mir-30a relieves migraine by degrading calca€¦ · mir-30a relieves migraine by degrading calca...

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2022 Abstract. OBJECTIVE: To investigate both the relationship and underlying mechanism be- tween miR-30a and migraine. PATIENTS AND METHODS: Peripheral blood samples were collected from migraine patients and healthy people to extract RNA for quantita- tive Real-time PCR (qRT-PCR). The relationship between mRNA expressions and patient da- ta was analyzed by t-test. Target genes of miR- 30a were predicted by the TargetScan. The bind- ing of miR-30a to the target gene was verified by dual fluorescein reporter assay. The pro- tein expression of calcitonin/alpha-CGRP gene (CALCA), the miR-30a target gene, was detected by Western blot. RESULTS: Expression levels of miR-30a in pe- ripheral blood of migraine patients were signifi- cantly lower than those of healthy controls de- tected by qRT-PCR, and the methylation level of miR-30a in promoter region was remarkably in- creased. In addition, expression levels of miR- 30a were significantly decreased in patients with bilateral seizures, persistent pain and high pain index. CALCA was found to be the target gene of miR-30a via bioinformatics analysis. We verified that miR-30a degrades CALCA by dual-lucifer- ase reporter assay. Western blot results showed that overexpression of miR-30a down-regulated the CALCA expression, and knockdown of miR- 30a upregulated the CALCA expression. CONCLUSIONS: Expression levels of miR-30a are significantly decreased in migraine patients and can relieve migraine through the degrada- tion of CALCA. Key Words: MicroRNA, MiR-30a, CALCA, Hypermethylation. Introduction Migraine is one of the most common primary headaches. It affects about 15% of the population; the prevalence is about 6-8% in male and 15-25% in female 1 . According to the epidemiology sur- vey of primary headache in 2012, the migraine prevalence in China was about 13%. The clinical manifestations of migraine are mainly recurrent moderate-severe pulsatile headache with nausea, vomiting, photophobia, phonophobia, and aggra- vating headache, which seriously affect the life quality and work ability of patients. Research on the burden of disease revealed that migraine costs about 331.7 billion RMB annually in our coun- try 2 . Migraine is listed as the sixth incompetent disease by WHO due to its heavy economic and social burdens 3 . Currently, migraine still lacks of available clinical markers, the diagnosis of which is highly dependent on clinical symptoms 4 and brings great difficulties in clinical diagnosis. MicroRNA is a kind of non-coding RNA, which is an essential molecule involved in epi- genetic regulation. It participates in physiologi- cal regulation of cell proliferation, differentiation and apoptosis in the body. Evidence indicated that miRNA is significant in the process of disease development 5 . It mainly forms RNA-induced si- lencing complex (RISC) by specifically binding to the complementary sequence of mRNA 3’- UTR, inhibiting mRNA translation or promoting its degradation, thus resulting in post-transcrip- tional silencing of gene. Highly expressed miR- NA leads to down-regulated target genes of the mRNA. Related research suggested that miRNA can also be served as a marker of painful diseas- es. Researches have shown that circulating miR- NAs can be served as potential markers, such as complex regional pain syndrome (CRSP) and fibromyalgia syndrome 6 . The first clinical study focused on miRNAs in migraine was reported in Molecular Neurobiology 7 . The report demon- strated that differentially expressed miRNA in serum was screened by high-throughput miRNA microchips, 32 out of 37 miRNAs were screened out and differentially expressed in migraine head- aches. Among them, the expression levels of miR- European Review for Medical and Pharmacological Sciences 2018; 22: 2022-2028 Y. ZHAI 1 , Y.-Y. ZHU 2 1 Health Management Division, The People’s Hospital of Weifang, Weifang, Shandong, China 2 Department of Spine Surgery, The People’s Hospital of Weifang, Weifang, Shandong, China Corresponding Author: Yanyan Zhu, BM; e-mail: [email protected] MiR-30a relieves migraine by degrading CALCA

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Page 1: MiR-30a relieves migraine by degrading CALCA€¦ · MiR-30a relieves migraine by degrading CALCA 2023 30a were significantly reduced in the serum of migraine patients. Therefore,

2022

Abstract. – OBJECTIVE: To investigate both the relationship and underlying mechanism be-tween miR-30a and migraine.

PATIENTS AND METHODS: Peripheral blood samples were collected from migraine patients and healthy people to extract RNA for quantita-tive Real-time PCR (qRT-PCR). The relationship between mRNA expressions and patient da-ta was analyzed by t-test. Target genes of miR-30a were predicted by the TargetScan. The bind-ing of miR-30a to the target gene was verified by dual fluorescein reporter assay. The pro-tein expression of calcitonin/alpha-CGRP gene (CALCA), the miR-30a target gene, was detected by Western blot.

