MiR-30a relieves migraine by degrading CALCA

Transcription

1 European Review for Medical and Pharmacological Sciences 2018; 22: MiR-30a relieves migraine by degrading CALCA 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 Abstract. OBJECTIVE: To investigate both the relationship and underlying mechanism between mir-30a and migraine. PATIENTS AND METHODS: Peripheral blood samples were collected from migraine patients and healthy people to extract RNA for quantitative Real-time PCR (qrt-pcr). The relationship between mrna expressions and patient data was analyzed by t-test. Target genes of mir- 30a were predicted by the TargetScan. The binding of mir-30a to the target gene was verified by dual fluorescein reporter assay. The protein expression of calcitonin/alpha-cgrp gene (CALCA), the mir-30a target gene, was detected by Western blot. RESULTS: Expression levels of mir-30a in peripheral blood of migraine patients were significantly lower than those of healthy controls detected by qrt-pcr, and the methylation level of mir-30a in promoter region was remarkably increased. 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-luciferase 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 degradation 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 survey 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 aggravating headache, which seriously affect the life quality and work ability of patients. Research on the burden of disease revealed that migraine costs about billion RMB annually in our country 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 epigenetic regulation. It participates in physiological 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 silencing complex (RISC) by specifically binding to the complementary sequence of mrna 3 - UTR, inhibiting mrna translation or promoting its degradation, thus resulting in post-transcriptional 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 diseases. 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 demonstrated 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 headaches. Among them, the expression levels of mir Corresponding Author: Yanyan Zhu, BM; @163.com

2 MiR-30a relieves migraine by degrading CALCA 30a were significantly reduced in the serum of migraine patients. Therefore, we supposed whether mir-30a can affect migraine 8. In rodent animal experiment, inflammatory soup can stimulate the release of CALCA from trifacial nerves. When migraine patients are in an attack, CALCA level in jugular venous blood is significantly increased, and is reduced after headache onset Intravenous injection of CALCA induces delayed migraine-like attacks in migraine patients. It remains unclear whether increased CALCA level in blood is a complication of spontaneous migraine attacks or the direct cause of the migraine 12. Undoubtedly, CALCA is a key link in the pathogenesis of migraine. Several clinical observational studies have reported that plasma concentration of CALCA is increased not only during the episode but also during the non-episode 13-15, suggesting persistent CALCA abnormality is existed 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 migraine. Patients and Methods Patients Migraine patients were selected from the International Headache Center of the People s Hospital 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 Disorder (P version); (2) Absence of systemic diseases such as blood pressure, diabetes, heart disease, nephropathy, liver disease, etc.; (3) Acute phase drugs can be used during the attack, but no longterm 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 elbow vein in the fasting state of all subjects were extracted and placed in the pro-coagulant drying tube at room temperature for 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 inverted into cdna using the PrimeScript RT reagent Kit (TaKaRa, Otsu, Shiga, Japan). QRT-PCR was performed on a 7500 Real-time PCR System (Applied Biosystems, Foster City, CA, USA) with mixed primers and RNA by SYBR Primix Ex Taq II kit (TaKaRa, Otsu, Shiga, Japan). Each experiment was repeated for three times. Cell Culture and Transfection 293T cells were purchased from the American 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% CO 2 incubator. The mimic, inhibitor and negative control (NC) of mir-30a, as well as wild type and mutation type of CALCA, were synthesized by Genepharma (Shanghai, China) and transfected into cells by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Cells were cultured in 24-well plates and placed in an incubator at 5% CO 2 and 37 C. After 6 h of cultivation, freshly prepared medium was changed. 24 h later, cells were harvested for the following cell experiments. Dual Luciferase Reporter Gene Through 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, respectively. 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, Madison, WI, USA). Western Blot Cells were lysed for protein extraction. The concentration of the cellular protein was measured by Bio-Rad Protein assay (Bio-Rad Laboratories, Hercules, CA, USA). An equal amount of protein from each sample was added for separation in 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transfected onto polyvinylidene difluoride (PVDF) 2023

3 Y. Zhai, Y.-Y. Zhu 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 antibodies for 1 h at room temperature. The membrane 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 analyzed by 2 - CT method. CT target mirna = CT - CT. CT = CT target mirna reference mirna target mirna target mrna - CT reference mrna. Differences between two groups were compared using t-test, correlation analysis was performed using Pearson test. p<0.5 was considered 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 Magazine 7. The report demonstrated that differentially expressed mirna in serum was screened by high-throughput mirna microchips 16. 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 increased (Figure 1B). Relationship Between the Expression Levels of mir-30a and CALCA and the Patient s Condition To investigate the relationship between the expression level of mir-30a and CALCA and the patient s condition, we calculated the expression level of mir-30a. The relationship between CALCA expression level and the patient s pain characteristics, pain duration, and pain score were, also explored. The results demonstrated that the mir-30a level in bilateral migraine headache was significantly 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 migraine 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 significantly. 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. 2024

