Hsa_circ_0000345 regulates the cellular development of ASMCs in response to oxygenized low‐density lipoprotein

Abstract The interaction between circRNAs and atherosclerosis has been extensively studied. However, more novel circRNAs need to be explored to help establish a perfect regulatory network. In the present research, hsa_circ_0000345 was demonstrated to regulate cellular development of oxygenized low‐density lipoprotein (ox‐LDL)‐treated aortic smooth muscle cells (ASMCs), which was closely related to the occurrence and progress of atherosclerosis. Ox‐LDL exposure remarkably decreased hsa_circ_0000345 expression in ASMCs. Transfection‐induced hsa_circ_0000345 overexpression activated cell viability (detected by an MTT assay) and restrained cellular apoptosis (analysed by flow cytometry) in the atherosclerosis cellular model. While down‐regulation of hsa_circ_0000345 reduced cell viability and promoted cell apoptosis. In addition, the data of the cell cycle distribution analysis and trans‐well assay indicated that cell cycle progression was arrested at the G1 phase while cell invasion was enhanced in ASMCs following treatment of ox‐LDL in the context of hsa_circ_0000345 OE plasmids. In addition, up‐regulation of hsa_circ_0000345 supported HIF‐1α at both the mRNA and protein level, and down‐regulation of hsa_circ_0000345 reduced HIF‐1α expression. Overall, the above findings revealed that hsa_circ_0000345 was a dramatic regulator of ASMCs proliferation, apoptosis and invasion in response to ox‐LDL treatment. Hsa_circ_0000345 was identified as a protector of cell viability during ox‐LDL induced cell development.

hsa_circ_0000345 expression in ASMCs. Transfection-induced hsa_circ_0000345 overexpression activated cell viability (detected by an MTT assay) and restrained cellular apoptosis (analysed by flow cytometry) in the atherosclerosis cellular model.
While down-regulation of hsa_circ_0000345 reduced cell viability and promoted cell apoptosis. In addition, the data of the cell cycle distribution analysis and transwell assay indicated that cell cycle progression was arrested at the G1 phase while cell invasion was enhanced in ASMCs following treatment of ox-LDL in the context of hsa_circ_0000345 OE plasmids. In addition, up-regulation of hsa_circ_0000345 supported HIF-1α at both the mRNA and protein level, and down-regulation of hsa_ circ_0000345 reduced HIF-1α expression. Overall, the above findings revealed that hsa_circ_0000345 was a dramatic regulator of ASMCs proliferation, apoptosis and invasion in response to ox-LDL treatment. Hsa_circ_0000345 was identified as a protector of cell viability during ox-LDL induced cell development.

K E Y W O R D S
aortic smooth muscle cells, atherosclerosis, HIF-1α, hsa_circ_0000345 of ASMCs is an important part of monitoring the development of atherosclerosis.
As an innovative classification of non-coding RNAs, circRNA biogenesis relies on a canonical splicing mechanism and is affected by a combination of cis-acting elements and trans-splicing factors, such as heterogeneous nuclear ribonucleoprotein (hnRNPs) and SR proteins. Moreover, the functions of circRNA depend on acting as 'sponges' and regulating protein functions and, therefore, circRNAs participate in various physiological and pathological processes. 5 In the cardiovascular system, it has been demonstrated that cir-cRNAs have vital regulatory roles in angiogenesis, smooth muscle cell functions and cardiac injury responses. 6,7 Circ_ANRIL prevented ribosome biogenesis and pre-rRNA processing by binding to PES1 (which is an essential 60S-preribosomal assembly factor) and simultaneously activated P53 activity, eventually leading to the inhibition of vascular smooth muscle cell (VSMCs) proliferation and enhancement of cellular apoptosis. Therefore, circ_ANRIL was considered an anti-atherosclerosis factor. 8 Circ_Lrp6 was confirmed to be highly expressed in blood vessels and associated with vascular lesions. In addition, recent studies have shown that circ_Lrp6 regulated the proliferation and migration of VSMCs by sponging miR-145. 9 More importantly, circRNAs also participated in modulating cell fates in VMSCs under hypoxic conditions. For instance, silencing of circ_000595 decreased hypoxia-induced VSMCs apoptosis by augmenting miR-19a, which has been shown to arrest the cell cycle by impeding cyclinD1 expression. 10,11 cZNF292, another hypoxia-induced circRNA, was shown to stimulate cell proliferation. 12 All of the above data suggest the possibility of circRNA of atherosclerosis regulation.
Systemically expressed hypoxia inducible factors (HIFs) critically influence adaptive physiological responses including vascular remodelling. Many target genes are transcriptionally activated by HIF-1α/HIF-1β heterodimers containing vascular endothelial growth factor receptors. Complete knockout of HIF-1α led to vascular regression and embryonic lethality. Studies have indicated that rescue of HIF-1α expression could restore hind limb ischaemia in rats with diabetes and atherosclerosis. 13 A recent phase I trial shown that patients with limb ischaemia might benefit from a constitutively active HIF-1α hybrid. 14 Therefore, increasing HIF-1α expression may be capable of promoting vascular remodelling and improving ischaemic symptoms in atherosclerosis. Last but not least, circRNAs and HIF-1α also closely interact. In hepatocellular carcinoma, has-circ-0046600 enhanced the expression of HIF-1α through competitive binding with miR-640. 15 In addition, knockdown of circ_PIP5K1A suppressed HIF-1α by promoting the sponging of miR-600. 16 In summary, cir-cRNAs exerted dramatic effects on hypoxia and atherosclerosis; however, the role of circRNAs/HIF-1α in atherosclerosis needs to be further explored.
Low-density lipoprotein (LDL) is an important risk factor for atherosclerosis, and oxidative modified LDL (oxLDL) promotes atherosclerosis. 17,18 CircRNA microarray revealed hsa_circ_0000345 (chr11:77409531-77413540) was down-regulated in oxLDL induced human umbilical vein endothelial cells (HUVECs). 19 In the current research, we investigated the role of hsa_circ_0000345 in the cellular development of oxLDL induced ASMCs to clarify potential mechanisms of atherosclerosis.

