Correlation between FRA-2 mRNA expression, DNA methylation level and metabolic syndrome

doi:10.3969/j.issn.1674-8115.2022.03.013

Abstract

Abstract: Objective

·To investigate the relationship between Fos-related antigen-2 (FRA-2) mRNA, DNA methylation levels and metabolic syndrome (MS).

Methods

·A case-control study was used to include 160 patients with MS risk factors (obesity, hyperlipidemia, hypertension and hyperglycemia) who were admitted to the Department of Endocrinology and Metabolism of the First Affiliated Hospital of Shihezi University School of Medicine from June 2020 to June 2021. They were divided into MS group (number of MS risk factors ≥3, n=80) and non-MS group (number of MS risk factors 1?2, n=80). In addition, healthy subjects at the same period were included in the control group (0 risk factor, n=80). The age, body mass index (BMI), waist circumference (WC), diastolic blood pressure (DBP), and systolic blood pressure (SBP) were collected and recorded; Fasting plasma glucose (FPG), triacylglycerol (TAG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), fasting serum insulin (FINS) and homeostasis model assessment-insulin resistance (HOMA-IR) were determined. FRA-2 mRNA expression level was detected by reverse transcription-polymerase chain reaction (RT-PCR), and FRA-2 DNA methylation level was determined by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). Kruskal-wallis H test was used for gender comparison, ANOVA was used for age comparison, and covariance analysis was used for comparison of clinical biochemical indicators, FRA-2 mRNA expression level and FRA-2 DNA methylation level. Spearman method was used to analyze the correlation between the methylation level of FRA-2 DNA CpG unit and MS-related clinical biochemical indexes, and between the expression level of FRA-2 mRNA and the methylation level of FRA-2 DNA. The relationship between FRA-2 DNA methylation level and the number of MS risk factors was analyzed by linear regression analysis.

Results

·Compared with the control group and the non-MS group, the age, BMI, WC, DBP, SBP, FPG, TAG, TC, LDL-C and HOMA-IR in the MS group were increased (all P<0.05), while HDL-C was decreased (P=0.000); the expression of FRA-2 mRNA in the MS group was decreased (F=49.155, P=0.000). Compared with the control group, the methylation levels of four FRA-2 DNA CpG units in the non-MS group and the MS group were increased (all P<0.05). Compared with the non-MS group, the methylation levels of five FRA-2 DNA CpG units in the MS group were increased (all P<0.05). The results of linear regression analysis showed that the number of MS risk factors was positively correlated with the level of FRA-2 DNA methylation (β=0.012, P=0.000). Spearman method analysis results showed that among the 10 detected FRA-2 DNA CpG units, CpG 3, CpG 8, CpG 9.10, CpG 12.13.14 and CpG 19 were correlated with WC, SBP, DBP, FPG, TAG and HDL-C (all P<0.05). The mRNA expression level of FRA-2 was negatively correlated with the DNA methylation level of FRA-2 (r=-0.607, P=0.000).

Conclusion

·There is a certain correlation between the methylation level of FRA-2 gene and the occurrence of MS. Increasing the methylation level of FRA-2 DNA and thus down-regulating the mRNA expression may play a role in the occurrence of MS.

Key words: metabolic syndrome (MS), Fos-related antigen-2 (FRA-2), methylation, correlation

CLC Number:

R589.1

DU Qingqing, HOU Zexin, LI Jun, HU Ying, CAO Guolei, LI Siyuan. Correlation between FRA-2 mRNA expression, DNA methylation level and metabolic syndrome[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(3): 350-356.

Figures/Tables 8

TrendMD

References 19

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Target gene	Primer sequence（5′→3′）
RT-PCR
FRA-2	up-stream：CCAGATGAAATGTCATGGC
	down-stream：CTCGGTTTGGTAGACTTGGA
β-actin	up-stream：CCCAGCACAATGAAGATCAAGATCAT
	down-stream：ATCTGCTGGAAGGTGGACAGCG
MALDI-TOF-MS
FRA-2	up-stream：AGGAAGAGAGGTAGGTTTAGGAGAGGGGTGTG
	down-stream：CAGTAATACGACTCACTATAGGGAGAAGGCTACAACCCCCAAAACTTAACTAAAAC

