance of the product was read at 420 nm.

Coupled In Vitro Transcription/Translation of Each 3′-UTR/C+87T Allele in Full-Length Human CHGA cDNA

T7 promoter-driven full-length human CHGA cDNA (including the 3′-UTR in two versions; C+87T) was transcribed (T7 promoter) and translated in vitro by the TNT Quick Coupled Transcription/Translation System (L1170; Promega) using a rabbit reticulocyte lysate. Newly synthesized proteins were detected by chemiluminescence with anti-CHGA antibody in the Transcend Non-Radioactive Translation Detection System (Promega).

Immunoblot Analyses

Proteins from cell or reticulocyte lysates were separated in 10% SDS-PAGE (Novex precast gel; Invitrogen) and electrophoretically transferred to nitrocellulose membranes (Protran, BA85; Whatman Inc., Florham Park, NJ). The membrane was blocked with 5% (wt/vol) powdered dry milk in Tris-buffered saline with 0.1% Tween-20. After incubation with primary antibody (rabbit anti-human catestatin region), the membrane was washed and incubated with secondary antibody (horseradish peroxidase-conjugated donkey anti-rabbit). The membrane was then developed by the Supersignal West picochemiluminescent substrate (Pierce, Rockford, IL). Anti-GAPDH was used as an internal control. Immunoreactive band quantification was done on the software ImageJ (http://rsbweb.nih.gov/ij/).

Evaluation of miR Function in Cells

Strategies of both knockdown and overexpression were adopted to explore miR function in cells. For knockdown, the Dharmacon miRIDIAN Hairpin Inhibitor (antagomir) hsa-miR-107 (hsa-miR-107 inhibitor; IH-300527–05) specific for miR hsa-miR-107 was used in an attempt to decrease the signal from that miR, and a predesigned negative control inhibitor (no. 1; IN-001005–01–05) was used as a control. The identity of each synthetic oligonucleotide was verified by mass spectrometry. For overexpression, a Dharmacon (Lafayette, CO) miRIDIAN 23-mer (5′-AGCAGCAUUGUACAGGGCUAUCA-3′) double-stranded RNA mimic for human miR-107 (C-300527–03) was used to increase that miR abundance, whereas a predesigned negative control (no. 1; CN-001000–01–05) was used to control for off-target effects.

In Vivo (Mouse) Studies

Effects of hsa-miR-107 on the Human CHGA 3′-UTR: Mouse Strains

We developed a humanized CHGA mouse strain as described previously.^14,15 We used a BAC vector carrying a 211-kbp human genomic insert (clone RP11–862G15) spanning the 12.1-kbp human CHGA locus^14,15 to generate mice with a functional human CHGA allele. The founder strain contained the entire 211-kbp insert as the transgene. It was bred with Chga−/− (knockout) mice, and the progeny were subsequently brother–sister mated to generate HumCHGA mice (CHGA+/+;Chga−/−): homozygous for the transgene and lacking the mouse Chga allele. The genetic background for this strain was mixed (at approximately 50% B6/approximately 50% 129 SVJ) after seven generations of inbreeding. The control mouse (wild-type mouse Chga +/+;CHGA−/−) for these experiments was on the same mixed background. In the humanized strain, the genotype at C+87T was T/T, whereas in the control mouse strain, the genotype at the C+87T site was also T/T.

Effects of hsa-miR-107 on the Human CHGA 3′-UTR: Continuous Telemetric BP and HR Monitoring of Wild-Type (Mouse Chga Gene) and Humanized CHGA Mice

