Detection of miRNAs in HPA Axis Function in Healthy Subjects
Phase: PHASE1
Conditions: Dexamethasone Effect on miRNA Profile in Healthy Humans
Sponsor: Cedars-Sinai Medical Center

06.06.19, Anat Ben-Shlomo 
Detection of miRNAs in HPA axis function in healthy subjects 
 
Detection of miRNAs in HPA axis function in healthy subjects 
Background 
Cortisol is a steroid hormone involved in metabolism, inflammation and stress response (1). 
It is secreted by the adrenal glands in a pulsatile fashion in response to a feedback loop involving 
the hypothalamus-pituitary-adrenal (HPA) axis. Corticotropin-releasing hormone (CRH) is 
secreted from the hypothalamus in response to circadian rhythm changes and in response to 
stressors. CRH stimulates corticotroph cells in the anterior pituitary gland to secrete 
adrenocorticotropic hormone (ACTH), which, in turn, promotes synthesis of corticosteroids such 
as cortisol in the adrenal glands. Secretion of cortisol from the adrenal gland then downregulates 
hypothalamic CRH and pituitary ACTH secretion through feedback inhibition.  
Cortisol stimulation is used to assess the adequacy of HPA axis functional response in 
humans. However, accurate cortisol measurements can be misleading, especially in the presence 
of pituitary and hypothalamic diseases affecting HPA axis response. New biomarkers for HPA 
axis function are needed.   
We recently showed significant changes in miRNA expression profiles in the serum of 
humans and mice during HPA axis regulation and identified miRNA that might be useful as 
biomarkers of HPA axis function. We found that miRNA levels are significantly decreased 30 
minutes after ACTH stimulation in mice and humans, while dexamethasone increases serum 
miRNAs in mice.  
The proposed study will test dexamethasone effect on miRNA profile in healthy humans. As 
our data indicate that miRNA levels change over time, we will study the time course of miRNA 
response to 1 mg IV dexamethasone within 60 minutes after injection.  
Dexamethasone is used routinely in human subjects for the treatment of inflammatory 
conditions and as a diagnostic tool in the evaluation of excess cortisol secretion (Cushing’s 
syndrome). For the latter, 1 mg dexamethasone is given orally at midnight. However, oral 
formula absorption in the gut can range from 20 to 60 minutes due to the presence of food and 
other factors. IV administration will bypass this variability and ensure an accurate and 
reproducible time-course study. 
We will compare miRNA expression before and after dexamethasone treatment. We will 
follow miRNA expression dynamics over a 1-hour time course to identify peak expression levels 
and to correlate miRNA expression with circulating dexamethasone levels. 
Dexamethasone and miRNA levels will be checked at baseline just before injection and then 
at 15, 30, 45, and 60 minutes after injection. Dexamethasone level is taken to verify treatment 
and to correlate it with miRNA expression. 
 
Definitions 
• miRNA (also called microRNA or miR), small sequences of RNA that bind and inhibit 
specific areas in the gene and regulate gene expression. Most of the miRNAs are 
intracellular, but some can be found in the circulation.  
• mRNA (messenger RNA), a nucleic acid polymer molecule transcribed from DNA that 
serves as a template for the synthesis of the specified protein. The level of mRNA 
indicates changes in gene expression. 
• Adrenal gland, an endocrine gland located above the kidney. It produces multiple steroid 
hormones, most importantly cortisol (or corticosterone in rodents) and aldosterone. Its 
inner core produces epinephrine and norepinephrine. 

06.06.19, Anat Ben-Shlomo 
Detection of miRNAs in HPA axis function in healthy subjects 
 
• Serum, the liquid portion of the blood after it has been allowed to clot. It is free of cells 
and platelets. 
• Plasma, the liquid portion of blood that has been prevented from clotting. It includes 
cells and WBC can be extracted from it. 
• Dexamethasone, a steroid hormone that belongs to the glucocorticoid steroid hormone 
group that also includes cortisol. It binds specifically to the glucocorticoid receptor to 
induce cellular and tissue outcomes similar to those seen with cortisol. It is use clinically 
to reduce inflammation and as a diagnostic tool for Cushing’s syndrome.  
• Cushing’s syndrome, a disorder caused by increased production of cortisol. Symptoms 
include increased appetite and weight gain, mood changes, trouble sleeping, nausea, 
upset stomach, easy bruising, swelling, hypertension, hyperglycemia, osteoporosis.   
 