Target gene	Primer sequence（5′→3′）
RT-PCR
FRA-2	up-stream：CCAGATGAAATGTCATGGC
	down-stream：CTCGGTTTGGTAGACTTGGA
β-actin	up-stream：CCCAGCACAATGAAGATCAAGATCAT
	down-stream：ATCTGCTGGAAGGTGGACAGCG
MALDI-TOF-MS
FRA-2	up-stream：AGGAAGAGAGGTAGGTTTAGGAGAGGGGTGTG
	down-stream：CAGTAATACGACTCACTATAGGGAGAAGGCTACAACCCCCAAAACTTAACTAAAAC

Clinical data	Control group （n=80）	Non-MS group （n=80）	MS group （n=80）	P value
Gender（male/female）/n	41/39	40/40	41/39	0.984
Age/year	49.81±9.13	50.08±15.10	55.90±7.96	0.001
BMI/（kg·m^-2）	22.36±1.41	24.94±2.83	29.15±3.19	0.000
WC/cm	80.91±4.34	92.91±10.28	103.82±9.14	0.000
DBP/mmHg	72.28±4.01	80.71±15.00	93.45±14.68	0.000
SBP/mmHg	118.90±7.10	130.34±20.22	144.08±18.02	0.000
FPG/（mmol·L^-1）	5.29±0.48	5.88±1.92	10.40±3.74	0.000
TAG/（mmol·L^-1）	1.15±0.17	1.54±1.04	2.18±0.77	0.000
TC/（mmol·L^-1）	3.92±0.51	3.90±0.76	5.12±1.04	0.000
LDL-C/（mmol·L^-1）	1.68±0.64	2.05±0.61	2.52±0.88	0.000
HDL-C/（mmol·L^-1）	1.35±0.31	1.18±0.31	1.00±0.19	0.000
FINS/（mU·L^-1）	1.35 （1.13， 2.30）	1.41 （0.98， 2.36）	1.91 （1.14， 2.84）	0.016
HOMA-IR	2.38 （1.63， 3.91）	2.55 （1.54， 4.45）	5.83 （3.05， 10.28）	0.000

Clinical data	Control group （n=80）	Non-MS group （n=80）	MS group （n=80）	P value
Gender（male/female）/n	41/39	40/40	41/39	0.984
Age/year	49.81±9.13	50.08±15.10	55.90±7.96	0.001
BMI/（kg·m^-2）	22.36±1.41	24.94±2.83	29.15±3.19	0.000
WC/cm	80.91±4.34	92.91±10.28	103.82±9.14	0.000
DBP/mmHg	72.28±4.01	80.71±15.00	93.45±14.68	0.000
SBP/mmHg	118.90±7.10	130.34±20.22	144.08±18.02	0.000
FPG/（mmol·L^-1）	5.29±0.48	5.88±1.92	10.40±3.74	0.000
TAG/（mmol·L^-1）	1.15±0.17	1.54±1.04	2.18±0.77	0.000
TC/（mmol·L^-1）	3.92±0.51	3.90±0.76	5.12±1.04	0.000
LDL-C/（mmol·L^-1）	1.68±0.64	2.05±0.61	2.52±0.88	0.000
HDL-C/（mmol·L^-1）	1.35±0.31	1.18±0.31	1.00±0.19	0.000
FINS/（mU·L^-1）	1.35 （1.13， 2.30）	1.41 （0.98， 2.36）	1.91 （1.14， 2.84）	0.016
HOMA-IR	2.38 （1.63， 3.91）	2.55 （1.54， 4.45）	5.83 （3.05， 10.28）	0.000

CpG unit	Control group	non-MS group	MS group	P value
CpG 1	0.02±0.04	0.04±0.07	0.04±0.04	0.027
CpG 3	0.01±0.02	0.02±0.03	0.03±0.03	0.000
CpG 4.5	0.02±0.03	0.02±0.03	0.10±0.07	0.000
CpG 6.7	0.02±0.02	0.02±0.02	0.04±0.04	0.000
CpG 8	0.00±0.01	0.01±0.01	0.03±0.03	0.000
CpG 9.10	0.00±0.01	0.01±0.01	0.03±0.03	0.000
CpG 11	0.02±0.01	0.02±0.02	0.03±0.03	0.026
CpG 12.13.14	0.00±0.01	0.03±0.05	0.10±0.04	0.000
CpG 15.16.17	0.04±0.03	0.05±0.05	0.07±0.04	0.000
CpG 19	0.01±0.02	0.04±0.05	0.10±0.06	0.000