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Alteration of RNA Demethylases by Cholesterol Crystals
Yakhak Hoeji 2024;68(1):56-61
Published online February 29, 2024
© 2024 The Pharmaceutical Society of Korea.

Tae Won Kim*,** and Gyeongmin Kim***,#

*College of Pharmacy, Kyungsung University
**Brain Busan 21 Plus Research Project Group, Kyungsung University
***Department of Biological Sciences of Companion Animals and Plants, Kyungsung University
Correspondence to: #Gyeongmin Kim, Department of Biological Sciences of Companion Animals and Plants, Kyungsung University, Busan 48434, Republic of Korea
Tel: +82-51-663-4644, Fax: +82-51-663-4089
Received August 2, 2023; Revised February 15, 2024; Accepted February 16, 2024.
Atherosclerosis is a chronic inflammatory disease of the arteries that can lead to cardiovascular diseases. Cholesterol crystals are one of the elements found in atherosclerotic plaques, and they play a significant role in the development and progression of the disease. In this study, we investigated the effects of cholesterol crystals on mRNA modification in macrophages, which are involved in plaque formation and inflammation. We used RAW 264.7 cells, a murine macrophage cell line, and exposed them to cholesterol crystals. We found that cholesterol crystals stimulated the production of pro-inflammatory cytokines, including IL-1β, IL-6, and TNFα. Furthermore, cholesterol crystals were taken up by the macrophages and were detected within the cells. Interestingly, cholesterol crystals upregulated demethylating enzymes FTO and ALKBH5, which are involved in RNA modification. However, the expression of methyltransferases, including METTL3, METTL14, NSUN2, and WTAP remained unchanged. These results suggest that cholesterol crystals can modulate mRNA modification by affecting demethylating enzymes and pro-inflammatory cytokines, potentially contributing to the development of atherosclerosis. Understanding the molecular mechanisms underlying cholesterol crystal-induced atherosclerosis may provide new insights into preventive and therapeutic strategies for cardiovascular diseases.
Keywords : Atherosclerosis, RNA modification, Cholesterol crystal, Pro-inflammatory cytokine

Atherosclerosis is a chronic inflammatory process of arteries that gradually grows and thickens, narrowing the arterial walls by dyslipidemia and other cellular abnormalities.1,2) It is a common type of cardiovascular disease and can lead to serious health problems such as heart attack, stroke, and peripheral artery disease.3) The underlying cause of atherosclerosis is inflammatory responses and the accumulation of plaques in the endothelium.4) The plaques in the endothelium are composed of cholesterol, calcium, and other substance, such as inflammatory macrophage foam cells.5) As the plaques develop, they can become unstable and prone to rupture and trigger an inflammatory response, leading to initiate blood clots or thrombi forming, which can further obstruct blood flow or completely block arterial walls.6)

Cholesterol crystals are a usual element that appears in atherosclerotic that, make more easily to understand the pathogenesis and manifestations of atherosclerosis. Cholesterol crystals are sharp, needle-like structures that are formed when excess cholesterol in the blood precipitates out and solidifies. In macrophages, the cholesterol concentration reaches a critical level, and it starts to crystallize and increase in size as more cholesterol accumulates. Cholesterol crystals stimulate inflammatory responses by activating NLRP3 inflammasome and releasing IL-1β.7-10) The atherosclerotic plaque has more cholesterol crystals that make the plaque grow faster, and it can easily occur erosion or rupture, leading to various cardiovascular diseases.11,12) Therefore, excessive levels of cholesterol in the blood can lead to atherosclerosis.

RNA performs spectrums of more than 100 chemical modifications in gene expression regulation, including RNA methylation.13) Modifying mRNA, especially N6-methyladenosine (m6A) modification, is eukaryotes’ most common internal modification.14) The function, localization, and metabolism of mRNA can be affected by the modification of RNA.15) Furthermore, recent studies have shown that modification of RNA act as a regulatory factor in atherosclerosis heart disease.16) m6A modification is important in endothelial diseases because it is reversible, allowing the regulation of m6A to perform after initial deposition. Protein complexes, including adenosine methyltransferases (writers), demethylating enzymes (erasers), and the m6A-binding proteins (readers), regulate the functions and changes of m6A modification such as occurrence, removal, and recognition, respectively.17) Same as m6A modification, 5-methylcytosine (5mC) modification is one of the most common internal modification and has gained increased attention in recent years. 5mC modification attach a methyl group to the fifth carbon of the cytosine ring in DNA and RNA molecules.18,19) These modification is a post-transcriptional modification that regulate important roles in many biological processes20). Although there are studies that cholesterol and mRNA modification can affect atherosclerosis, it is still unclear if cholesterol crystals affect protein complexes leading to mRNA modification. The purpose of this study is to determine the mechanism of cholesterol crystal on mRNA modification that can occur atherosclerosis in macrophage.


