Correlation between the microRNA-874-Sirtuin2-p53/nuclear factor-kappa B signaling pathway and depressive symptoms: a prospective multicenter study

Shan-Shan He, Hui-Duo Jiao, Qun-Qiang Wu, Chun-Hua Liao, Rui Liu

Department of Rehabilitation, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi Province, China

Abstract

Key words: depression; endogenous small RNA; inflammatory response; miR-874; neurogenesis; SIRT2

INTRODUCTION

Depression is a common mental disorder, with a prevalence of approximately 2.1% in China (National Health Commission of China, 2019), and is associated with an increased risk of suicide (Haroz et al., 2017). Depression has been shown to be associated with various factors, such as the social environment, psychology, neurobiochemistry, and genetics; however, the exact pathogenesis of depression remains to be determined, and no effective treatments are currently available. Studies have shown that impaired hippocampal neurogenesis and local inflammatory responses are important mechanisms associated with depression (David et al., 2009), and inflammatory responses can further reduce hippocampal neurogenesis and aggravate depressive symptoms (Kim et al., 2016; Tang et al., 2016).

Sirtuin2 (SIRT2) is involved in the regulation of apoptosis and inflammatory signaling pathways. Our preliminary study found that the sustained downregulation of SIRT2 in the rat hippocampus was associated with depressive symptoms, and the overexpression of SIRT2 promoted hippocampal neurogenesis and relieved depression-like behaviors in rats (Liu et al., 2015). Long-lasting increases in SIRT2 expression can enhance neuronal regeneration in the hippocampus and attenuate local inflammatory responses, which may represent a potential long-acting anti-depression strategy. However, the regulatory mechanisms associated with SIRT2-related signaling pathways remain unclear.

Endogenous, small RNAs, or microRNAs (miRs), are noncoding RNAs of less than 22 nucleotides in length. miRs can imperfectly complementarily bind to the 3′-untranslated regions (3′-UTRs) of target mRNAs and are capable of reducing target protein levels rapidly and persistently via translational repression and mRNA cleavage (Bushati and Cohen, 2007). miRs can be involved in nervous system development and plasticity (Fiore et al., 2008), and multiple clinical studies have shown that miRs are associated with depression (Fiori et al., 2017; Lopez et al., 2017; Wang et al., 2018) (Table 1). Therefore, we speculate that miR molecules in the brain may be associated with the persistent changes in SIRT2 protein levels that mediate hippocampal neurogenesis after depression.

Our preliminary study utilized bioinformatics software to predict the miR molecule that regulates SIRT2 protein levels and found that the 3′-UTR sequence of SIRT2 mRNA is rich in miR-874 binding sites, suggesting that miR-874 may act as an upstream regulatory molecule for SIRT2 protein expression. We also observed increased miR-874 expression levels in serum samples from patients with depression in a small sample pre-test, and the miR-874 expression level was directly proportional to the severity of depression, suggesting that miR-874 is very likely to participate in the pathogenesis of depression. However, how miR-874 affects hippocampal neurogenesis and whether miR-874 exerts its effects on depression through the regulation of SIRT2 levels remain unclear.

Studies have shown that SIRT2 can inhibit cellular oxidative stress via the regulation of p53/nuclear factor (NF)-κB expression (Yang et al., 2019), preventing high-glucose-induced vascular endothelial cell injury (Zhang et al., 2018) and disrupting neuroinflammation and blood-brain barrier destruction. Therefore, we hypothesize that miR-874-SIRT2-p53/NF-κB may participate in the pathogenesis of depression by regulating hippocampal neurogenesis and inflammation. This multicenter, prospective, clinical trial will explore the association between miR-874 expression and depressive symptoms in patients with depression, providing evidence for the development of antidepressants that target microRNAs in the future.

PARTICIPANTS AND METHODS

Study design

A multicenter, prospective, clinical trial (Figure 1).

