RNA-Sequencing Analysis Reveals L-theanine Regulating

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Bioactive Constituents, Metabolites, and Functions

RNA-Sequencing Analysis Reveals L-theanine Regulating Transcriptional Rhythm Alteration in Vascular Smooth Muscle Cells Induced by Dexamethasone zhongwen Xie J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05057 • Publication Date (Web): 27 Jan 2019 Downloaded from http://pubs.acs.org on January 27, 2019

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Journal of Agricultural and Food Chemistry

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TITLE: RNA-Sequencing Analysis Reveals L-theanine Regulating Transcriptional

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Rhythm Alteration in Vascular Smooth Muscle Cells Induced by Dexamethasone

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AUTHORSHIP:

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Ruru Wang † §, Menzhao Xiao † §, Yujing Zhang † §, Chi-Tang Ho§ ‡ , Xiaochun Wan † §

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Daxiang Li*,†,§,and Zhongwen Xie *,†,§

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†State Key Laboratory of Tea Plant Biology and Utilization, School of Tea and Food

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Sciences and Technology and §International Joint Laboratory on Tea Chemistry and

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Health Effects of Ministry of Education, Anhui Agricultural University, Hefei, Anhui

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230036, PR China

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‡Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick,

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New Jersey 08901-8520, United States

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*

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+86-551-65786153

Correspondences:

[email protected]

or

[email protected];

ACS Paragon Plus Environment

Tel.:


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ABSTRACT: L-theanine, a unique amino acid in tea leaves, is known to have

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beneficial effects on stress relief, tumor suppression, prevention of hypertension

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and cardiovascular diseases (CADs). The disruption of the circadian rhythm has been

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implied in the pathogenesis of CADs. However, it is unknown whether the

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L-theanine has the modulatory effect on the vascular circadian rhythm. In this

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research, we have established a circadian gene expression model in rat vascular

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smooth muscle cells (VSMCs) by dexamethasone induction. L-Theanine treatment

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enhanced the expression amplitude of clock genes including Bmal1, Cry1, Rev-erbα

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and Per2. Moreover, pairwise comparisons of the RNA-seq data showed that

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L-theanine is able to up-regulate a ray of the rhythm genes, and differentially

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expressed genes (DEGs) that are involved in vasoconstriction and actin cytoskeleton

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regulation pathways. Our data suggest that L-theanine changes the circadian gene

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rhythm involving in the process of vascular smooth muscle restructure.

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KEYWORDS: L-theanine, Biological rhythms, RNA-seq, Circadian genes, VSMCs

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INTRODUCTION

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L-Theanine is a unique non-protein amino acid naturally found in tea plants. It

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supplies the umami taste of green tea infusion.1,

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reported that L-theanine has beneficial effects on attenuating the brain and liver

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damage, improving the immune ability of immune cells, reducing the apoptosis rate

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of nerve cells, inhibiting the tumor formation and cancer cell proliferation, and

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reducing blood pressure.3,4 However, little is known whether L-theanine regulates

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biological clock of smooth muscle cells.

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Biological clock, which is also known as circadian rhythm, is the cyclical fluctuation

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of biological behavior and physiological phenomena around 24 hours in organism. In

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mammals, the central clock is located in the suprachiasmatic nucleus (SCN) of the

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brain hypothalamus, and peripheral clocks are located in most mammalian

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peripheral cells. The central clock is entrained by light/dark cycles, whereas

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peripheral clocks are entrained by feeding cycles. Peripheral organs have their

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independent autonomous circadian clocks but are synchronized by the SCN via

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neural and endocrine signaling pathways. The Clock and Bmal1 are core circadian

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genes, and also are transcriptional factors, which hetero-dimerize together to

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activate expression of Period (Per1/2/3) and Cryptochrome (Cry1/2) via the E/E’box

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sequence. PER/CRY protein complexes then inhibit the transcription-activating role

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of ClOCK/BMAL1, decreasing Per and Cry expression, thereby forming the negative

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feedback loop.5 Additional circadian clock components such as Rev-erbα and Rorα

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Previous researches have


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also contribute to maintaining proper circadian rhythms and are thought to have a

