Hsa_circ_0000437 upregulates and promotes disease progression in rheumatic valvular heart disease

Abstract Background Currently, the diagnosis and outcome of rheumatic valvular heart disease (RVHD) are less than ideal, and there are no accurate biomarkers. Circular RNA (circRNA) might participate in the occurrence and development of RVHD. Materials and methods We use circRNA microarray to filter out the target has_circ_0000437. qRT‐PCR was used to measure the expression levels of hsa_circ_0000437 in RVHD plasma samples. We assessed the diagnostic value of hsa_circ_0000437 in RVHD. Cell function in vitro experiment was to explore the effect of has_circ_0000437 on RVHD. Results Has_circ_0000437 is highly expressed in RVHD (p < 0.001). has_circ_0000437 has the diagnostic value in RVHD. In RVHD, hsa_circ_0000437 can promote cell proliferation and migration but inhibits its apoptosis. This may be due to the combination of has_circ_0000437 and target miRNA in the cytoplasm that affects the progress of RVHD. Conclusions Has_circ_0000437 can promote the process of RVHD and may be a potential for the diagnosis and treatment of RVHD.

with mechanical valve replacement require life-long anticoagulation therapy after surgery, all of which severely affect the quality of life. 1 At present, the essential examination method for RVHD is transthoracic echocardiography (TTE); however, the detection accuracy of TTE is affected by the operator, and the technique is prone to errors. The test indicators that assist in diagnosing RVHD include erythrocyte sedimentation rate, C-reactive protein, mucin, and protein electrophoresis. There are also immune indicators such as circulating immune complexes, total serum complement and complement C3, immunoglobulins IgG, IgM, IgA, B lymphocyte detection, and anti-myocardial antibodies. 7,8 The sensitivity and specificity of these tests are not high, and they may not remain elevated during the chronic phase of the disease. Therefore, it is critical to identify biomarkers of RVHD, clarify the biological function and mechanism of RVHD valve disease, and provide adequate tools for the diagnosis and treatment of RVHD.
Circular RNA (circRNA) is a circular molecule formed by lassodriven circularization or circularization driven by intron pairing. 9 On the other hand, circRNAs can be divided into noncoding circRNAs and coding circRNAs. 10,11 Unlike linear RNA, circRNA has a stable circular structure, so nucleases do not quickly degrade it. Although the earliest circRNAs were found in plant viruses in 1976, they were considered byproducts of transcription and were mistakenly believed to have no critical biological functions. 12 However, recent studies have shown that circRNAs regulate gene expression in a variety of ways. The biological functions of circRNA include miRNA molecular sponges, regulation of gene transcription, interaction with RNA-binding protein, and translating proteins. [13][14][15][16][17] RNA-seq data shows that more than 1,000 types of circRNA have been detected in adult tissues and blood samples such as the colon, heart, kidney, liver, lung, stomach, and brain. Most circRNA has tissue and blood specimen specificity, and circRNA is involved in regulating these diseases. [18][19][20][21][22] Therefore, in combination with these characteristics, circRNA might help detect disease occurrence, development, diagnosis, and treatment.
There are many studies on circRNA in cardiovascular diseases (CVD) such as coronary heart disease, hypertension, atherosclerosis, and cardiomyopathy. [23][24][25][26] However, there are few studies of circRNA in RVHD, and it is urgent to explore its role in RVHD.
For this reason, it is critical to identify effective test indicators for early disease prediction, outcome prediction, diagnosis, and treatment. In the present study, the expression profile of circRNA in the plasma of three RVHD patients and three control patients was analyzed using circRNA microarrays. Differentially expressed circRNA was identified (GSE16 8932, https://www.ncbi.nlm.nih. gov/geo/query/ acc.cgi?acc=GSE168932)when fold change >2 and p < 0.05. We combined circBase and GEO database to identify the target circRNA has_circ_0000437 that was not previously studied in the literature and then expand the sample size to verify the expression of the target has_circ_0000437 and explore its possibility as a biomarker for RVHD. The influence of has_circ_0000437 on the progression of RVHD was also explored using in vitro experiments.

| Collection and storage of plasma samples
We drew 3 ml of peripheral venous blood into EDTA-containing anticoagulation tubes, centrifuged at 3000 rpm for 15 min, removed the upper light yellow plasma layer in 2 ml RNase-free eppendorf tubes, and stored the samples at −80°C until use.