RESULTS: Expression levels of miR-30a in pe-ripheral blood of migraine patients were signifi-cantly lower than those of healthy controls de-tected by qRT-PCR, and the methylation level of miR-30a in promoter region was remarkably in-creased. In addition, expression levels of miR-30a were significantly decreased in patients with bilateral seizures, persistent pain and high pain index. CALCA was found to be the target gene of miR-30a via bioinformatics analysis. We verified that miR-30a degrades CALCA by dual-lucifer-ase reporter assay. Western blot results showed that overexpression of miR-30a down-regulated the CALCA expression, and knockdown of miR-30a upregulated the CALCA expression.

CONCLUSIONS: Expression levels of miR-30a are significantly decreased in migraine patients and can relieve migraine through the degrada-tion of CALCA.

Key Words:MicroRNA, MiR-30a, CALCA, Hypermethylation.

Introduction

Migraine is one of the most common primary headaches. It affects about 15% of the population; the prevalence is about 6-8% in male and 15-25% in female1. According to the epidemiology sur-

vey of primary headache in 2012, the migraine prevalence in China was about 13%. The clinical manifestations of migraine are mainly recurrent moderate-severe pulsatile headache with nausea, vomiting, photophobia, phonophobia, and aggra-vating headache, which seriously affect the life quality and work ability of patients. Research on the burden of disease revealed that migraine costs about 331.7 billion RMB annually in our coun-try2. Migraine is listed as the sixth incompetent disease by WHO due to its heavy economic and social burdens3. Currently, migraine still lacks of available clinical markers, the diagnosis of which is highly dependent on clinical symptoms4 and brings great difficulties in clinical diagnosis.

MicroRNA is a kind of non-coding RNA, which is an essential molecule involved in epi-genetic regulation. It participates in physiologi-cal regulation of cell proliferation, differentiation and apoptosis in the body. Evidence indicated that miRNA is significant in the process of disease development5. It mainly forms RNA-induced si-lencing complex (RISC) by specifically binding to the complementary sequence of mRNA 3’-UTR, inhibiting mRNA translation or promoting its degradation, thus resulting in post-transcrip-tional silencing of gene. Highly expressed miR-NA leads to down-regulated target genes of the mRNA. Related research suggested that miRNA can also be served as a marker of painful diseas-es. Researches have shown that circulating miR-NAs can be served as potential markers, such as complex regional pain syndrome (CRSP) and fibromyalgia syndrome6. The first clinical study focused on miRNAs in migraine was reported in Molecular Neurobiology7. The report demon-strated that differentially expressed miRNA in serum was screened by high-throughput miRNA microchips, 32 out of 37 miRNAs were screened out and differentially expressed in migraine head-aches. Among them, the expression levels of miR-

European Review for Medical and Pharmacological Sciences 2018; 22: 2022-2028

Y. ZHAI1, Y.-Y. ZHU2

1Health Management Division, The People’s Hospital of Weifang, Weifang, Shandong, China 2Department of Spine Surgery, The People’s Hospital of Weifang, Weifang, Shandong, China

Corresponding Author: Yanyan Zhu, BM; e-mail: [email protected]

MiR-30a relieves migraine by degrading CALCA

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MiR-30a relieves migraine by degrading CALCA

2023

30a were significantly reduced in the serum of migraine patients. Therefore, we supposed wheth-er miR-30a can affect migraine8. In rodent ani-mal experiment, “inflammatory soup” can stimu-late the release of CALCA from trifacial nerves. When migraine patients are in an attack, CALCA level in jugular venous blood is significantly in-creased, and is reduced after headache onset9-11. Intravenous injection of CALCA induces delayed migraine-like attacks in migraine patients. It re-mains unclear whether increased CALCA level in blood is a complication of spontaneous migraine attacks or the direct cause of the migraine12. Un-doubtedly, CALCA is a key link in the pathogen-esis of migraine. Several clinical observational studies have reported that plasma concentration of CALCA is increased not only during the ep-isode but also during the non-episode13-15, sug-gesting persistent CALCA abnormality is exist-ed in migraine patients. Therefore, the study of the relationship between miRNA and CALCA in migraine may provide a better understanding of the mechanism of migraine, further providing an approach for the diagnosis and treatment of mi-graine.

Patients and Methods

PatientsMigraine patients were selected from the Inter-

national Headache Center of the People’s Hospi-tal of Weifang and matched controls were selected from hospital staff and non-immediate relatives. Inclusion criteria were: (1) Diagnosis of migraine with or without aura preceded based on the first edition of the IHS Classification of Headache Dis-order (P version); (2) Absence of systemic diseas-es such as blood pressure, diabetes, heart disease, nephropathy, liver disease, etc.; (3) Acute phase drugs can be used during the attack, but no long-term preventive drugs were applied; (4) Pregnant and lactating women were excluded; (5) Subjects younger than 14 years old, or older than 60 years of age were excluded. All participants signed a written informed consent form according to the requirement of the Hospital Ethics Committee of the People’s Hospital of Weifang.

