Oxidized low-density lipoprotein is a common risk factor for cardiovascular diseases and gastroenterological cancers via epigenomical regulation of microRNA-210

Hyperlipidemia, including the oxidized low-density lipoprotein (oxLDL) accumulation, is a risk and highly associated with the development of cancers and cardiovascular diseases. microRNA-210 (miR-210), a hypoxia-responsive microRNA regulated by HIF-1α, has been implicated in cancer and cardiovascular disease formation. Furthermore, Bioinformatics analysis revealed that the promoter of the miR-210 gene contains CpG-rich regions. It is unclear whether miR-210 expression could be epigenetically regulated in these disease progresses. The study aimed to explore the relationships between lipid and miR-210 in the context of cardiovascular disease and gastrointestinal cancer. We demonstrated oxLDL can decrease methylation in the miR-210 promoter to up-regulate miR-210. HIF-1α can bind to miR-210 promoter, but this HIF-1α binding site can be blocked by methylation. We showed that subjects of carotid atherosclerosis, stroke patients and cancer patients had hypomethylation in the miR-210 promoter, especially the HIF-1α binding site. Furthermore, miR-210 can directly inhibit sprouty-related EVH1 domain 2 (SPRED2) expressions, and SPRED2 reduces cell migration via ERK/c-Fos/MMPs pathways. Increased miR-210 and reduced SPRED2 levels were found in aorta of mice under high-fat diet and tumor tissues, which implied that miR-210 can be an underlying mechanism to explain oxLDL as a common risk factor for cardiovascular disease and gastrointestinal cancer.


RNA isolation, quantitative real-time reverse transcription-polymerase chain reaction (PCR), and immunoblotting analysis
Total RNA extraction from cell culture or mouse aorta was carried out using Trizol ® according to the manufacturer's instructions. Complementary (c)DNA was synthesized from 1 μg of total RNA using a random primer and the MultiScribe(tm) Reverse Transcriptase Kit. For miR-210 and U44 detection, cDNA was synthesized using the TaqMan ® MicroRNA Assay. For the quantitative real-time PCR, specific primers for human HIF-1α, SPRED1, SPRED2, and SPRED3, and GAPDH were designed and are listed in Suppl. Table 6. Gene expression was measured with preoptimized conditions using an ABI 7900 real-time PCR machine (Applied Biosystems, Waltham, MA, USA). For detecting protein expression levels, total proteins were collected from cells and tissues. The methods for quantitative real-time PCR and immunoblotting assay were detailed as previously (1).

Transwell migration assays and Wound healing assay
To conduct the transwell migration assays, 5 × 10 4 cells were placed in the top chamber of a transwell migration chamber (8 μm; Millipore, Billerica, MA, USA). The detail methods of transwell migration assays and wound healing assay were carried out as previously described (2).

Construction of the SPRED2 3'UTR reporter plasmid and mutagenesis
A PCR was performed using the primers (shown in Suppl. Table 6) specifically for the SPRED2 3'UTR, of which the forward primer was SpeI-site-linked and the reverse primer was MluI-site-linked. HASMC genomic DNA was used as the template. PCR products were digested with SpeI/MluI and cloned downstream of the luciferase gene in the pMIR-REPORT luciferase vector (Ambion, Waltham, MA, USA). This vector was sequenced and named pMIR-SPRED2-3UTR. Sitedirected mutagenesis of the miR-210 target-site in the SPRED2 3'UTR was carried out using a QuikChange® Site-Directed Mutagenesis Kit (Stratagene, Heidelberg, Germany) and named pMIR-SPRED2-mutant 3UTR, in which pMIR-SPRED2-3UTR was used as a template. The methods for reporter assay were detailed as previously (1).

Construction of the miR-210 promoter reporter plasmid
To construct the miR-210 promoter reporter plasmid, a 550-bp fragment of the miR-210 promoter containing the HIF-α-binding site was isolated by a PCR (the primers are listed in Suppl. Table 6). After digestion of the PCR product with XhoI and HindIII, the insert was cloned into the pGL3 reporter vector (Promega, Madison, WI, USA) to create the pGL3-miR-210 promoter vector.

SUPPLEMENTARY DATA
www.impactjournals.com/oncotarget/ An overlapping PCR was carried out to construct the miR-210 promoter reporter plasmid where the HIF-1αbinding site was truncated. In this procedure, the pGL3-miR-210 promoter vector was used as a template with suitable primers listed in Suppl. Table 6. All plasmid sequences were confirmed by DNA sequencing.

Statistical analysis
Student's t-test was used to compare all experimental results. A p value of < 0.05 was considered significant. Methylation levels of miR-210 promoter in atherosclerosis and stroke patients than controls. Genomic DNA was collected from the whole blood of control subjects (n = 10), and atherosclerosis (n = 10) and stroke (n = 47) patients. After bisulfite treatment and pyrosequencing, methylation levels were determined by PyroMark Q24 (Qiagen). Data are means ± SD of three experiments. *P < 0.05. C to E. Methylation levels of the miR-210 gene promoter in different cancer samples. Genomic DNA was collected from patients' cancer tissues. After bisulfite treatment and pyrosequencing, methylation levels were determined by PyroMark Q24 (Qiagen). (C) Non-early relapse (n = 30) vs. early relapse (n = 8) in colorectal cancer patients, (D) non-recurrence (n = 11) vs. recurrence (n = 10) in oral cancer patients, and (E) stage II (n = 11) vs. stage III (n = 9) gastric cancer. Data are means ± SD of three experiments. *P < 0.05. Any outlier > mean ± 3 SD needs to be removed. Mice with HT-29 xenografts were injected every 3 days with PBS (n = 3) and 5 mg/kg AZA (n = 4). The tumor volume and mouse weight are respectively measured everyday. After treatment for 3 weeks, total RNAs were extracted from tumor tissue. The HIF-1αgene expression levels were determined by quantitative real-time PCR. Data are means ± SD of three experiments. *P < 0.05. Figure S7: The expression levels of miR-210, SPRED2, and HIF-1α in colorectal cancer, oral cancer and gastric cancer patients. Total RNA was extracted from tumor tissues. The relative expression levels of miR-210, SPRED2, and HIF-1α were respectively measured by quantitative real-time PCR. A. non early relapse (n = 38) V.S. early relapse (n = 14) in colorectal cancer patients. B. non-recurrence (n = 16) V.S. recurrence (n = 13) in oral cancer patients. C. stage II (n = 11) V.S. stage III (n = 9) in gastric cancer patients. D. The correlation between miR-210 and SPRED2 levels. Data are means ± SD of three experiments. *P < 0.05.