Study setting

The trial will be completed at the Department of Rehabilitation at Tangdu Hospital and the Department of Psychosomatic Medicine at Xijing Hospital of the Fourth Military Medical University in Xi'an, Shaanxi Province, China.

Table 1: Clinical studies on endogenous small RNAs and depressive symptoms in recent years

Figure 1: Trial flow chart.

Clinician qualifications

The Department of Psychosomatic Medicine at Xijing Hospital of the Fourth Military Medical University was formerly known as the Department of Neuropsychiatry at Xijing Hospital of the Fourth Military Medical University. It was founded in 1949, by the famous psychiatrist Professor Qing-Han Yu, and was the first psychiatric department in Northwest China. The Department of Rehabilitation at Tangdu Hospital of the Air Force Military Medical University is also a well-known department for rehabilitative medicine in Shaanxi Province.

The members of our team have obtained at least a master's degree in psychiatry, have obtained at least the title of attending physician, and have at least 5 years of clinical experience in psychiatry.

Participants

Recruitment

A recruitment message will be advertised to patients using posters in the Department of Psychosomatic Medicine at Xijing Hospital of the Fourth Military Medical University, in the Department of Rehabilitation at Tangdu Hospital of the Air Force Military Medical University, and in the admission offices of both hospitals. Recruitment will be also conducted through WeChat, academic conferences, small patient education sessions, and expert consultations. Interested patients or their legal guardians will be able to contact the principal investigator via phone, email, or WeChat.

Selection of participants

· The depression group will be composed of patients who are diagnosed with depression and who are hospitalized in the Department of Rehabilitation at Tangdu Hospital of the Air Force Military Medical University and in the Department of Psychosomatic Medicine at Xijing Hospital of the Air Force Military Medical University in Xi'an, Shaanxi Province, China.

Inclusion criteria were collectively identified by the research team experts, and those patients who meet all of the following criteria will be recruited:

(1) Meet the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) criteria for the diagnosis of depression (American Psychiatric Association, 2013);

(2) Hamilton Depression Rating Scale 24-Item (HAMD-24) total score ≥ 20 points (Hamilton, 1960);

(3) Aged 18-65 years, irrespective of gender; and

(4) Signs a written informed consent form.

Exclusion criteria were jointly determined by the research team experts, and patients will be excluded if they meet any of the following criteria:

(1) Other concurrent mental disorders that meet the diagnostic criteria defined by the DSM-5;

(2) Use of psychotropic drugs, except for benzodiazepines and Z-type sleep aids, during the 2 weeks prior to admission;

(3) Pregnant or lactating; and

(4) Participation in other clinical drug trials.

· The healthy control group will be composed of healthy volunteers who obtain a physical examination at the Department of Rehabilitation at Tangdu Hospital of the Air Force Military Medical University and the Department of Psychosomatic Medicine at Xijing Hospital of the Air Force Military Medical University in Xi'an, Shaanxi Province, China.

Inclusion criteria were collectively identified by the research team experts, and healthy volunteers who meet all of the following criteria will be recruited:

(1) Aged 18-65 years, irrespective of gender; and

(2) Signs a written informed consent form.

Exclusion criteria were collectively determined by the research team experts, and healthy volunteers will be excluded if they meet any of the following criteria:

(1) Use of psychotropic drugs, except for benzodiazepines and Z-type sleep aids;

(2) Pregnant or lactating; and

(3) Participation in other clinical drug trials.

· Withdrawal criteria include the withdrawal of informed consent forms or voluntary withdrawal from the trial.

· The criteria for the termination of the trial are as follows:

(1) Serious safety issues identified by the investigator;

(2) Aggravation of patient conditions;

(3) Poor compliance or inability to complete the study; and

(4) Revocation of the study by administrators.

All enrolled patients with depression will be newly diagnosed, characterized by experiencing their first attack of depression, and with no history of antidepressant use. Moreover, no drug interventions will be included in the trial, and no medications will be given to the healthy control group.