Serum miRNA and mRNA Extraction, and qRT-PCR

3 mL of the whole blood injected from the el-bow vein in the fasting state of all subjects were extracted and placed in the pro-coagulant drying

tube at room temperature for 30-60 min. After clot formation, the blood sample was centrifuged at 3000 rpm for 10 min. Serum samples were then harvested and stored in -80°C for further usage. Serum miRNA and mRNA were extracted via TRIzol reagent. The extracted RNA was invert-ed into cDNA using the PrimeScript RT reagent Kit (TaKaRa, Otsu, Shiga, Japan). QRT-PCR was performed on a 7500 Real-time PCR System (Ap-plied Biosystems, Foster City, CA, USA) with mixed primers and RNA by SYBR Primix Ex Taq II kit (TaKaRa, Otsu, Shiga, Japan). Each experi-ment was repeated for three times.

Cell Culture and Transfection293T cells were purchased from the Ameri-

can Type Culture Collection (ATCC, Manassas, VA, USA), cultured in Dulbecco’s modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and placed in a 37°C, 5% CO2 incu-bator. The mimic, inhibitor and negative control (NC) of miR-30a, as well as wild type and mu-tation type of CALCA, were synthesized by Ge-nepharma (Shanghai, China) and transfected into cells by Lipofectamine 2000 (Invitrogen, Carls-bad, CA, USA). Cells were cultured in 24-well plates and placed in an incubator at 5% CO2 and 37°C. After 6 h of cultivation, freshly prepared medium was changed. 24 h later, cells were har-vested for the following cell experiments.

Dual Luciferase Reporter GeneThrough online target scan bioinformatics

analysis software, we found that CALCA may be the target gene of miR-30a. The wild type and mutation type sequences that may bind to the sequences were loaded into the PGL3 vector, re-spectively. The mimic, inhibitor, NC of miR-30a, as well as wild type and mutation of CALCA, were transfected into cells by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 24 h, cell fluorescence was measured using the Dual Luciferase Reporter Assay Kit (Promega, Madi-son, WI, USA).

Western BlotCells were lysed for protein extraction. The

concentration of the cellular protein was mea-sured by Bio-Rad Protein assay (Bio-Rad Labora-tories, Hercules, CA, USA). An equal amount of protein from each sample was added for separa-tion in 10% sodium dodecyl sulphate-polyacryl-amide gel electrophoresis (SDS-PAGE) and trans-fected onto polyvinylidene difluoride (PVDF)

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membranes (Millipore, Billerica, MA, USA). Primary antibodies (CALCA and GAPDH) were incubated overnight at 4°C. After incubation, the membrane was incubated with the secondary an-tibodies for 1 h at room temperature. The mem-brane was washed six times with tris-buffered saline and tween (TBST) and protein bands were analyzed by Find-do × 6 Tanon (Tanon, Shanghai, China).

Statistical Analysis The relative quantification of miRNA was an-

alyzed by 2-∆CT method. ∆CTtarget miRNA = CTtarget

miRNA - CTreference miRNA. ∆CTtarget miRNA = CTtarget mRNA - CTreference mRNA. Differences between two groups were compared using t-test, correlation analysis was performed using Pearson test. p<0.5 was con-sidered statistically significant. We used statistical product and service solutions (SPSS) 20.0 (IBM, Armonk, NY, USA) software for data analysis.

Results

MiR-30a Levels in Migraine Patients Were Significantly Decreased, and the Methylation Level in Promoter Region Was Significantly Increased

The first clinical study focused on miRNAs in migraine was reported in Molecular Neurobiology Magazine7. The report demonstrated that differ-entially expressed miRNA in serum was screened by high-throughput miRNA microchips16. Results indicated that the serum levels of miR-30a were lower in migraine patients (Figure 1A). At the

same time, we also found that the methylation level of miR-30a promoter was significantly in-creased (Figure 1B).

Relationship Between the Expression Levels of miR-30a and CALCA and the Patient’s Condition

To investigate the relationship between the ex-pression level of miR-30a and CALCA and the pa-tient’s condition, we calculated the expression lev-el of mir-30a. The relationship between CALCA expression level and the patient’s pain character-istics, pain duration, and pain score were, also ex-plored. The results demonstrated that the miR-30a level in bilateral migraine headache was signifi-cantly increased (Figure 2A). Similarly, the miR-30a levels were significantly increased in patients with persistent pain and pain index over 7 (Figure 2B-C). Besides, the expression levels of CALCA were significantly down-regulated in bilateral mi-graine headache, persistent pain and pain index over 7 (Figure 2D-F). These results illustrated that miR-30a and CALCA may be involved in the progression of migraine.

