LncRNA TARID induces cell proliferation through cell cycle pathway associated with coronary artery disease

Long non-coding RNA TARID (lncRNA TARID) can activate the tumor suppressor TCF21 in tumorigenesis by inducing promoter demethylation. However, the impact on lncRNA TARID and its variants of coronary artery disease (CAD) are poorly understood. We performed a case–control study enrolling 949 cases and 892 controls to assess genotype. Five variants were genotyped by TaqMan assay. 20 cases and 20 controls were used to evaluate the expression of lncRNA TARID. The cell proliferation rate was evaluated by CCK-8. The RT-qPCR and cell cycle analysis were applied to examine cell proliferation-related mRNA and cell distribution. This study indicated that rs2327433 GG genotype was associated with CAD risk adjusting for traditional risk factors (OR = 2.74, 95%CI: 1.10–6.83, P = 0.03). Our results analyses revealed that the genotype of rs2327433 was related to the proportion of CAD patients with left anterior descending artery disease and left circumflex artery disease (P = 0.025 and P = 0.025, respectively). The results showed that the minor allele frequency of rs2327433 was significantly correlated with the severity of the disease (P = 0.029). The eQTL analysis showed that rs2327433 may affect the transcription factors TCF21 regulated by lncRNA TARID. We found that TARID silencing regulated cell proliferation and altered cell cycle progression by induced upregulation of CDK1 and PCNA. SNP rs2327433 in lncRNA TARID was associated with CAD risk and the severity of CAD in the Chinese Han population. Furthermore, SNP rs2327433 may affect the expression of atherosclerosis-related transcription factor TCF21 regulated by lncRNA TARID. Finally, our study provided a new lncRNA-dictated regulatory mechanism participating in cell proliferation.


Introduction
Coronary artery disease (CAD) is a major cause of morbidity and mortality worldwide [1]. This disease is caused by the interaction of genetic and environmental factors [2]. Its etiology and pathogenesis have not yet been fully elucidated. Genetic factors play a decisive role in the pathogenesis of CAD.
In recent years, CAD-related single nucleotide polymorphism (SNP) sites confirmed by GWAS are located in non-protein-coding genes [3][4][5]. Long non-coding RNA (lncRNA), a type of transcript comprising > 200 nucleotides, was involved in the etiology of diverse human disorders through epigenetic, transcriptional, and post-transcriptional regulations [6]. Interestingly, it appeared that lncRNA also participated in the development of cardiovascular diseases, including heart failure, cardiac hypertrophy, cardiac metabolic diseases, and myocardial infarction [7]. The well-known 9p21 chromosome region has been reported [8]. The SNP in the lncRNA (Long non-coding RNA) ANRIL of this region is significantly related to CAD risk [9]. In our previous study, we also found that the SNP on lncRNA ANRIL is not only significantly related to the susceptibility of CAD [10], but also significantly related to the severity of CAD [11]. A large number of lncRNA have been found to be abnormally expressed in CAD [12] or myocardial infarction [13], which indicates that this noncoding RNA molecule may play a very important role in the occurrence of CAD. Therefore, the impact of lncRNA and its genetic variation on the sensitivity and severity of CAD needs further study.
Mechanistically, long non-coding RNA (lncRNA) are large intergenic noncoding RNAs lacking protein-coding function which are involved in diverse cell functions, such as proliferation, apoptosis, and migration [14]. LncRNA TARID is located in the 6q23.2 region of the chromosome. The GWAS study found that the chromosome region 6q23.2 is significantly related to CAD susceptibility [15], which indicates that TARID may be related to the occurrence and development of coronary heart disease [16]. Studies have found that this lncRNA is involved in the demethylation of TCF21 promoter to promote its expression [17]. In the latest research, the protein-coding gene TCF21 was discovered to regulate smooth muscle cell phenotypic transformation and inhibit atherosclerosis [18]. Accordingly, the function of lncRNA TARID, and the relationship between the TARID variants and the CAD susceptibility in the Chinese Han population deserves further exploration.