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role in establishing the night-time transcriptome.6

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Many cardiovascular physiological functions have obvious circadian rhythm, and

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many cardiovascular diseases, such as myocardial ischemia, myocardial infarction,

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also show rhythmically and these diseases usually occur with high frequency in the

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morning.7 This suggests that cardiovascular physiology and pathology are closely

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related to circadian clock.8, 9 Indeed, clock rhythm is found in mainly all tissues and

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various types of cells including endothelial cells, smooth muscle cells and vascular

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stem cells in the vascular system. This vascular clock rhythm physiologically

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contributes to regulate the daily vascular function, and plays a pivotal role in the

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pathophysiology of vascular diseases.8 Moreover, vascular circadian rhythm inserts

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regulatory effects on vessel contraction, haemodynamics, inflammatory response

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and endothelium-derived NO synthesis and release, resulting in plaque formation

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and instability which participated in CADs. 10,11

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Recent findings showed that nutrients reset peripheral circadian oscillation and the

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local clock genes govern downstream metabolic pathways.12, 13 A number of studies

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also showed that consumption of beneficial components at right times would have

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similar health effects as medication administered at specific times in

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chronopharmacology. 14,15 Nutrients can modulate circadian rhythmicity, and some

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studies reported that natural components administration changes biological rhythm,

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and increasing the amplitude of rhythm leads to resist obesity, strengthen immunity

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and make the body better adapt to the environment.16,17 These natural components 3


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are able to alter signaling pathways that impact the molecular oscillator in

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peripheral cells. The molecular clock inside the cells of the cardiovascular system

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allows it to respond appropriately to the stimulation of the external nutrition.18 The

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attenuation of this molecular clock's oscillation can affect the timely and effective

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reaction of the cardiovascular to the external environment, thus affecting the

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occurrence and development of some CADs.19 Previous studies majorly focused on

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the effects of drugs and bioactive substances on the binding of Clock/Bmal1

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heterodimer to E-box of target genes promoter region and insert the effects of

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nutrients on the biological clock. 20,21

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Changes in lifestyle, dietary habits and increased life expectancy have led to

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increase

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hypercholesterolemia, obesity, diabetes and hypertension over recent decades.

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According to the World Health Organization (WHO), CADs are the leading cause of

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death worldwide. Growing evidence links circadian alterations to metabolic

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disorders. And clock mutation reduces circadian pacemaker amplitude in mice.

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And alteration of cardiovascular circadian genes results in CADs. Disruption of

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smooth muscle Bmal1 leads to alteration in rhythmic blood pressure and protection

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of abdominal aortic aneurysm,23,24 endothelium specific disruption of the circadian

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clock exacerbates the thrombogenic response and changes blood pressure, and

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vascular transplantation of Bmal1-KO mice to WT mice leads to atherosclerosis.25

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These results indicate that modulation of circadian rhythms in vascular system

widespread

presence

of

metabolic

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syndrome,

such

as

22


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might be a new strategy to prevention of CADs. In addition, L-theanine treatment

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decreases blood pressure in human study.4

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Furthermore, VSMCs are the major components in a vessel wall, and play an

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important role in cardiovascular pathophysiology. However, little is known about

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whether L-theanine modulates circadian rhythm in vasculature and if so, how the

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L-theanine alters circadian genes expression.

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In the present study, a circadian gene expression model using rat VSMCs by

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dexamethasone induction was established. We then investigated how L-theanine

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addition changed the rhythmic expression of major clock genes. A RNA-seq analysis

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was employed on VSMCs to dissect the transcriptomic profiles in response to the

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L-theanine treatments at different Zeitgeber times. Gene ontology enrichment and

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KEGG pathway analysis of RNA-seq data were employed in deciphering the network

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of metabolic and signaling pathways involved in the regulation of vascular smooth

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function to L-theanine induction, with specifically focused on vascular clock gene

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expression profiles. To the best of our knowledge, this is the first RNA-seq-based

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study to assess the effect of L-theanine on the vascular circadian rhythms and

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circadian gene expression profiles in primary cultured VSMCs. Our next-generation

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sequencing data contributed to the molecular effects exerted by L-theanine on

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vascular circadian rhythms and circadian gene expression profiles .