The blood sample collections and laboratory tests associated with this trial will be free to participants. During the trial, each patient will receive a scientific and professional assessment of his or her mental health and psychological characteristics, which will be provided by a psychiatrist with a title of at least attending physician. Because this trial does not involve invasive treatments, except for blood drawing, the patient will be highly unlike to be at any risk. If blood drawing results in skin damage, the research team will compensate the patient by covering the costs of antibiotics and local disinfection.

Outcome measures

· The primary outcome mea sures include the expression levels of miR-874 and SIRT2 mRNA in the blood. A 5 mL blood sample, taken from the right elbow vein, will be extracted from each participant and centrifuged. After removal of the supernatant, the precipitate will be retained, and the miR-874 and SIRT2 expression levels will be determined using a nucleic acid analyzer.

· The secondary outcome measures are as follows:

(1) Levels of inflammatory factors: the levels of interleukin-1β, interleukin-6 and tumor necrosis factor-α will be examined by enzyme-linked immunosorbent assays (ELISA), using blood samples collected from the patient's right elbow vein.

(2) HAMD-24 scores: the 24 items include depressed mood, guilt, suicidal thoughts, difficulty falling asleep, lack of sleep, early awakening, work and interests, psychomotor retardation, psychomotor agitation, psychic anxiety, somatic anxiety, gastrointestinal symptoms, general somatic symptoms, sexual disturbances, hypochondriasis, insight, weight loss, diurnal variations, depersonalization and derealization, paranoid symptoms, obsessional and compulsive symptoms, helplessness, hopelessness, and worthlessness. A higher score indicates more severe depression (Hamilton, 1960).

(3) Hamilton Anxiety Rating Scale (HAMA) score, consisting of the following seven items: anxious mood, tension, fears, insomnia, cognition, depressed mood, and behavior at interview. A higher score indicates more severe anxiety (Hamilton, 1959).

(4) Global Assessment of Functioning (GAF) score, which reflects the severity of depression. A higher score indicates better conditions for the patient (Pedersen and Karterud, 2012).

(5) The incidence of overall adverse events: we will identify adverse events, analyze the causes of adverse events, make judgments in response to adverse events, and track observations and recordings associated with adverse events. Data associated with adverse events related to the trial, including symptoms, symptom severity, time of occurrence, symptom duration, measures taken, and processing, should be recorded on a case report form. The relevance of adverse events to the treatment will be evaluated and recorded in detail on forms that will be signed and dated. When an adverse event occurs, the clinician, as an investigator, can decide whether to terminate the study based on the patient's condition. The investigator should follow up on adverse events until the symptoms disappear or until the symptoms are stabilized. Adverse events should be well-documented, and in the event of serious adverse events, such as hospitalization, prolonged hospital stays, disability, the inability to work, life-threatening events or death, and congenital malformations, the clinician will promptly take appropriate treatment measures to ensure the patient's safety. In the event of a serious adverse event, follow-ups will be continued for those patients who can be followed on schedule, and the patient's condition should be monitored closely. In the event of a serious adverse event during the clinical trial, the investigator should immediately take appropriate measures for the patient's benefit, and these measures should be recorded on case report forms and promptly reported to the principal investigator and to the institutional ethics committee. Investigators must carefully report any serious adverse events, on a case-by-case basis, and sign and date the report.

Timing of outcome measure evaluation is shown in Table 2.

Table 2: Schedule of outcomes

Sample size calculation

Based on a series of genetic tests reported in previous literature (Liu et al., 2015) and the actual conditions of depression patients admitted at our hospital, the sample size of this trial was determined to be 50 cases, and 50 healthy subjects will be recruited concurrently as controls.

Blindness

Due to the lack of drug administration or the use of special treatments, no blinded design was determined to be necessary when drawing blood samples.