Study population
We performed a two-stage case-control study to examine the effects of genetic variants on CAD and to detect the expression level of lncRNA TARID. The first stage consisted of 949 case patients and 892 age-and sex-frequency-matched controls. The second stage, which was collected to verify the difference in lncRNA TARID expression level, was made up of 20 patients and 20 controls. All the enrolled subjects were from Chongqing City. Approval was obtained from the ethics committee of Chongqing Medical University in Chongqing, China (No. 2021043). The procedures used in this study adhere to the tenets of the Declaration of Helsinki. All subjects signed written informed consent.
Inclusion criteria for CAD cases have been described elsewhere [10]. The control group chooses people who have undergone health examinations during the same period. Structured questionnaires were accomplished by trained interviewers. The questionnaire collected information about demographic variables, medical history, drug use, and lifestyle factors.

SNP selection
According to the tag SNP information in Hapmap and 1000 genome databases, the previous CAD-related research of the Chongqing population, and the results of GWAS research reported at home and abroad, this study selected five noncoding genes TARID SNPs. Five SNPs were selected on the TARID gene located on human chromosome 6, namely rs1966248, rs2327429, rs2327433, rs12190287, and rs6569912.
The selected SNPs met the following inclusion criteria: (1) minor allele frequency (MAF) ≥ 0.1 in the Chinese Han population of the HapMap project (the Phase II database); (2) Hardy Weinberg equilibrium (HWE) test P value ≥ 0.05.

Genotyping
Fasting venous blood was collected from each participant in 5-ml EDTA tubes, and the genomic DNA was isolated with a Tiangen DP319-02 kit (Tiangen Company, Beijing, China). Genotyping was performed with the TaqMan Genotyping Assay Systems and ABI PRISM 7900HT (Applied Biosystems, Foster City, CA, USA), in 384-well format. Genotypes were automatically detected by analysis software version 2.2.1 (SDS 2.2.1).

Cell proliferation assay
Cell proliferation was detected by the Cell Counting Kit-8 (CCK-8) assay according to the manufacturer's instructions. 5000 cells per well were seeded into 96-well plates and cultured at 37 °C in 5% CO 2 . After the indicated treatments, each well was added with 10ul CCK-8 reagent (HY-K0301, MCE) and incubated for 1 h at 37 °C. The absorbance at 450 nm was recorded by a microplate reader. Cell viability was calculated after 12 h, 24 h, 48 h, and 72 h according to the relative optical density. Five independent replicates were performed for each treatment and control.

Cell cycle analysis
DNA analysis by flow cytometry was employed to measure cell distribution [19]. The cells were seeded in 6-well plates at a density of 70,000 cells/well and cultured at 37 °C overnight. At 48 h after transfection, viable cells were gathered and fixed at 4 °C overnight. The fixed cells were then stained with PI at 37 °C for 30 min in the dark. The cell cycle analysis of different groups was evaluated using a flow cytometer (CytoFLEX, American). All experiments were carried out with three independent replicates.

RT-qPCR
The total RNA was extracted from cells using RNAi plus Reagent (TaKaRa, Japan) and the cDNA was reverse transcribed by using 2×Fast PCR Master Mix (MedChemExpress lnc.) or PrimeScript™ RT Master Mix (TaKaRa, Japan). qPCR was performed using random primers and SYBR Green qPCR Master Mix Kit (MedChemExpress lnc.) on the BioRad ® 96 instrument Real-Time PCR System. The PCR reaction condition was as follows: denaturation at 95 °C for 3 min, followed by 40 cycles of 95 °C for 5s, annealing at 60 °C for 10s, and a final extension at 72 °C for 30s. The sequences of primers were all listed in supplementary 1. GAPDH was used as a reference internal control. The fold change of gene expression was calculated as the 2 −△△CT method.

