Up‐regulation of circular RNA hsa_circ_0037909 promotes essential hypertension

Aims Essential hypertension (EH) is a high prevalence disease facing a public health challenge. People were little known about the genetics of diagnosing the cause of EH. Circular RNAs that have a continuous cycle of covalent closure, without affected by RNA exonuclease, and are more stable and hard to degrade may involve into the molecule regulation mechanism of EH as an important biomedical. Methods qRT‐PCR was used to analyze circRNAs in total volume of human blood and the induced human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs). Our case‐control study was involved with 48 pairs of case controls with sex and age (±3 years) match. We conducted t test, Pearson's χ2 test, and receiver operating characteristics (ROC) curve analysis for the corresponding analysis. Results The expression level of hsa_circ_0037909 in EH patients was significantly higher than that in the healthy controls (P = 0.007), and the expression level of hsa‐miR‐637 in EH patients was significantly lower in than that in the healthy controls (P = 0.039); the same result appears in the HAECs and HUVECs. Hsa‐miR‐637 (adjusted P = 0.018), hsa_circ_0037909 (adjusted P = 0.005), HDL (adjusted P = 0.024), and serum creatinine (adjusted P = 0.014) were brought into the model which performed logistic regression analysis. The combination of two RNAs was excellent (P < 0.001) through ROC curve analysis. Hsa_circ_0037909 was significantly positively correlated with serum creatinine (P < 0.001) and low‐density lipoprotein (LDL) (P = 0.017). Conclusions Our findings suggested that the combination of hsa_circ_0037911 and hsa‐miR‐637 may be a significant important biomarker for early diagnosis of EH. Hsa_circ_0037909 may affect serum creatinine or LDL leading to the formation of EH.

test, and receiver operating characteristics (ROC) curve analysis for the corresponding analysis.

Results:
The expression level of hsa_circ_0037909 in EH patients was significantly higher than that in the healthy controls (P = 0.007), and the expression level of hsa-miR-637 in EH patients was significantly lower in than that in the healthy controls (P = 0.039); the same result appears in the HAECs and HUVECs. Hsa-miR-637 (adjusted P = 0.018), hsa_circ_0037909 (adjusted P = 0.005), HDL (adjusted P = 0.024), and serum creatinine (adjusted P = 0.014) were brought into the model which performed logistic regression analysis. The combination of two RNAs was excellent (P < 0.001) through ROC curve analysis. Hsa_circ_0037909 was significantly positively correlated with serum creatinine (P < 0.001) and low-density lipoprotein (LDL) (P = 0.017).

Conclusions:
Our findings suggested that the combination of hsa_circ_0037911 and hsa-miR-637 may be a significant important biomarker for early diagnosis of EH. Hsa_ circ_0037909 may affect serum creatinine or LDL leading to the formation of EH.

K E Y W O R D S
biomarker, essential hypertension, hsa_circ_0037909, mir-637

| INTRODUC TI ON
Essential hypertension (EH), with age-related, chronic, and frequent disorder, is prevalent in 40% of adults aged 25 and over as a public health problem. There are many complications of EH, such as hypertensive heart disease, peripheral vascular disease, and retinopathy. 1 Since EH is a complex multifactorial disease, patients who diagnosed with EH are little known about the etiology. Unraveling the complete etiology to EH is proved to be challenging.
Circular RNAs (circRNAs) have a continuous cycle of covalent closure and without affected by RNA exonuclease. 2 circRNAs, different from traditional linear RNA, are more stable and not easy to degrade with neither 5' to 3' polarity nor polyadenylated tail. 3 Functionally, in recent studies, circRNAs are able to act as miR-NAs sponges in cells due to the rich binding sites of microRNAs (miR-NAs). Thus, the inhibition of miRNA on target was eliminated and the number of target genes was increased. This mechanism is called the competitive endogenous RNA (ceRNA) mechanism. 4 The RNAs research found that circRNAs play a key role in the development of the disease like EH.

Hsa_circ_0037909
is located in the fragment 11 984 918-11 990 642 bp on chromosome 16 with 340nt sequence length that included three exons, and the association gene symbol is GSPT1. According to the bioinformatics analysis, it may be associated with EH and become an accurate prediction for it.
In the current study, combination of circBase database and bioinformatics analysis, we tested whether the expression levels of hsa_circ_0037909 were related to EH risk. In addition, the sensitivity of hsa_circ_0037909 analysis was carried out by receiver operating characteristics (ROC) graphs. According to the results, hsa_circ_0037909 may be a potential biomarker for EH as a screening and prevention tools.

| Study population
In the circRNA microarray step, we collected five newly diagnosed All research subjects had following 12-hour overnight fasting, using ethylenediaminetetraacetic acid to gather venous blood and store at −80℃. All participants' written informed consents were obtained before being enrolled in the study.