MiR-30a Relieved Migraine by Degrading CALCA

Studies have pointed out that serum levels of CALCA in migraine patients decreased sig-nificantly. In the present work, serum levels of CALCA in migraine patients were decreased and inversely proportional to the expression of miR-30a (Figure 3A). In 293T cells, the expression of CALCA was significantly decreased after miR-30a overexpression (Figure 3B), and the expres-

Figure 1. MiR-30a is downregulated and CALCA is upregulated. A, MiR-30a was downregulated in migraine patients. B, CALCA was downregulated in migraine patients.

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sion of CALCA was significantly increased after interfering with miR-30a (Figure 3C). To explore the mechanism of miR-30a in migraine, we pre-dicted the target gene of miR-30a via the Target-Scan and found that CALCA binds to miR-30a (Figure 3D). We constructed the wild type and mutation type sequences of the binding region and examined whether they bind to miR-30a via dual luciferase reporter assay. The results considered that wild type of CALCA and miR-30a mimic had the lowest luciferase activity, indicating that miR-30a can bind to 3’-UTR of CALCA to degrade CALCA (Figure 3E). Western blot results point-ed out that the protein expression of CALCA was significantly decreased after overexpression of miR-30a (Figure 3F). On the contrary, the protein level of CALCA was upregulated after inhibition of miR-30a (Figure 3G). The above data demon-strated that miR-30a may alleviate migraine by downgrading CALCA.

Discussion

Migraine can occur at any age, the first onset begins mostly in adolescence. The prevalence of females after adolescence is much higher than that of males. The peak onset of migraine is around 40 years old is. About 50% of female migraine patients could be related to the menstrual cycle. Migraine without aura is the most common type, accounting for about 80%, migraine with aura accounts for 10%. Migraine triggers are diverse, including a family history of food (e.g., cheese, wine), medications (e.g., vasodilators), stress, weather changes, reduced eating, nervousness, anxiety, etc.17-19. About 50% of migraine patients have a family history, pathogenic genes are found only in some very rare familial migraines, where-as no exact genetic susceptibility loci have been found in the common types of migraines20,21. As a result, migraine is also thought to be the result of

Figure 2. The correlation between miR-30a and CALCA, and clinical data. (A) Expression levels of miR-30a in patients with bilateral migraine headache, persistent pain (B) and pain index over 7 (C) were significantly increased. (D) Expression levels of CALCA in patients with bilateral migraine headache, persistent pain (E) and pain index over 7 (F) were significantly decreased.

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put miRNA microchips for migraine26. Serum levels of miR-30a were significantly increased in migraine patients, and the expression levels of CALCA were significantly lower in migraine patients. Increased levels of miR-30a were found in patients with bilateral, persistent pain and pain index over 7. Opposite results were observed in the expression level of CALCA. These results demonstrated that miR-30a may be involved in the progression of migraine. We verified that CALCA expression was significantly elevated in the serum of migraine patients. At the same time, the expression of CALCA was inversely propor-

interactions between multiple genes and genetic factors22.

There are about 1500 human miRNAs discov-ered so far23. MiRNAs in the blood circulation bind to protein-lipoprotein carriers, which are tolerant to temperature and freeze-thaw repeatedly, and are not easily to be degraded24,25. Potential hor-monal effects may be involved in the regulation of distant tissues and cells25. Many miRNAs in plas-ma/blood are associated with disease status and prognosis, which could be served as biomarkers of disease25. This study screened differentially ex-pressed miRNAs based on the first high-through-

Figure 3. The mechanism of miR-30a in migraine. (A) CALCA was conversely related with miR-30a. (B) CALCA was downre-gulated in miR-30a mimic group (C). CALCA was upregulated in miR-30a inhibitor group. (D)The binding sites of miR-30a and CALCA. (E) The luciferase activity was downregulated in the CUCLA WT and miR-30a group. (F) Protein level of CALCA was downregulated in miR-30a mimic group. (G). Protein level of CALCA was upregulated in miR-30a inhibitor group.

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tional to the expression of miR-30a. In addition, the expression of CALCA was significantly de-creased after overexpressing miR-30a, and sig-nificantly increased after interfering with miR-30a. We predicted the target gene of miR-30a by the TargetScan and found that CALCA can bind to mir-30a. The luciferase activities of wild type of CALCA and miR-30a mimic were found to be the lowest by dual luciferase reporter assay. This suggested that miR-30a can bind CALCA’s 3’-UTR to degrade CALCA. CALCA protein ex-pression was found to be decreased significantly after overexpression of miR-30a. We first specu-lated that miR-30a could suppress the progression of migraine through the degradation of CALCA.

Conclusions

We showed that the expressions of miR-30a in migraine patients are significantly lower, which alleviate migraine by degradation of CALCA.

Conflict of InterestThe Authors declare that they have no conflict of interest.

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