Ethical approval

This study will be performed in accordance with the Declaration of Helsinki developed by the World Medical Association and was approved by Hospital Ethics Committee, Tangdu Hospital of Fourth Military Medical University (approval No. K201806-03) on June 1, 2018 (Additional file 1). Any modification to the study protocol or informed consent will be ratified by the Ethical Committee. The study was registered with the Chinese Clinical Trial Registry (registration No. ChiCTR1800018933) on October 17, 2018. Version of study protocol refers to 1.0. The study follows the STrengthening the Reporting of OBservational studies in Epidemiology (STROBE) statement (Additional file 2).

Informed consent

Participants will be provided with complete and comprehensive information regarding the purpose, the planned procedures, and the possible benefits and risks of this study, after which they will be asked to sign an informed consent form (Additional file 3) indicating that they understand the risks associated with the study and that they have the right to withdraw from the study at any time. Informed consent forms will be maintained as clinical documents for future reference. Participants' personal privacy and confidentiality will be protected during the study. Any modifications made to the informed consent form must be confirmed, and the modified forms must be re-signed by the participants or their legal representatives.

Statistical analysis

All statistical analyses will be calculated using SPSS 19.0 (IBM, Armonk, NY, USA). All statistical tests will be twosided tests, and a P value of less than or equal to 0.05 will be considered statistically significant. Multiple linear regression analyses will be used to analyze the correlation between gene/molecule expression levels and the severity of depressive symptoms, inflammatory factor levels, and outcomes.

Data collection

The investigators participating in the clinical research will participate in training sessions regarding the unified criteria for data judgment and data recording. The investigators will be instructed to itemize the case report forms, term by term, using a black pen, in a timely, truthful, detailed, and serious manner.

Data management

All laboratory data related to the study will be recorded on the case record report form in a timely and truthful manner, and the original report or a copy of the original report will be attached to the research medical record. All observations and findings during the trial should be verified to ensure data reliability. The contents of the research medical record are generally not subject to change. If necessary, any corrections made to the record should be marked using a horizontal line, and the original record data should continue to be clearly visible. Moreover, the clinician who makes the correction will indicate the reason for the correction and sign and date the corrected version.

Quality control

Inspectors appointed by the sponsor will be responsible for quality control. The specific duties of these inspectors will be to ensure that the investigators strictly follow the study protocol, the relevant standard operating procedures, the guiding principles, and the regulatory requirements throughout the clinical trial. The inspector should also strictly follow the study protocol, relevant standard operating procedures, guiding principles and regulatory requirements throughout the clinical trial. The inspector should communicate with the research center throughout the trial. Before the trial is officially launched, the inspector should convene a kick-off meeting regarding the study protocol and the standard operating procedures for the primary researchers and all the staff participating in the trial. During the trial, the auditor should regularly monitor the progression of the trial and examine original records and case report forms, to verify that the investigators comply with the study protocol, trial flow, and relevant regulations and to ensure that the obtained data are objective, true, and legal. At the end of the study, the inspector should verify and archive all documents in the research center.

Each sub-center should establish an internal quality assurance system to strictly supervise and control the quality of the research performed at the center.

Modifications to the protocol

The study protocols are generally not subject to change after discussions by the study team members and approval by the Ethics Committee. The investigator should apply for reapproval from the Ethics Committee if it becomes necessary to modify the protocol, with reference to new data or new laws and regulations.

Audits

During the trial and after the completion of the trial, the inspector will strictly audit the implementation of the study protocol and data recording, and whether the trial management was performed in accordance with the established plan. Clinical inspectors will perform clinical audits every 2 months and will report to the Ethics Committee regarding the trial process. Trial registration will be updated simultaneously. The research team should take corrective measures to fix any problems identified by audits and institute preventive measures to avoid similar problems in the future.

Confidentiality

All clinical data will be limited to research use and commercial use will not be permitted. Patient information will be encoded prior to entry into the database, and these codes will not contain any personally identifiable information. The personal information of patients and their families will be confidential. Patient data will be monitored by the Ethics Committee and the Food and Drug Administration but will not be disclosed.