| CircRNA microarray assays
We used Arraystar Human circRNA microarray (Arraystar) to test 3 pairs of RVHD and normal control samples. We analyzed and processed the detected data with R software package and uploaded the original data to the GEO database. Finally, circRNAs with a fold change of >2.0 and p-value <0.05 were selected as candidates for subsequent research.

| Total RNA extraction and reverse transcription
According to the manufacturer's instructions, the total RNA in plasma was extracted with TRIzol LS reagent (Invitrogen). We then used a DS-11 spectrophotometer machine (DeNovix, Germany, USA) to measure the quality and concentration of total RNA. The ratio of A260/A280 absorbance was used to evaluate the RNA quality. For qualified samples, the absorbance range was between 1.8 and 2.1.
We used the reverse transcription GoScript RT system (Promega) to synthesize cDNA with total RNA and random primers according to the manufacturer's instructions.

| qRT-PCR
We used the Mx3005P qRT-PCR PCR system (Stratagene) to amplify hsa_circ_0000437 GoTaq using qPCR Master Mix (Promega) according to the kit instructions. The specific convergent primer sequences of hsa_circ_0000437 are shown in Table S2. We use glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an external reference, and its primer sequence is shown in Table S2. The primers were synthesized by BGI tech. We expressed data using 2 −ΔCq , which is directly proportional to expression data.

| Cell culture and transfection
The immortalized primary cell hVICs cell line constructed from the heart valve interstitium of patients with RVHD was purchased from iCell (iCell,). hVICs cell line was cultured in basic medium for primary mesenchymal cells (iCell,), containing 1% primary mesenchymal cell culture additive (iCell,) and 10% fetal bovine serum

| Cell counting kit 8 (CCK-8) assay
The transfected cells were seeded into 96-well plates at various times (1, 2, 3, 4, and 5 days) at 5 × 10 3 per well. Then, 10 μl of CCK-8 reagent (Dojindo,) was added to each well, and cells were incubated at 37°C for 3 h. Finally, a SpectraMax M5 Microplate Reader (Molecular Devices) was used to measure the absorbance (OD) value at 450 nm to determine the degree of cell growth.

| Wound healing assay
After the transfected cells are cultured to confluence, we scraped the cells with a 200 µl pipette tip to create an artificial wound. We washed the cells with phosphate-buffered saline (PBS) to remove floating cells, obtained photographs at 0 h, and then again at 48 h.
Image Pro Plus v6.0 software package (Media Cybernetics Inc.,) was used to measure the distance of cell migration.

| Transwell migration assay
We used 24-well Transwell chambers (Costar,) to estimate cell migration. We collected transfected cells and resuspended them in a serum-free medium. We then seeded 200 µl of 8 × 10 4 cells into the upper chamber. We added 500 µl of medium containing 20% FBS to the lower chamber. After incubating for 24 h, cells were fixed with 4% paraformaldehyde, then stained with 0.1% crystal violet, and finally counted at 40× magnification with an optical microscope.

| Cell apoptosis assay
The transfected cells were removed using EDTA-free trypsin, and the cells were resuspended in binding buffer. According to the instruction manual, cells were stored at room temperature, stained with Annexin V-FITC/PI Apoptosis Kit (Multi Sciences, China) according to the instruction manual, and stored in the dark for 15 min.
A FACSCalibur flow cytometer (Becton Dickinson Co.,) was used to identify apoptotic cells.

| Cell-cycle assay
First, we starved cells with a serum-free medium to synchronize the cell cycle. We collected the transfected cells, washed them in PBS, and fixed them in 70% ethanol at -20°C overnight. We washed the cells with precooled PBS, added 1 ml PI /RNase staining buffer (Multi Sciences,) for staining, incubated for 30 min in the dark, and measured the cell cycle fraction using the FACSCalibur flow cytometer (Becton Dickinson Co.,).

| Statistical analysis
The data were analyzed using Statistical Program for Social Sciences 20.0 software (SPSS, IBM, USA), and the data were expressed as the mean ± standard deviation. We used the two-sided Student's t test to calculate the differences between groups. p < 0.05 was considered statistically different.