 
Study Protocol 
 
Subjects 
Twenty subjects will be recruited from CSMC employees. No information will be gathered 
from subjects’ medical files. An email will be sent out to all members of the Endocrinology 
department, the Pituitary Center, and affiliated offices (physicians, scientists, technicians, nurses, 
administrators). Employee email addresses will be included as BCC so as not to reveal the 
distribution list. In this email, study staff will detail all planned procedures, inclusion/exclusion 
criteria, and potential side effects. Those who express interest in participating will be contacted 
by study staff, who will review with them the protocol and informed consent and confirm 
eligibility for enrollment. Study staff will not enroll subjects who directly report to them to 
mitigate the potential for coercion. 
 
Inclusion Criteria 
• Adults age ≥ 18 
 
Exclusion Criteria 
• Pregnancy 
• Presence of a disease that affects HPA axis function 
• Current use of medications that affect cortisol secretion, such as opioid pain medication, 
anti-fungal agents, anti-epileptic agents, and glucocorticoids, including hydrocortisone, 
prednisone, dexamethasone, steroid-containing inhalers, and steroid injected into the 
joints 
• History of adverse event related to use of glucocorticoids 
 
Procedures 
• All procedures will be performed at the CTRC by staff nurses. 
• A vein line will be inserted to allow for dexamethasone injection and for easier blood 
draws during the study. 
• A total of 60 mL will be taken from each subject, drawn into 10 red-cap tubes (6 mL 
each) at 5 time points over the course of the study. 

06.06.19, Anat Ben-Shlomo 
Detection of miRNAs in HPA axis function in healthy subjects 
 
• At baseline, before dexamethasone injection, blood will be drawn into 2 red cap tubes: 
one tube will be used to assess dexamethasone levels and one tube will be used to assess 
for miRNAs. 
• Immediately after baseline blood collection, 1 mg dexamethasone will be administered 
intravenously at a rate of 4 mg/min. 
• After injection, blood will be drawn at 15, 30, 45, and 60 minutes into 2 red cap tubes: 
one tube will be used to assess dexamethasone levels and one tube will be used to assess 
for miRNAs. 
• After the 60-minute blood draws, the IV line will be removed, and the subject will be free 
to leave the facility. 
 
Adverse Effects 
We have extensive experience with the oral dexamethasone 1 mg diagnostic test for 
Cushing’s syndrome. We expect no new side effects with this same single dose administered 
intravenously. Side effects commonly associated dexamethasone are observed with higher doses 
and longer use of this medication. These effects mimic symptoms of Cushing’s syndrome and 
include swelling, increased appetite, weight gain, mood changes, trouble sleeping, nausea, upset 
stomach, easy bruising, hypertension, and hyperglycemia. 
We expect no adverse events from the blood draws. 
 
Blood Processing 
• Red-cap tubes used to collect blood to assess dexamethasone levels will be sent to the 
CSMC Quest laboratory. 
• Red-cap tubes used to collect blood to assess for miRNAs will be put on ice and hand-
delivered by study staff to Dr. Shlomo Melmed’s laboratory (Davis 3024) for serum 
extraction. 
o Serum will be stored in -80°C freezer. 
o Analysis of miRNAs will be done as one batch on all samples collected. 
 
 
Data Collection and Storage 
• Subject name, age, and sex will be recorded by study staff. 
• At CTRC, blood draw tubes will be coded using the subject’s initials and the day of 
collection. For example, blood from John Adam Smith collected on April 24, 2018, 
would be coded as J04A24S18. If the middle name is not available, there will be no letter 
in the fourth position. 
• This same code will also be used during sample processing and analysis. 
• Subjects will be identified only on the PI’s personal computer at CSMC. 
• Data will be stored for 5 years and will be accessible only to the PI. 
 
Compensation 
Subjects will be compensated with a $25 Target gift card. 
 
 
REFERENCES 

06.06.19, Anat Ben-Shlomo 
Detection of miRNAs in HPA axis function in healthy subjects 
 
1. 
Stewart P, Newell-Price, JDC. The adrenal cortex. In: Melmed S, et al, eds. Williams 
Textbook of Endocrinology, 13th ed. Philadelphia, PA: Elsevier; 2016. 
2. 
Yamamoto M*, Ben-Shlomo A*, Kameda H, et al. Somatostatin receptor subtype 5 
modifies hypothalamic-pituitary-adrenal axis stress function. JCI Insight. 2018;3:e122932. 
*Equal contribution. 
3. 
Altuvia Y, Landgraf P, Lithwick G, et al. Clustering and conservation patterns of human 
microRNAs. Nucleic Acids Res. 2005;33:2697-2706.