Cell culture

RAW 264.7 cells, a murine macrophage cell line, were purchased from Korea Cell Line Bank (Seoul, Korea). Cells were grown in 1 g/L D-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco BRL, Karlsruhe, Germany) supplemented with 10% fetal bovine serum and penicillin-streptomycin (10,000 unit). Before experiment use, cells were incubated under 37oC with 5% CO2 and seeded in a 12-well plate.

Gene expression analysis

Total RNAs from RAW 264.7 cells were extracted using a total RNA Extraction kit (SJ BioScience, Daejeon, Korea), including DNase I (SJ BioScience, Daejeon, Korea). Nanodrop (MicroDigital Co., Ltd., Gyeonggi-do, Korea) was used to quantify RNA concentration. Then quantitative real-time PCR was performed using SYBR Green PCR Master Mix (SJ BioScience, Daejeon, Korea). In the light of the SYBR Green protocol, 20 μL actions were run with 2μL of cDNA. The QuantStudioTM 1 Real-Time PCR Instrument (Thermo Fisher Scientific Inc., USA) was performed in RT-PCR experiments. PCR steps follow below: first hot start at 95oC for 10 min, subsequent cycles of 95oC for 15 sec, and 60oC for 1 min during the fluorescence was measured. All samples were normalized to 18s, and the levels of relative gene expression were calculated by the 2-(ΔΔCt) method. Beta-actin used the internal reference. The primer sequences are listed in Table 1. Each sample and all quantitative real-time PCRs were done in triplicate.

Primer sequences used for RT-PCR analysis

Gene Forward primer Reverse primer

Fluorescence microscopy

RAW 264.7 cells were seeded in a 35-mm dish at 3.5×104 cells/well and incubated for 24 hr. Then cells were treated with 100 μg/mL of cholesterol crystal. After 24 hr, cells were washed with PBS, and the cell nuclei were visualized by staining DAPI. The cholesterol crystals were detected by the reflection mode of fluorescence microscopy (Zeiss, Jena, Germany) using autofluorescence mode.

Statistical analysis

The statistical analyses were carried out using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA). The results were determined using one-way analysis of variance (ANOVA) and followed by Dunnett's multiple comparisons test, where p<0.05 is considered significant. Data are shown as mean±SEM.

Result and Discussion

Effects of cholesterol crystal on pro-inflammatory cytokines

To determine whether cholesterol crystal can stimulate proinflammatory cytokines genes such as IL-1β, IL-6, and TNFα, we performed quantitative real-time PCR in RAW 264.7 cells. Interestingly, when 10 and 100 μg/mL of cholesterol crystals were exposed for 24 hr in RAW 264.7 cells, IL-1β, IL-6, and TNFα were significantly increased (Fig. 1). Furthermore, IL-1β and TNFα were increased in a dose-dependent manner (Fig. 1A and C). These results indicate that cholesterol crystals can activate inflammatory responses by producing pro-inflammatory cytokines that mainly cause atherosclerosis.

Fig. 1. Effects of cholesterol crystal on pro-inflammatory cytokines expression in RAW 264.7 cell.
RAW 264.7 cells were incubated with normal medium and then treated 10 and 100 μg/mL of cholesterol crystal (CC). Expression of (A) IL-1β, (B) IL-6 and (C) TNF-α, proinflammatory cytokines, (n=4 in each group) were measured by quantitative real-time PCR. Data are presented as mean±SEM (*p<0.05, **p<0.01 compared to empty cholesterol crystal vector, respectively).

Detection of Cholesterol crystal

Microscopy was used to determine whether cholesterol crystal can be uptaken by the RAW 264.7 cells. As shown in Fig. 2, apparent fluorescence signals were observed for nuclei of RAW 264.7 cells that DAPI stained, and cholesterol crystal was not found in the cytoplasm of RAW 264.7 cells (Fig. 2A). However, after cholesterol crystal was treated in RAW 264.7 cells, cholesterol crystal was detected in cells (Fig. 2B). This result suggests that cholesterol crystals can be uptaken by macrophages and exist in the cells.

Fig. 2. Cholesterol crystal affects RAW 264.7 cell nuclei.
264.7 cells were incubated with normal medium and then treated 100 μg/mL of cholesterol crystal for 24 hr. Cells were immunostained using DAPI. Empty cholesterol crystal treated cells shown in (A) and 100 μg/mL of cholesterol crystal treated cells shown in (B).

Effects of cholesterol crystal on demethylating enzymes

Demethylating enzymes, the sixth nitrogen atom of the adenylate to erase the m6A methylation of the RNA molecule, which is also a key step in making the m6A modification reversible.21) To confirm the effect of cholesterol crystal on demethylating enzymes, including fat-mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5), we performed quantitative real-time PCR in 10 and 100 μg/mL of cholesterol crystal-exposed RAW 264.7 cells for 24 hr. Interestingly, FTO and ALKBH5 were significantly increased by cholesterol crystal in a dose-dependent manner (Fig. 3). These results indicate that demethylating enzymes can be modulated by cholesterol crystal in the RAW 264.7 cells.