Statistical analysis
The measurement data is expressed as the mean ± standard deviation (mean ± SD), and the count data is expressed as the percentage of the number of cases. The 1-Sample K-S test was used to test the normality of continuous variables. The data that obeyed the normal distribution was analyzed by the F test. The data that did not meet the normal distribution were analyzed by the Kruskal-Wallis rank sum test. The Chi-square test was used to the Hardy-Weinberg equilibrium test of each SNP and the count data.
Binary unconditional logistic regression was used to analyze the relationship between the genotype of each SNP and the risk of CAD. All statistical analysis tests are two-sided tests. P < 0.05 is considered statistically significant. Haploview 4.2 software was used to map the linkage relationship between each SNP. SHEsis (http:// analy sis. bio-x. cn/ myAna lysis. php) online tool was used to perform haplotype frequency estimation of SNP.

Characteristics of the subjects and variants
This study adopted the method of a case-control study. This study included 949 CAD cases with a mean age of 66.2 ± 10.7 years and 892 controls with a mean age of 65.6 ± 12.7 years. Cases and controls were frequency matched according to age and gender. The basic clinical data of the study subjects were shown in Table 1. The pairwise LD of the five SNPs was calculated for the 949 casepatients and 892 age-and sex-frequency-matched controls. The haplotype block was generated from the Haploview software (Supplementary 2).

Association between SNPs and CAD risk
The distribution of genotype and the allele frequency in the case group and the control group were shown in Table 2.
The results of the univariate analysis showed that there was no statistical difference in the frequency distribution of the five SNPs between the case-control groups. Considering that confounding factors may bias the results of the study, we further adopted a binary unconditional logistic regression model. As represented in the table, SNP rs2327433 in TARID was associated with CAD after adjusting for sex, age, smoking, drinking, diabetes, and five SNPs. The rs2327433 GG genotype was found as a risk factor for CAD in adults (OR = 2.74, 95%CI: 1.10-6.83, P = 0.03). As shown in Supplementary 3, the stratified analysis indicated a significant association between SNPs (rs2327433 and rs1966248) and the risk of CAD. Our results showed that genotype GG of the SNP rs2327433 was associated with risk of CAD in the male's group and the drinking group (OR = 3.37, 95%CI: 1.22-9.32, P = 0.02 and OR = 9.72, 95%CI: 1.14-82.83, P = 0.04, respectively). The results indicated that genotypes AA carriers of rs1966248 had a significant association with CAD risk in non-diabetes subjects (OR = 1.45, 95%CI: 1.04-2.03, P = 0.03).
The results of GMDR model analysis for the entire case-control data were shown in Table 3, which showed that the best model was the four-factor model. This model was adjusted for smoke. There were significant interactions with polymorphisms in the four genes in TARID.
We used the eQTL research platform provided by the GTEx (version 8) database to analyze the relationship between SNP rs2327433 and the expression levels of host genes and adjacent CAD-related genes. The eQTL analysis can confirm SNP-related gene expression differences [20]. We have listed the existing organizational data in Supplementary 4. The results showed that the different genotypes of rs2327433 were significantly correlated with the expression level of the TCF21 gene. The results reveal that in the esophageal mucosa (enriched endothelial cells) the expression of TCF21 is lower in the GG genotype of SNP rs2327433. TCF21 is a protein-coding gene adjacent to the downstream of TARID. By performing eQTL analysis in aortic tissue from the STARNET database25 on this larger set of SNPs, studies found that the CAD risk imparted by rs2327433 was also correlated with lower TCF21 expression from that allele [21]. Reduced TCF21 expression is associated with increased coronary disease risk [21].

Association between CAD-related variant rs2327433 and the severity of CAD
The binary logistic regression analyses revealed that the genotype of rs2327433 was related to the proportion of CAD patients with left anterior descending artery disease (LAD) (Fig. 1a) and left circumflex artery disease (LCX) (Fig. 1b) after adjusting for traditional risk factors such as age, smoking, gender, drinking (P = 0.025 and P = 0.025, respectively). We further assessed the effect of SNPs on the development of CAD. The results showed that the minor allele frequency of rs2327433 was significantly correlated with the severity of the disease (P = 0.029) (Fig. 1c).