Results dissemination

Data will be available immediately following publication, with no end date. Results will be disseminated through presentations at scientific meetings and/or by publication in a peer-reviewed journal.

DISCUSSION

Limitations

Further pathophysiological studies are warranted, as no direct analyses of the p53/NF-κB signaling pathway are included in the present trial. The use of different medications for the treatment of depression is likely to result in changes to miR-874 or SIRT2 expression levels, and further research examining such changes will be necessary in the future.

Generalizability

If we are able to identify an association between the miR-874-SIRT2-p53/NF-κB signaling pathway and depressive symptoms, these findings will contribute to the explorations of the pathogenesis of depression and will provide potential therapeutic targets for the treatment of depression.

Explanatio n

miRs can regulate neural development in many ways. miR-124a and miR-125b have been shown to be involved in the growth of neuronal axons (Dai and Ahmed, 2011; Boese et al., 2016), and miR-132 and miR-134 have been associated with neuronal synapse formation and can affect memory and neural plasticity (Pathania et al., 2012; Fan et al., 2018). Studies have shown that the levels of a variety of miRs, such as miR-451a, miR-34a-5p, miR-221-3p, miR-128a, miR-24a, miR-16, miR-34a, and miR-132, change markedly in the serum of patients with depression (Bocchio-Chiavetto et al., 2013; Kuang et al., 2018; Yuan et al., 2018). However, few studies have examined the underlying mechanism through which miRs influence depression.

Low levels of SIRT2 expression have been associated with the development of depression, and SIRT2 influences neuroinflammation through the p53/NF-κB pathway. Our preliminary study found that miR-874 can inhibit the expression of SIRT2. We, therefore, hypothesize that the miR-874-SIRT2-p53/NFκB may be involved in depression via effects on hippocampal neurogenesis and inflammation. This assumption will be verified in the present clinical trial.

TRIAL STATUS

Registration time:Octorber 17, 2018.

Recruitment time:October 31, 2018 to February 28, 2021.

Study completed:September 1, 2021.

Current status:Recruiting.

Additional files

Additional file 1: Hospital ethics approval document (Chinese).Additional file 2: STROBE checklist.

Additional file 3: Model consent form (Chinese).

Author contributions

Study design: RL; implementation of the trial: QQW, HDJ, CHL; data analysis: RL, SSH; manuscript writing: RL and SSH. All the authors approved the final manuscript for publication.

Conflicts of interest

The authors have no conflicts of interest to declare.

Financial support

This work was supported by the National Natural Science Foundation of China, No. 81771469 (to RL). The funder had no role in the study design, data collection, management, analysis, and interpretation; paper writing; or decision to submit the manuscript for publication.

Institutional review board statement

The study was approved by the Hospital Ethics Committee, Tangdu Hospital of Fourth Military Medical University, Xi'an, Shaanxi Province in China (approval No. K201806-03) on June 1, 2018. This study will be performed in strict accordance with the Declaration of Helsinki.

Declaration of participant consent

The authors certify that they will obtain all appropriate consent forms from the participants or their legal guardians. In the forms, the participants or their legal guardians will give their consent for participants' images and other clinical information to be reported in the journal. The participants or their legal guardians understand that the participants' names and initials will not be published and due efforts will be made to conceal their identity.

Reporting statement

This study followed the STrengthening the Reporting of OBservational studies in Epidemiology (STROBE) statement.

Biostatistics statement

The statistical methods of this study were reviewed by the biostatistician of Tangdu Hospital, Fourth Military Medical University, China.

Copyright license agreement

The Copyright License Agreement has been signed by all authors before publication.

Data sharing statement

Individual participant data that underlie the results reported in this article, after deidentification (text, tables, figures, and appendices) will be available immediately following publication, with no end date. Results will be disseminated through presentations at scientific meetings and/or by publication in a peer-reviewed journal. Anonymized trial data will be available indefinitely at www.figshare.com.

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Externally peer reviewed.

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