Fig. 3. Cholesterol crystal upregulates RNA demethylases expression in RAW 264.7 cell.
RAW 264.7 cells were incubated with normal medium and then treated 10 and 100 μg/mL of cholesterol crystal (CC). Expression of (A) ALBKH5 and (B) FTO, RNA demethylases, (n=4 in each group) were measured by quantitative real-time PCR. Data are presented as mean±SEM (**p<0.01, ***p<0.001 compared to empty cholesterol crystal vector, respectively).

Effects of cholesterol crystal on methyltransferases

Methyltransferases catalyze m6A and 5mC methylation that the progress increases the level of RNA methylation using an active methyl group. 10 and 100 μg/mL of cholesterol crystals were exposed to RAW 264.7 cells to confirm whether cholesterol crystals increased methyltransferase-related genes by quantitative real-time PCR. As shown in Fig. 4, the cholesterol crystal did not change the expression of methyltransferase-related genes, including METTL3, METTL14, NSUN2, and WTAP. These results mean cholesterol crystals can not alter adenosine methyltransferases.

Fig. 4. Effects of cholesterol crystal on methyltransferases expression in RAW 264.7 cell.
RAW 264.7 cells were incubated with normal medium and then treated 10 and 100 μg/mL of cholesterol crystal (CC). Expression of (A) METTL3, (B) METTL14, (C) NSUN2 and (D) TRDMT1, pro-inflammatory cytokines, (n=4 in each group) were measured by quantitative real-time PCR. Data are presented as mean±SEM (*p<0.05, **p<0.01 compared to empty cholesterol crystal vector, respectively).

Atherosclerosis is a disease linked to inflammation, and its incidence is on the rise in modern society. As a result, research on cholesterol crystals has become increasingly significant, as studies suggest that they can induce inflammation, a key factor in atherosclerosis, and contribute to plaque rupture.7,11,22) Inflammation induced by activation of NLRP3 inflammasome that is priming with endogenous molecules such as IL-1β and TNF-α through activation of NF-κb.23,24) Previous studies have shown that cholesterol crystals trigger IL-1β production in macrophages, and IL-1β affects atherosclerosis via NLRP3 inflammasome.8,9) Additionally, alterations in RNA methylation of pro-inflammatory genes such as TNF-α and IL-6 can activate and stimulate plaque formation.25) Futhermore, m6A RNA modification can regulate inflammatory gene expression that has important role in diseases such as cardiovascular disease, cancer and metabolic disorders.26) Consequently, our study confirms that cholesterol crystals can affect pro-inflammatory genes, promoting plaque formation and rupture through RNA methylation changes.

Compelling evidence shows that m6A and 5mC RNA modification in genes plays crucial roles in inflammatory responses and lipid metabolism, which are critically affecting atherosclerosis. Dysregulation of m6A methyltransferases such as METTL3 and METTL14 can be critical events in atherosclerosis development. METTL3 regulates macrophage polarization and foam cell formation contributing to the development of atherosclerotic plaques,27) and METTL14 regulates gene expression, such as endothelial inflammation and dysfunction.28) Similarly, inhibition of NSUN2, 5mC methyltransferase, increases vascular endothelial inflammatory response by interrupting leukocyte adherence to endothelial cells.29) Conversely, increasing the expression of demethylases like FTO and ALKBH5, both belonging to the alpha-ketoglutaratedependent dioxygenase family, affects atherosclerosis development. When FTO demethylates m6A, it suppresses PPARγ, leading to downregulation of CD36 expression and inhibiting lipid uptake in macrophages. Furthermore, activated FTO by AMPK increases the expression of ABCA1, facilitating intracellular cholesterol efflux.30) Although several studies have shown the effect of m6A and 5mC RNA modification in atherosclerosis, there has not been any prior evidence of the molecular mechanism connecting cholesterol crystal and m6A and 5mC RNA modification.

Previous evidence was consistent with our result that macrophages can take up cholesterol crystals and induce IL-1β production.8) Interestingly, here we provide evidence for the first time that cholesterol crystal upregulated demethylases, including FTO and ALKBH5, in a dose-dependent manner. Only TRDMT1, however, was upregulated by cholesterol crystal among the methyltransferase. In previous studies, pro-inflammatory cytokines and RNA demethylases play a role in plaque formation and lipid metabolism.25,30) Therefore, we speculate that cholesterol crystal stimuli atherosclerosis by upregulating inflammatory response and RNA demethylation.

In conclusion, our study showed for the first time that cholesterol crystals can alter the expression of pro-inflammatory cytokines and RNA demethylases, thereby promoting atherosclerosis. These finding expands our understanding of the progression mechanism of cholesterol crystal in atherosclerosis and provides potential prevention.


This work was supported by Kyungsung University Research Grants in 2021.

Conflict of Interest

All authors declare that they have no conflict of interest.

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