BP and HR in mice were continuously monitored by intra-arterial telemetry using the Data Sciences International (DSI; Transoma Medical, St. Paul, MN) PhysioTel telemetry system. Adult male mice (16–18 weeks of age) at approximately 28 g body weight were anesthetized with isoflurane (5% for induction and 2% for maintenance) and implanted with a catheter coupled to a TA11PA-C20 (DSI) transmitter in the left carotid artery. Telemetry signals were received by an antenna below the cage that relayed the data to a signal processor (DataQuest A.R.T. Gold, version 2.3; DSI) connected to a desktop personal computer (Hewlett-Packard, Portland, OR). After the implantation surgery, the animals were rested for 10 days for normalization of the diurnal pattern of BP before recording BP in these conscious mice fitted with DSI transmitters. Baseline BP and HR (beats per minute) were determined by averaging 10 consecutive seconds of data every 5 minutes for 24 hours. The mice were housed in a 12-hours light (06:00–18:00 hours) and 12-hours dark cycle.

Administering an hsa-miR-107 Inhibitor In Vivo

An inhibitor (antagomir) of hsa-miR-107 was custom synthesized with the sequence 5′-C_sU_sGUACAAUGCUG_sC_sU-3′, where the subscript s indicates a phosphorothioate linkage to stabilize the oligonucleotide against degradation in vivo (Valuegene, San Diego, CA). The inhibitor was reconstituted in saline and injected intraperitoneally at a single dose of 3.3 µg/g body wt at approximately 10:00 hours. Telemetric BP and HR results were evaluated over 24 hours after antagomir injection by linear mixed effects model for repeated measures in SPSS factoring for time (after antagomir), strain, and strain-by-time interaction. The effect of in vivo antagomir treatment on expression of CHGA mRNA in adrenal and brainstem tissues was monitored by qRT-PCR 5 hours postintraperitoneal injection.

Renal Effects of Chga Gene Deletion

Targeted ablation of the Chga locus was achieved as previously described.³ Blood was obtained from the homozygous Chga(−/−) deletion animals (and comparable genetic background controls) by tail bleeding, anticoagulated on EDTA, and centrifuged. The supernatant plasma was frozen at −70°C for later assay of plasma creatinine by HPLC with cation exchange (with calibration as described³⁹) to avoid interference by endogenous no creatinine plasma chromogens. GFR was then estimated by regression on the basis of the mouse data tables in ref. 39.

Computation

RNA sequence alignments between the human CHGA 3′-UTR and complementary candidate miRs as well as free energy of binding estimates were performed at the RegRNA interface (http://regrna.mbc.nctu.edu.tw/) using the miRanda algorithm⁴⁰ at miRBase-21 (www.mirbase.org/), querying n=1919 human miR motifs, with software available at http://www.microrna.org/microrna.

A match score for each miR:mRNA complementary region is calculated first (on the basis of a modified Smith–Waterman scoring algorithm for A:U and G:C matches during dynamic program alignment with a default cutoff≥80). G:U wobble pairs are allowed but at reduced scores. Thus, the alignment score was calculated as +5 for G:C or A:T pairs and +2 for G:U wobble pairs.

Then, the ΔG (free energy of binding or minimum free energy in kilocalories per mole) for high-scoring (cutoff≥80) alignments is calculated using RNAlib in the ViennaRNA package, with a default cutoff≤−14 kcal/mole. The miRanda scores for hsa-miR-107:human_CHGA_3′-UTR in this study exceeded both of these criteria.

Candidate miR:mRNA matches were then tested experimentally for perturbation by either the miR itself or its antagomir (antisense version).

Statistical Analyses

Values are given as means±SEMs. Numbers of experimental replicates are given in the figures. Data shown are representative of typical experiments. Statistical analyses were performed with SPSS-17 (SPSS Inc., Chicago, IL) by t test (two-tailed) or two-way ANOVA as appropriate after inspection of the data distribution. Prolonged longitudinal responses of cardiovascular traits to interventions in the mouse were evaluated by repeated measures linear mixed models. When data were not normally distributed, we used either data transformation (e.g., log₁₀) or nonparametric tests (e.g., Mann–Whitney U tests). Differences were considered significant at P<0.05.

Study Approval

The animal study protocol was approved by the University of California at San Diego Institutional Animal Care and Use Committee.

Disclosures

None.