| Cell culture
Human aortic smooth muscle cells (ASMCs) were obtained from KeyGEN Bio TECH (Nanjing, China) and cultured in 10% foetal bovine serum (FBS)-containing F12K medium with penicillin and streptomycin. The cell culture incubator chamber was maintained at 5% CO 2 and 37°C. The cell culture medium was replaced every 2 days, and all experiments used cells within 15 passages. medium, and we mixed the liquids 5 minutes later. Subsequently, 400 µL of the mixture was added to each well after 20 minutes. After transfection for 6-8 hours, the medium was replaced with normal medium. All experiments were performed the day after transfection.

| RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from cells using TRIzol (Invitrogen) following the manufacturer's instructions. First-strand cDNA was synthesized from total RNA using a reverse transcription system (Takara, China).
The Sangon Biotech (Shanghai, China) provided all primers and GAPDH served as the reference gene. The primers used for qRT-PCR were as follows: hsa_circ_0000345, 5'-GTGGCAATTATCCCCAAACTGT-3'

| MTT assays
MTT assays were used to measure cell viability. ASMCs were digested, resuspended and seeded in 96-well plates. After different treatments for the indicated days, cells were incubated with 40 µL of MTT (5 mg/mL) in each well, and the supernatant was discarded before 150 µL DMSO was added. After shaking for 10 minutes in the dark, the crystals were completely dissolved. Finally, the absorbance was detected using a microplate reader at 490 nm.

| Flow cytometry for cellular apoptosis
Cellular apoptosis was detected using the Annexin V-FITC/PI apoptosis detection kit (Bestbio, Shanghai, China) and flow cytometry (Beckman Coulter, Brea, CA, USA). After transfection and the indicated treatments, EDTA-free trypsin (Beyotime, Shanghai, China) was used to digest the cells. We washed and collected cells twice by centrifugation at 225 g for 5 minutes. We then discarded the supernatants and resuspended cells with binding buffer (500 µL).
Subsequently, 5 µL of Annexin V and 5 µL of propidium iodide (PI) were added into the cell suspension and incubated for 10 minutes.
Finally, the percentage of specific cell populations was measured by flow cytometry to analyse the cellular apoptosis rate.

| Trans-well assays
Pre-treated ASMCs were collected and resuspended with non-serum

| Protein extraction and Western blot
ASMCs were collected after each treatment, and then, cold PBS and lysis buffer (PMSF: RIPA = 1:100) were applied to wash and lyse the cells, respectively. After 30 minutes, cell lysates were collected and centrifuged at 22 500 g for 30 minutes, followed by transferring the supernatants. Then, total protein was mixed with SDS-PAGE loading buffer and separated with a 10% SDS-PAGE gel. Subsequently, we electric-blotted the target protein band onto PVDF membranes (Millipore Corp, Bedford, MA, USA). The membranes were blocked for 2 hours, washed with TBST and then incubated with diluted antibody against HIF-1α (1:1000) and GAPDH (1:1000) overnight in a refrigerator (4°C). Finally, HRP-conjugated secondary antibody (ZSGB-Bio, Beijing, China) was added to the membranes for 1 hour at room temperature, and an ECL-chemiluminescent kit (ECL-plus, Hanover, NH, USA) was used to expose the immunoreactive blots.
The relative protein expression was quantified by ImageJ 1.49 (National Institutes of Health).