| circRNAs and miRNAs extract and reverse transcription
Total RNAs were extracted from peripheral blood samples using

| Validation of circRNAs and miRNAs expression by qRT-PCR
According to expression profile analysis, 5

| Cell culture
Human aortic endothelial cells (HAECs) were cultured in RPMI 1640 medium that is 10% of fetal bovine serum. Human umbilical vein endothelial cells (HUVECs) were cultured with Dulbecco's modified Eagle's medium. At 37°C, two cells were included 5% of carbon dioxide. Each orifice cell was divided into experimental and control groups. The control groups were routinely cultured, and the experimental groups were cultured with TNF-α 1 μmol/L 24 hours, 1 μmol/L 48 hours, 10 μmol/L 24 hours, and 10 μmol/L 48 hours.
Then, circRNAs and miRNAs of cells' extract, reverse transcription, and validation are the same as the population samples. Three samples were tested at a time, and each cell line was repeated at least three times. SPSS 19.0 software package (IBM Corp., Armonk, NY, USA) was used to analyze experimental data. The expression of circRNAs and miRNAs was calculated according to

| Baseline data of study subjects
Our study aimed to found the circRNAs signature to understand the EH mechanisms underlying the pathogenesis in EH patients; 48 pairs of case controls with sex and age (±3 years) match were enrolled from Yinzhou People's Hospital. In Table 1, all participants were within normal ranges in the aspect of baseline data. As expected, there were no statistically significant differences in gender and age.

| Validation of gene expression in blood samples
According to microarray data, 5 we selected one up-regulated cir-cRNA and one miRNA that corresponds circRNA. In particular, the GAPDH was considered to be an internal control for normalizing circRNAs and the cel-miR-39 was considered to be an external control for normalizing miRNAs. 5 As shown in Table 1 and Figure 1A,

| Diagnostic accuracy of RNAs
After logistic regression analysis adjusted, we found that some Further data analysis showed that hsa_circ_0037909 has a significant positive correlation with serum creatinine (Figure 2A; r = 0.396) and low-density lipoprotein (LDL) ( Figure 2B; r = 0.246, P = 0.017).

| Effect of gene expression in endothelial cells
Since endothelial cells are related to the pathology of target tissue damage of hypertension, we used TNF-α to induce the formation of EH mimics and examined changes about hsa_circ_0037909 and hsa-miR-637. We detected HAECs and HUVECs, the predicted target gene expression in both experimental and negative control groups. qRT-PCR analysis showed that two kinds of endothelial cells

| D ISCUSS I ON
CircRNAs in great part are derived from exons, and a few RNAs are formed by direct cyclization of introns. 6 Increasing circRNAs has been found in a variety of species over time, including humans. 7 The increasing molecular functional circRNAs were revealed. 8  Besides its action mechanism and clinical application value are unclear yet.
According to the results of microarray, we found that hsa_ circ_0037909 regulated hsa-miR-637, and hsa_circ_0037909 was up-regulated and hsa-miR-637 was down-regulated. In validation step, the finding was consistent with microarray (Figures 1 and 3).
This is the first time that hsa_circ_0037909 acted as higher regulated and hsa-miR-637 acted as lower regulated in the whole blood and endothelial cells simultaneously.
Hsa-miR-637 levels decrease with tumor that highly correlated.
It could act as a suppressor regulated on follicular thyroid carcinomas, 13 ovarian cancer, 14 and hepatocellular carcinoma, 15 and it also participated in the differentiation of cells, such as human mesenchymal stem cells, 15 migration and invasion of glioma cell, and human pancreatic ductal adenocarcinoma cells via suppressing Akt1 expression. 16 At the same time, hsa-miR-637 can performed as a marker of follicular thyroid cancer, 17 even hypertension. 18 In the ATP6V0A1 polymorphism, the miR-637 expression has been associated with EH development. 19 In another study, down-regulation of hsa-miR-637, which regulates the CDK6 expression from lung smooth muscle cells, increases the risk of hypoxia-induced pulmonary hypertension. 20  Some scientists discovered that hsa_circ_0003575 was a higher expression in HUVECs, which induced by oxidative modified LDL (oxLDL), and promoted the HUVECs proliferation and angiogenesis. 23 The mechanism is explained by the circRNAs, as a miRNAs sponge, with an indeterminate amount of miRNA response elements in its cyclic structure. Therefore, circRNAs may absorb the number of miRNAs and promote the expression of mRNAs. 24 In the current study, we discovered that hsa_circ_0037909 was up-regulated not only in whole blood, but also in both HAECs and HUVECs. The corresponding hsa-miR-637 is down-regulated. Meanwhile, there is a positive correlation between hsa_circ_0037909 and LDL. The relationship between serum creatinine and hypertension has been released. 25 Luan et al also found that circRNAs have a positive correlation with serum creatinine and act as a sponge for hsa-miR-150. 26 Our results have shown the same tendency in hsa_circ_0037909 expression levels and serum creatinine concentration. Taken together, we have enough reason to prove that the combination of hsa_circ_0037909 and hsa-miR-637 will affect biochemical indica-  bioinformatics analyses indicated that circRNA is a risk factor for hypertension. 30 Taken together, this result presents that hsa_circ_0037909 was significantly increased and hsa-miR-637 was significantly decreased in the EH group and TNF-α-induced HAECs and HUVECs. Their expressions are interrelated with LDL and serum creatinine concentrations. Also, hsa_circ_0037909 may play a meaningful part about EH pathogenesis, which has been suggested. Next, we will build circRNA-miRNA-mRNA network models and seek circRNAs functional mechanism with more depth.