| Characterization and specific primers of hsa_ circ_0000437
Hsa_circ_0000437 was encoded from the 12q24.11 region. In this chromosomal region, the typical transcript is coronin 1C (CORO1C) mRNA, consisting of 19 exons. hsa_circ_0000437 is composed of exon 7 to exon 8 ( Figure 1A). We amplified hsa_circ_0000437 by designing specific convergent hsa_circ_0000437 primers. To verify the correctness of the qRT-PCR primers, we first obtained melting curves. We found that the amplified product produced only one peak ( Figure 1B), suggesting no nonspecific amplification and no primer dimers. The qRT-PCR product was also analyzed using the Sanger sequencing method. We found that the sequence of the product after hsa_circ_0000437 amplified by qRT-PCR contained a circularization site ( Figure 1C). This sequence was precisely the same as listed in hsa_circ_0000437 on the circBase website (http://circr na.org/).

| Hsa_circ_0000437 is increased in the plasma of RVHD patients relative to NRVD and normal controls
We measured the expression levels of hsa_circ_0000437 in plasma samples of 42 RVHD patients and 42 NRVD patients and normal controls. We found that the levels of hsa_circ_0000437 were significantly higher in RVHD patients than in normal controls and NRVD patients (Figure 2A), consistent with the results of the microarray analysis.

| Relationship between the expression level of has_circ_0000437 and clinicopathological factors
The Spearman and Pearson correlation analysis methods were used to analyze the correlation between the expression levels of has_ circ_0000437 and clinical parameters. Expression of has_circ_0000437 correlated with multivalvular disease (p = 0.013). Because the MV is diseased in virtually all patients with RVHD, it was impossible to simply analyze its correlation with circRNA expression levels ( Table 1).

| Relationship between has_ circ_0000437 and RVHD
We analyzed the relationship between has_circ_0000437 and RVHD by combining the RVHD and NRVD groups' clinical parameters. Single-factor logistic regression analysis showed that We analyzed the relationship between has_circ_0000437 and RVHD by combining age and gender of the RVHD and the control groups. Univariate logistic regression analysis showed that has_circ_0000437 correlated with RVHD (OR = 0.021, 95% CI: 0.003-0.128, p < 0.001). After adjusting for age and gender factors, has_circ_0000437 remained associated with RVHD (OR = 0.016, 95% CI: 0.002-0.124, p < 0.001) (Tables 4,5). These results suggest that has_circ_0000437 is an independent risk factor for RVHD.

| Diagnostic efficacy of has_circ_0000437 for RVHD
ROC curve analysis was used to determine the diagnostic efficacy of has_circ_0000437 for RVHD. The area under the ROC curve of the RVHD group compared with the NRVD group was 0.930 (p < 0.001).
The sensitivity was 0.962, and the specificity was 0.810 ( Figure 2B).
The area under the curve (AUC) of the RVHD group compared with the normal control group was 0.952 (p < 0.001). The sensitivity was 0.976, and the specificity was 0.857 ( Figure 2C). The combined AUC was 0.957 (p < 0.001). The sensitivity was 0.978, and the specificity was 0.863 ( Figure 2D). We also compared the diagnostic efficacy of electrocardiogram (ECG) and TTE with has_circ_0000437 ( Table 6).
The sensitivities of the two were 0.70 and 0.62, respectively, and the specificities were 0.62 and 0.74, respectively. 28 The diagnostic efficiency of has_circ_0000437 in the RVHD group compared with the NRVD and the normal control groups were higher than that of ECG and TTE.

| Hsa_circ_0000437 promotes proliferation of hVICs cells
Hsa_circ_0000437 overexpression plasmid and blank negative control (NC) vector pcDNA3.1 were transfected into hVICs cells. siRNA and NC of hsa_circ_0000437 were also transfected into hVICs cells and were found to upregulate and downregulate the expression levels of hsa_circ_0000437 in hVICs cells ( Figure 3A,B). Using the CCK-8 assay, we found that hsa_circ_0000437 siRNA significantly inhibited the proliferation of hVICs cells ( Figure 3C). When F I G U R E 2 Expression levels of hsa_ circ_0000437 in RVHD plasma samples and their diagnostic value. (A) Expression levels of hsa_circ_0000437 in the RVHD group were significantly higher than those in the NRVD and the normal control groups (n = 42). (B) AUC of the RVHD group compared with the NRVD group. (C) AUC of the RVHD group compared with the normal control group. (D) AUC after the combination of the RVHD, NRVD, and normal control groups. AUC: the area under the curve, ***p < 0.001 hsa_circ_0000437 was overexpressed, it promoted the proliferation of hVICs ( Figure 3D).