| Statistical analysis
All experiments were performed in triplicate. Statistical analyses were performed using Student's t test, one-way ANOVA followed by Bonferroni's multiple comparison tests, as appropriate. Statistical data were performed using GraphPad Prism 6. P < 0.05 was considered statistically significant.

| Ox-LDL decreases hsa_circ_0000345 level
In the present research, we conducted ox-LDL to development an atherosclerosis cellular model. 19,20 To investigate whether hsa_ circ_0000345 played a role in the response of ASMCs to atherosclerosis, we examined whether hsa_circ_0000345 level was altered in response to ox-LDL. ASMCs were exposed to ox-LDL at gradient concentrations (0, 40, 60, 80 or 100 mg/L). The RNA expression of hsa_circ_0000345 was decreased by ox-LDL exposure ( Figure 1A).
Hsa_irc_0000345 had a concentration-dependent reduction in expression ( Figure 1B). Thereafter, 100 mg/L was used as an optimal concentration, and the level of hsa_circ_0000345 was measured at the indicated time points (0, 6, 12, 24 or 48 hours). As expected, a time-dependent decline in hsa_circ_0000345 was observed in response to ox-LDL ( Figure 1C). The reduction of hsa_circ_0000345 started at 6 hours, which indicated that hsa_circ_0000345 was sensitive to ox-LDL exposure in ASMCs and might play a key role in the early stage of atherosclerosis. In conclusion, hsa_circ_0000345 expression is inhibited by atherosclerosis. Therefore, it was necessary to explore the functions of hsa_circ_0000345.

| Overexpression of hsa_circ_0000345 activates cell viability and restrains cell apoptosis
To determine whether a specific role for hsa_circ_0000345 exists in modulating the destiny of ASMCs in response to ox-LDL exposure, we constructed hsa_circ_0000345 OE plasmids and transfected them into ASMCs. As Figure 2A

| Overexpression of hsa_circ_0000345 induces G1 phase cycle arrest and strengthens cell invasion
To further explore the effects of hsa_circ_0000345 in atherosclerosis, we assessed whether cellular proliferation or invasion was affected by hsa_circ_0000345 overexpression in ox-LDL pre-treated ASMCs. We utilized an analysis of cell cycle distribution to evaluate the cell proliferation. Compared with the control or vector, the proportion of cells in the G1 phase increased and the proportion of cells in the S or G2 phase decreased significantly after plasmid transfection ( Figure 3A and B), indicating that hsa_circ_0000345 overexpression induced the cycle arrest at G1 phase. We also determined whether hsa_circ_0000345 overexpression via OE plasmids would have effects on cellular invasion.
We conducted trans-well assays in ASMCs and found that hsa_ circ_0000345 overexpression significantly enhanced cell invasion ( Figure 3C and D). These data suggest that hsa_circ_0000345 represses cellular proliferation and facilitates cellular invasion in ox-LDL-treated ASMCs.

| Overexpression of hsa_circ_0000345 increased the protein expression levels of HIF-1α
To study the molecular basis that underlies the hsa_circ_0000345-

