Long Noncoding RNA AF131217.1 Regulated Coronary Slow Flow-Induced Inflammation Affecting Coronary Slow Flow via KLF4

Introduction This study investigated the correlation between the levels of long noncoding ribonucleic acids (lncRNAs) AF131217.1 and coronary slow flow (CSF). Methods A total of 22 patients in the high-sensitivity C-reactive protein (hsCRP) group diagnosed with CSF from January 2018 to December 2018 were enrolled in this study. Coronary flow velocity was determined using the thrombolysis in myocardial infarction frame count (TFC) method. Results: LncRNA AF131217.1 expression in the CSF model was activated. Mean TFC was positively correlated with lncRNA AF131217.1 levels and hsCRP levels. LncRNA AF131217.1 induced inflammation factor levels in the in vitro model. Micro ribonucleic acid (miR)-128-3p is a target spot of lncRNA AF131217.1 on the inflammation in vitro model via Kruppel-like factor (KLF) 4. MiR-128-3p reduced inflammation factor levels (tumor necrosis factor alpha, interleukin [IL]-6, IL-1β, and IL-18). Conclusion: Thus, lncRNA AF131217.1 promoted inflammation in the regulated CSF via KLF4 by miR-128-3p.


INTRODUCTION
The coronary slow flow (CSF) phenomenon is defined as the presence of delayed perfusion of peripheral coronary artery identified by coronary arteriography after excluding coronary artery disease, cardiomyopathy, valvular disease, congenital heart disease, connective tissue disease, etc. Although coronary angiography in patients with slow blood flow shows no stenosis of coronary artery, recurrent cardiovascular events still occur, generally clinically manifested by angina, arrhythmia, or acute coronary syndrome (ACS), which may be associated with CSF and usually requires emergency admission [1,2] . The exact pathological using a cineangiography. CSF was diagnosed if TFC > 27 in at least one coronary artery. Three cardiologists who were blinded to the clinical findings independently assessed the TFC.

Real-Time Reverse Transcription Polymerase Chain Reaction
Total RNA was isolated with the RNeasy Micro Kit (Qiagen) and cDNA was synthesized with the Maxima First Strand cDNA Synthesis Kit. Quantitative real-time polymerase chain reaction was performed using SYBR Green Master Mix. Relative fold changes were calculated using the 2 -△△Ct method.

Western Blots
Cells were lysed with radioimmunoprecipitation buffer, and protease and phosphatase inhibitors (Sigma-Aldrich) were added. Protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime, Jiangsu, China). Equal amounts of proteins were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking with no-fat skim milk, the membranes were incubated with primary antibodies (KLF4, RhoF, nuclear factor kappa B [NF-κB], and glyceraldehyde-3-phosphate dehydrogenase) diluted in Tris Buffered Saline with Tween 20 overnight at 4°C and then with mechanism of CSF is still unclear. At present, there is no standardized therapeutic measures for CSF in clinical practice, with inconsistent reported efficacy of drug treatment [2] . Studies have shown that CSF is strongly associated with cardiovascular adverse events, such as arrhythmia, ACS, and sudden cardiac death. In addition, the levels of high-sensitivity C-reactive protein (hsCRP), vascular cell adhesion molecules, and intercellular adhesion molecules are significantly increased in CSF patients than in healthy individuals, which is also positively correlated with thrombolysis in myocardial infarction frame count (TFC), indicating endothelial activation and inflammatory response in CSF patients [2,3] .
Kruppel-like factors (KLF) are a type of transcription factors with zinc-finger structure, which is characterized by the three C2H2 zinc-finger structures at the carboxyl terminal [4] . KLF family members are widely involved in the regulation of multiple life activities, including cell proliferation, apoptosis, differentiation, and embryonic development [5] . The abnormal function of KLF family members is strongly associated with metabolic diseases, cardiovascular diseases, and cancer. KLF4, originally isolated from the gastrointestinal tract, is one of the transcriptional regulators required for the early differentiation of adipocytes [6] . It activates the expression of the CCAAT/enhancer binding protein beta (C/ EBPβ) gene by binding itself to the promoter of C/EBPβ, thereby activating the downstream cascade of fat cell differentiation to promote this differentiation [6,7] . In recent years, KLF4 has become a research hotspot in relevant fields due to its regulatory roles on chronic inflammatory responses of various cells [4] .
Long noncoding ribonucleic acids (lncRNAs) are a type of noncoding RNA with over 200 bp in length [8] . To date, hundreds of thousands of eukaryotic lncRNAs have been found, most of which are lowly conservative and involved in the pathogenesis and progression of a series of diseases, including tumors, nervous system disorders, metabolic diseases, reproductive development, and cardiovascular diseases [9] . Long intergenic noncoding RNA-p21, a p53-induced lncRNA, is able to inhibit cell proliferation and promote apoptosis during atherosclerosis [9,10] . This study investigated the correlation between the levels of lncRNA AF131217.1 and CSF.