TARID silencing affected cell cycle progression and the expression of cell cycle-related genes
LncRNA TARID activates the expression of TCF21 by inducing promoter methylation [17]. The SNP site rs2327433 of lncRNA TARID is related to the expression of TCF21. On the esophageal mucosa, the expression of TCF21 is lower in the GG genotype of SNP rs2327433. We speculate that the GG genotype of SNP rs2327433 affects the expression of lncRNA TARID, preventing it from activating TCF21. Then the expression of lncRNA TARID in patients with coronary heart disease is not clear, we use human peripheral blood mononuclear cells to detect the expression level of lncRNA TARID. The results showed that after normalization with housekeeping gene (18s), compared with healthy subjects, the expression level of TARID in peripheral blood mononuclear cells of CAD cases was significantly reduced (P < 0.01) (Fig. 2a). Studies have shown that TCF21 affects the cell cycle progression of cells [22]. However, it is not clear whether the silencing of lncRNA TARID can affect the cell cycle through the TCF21 pathway. As displayed in Fig. 2b, the expression of TARID RNA in HUVEC cells transfected with si-TARID-1 or si-TARID-2 was significantly lower than in the NC group, respectively.
We spontaneously want to know whether si-TARID-1 contributes to cell proliferation. In HUVEC cells, the absorbance of the si-TARID-1-treated group was significantly Fig. 2 lncRNA TARID-mediated cell cycle pathway associated with CAD. a The expression of lncRNA TARID between patients and controls. peripheral blood mononuclear cell was used to measure the expression level of lncRNA TARID. b Detection of TARID silencing (si-TARID-1, si-TARID-2) efficiency in HUVEC. c CCK-8 assays were performed to detect HUVEC cell proliferation. NC is the blank vector control of the silenced TARID. The graphs show the mean ± SD (n = 5 per group). d Detection of TARID silencing (si-TARID-1) efficiency in THP-1. e and f TARID silencing affected cell cycle progression of THP-1. Cell cycle distribution of THP-1 cells after THP-1 silencing was analyzed and quantified by ModFit (LT 4.1). Experiments were performed in triplicate. The data are expressed as the means and SD from three replicate samples, and the statistical comparisons were analyzed by two-sided t-test. g, h TARID regulated the expression of cell cycle-related genes THP-1 or HUVEC. THP-1 or HUVEC were transfected with NC, si-TARID-1 for 48 h. The RNA level was evaluated by RT-qPCR. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, compared with the NC group. The fold change of gene expression was calculated as the 2 − ΔΔCT method increased compared with the control group, indicating that TARID silencing promotes cell proliferation (Fig. 2c). The results showed that after si-TARID-1 was transfected into HUVEC cells, compared with the control group, the proliferation of cells in the transfected si-TARID-1 group was significantly different at 48 h (P = 0.022) and 72 h (P = 0.002). There was no statistical difference in the transfected si-TARID-2 group. We found that si-TARID-1 was more effective, and we transfected si-TARID-1 into THP-1 cells to test the transfection efficiency (P = 0.0065) (Fig. 2d). To assess how TARID silencing affects cell cycle progression, we conducted flow cytometry to analyze the distribution of cells across the major phases of the cell cycle in THP-1 cells transfected with si-TARID-1. The results revealed that TARID silencing resulted in a decrease of THP-1 cells in the S-phase from 41.37 ± 1.38% to 37.25 ± 0.85% (P = 0.0116), and increased accumulation in the G2 phase from 1.497 ± 0.741% to 4.137 ± 0.717% (P = 0.0114), relative to the control group ( Fig. 2e and f). To further study the effect of TARID in cell cycle regulation, RT-qPCR was performed to determine cell cycle-related genes. As shown in Fig. 2g and h, TARID silencing up-regulated CDK1 and PCNA in THP-1 cells or HUVECs.