| Hsa_circ_0000437 promotes migration of hVICs cells
Wound healing assay revealed that hsa_circ_0000437 siRNA significantly reduced cell migration ( Figure 4A). When hsa_circ_0000437 was overexpressed, cell migration was increased ( Figure 4B). The Transwell assay revealed that hsa_circ_0000437 siRNA significantly reduced the number of cells migrating to the lower chamber ( Figure 4C). When hsa_circ_0000437 was overexpressed, the number of cells migrating to the lower chamber increased ( Figure 4D).
These findings suggest that hsa_circ_0000437 promotes the migration ability of hVICs.

| Hsa_circ_0000437 inhibits apoptosis and affects cell cycle in hVICs cells
Using flow cytometry analysis, we found that hsa_circ_0000437 siRNA significantly increased the number of apoptotic cells ( Figure 5A). When hsa_circ_0000437 was overexpressed, the number of apoptotic cells was reduced ( Figure 5B). These findings suggest hsa_circ_0000437 inhibits apoptosis in hVICs.
Also using flow cytometry analysis, we found that hsa_ circ_0000437 siRNA blocked the cell cycle in the G0/G1 phase ( Figure 5C). When hsa_circ_0000437 was overexpressed, the cell cycle was blocked in G2/M phases ( Figure 5D). These findings suggest that hsa_circ_0000437 promoted cell cycle progression.

| Positioning and sponge function of hsa_ circ_0000437
The location of hsa_circ_0000437 in hVICs cells was confirmed using FISH. We found that hsa_circ_0000437 (red fluorescence) was located in the cytoplasm ( Figure 6A). Based on these results, we conclude that hsa_circ_0000437 is a circRNA from the CORO1C locus, located in the cytoplasm. We used circbank and circular RNA interaction software to predict the miRNAs bound to hsa_ circ_0000437. After taking the intersection, we found that they were hsa-miR-502-5p, hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, and hsa-let-7f-5p ( Figure 6B-F). These miRNAs may co-localize with hsa_circ_0000437 and act as sponges in the cytoplasm. of the RVHD group was significantly different from that of the NRVD group. Multivariate logistic regression analysis was used to compare the expression levels of has_circ_0000437 between RVHD, NRVD, and the control group; we found that has_ circ_0000437 was a significantly independent influencing factor of RVHD. We also found no significant difference in the expression of has_circ_0000437 between the NRVD and control groups, suggesting that has_circ_0000437 is independent of RVHD and its underlying pathogenesis is different from NRVD. We found that the  The role of miRNA sponges in the biological function of circRNA is an intense area of research. MiRNA is an essential regulator of gene expression that targets mRNA to prevent its translation or promote its degradation. There are several miRNA-binding sites on cir-cRNA. In the cytoplasm, they bind miRNA to inhibit miRNA activity and enhance the expression of target genes. 34 In the present study, we found that has_circ_0000437 affected the proliferation, migra- and aggravates the development of the disease. In chronic RVHD patients, there is an increase in some inflammatory indicators in the serum, especially IL-6, suggesting that it plays a role in chronic valve changes in RVHD. 38 Based on our findings, has_circ_0000437 can be used as a biomarker for the diagnosis of RVHD. It can also affect the biological function of immortalized primary cell hVICs constructed from heart valve tissue of patients with RVHD. These findings suggest that has_circ_0000437 promotes the progression of RVHD and may participate in the pathogenesis of RVHD through sponge absorption of hsa-let-7c-5p and hsa-let-7f-5p. These findings provide a basis for the discovery of potential therapeutic targets for RVHD. The specific mechanism of has_circ_0000437 to promote the occurrence and progression of RVHD needs to be further explored. Of course, the mechanism that has_circ_0000437 promotes the progress of RVHD has yet to be studied. Has_circ_0000437 may affect the development of RVHD by regulating or targeting binding proteins or RNA. These mechanisms need to be further studied. In addition, we need to use animal experiments to verify our findings. For the feasibility of clinical application, further clinical trials and ethical approval are needed.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets analyzed during the current study are available from the corresponding author upon reasonable request.