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■ MATERIALS AND METHODS

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Cell Culture and Samples Collection

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VSMCs were isolated from the rat thoracic aorta by enzymatic dissociation, as

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described previously.26,27 Briefly, the VSMCs were cultured in Dulbecco’s Modified

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Eagle’s Medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplied

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with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA),

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100 U/mL penicillin and 100 μg/mL streptomycin (Thermo Fisher Scientific, Waltham,

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MA, USA) at 37 °C with 5% CO2. When reaching 70-80% confluence, the cells were

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trypsinized with Trypsin-EDTA Solution (Thermo Fisher Scientific, Waltham, MA, USA)

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for 2 min, then washed in PBS and resuspended in DMEM growth medium. Finally,

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the cells were passages with one to three ratio for subculture. The cells between the

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fifth and tenth passages were used in all experiments. The cells were plated in 35

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mm dishes for growth. The day before the experiment, cells at approximately 90%

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of confluence were incubated with serum free starvation DMEM for 24 hours.

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Subsequently, the cells were treated with medium containing dexamethasone (100

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nM, Sigma, St. Louis, MO, USA) for 15 min and then changed back to a serum free

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DMEM with or without L-theanine (40 µM, Sigma, St. Louis, MO, USA) for the

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various time points when the cells samples were collected. The timing of the

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beginning dexamethasone induction was defined as Zeitgeber time 0 (ZT0), and cells

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were harvested for RNA extraction with 4 hour interval for two days at ZT0, ZT4, ZT8,

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ZT12, ZT16, ZT20, ZT24, ZT28, ZT32, ZT36, ZT40, ZT44 and ZT48. And the cells were

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immediately fixed with TRIzol™ (Thermo Fisher Scientific, Waltham, MA, USA). 6



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RNA Isolation, cDNA Synthesis, and Real Time PCR

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Total RNA was extracted using RNA isolator according to the protocols of

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manufacturers (Vazyme Biotech, Nanjing, China). Reverse transcription was

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conducted using first strand cDNA synthesis kit (Vazyme Biotech), and the Real-time

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PCR was performed using qPCR SYBR Green Master Mix kit (Vazyme Biotech)

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following the method described previously.27 Primer sequences were designed for

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rat and listed in Supplementary Table S1.

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RNA Sequencing

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Two groups, control (C) and L-theanine treatment (T) were included for RNA-seq

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analysis, Four time points (ZT0, ZT12, ZT24, ZT36,) were chosen for each of group

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and three biological replicates (RI, RII, and RIII) were performed. We used 2 µg of

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total RNA from each of samples to prepare the TruSeq library. The NEB Next TruSeq

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RNA Sample Preparation Kit was employed to prepare barcoded cDNA (E7530S;

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E7490S; E7335S; E7500S, New England Biolabs, Ipswich, MA, USA).28 The fragments

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per Kilobase of transcript per Million fragments mapped (FPKM) was calculated by

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dividing the read count of each transcriptional model with its length and scaling the

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total per sample to one million, and was used to indicate the expression levels of

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each sample. A gene expression value in FPKM equal or greater than 1 was

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considered to be expressed in the sample.

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Transcriptome Data Analysis

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The aligning of the raw sequences data was analyzed by proper quality control.

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FastQC and Trimmomatic v. 0.33. were used for Quality control (QC) and trimming. 7


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TopHat/Bowtie2 was employed to map quality checked reads after QC . DESeq R

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package (1.10.1) was applied to analyze differential expression of two groups.

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DESeq provides statistical tools for evaluating differential expression in digital gene

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expression data using a model based on the negative binomial distribution. The

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Benjamini and Hochberg’s approach was used to adjust the resulting P values for

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controlling the false discovery rate. Genes with a DESeq adjusted P-value < 0.05

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were assigned as differentially expressed.29,30

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Real Time -PCR

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To validate the reliability of RNA-seq results, core genes (Bmal1, Npas2) and clock

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target genes (Cry1, Per2, RORα, Dbp) and related to vascular contraction genes

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(Rock2, CPI-17) were selected for real time-PCR validation.