Discussion
In this study, we selected five SNPs (rs1966248, rs2327429, rs2327433, rs12190287, and rs6569912) in the lncRNA TARID gene for the association study with CAD. We used a case-control study method to reveal the relationship between five SNPs and CAD risk in the Chinese Han population. We found that SNP rs2327433 in TARID was associated with CAD risk in the Chinese population. This SNP was also related to the severity of coronary heart disease. SNP rs2327433 is located in the promoter region of lncRNA TARID. Our results suggested that the genotype of rs2327433 was related to the proportion of CAD patients with LAD and LCX disease. We further assessed the effect of SNPs on the development of CAD. The results showed that the minor allele frequency of rs2327433 was significantly associated with the severity of the disease. In the stratified analysis, we found that the G allele of rs2327433 has an increased risk of CAD in male subgroups and drinking conditions.
To investigate the relationship between SNP genotype and gene expression, we conducted the eQTL analysis. The eQTL analysis results showed that the SNP in the TARID gene region was related to the expression levels of TARID and TCF21. SNP rs2327433 is located in the promoter region of lncRNA TARID. TARID is a non-proteincoding gene located in the 6q23.2 region of the chromosome [23]. Studies have found that TARID can activate TCF21 expression by inducing promoter demethylation [17]. It also further clarified the specific mechanism of TARID regulating TCF21 [24]. TCF21 can regulate the phenotypic transition of vascular smooth muscle to play an anti-atherosclerotic effect [18]. The results showed that the different genotypes of rs2327433 were significantly correlated with the expression level of the TCF21 gene in skeletal muscle or esophageal mucosa. The results showed that the expression of TCF21 is lower in the GG genotype of SNP rs2327433 in the esophageal mucosa (enriched endothelial cells). Therefore, we speculate that this SNP rs2327433 may play an important role in coronary heart disease by affecting the pathway that TARID regulates TCF21 function.
The SNP rs12190287 is significantly related to myocardial infarction in the Chinese population [25]. It has been reported that rs12190287 is significantly related to the expression level of the adjacent gene TCF21 [15]. Studies have further verified the association between rs12190287 and the risk of CAD [26]. These studies indicated that rs12190287 is involved in the miRNA-dependent regulation of TCF21 [16]. But rs12190287 is not statistically significant in Japanese patients with coronary heart disease [27]. This is an SNP reported in other populations, which is inconsistent with our study. Our study did not find that this SNP rs12190287 is directly related to the risk of CAD in the Chinese Han population. This may be attributed to the synergy of multiple parts of coronary heart disease (rs1966248 rs2327433 rs12190287 rs6569912) or the ethnic differences. We further investigated the expression level of lncRNA TARID in the case-control group. The data of expression level in peripheral blood mononuclear cells indicated that knockdown of TARID is an association with coronary heart disease. LncRNA is related to many cell functions, such as cell proliferation. Our data suggested that TARID silencing could increase the cell proliferation ability. Cell proliferation depends on the cell cycle, involving multiple checkpoints [28]. In our study, the increase in the S phase and decrease in the G2 phase suggested that the cell cycle was interrupted in the condition of TARID-silencing. Although the S phase and the G2 phase of the cell cycle were less altered after TARID silencing, possibly due to the timing of detection of cell cycle distribution after silencing lncRNA TARID [29], the observed results were still statistically significant meaningful. To identify TARID could affect cell proliferation by regulating cell proliferation-related genes. Hence, we checked a series of cell cycle-related genes and found that PCNA and CDK1 were up-regulated. Several studies suggested that up-regulated PCNA is related to promoting cell proliferation [30]. PCNA is highly expressed in the S phase of the cell cycle and is an important node for cell inspection [31,32]. One of the genes essential for cell proliferation and cell cycle regulation is CDK1 [33].
There are several limitations of this study that must be admitted. First, the sample size of CAD patients with angiographic evidence is relatively small, thus they may not be representative of the general population. Second, a prospective cohort study with a larger sample size is required to confirm our results.

Conclusions
In conclusion, we found that lncRNA TARID rs2327433 was significantly related to the severity of coronary artery disease. This finding implies that the rs2327433 may strongly influence the development of CAD. our experimental results indicated that TARID knockdown in the human myeloid leukemia mononuclear cell THP-1 regulated proliferation and altered cell cycling by regulating the PCNA/CDK1 pathways.