4 MiR-30a relieves migraine by degrading CALCA 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. sion of CALCA was significantly increased after interfering with mir-30a (Figure 3C). To explore the mechanism of mir-30a in migraine, we predicted 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 pointed 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 demonstrated 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 About 50% of migraine patients have a family history, pathogenic genes are found only in some very rare familial migraines, whereas no exact genetic susceptibility loci have been found in the common types of migraines 20,21. As a result, migraine is also thought to be the result of 2025

5 Y. Zhai, Y.-Y. Zhu put mirna microchips for migraine 26. 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 proporinteractions between multiple genes and genetic factors 22. There are about 1500 human mirnas discovered so far 23. 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 degraded 24,25. Potential hormonal effects may be involved in the regulation of distant tissues and cells 25. Many mirnas in plasma/blood are associated with disease status and prognosis, which could be served as biomarkers of disease 25. This study screened differentially expressed 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 downregulated 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. 2026

6 MiR-30a relieves migraine by degrading CALCA tional to the expression of mir-30a. In addition, the expression of CALCA was significantly decreased after overexpressing mir-30a, and significantly 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 expression was found to be decreased significantly after overexpression of mir-30a. We first speculated 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 Interest The Authors declare that they have no conflict of interest. References 1) Kaiser EA, Russo AF. CGRP and migraine: could PACAP play a role too? Neuropeptides 2013; 47: ) Yu S, Liu R, Zhao G, Yang X, Qiao X, Feng J, Fang Y, Cao X, He M, Steiner T. The prevalence and burden of primary headaches in China: a population-based door-to-door survey. Headache 2012; 52: ) Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, : a systematic analysis for the global burden of disease study Lancet 2017; 390: ) Durham P, Papapetropoulos S. Biomarkers associated with migraine and their potential role in migraine management. Headache 2013; 53: ) Liu ZH, Sun XP, Zhou SL, Wang HX. Research on the relations between the variation of mirna-184 before and after treatment of acute myocardial infarction and prognosis. Eur Rev Med Pharmacol Sci 2017; 21: ) Murrof FI. Curettage--an enigma in periodontal therapy. J Dent Que 1990; 27: ) Andersen HH, Duroux M, Gazerani P. Serum MicroRNA and in Pain-Free periods. Mol Neurobiol 2016; 53: 8) Andersen HH, Duroux M, Gazerani P. Serum MicroRNA and in pain-free periods. Mol Neurobiol 2016; 53: 9) Fan PC, Kuo PH, Chang SH, Lee WT, Wu RM, Chiou LC. Plasma calcitonin gene-related peptide in diagnosing and predicting paediatric migraine. Cephalalgia 2009; 29: ) Goadsby PJ, Edvinsson L, Ekman R. Vasoactive peptide release in the extracerebral circulation of humans during migraine headache. Ann Neurol 1990; 28: ) Cady RK, Vause CV, Ho TW, Bigal ME, Durham PL. Elevated saliva calcitonin gene-related peptide levels during acute migraine predict therapeutic response to rizatriptan. Headache 2009; 49: ) Messlinger K. Migraine: where and how does the pain originate? Exp Brain Res 2009; 196: ) Ashina M, Bendtsen L, Jensen R, Schifter S, Olesen J. Evidence for increased plasma levels of calcitonin gene-related peptide in migraine outside of attacks. Pain 2000; 86: ) Fusayasu E, Kowa H, Takeshima T, Nakaso K, Nakashima K. Increased plasma substance P and CGRP levels, and high ACE activity in migraineurs during headache-free periods. Pain 2007; 128: ) Cernuda-Morollon E, Larrosa D, Ramon C, Vega J, Martinez-Camblor P, Pascual J. Interictal increase of CGRP levels in peripheral blood as a biomarker for chronic migraine. Neurology 2013; 81: ) Andersen HH, Duroux M, Gazerani P. Serum MicroRNA and in pain-free periods. Mol Neurobiol 2016; 53: 17) Goadsby PJ. Stress and migraine: something expected, something unexpected. Neurology 2014; 82: ) Wang J, Huang Q, Li N, Tan G, Chen L, Zhou J. Triggers of migraine and tension-type headache in China: a clinic-based survey. Eur J Neurol 2013; 20: ) Fraga MD, Pinho RS, Andreoni S, Vitalle MS, Fisberg M, Peres MF, Vilanova LC, Masruha MR. Trigger factors mainly from the environmental type are reported by adolescents with migraine. Arq Neuropsiquiatr 2013; 71: ) de Vries B, Frants RR, Ferrari MD, van den Maagdenberg AM. Molecular genetics of migraine. Hum Genet 2009; 126: ) Wan D, Wang C, Zhang X, Tang W, Chen M, Dong Z, Yu S. Association between angiotensin-converting enzyme insertion/deletion polymorphism and migraine: a meta-analysis. Int J Neurosci 2016; 126: ) Goadsby PJ, Holland PR, Martins-Oliveira M, Hoffmann J, Schankin C, Akerman S. Pathophysiology of migraine: a disorder of sensory processing. Physiol Rev 2017; 97:

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