Study Subjects and CSF Diagnosis
A total of 22 patients in the hsCRP group diagnosed with CSF at the Affiliated Chinese Medicine Hospital of Xinjiang Medical University, from January 2018 to December 2018, were enrolled in this study. All patients had stenosis of lumen diameter < 40% (no significant vascular lesion). The control group was 24 patients who did not meet the abovementioned criteria. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or National Research Committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. This study was approved by the Ethics Committee of the Affiliated Chinese Medicine Hospital of Xinjiang Medical University. Written informed consent was obtained from all individual participants included in the study. TFC was used to quantitatively measure coronary blood flow the horseradish peroxidase-conjugated secondary antibody for two hours at room temperature. Protein bland was detected by electrochemiluminescence chromogenic substrate kits and quantified by Image Lab 3.0 (Bio-Rad Laboratories, Inc.).

Statistical Analysis
All data are expressed as the mean ± standard error of mean. P<0.05 was considered statistically significant. Statistical significance between groups of data was calculated by Student's t-test or one-way analysis of variance and Tukey's post-test.

LncRNA AF131217.1 Expression in the CSF Model
Initially, we sought to validate lncRNA AF131217.1 expression in the CSF model. We found using gene chip that six genes were downregulated and 11 genes were upregulated in the CSF model ( Figures 1A and 1B). LncRNA AF131217.1 expression in the CSF model was activated, compared with the control group ( Figure 1C). Mean TFC was positively correlated with lncRNA AF131217.1 levels ( Figure 1D) and hsCRP levels ( Figure 1E).

DISCUSSION
CSF is defined as the presence of delayed perfusion of peripheral coronary artery but without obvious lesions in the coronary arteries diagnosed by coronary arteriography after excluding coronary spasm, coronary artery dilatation, coronary angioplasty, cardiomyopathy, heart valvular disease, autoimmune diseases, tumors, and other important organs or systemic diseases. More and more attention are being paid to the research of CSF, however, the etiology and pathogenesis of CSF remain unclear [11][12][13] . We showed that lncRNA AF131217.1 expression in the CSF model was activated. Mean TFC was positively correlated with lncRNA AF131217.1 levels and hsCRP levels. These characteristics suggest that lncRNA AF131217.1 participated in the pathophysiological process of CSF.
Lu et al. [14] showed that shear-sensitive lncRNA AF131217.1 reduced inflammation in HUVECs via regulation of KLF4. Cardiovascular disease is a common disease that threatens the health of human beings, especially the elderly [15] . The annual number of people dying from cardiovascular disease ranks the first globally [15] . LncRNAs are a type of noncoding RNAs with over 200 nt in length, which are also the most abundantly expressed RNA [16,17] . Accumulative studies have revealed that lncRNAs play an important regulatory role in the pathogenesis and development of cardiovascular diseases [16] . Interestingly, lncRNA AF131217.1 induced inflammation factor levels and played an important role in the inflammation of CSF.
Wang et al. [18] showed that miR-128-3p accelerates cardiovascular calcification in type 2 diabetes mellitus rats. Studies have shown that CSF is closely associated with cardiovascular adverse events, including arrhythmia, ACS, and sudden cardiac death [19,20] . When coronary angiography is performed on patients with suspected cardiovascular disease, the detection rate of CSF is approximately 1% [19] . Recent studies have demonstrated that inflammatory response plays an important role in the pathogenesis of the KLF family, KLF2 and KLF15 can also competitively bind with P300 [24] . It has been reported that KLF4 can bind to the active subunit P65 of NF-κB in vascular endothelial cells, to promote the binding of P65 to its downstream inflammatory factor vascular cell adhesion molecule 1, thereby playing a pro-inflammatory role [25] . Meanwhile, we found that KLF4 is a target spot of miR-128-3p on the inflammation in vitro model.

CONCLUSION
In summary, lncRNA AF131217.1 expression in the CSF model was activated and promoted inflammation by the suppression of miR-128-3p via KLF4/RhoF/NF-κB signal pathway, and this may of CSF [19,21] . Our study showed that miR-128-3p is a target spot of lncRNA AF131217.1 on the inflammation in vitro model. KLF4 has been reported to physically bind to the active subunit P65 of transcription factor NF-κB in vascular endothelial cells to prevent the nuclear translocation of P65, thereby exerting its antiinflammatory role by inhibiting the transcriptional activation on downstream inflammatory factors of P65 [22] . In endothelial cells and immune cells, P300 can bind with the active subunit P65 of NF-κB to induce acetylation of P65, thereby promoting the transcriptional activation on downstream inflammatory factors by P65 [23] . Overexpressed KLF4 can competitively bind to P300, inhibiting the binding of P300 with P65 to attenuate its transcriptional activity, subsequently exerting an anti-inflammatory effect [23] . As members play an important role in the pathogenesis of CSF. An elevated plasma lncRNA AF131217.1 level may indicate the presence of CSF. Further studies are needed to establish clinical significance of increased plasma lncRNA AF131217.1 levels and to investigate the therapeutic efficacy of targeting miR-128-3p/KLF4/RhoF/ NF-κB signal pathway.