| Down-regulation of hsa_circ_0000345 inhibits cell proliferation, strengthens cell apoptosis and decreased the protein expression levels of HIF-1α
We transfected si-circ_0000345-1 and si-circ_0000345-2 into ox-LDL treated ASMCs. As Figure 5A shows, the siRNAs significantly reduced the expression of hsa_circ_0000345. MTT assays showed down-regulation of hsa_circ_0000345 inhibited cell viability as compared to controls ( Figure 5B). Flow cytometry results showed hsa_circ_0000345 silencing promoted cellular apoptosis from approximately 60% to 70% in ox-LDL pre-treated ASMCs ( Figure 5C).
Relative BAX/Bcl-2 protein expression was also increased by downregulation of hsa_circ_0000345 ( Figure 5D-G). Down-regulation of hsa_circ_0000345 also reduced the mRNA and protein expression of HIF-1α ( Figure 5H-J). These results suggest that hsa_circ_0000345 silencing inhibited cell viability and HIF-1α, promoted cellular apoptosis in ox-LDL-treated ASMCs.  Control represents cells without any transfection (exposed to 100 mg/L ox-LDL for 24 h); vector represents cells transfected with empty vector (exposed to 100 mg/L ox-LDL for 24 h); OE circ_0000345 represents cells transfected with hsa_circ_0000345 OE plasmids (exposed to 100 mg/L ox-LDL for 24 h). Values were presented as the mean ± SD of three independent experiments. *P < 0.05, significance versus the control group. An empty vector was utilized as a negative control The distribution of each cell cycle was analysed by flow cytometry. (C and D) Cell migration was illustrated by trans-well assays. Control represents cells without any transfection (exposed to 100 mg/L ox-LDL for 24 h); vector represents cells transfected with empty vector (exposed to 100 mg/L ox-LDL for 24 h); OE circ_0000345 represents cells transfected with hsa_circ_0000345 OE plasmids (exposed to 100 mg/L ox-LDL for 24 h). Values were presented as the mean ± SD of three independent experiments. *P < 0.05, significance versus the control group F I G U R E 4 Expression of HIF-1α mRNA and protein in response to hsa_circ_0000345 overexpression. (A) The relative mRNA level of HIF-1α was measured by qRT-PCR. (B and C) The relative protein level of HIF-1α was detected through Western blot. Control represents cells without any transfection (exposed to 100 mg/L ox-LDL for 24 h); vector represents cells transfected with empty vector (exposed to 100 mg/L ox-LDL for 24 h); OE circ_0000345 represents cells transfected with hsa_circ_0000345 OE plasmids (exposed to 100 mg/L ox-LDL for 24 h). Values were presented as the mean ± SD of three independent experiments. *P < 0.05, significance versus the control group. GAPDH was utilized as a negative control such as the balance between apoptosis and survival, depend on cellular systems that are regulated by non-coding RNA. In our study, hsa_circ_0000345 was reduced after treatment with different concentrations of ox-LDL. Therefore, it will be interesting to determine whether the multiple effects of hsa_circ_0000345 participate in the dual role of ox-LDL.
The present work has shown that overexpression of hsa_ circ_0000345 induced a reduction of HIF-1α at both the transcriptional and translational level. The ability of HIFs to extensively modulate hypoxia has been appreciated for some time. Hypoxia is an inevitable manifestation of atherosclerosis due to the thickening of blood vessel walls. 27 The most fundamental function of HIFs is angiogenesis management, which is an important risk factor in the progression of atherosclerosis. The mechanisms of angiogenesis have also been extensively studied. Migration inhibitory factor (MIF) was involved in foam cell transformation and vascular remodelling. [28][29][30] In human umbilical artery smooth muscle cells si-NC represents cells transfected with NC siRNA (exposed to 100 mg/L ox-LDL for 24 h); si-circ_0000345-1 represents cells transfected with hsa_circ_0000345 siRNA-1 (exposed to 100 mg/L ox-LDL for 24 h); si-circ_0000345-2 represents cells transfected with hsa_ circ_0000345 siRNA-2 (exposed to 100 mg/L ox-LDL for 24 h). Values were presented as the mean ± SD of three independent experiments. *P < 0.05, significance versus the control group. si-NC was utilized as a negative control thrombospondin-1 (THBS1) could be neutralized by HIF-1 knockdown, and cell migration was simultaneously detained. The above data indicated the significant association between HIFs and the routine development of VSMCs. Meanwhile, many studies have documented a correlation between circRNAs and HIFs. In hepatocellular carcinoma, has_circ_0001730 inhibited tumorigenesis, development and metastasis by decreasing the expression of HIF-1α. 31 Has_circ_0010729 suppressed cell proliferation and promoted apoptosis, and has_circ_0010729 was coexpressed with HIF-1α in HUVECs. 32 Interestingly, our results confirmed that hsa_ circ_0000345 enhanced HIF-1α at the mRNA and protein level, showing an important connection between circRNAs and HIF. In addition, ox-LDL induced a variety of transcription factors, including HIF-1α, during the atherosclerosis development, which established the significance of ox-LDL/circ_0000345/HIF-1α signalling in the occurrence and development of atherosclerosis.
In summary, hsa_circ_0000345 could regulate the cellular development of ox-LDL induced ASMCs. However, a detailed study of hsa_circ_0000345 will still be the focus of subsequent research.