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Western Blot Analysis

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Western blot was performed following the method described previously.27 In brief,

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the frozen cells were homogenized using a 2×SDS homogenization buffer. Equal

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amounts of denatured proteins were loaded and separated by SDS-PAGE gels, and

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the proteins were transferred to nitrocellulose membranes with constant current

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model. The membranes were blocked with 5% skimmed milk in PBS-T buffer for 1 h

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and were further incubated with the first antibodies ROCK2, CPI-17 (Santa Cruz

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Biotechnology, Santa Cruz, CA, USA), and β-Actin (Proteintech, Wuhan, China) at 4

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℃ overnight, then incubated with appropriate secondary antibodies (Proteintech)

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for 1 h at room temperature. Enhanced chemiluminescent (ECL) reagent (Vazyme 8



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Biotech) was used to detect protein bands, which were captured and analyzed using

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the ChemicDoc Imaging System (Bio-Rad Laboratories, Hercules, CA, USA) with

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ImageLab system (Bio-Rad Laboratories), respectively.

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Statistical Analysis

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GraphPad PRISM 6 was used for statistical analyses and data visualization. Data are

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presented as mean ± SEM. Multiple group statistical comparisons were determined

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by one-way or two-way ANOVA with Turkey’s or Dunnett’ tests. P value less than

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0.05 was considered to be statistically significant. Peak times of circadian gene

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expression were calculated by fitting a sinusoidal function to the real time-PCR data.

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RESULTS

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Desamethasone Synchronizes Clock Genes mRNA Rhythmic Expression in VSMCs

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In the present study, the rat primary cultured VSMCs were used to determine

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whether several core circadian genes were induced rhythmic expression by

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dexamethasone. Our real time PCR data revealed that brief treatment of VSMCs

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with dexamethasone resulted in the robust rhythmic expression of Bmal1, Cry1,

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Rev-erbα, and Per2 for at least 2 circadian cycles. Bmal1 mRNA expression level

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initiated a short-term increase and then decrease showing an around 24 hour

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rhythm, which consisted of peaks at ZT12 and ZT36. The expression Levels of Cry1

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mRNA showed peaks at ZT8 and ZT32. Robust approximate cycling of Rev-erbα and

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Per2 mRNA expression was also observed, with mRNA expression levels

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accumulating antiphase to Bmal1 RNA cycles. However, another core clock gene

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Clock mRNA did not show rhythmic expression (Figure 1).

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Transcriptome Sequencing and Assembly

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Using this VSMCs circadian model, we attempted to figure out genes that responded

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to L-theanine induction. To obtain a comprehensive circadian gene transcriptional

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profile of the induced VSMCs, collecting sample at the right time point is crucial. In

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general, gene expressions and regulations precede the presence of enzymes and

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their products. Based on this rational and the several circadian genes expression

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pattern showing in Figure 1, VSMCs samples at ZT0, ZT12, ZT24, ZT36 with or

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without L-theanine induction were chosen to explore the potential rhythmic genes

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response to L-theanine by RNA-seq analysis. The cDNA libraries for each sample 10



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were constructed and sequenced. The sequencing data showed that around 25

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million reads were obtained for each sample. After data filtering and stringent

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quality control, we mapped clean reads to reference genome using Taphat2. Among

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all reads, 83~95% of reads are mapped to rat genome, and the uniformity of the

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mapping result for each sample suggests that the samples are reliable and

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comparable. The details of mapping data are shown in Table 1. Our data suggested

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that the assembled results were qualified for further analyses. The levels of gene

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expression were quantified and expressed as fragments per kilobase of transcript

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per million mapped reads (FPKM). Based on the gene expression information, we

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preformed histogram to show the distribution of the gene expression level of each

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sample. In addition, we observed the dispersion of the gene distribution. In order to

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show the gene expression level under different FPKM value, we calculated the gene

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expression level under eight different FPKM ranges (0 < FPKM < 0.1, 0.1=< FPKM< 1,

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1=< FPKM < 5, 5 =< FPKM

RNA-Sequencing Analysis Reveals L-theanine Regulating

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