A novel miR‐82 target PA2G4 and FHL3 checking myocardial ischemia

Dear Editor, The incidence and mortality of ischemic heart disease increase all over theworld, producing immense health and economic burdens.1 MicroRNAs (miRNAs) regulatemultiple biological processes.2 It has been identified thousands of distinct miRNAs in humans and model organisms. The expression of miRNAs in heart tissues has been studied.3 miRNAs can be potentially used as therapeutic targets and noninvasive clinical biomarkers.4 A novel miR-82 was discovered in amulticenter, doubleblind, randomized, large-scale, and placebo-controlled clinical study. The efficacy of Danhong injection (DHI, a Chinese materia medica standardized product, which is widely used to treat coronary heart disease) in the treatment of chronic stable angina was evaluated (ClinicalTrials.gov, NCT01681316).5 The homologous sequence ofmiR82 was not found in other species by sequence alignment in databases. The level of novel miR-82 was decreased in the therapeutically effective patients (Figure 1A) and increased in the therapeutically ineffective patients (Figure 1B) in the DHI group. It was indicated that miR-82 upregulation may be associated with the stable angina treatment of DHI and its downregulationmay have a protective effect onmyocardial ischemia. miR-82 may be a new biomarker and therapeutic target for myocardial ischemia. The structure and sequence of miR-82 were identified as follows (Figure 1C, completed by Shenzhen Huada Gene Technology Service Co., Ltd.). We experimentally confirmed the existence of miR-82 in AC16 and H9C2 cells with qRT-PCR and northern blot analysis (Figure 1D and E). The relative expression ofmiR-82 from six types of rat tissues is shown in Figure 1F. These results revealed the existence ofmiR-82 in diverse rat tissues. The level of miR-82 in blood was the most abundant, followed by the heart, which was nearly equivalent in liver, lung, and kidney, and least in the spleen. These results suggested that miRNA-82 was not human specific, which was also expressed in rats.

The miR-82 expression was upregulated in ischemic AC16 in a time-dependent manner ( Figure 1G). As AC16 exposed to ischemia for 24 hours resulted in approximately 50% cell death ( Figure 1H) and 9.4-fold miR-82 upregulation ( Figure 1G), AC16 cells were treated in trigas incubator for 24 hours in all subsequent ischemiarelated experiments. We transfected miR-82 mimic and miR-82 inhibitor into AC16 cells and explored the effect of miR-82 in myocardial ischemia in vitro. The transfection with miR-82 mimic decreased the cell viability ( Figure 1I), increased lactate dehydrogenase (LDH), and intracellular Malondialdehyde (MDA) content in AC16 cell supernatant and decreased intracellular Superoxide dismutase (SOD) content ( Figure 1J), and increased the apoptosis rate (Figure 1K). Although inhibition of miR-82 by transfection with miR-82 inhibitor led to the opposite results, which had protective effect on ischemic AC16 cells ( Figure 2C-F). These results indicated that the inhibition of miR-82 has protective effects on ischemic cardiomyocytes. Incubation of ischemic AC16 cells with DHI (80, 40, and 20 μL/mL) or salvianolic acid B (10 −5 , 10 −6 , and 10 −7 M) caused a dose-dependent increase ( Figure 1G) in the viability and decrease of the miR-82 expression ( Figure 1H). It was indicated that the miR-82 downregulation may be involved in the stable angina treatment of DHI.
In order to identify the target genes that may interact with miR-82, miRB, Targetscan, RNA22, and miRanda, four target prediction software were used. A total of 61 target genes were predicted by the four software. Wholegenome sequencing analysis with Illumina HiSeqTM 2000 indicated that elevated expression of miR-82 caused a total of 316 genes with significant expression differences, of which 256 were significantly downregulated. As shown in Figure Table S1. To confirm the targets genes of miR-82, we performed qRT-PCR and the luciferase binding assay. We detected the expression of the seven predicted target genes using qRT-PCR. The results are shown in Figure 2B. The transfection with miR-82 mimic could significantly inhibit the mRNA expression of PA2G4, TGFBR2, and FHL3 compared with the control group, and the transfection with miR-82 inhibitor resulted in an increase in the expression of these three mRNAs. These results indicated that miR-82 could regulate the expression of PA2G4, TGFBR2, and FHL3 mRNA in AC16 cells, suggesting that PA2G4, TGFBR2, and FHL3 may be target genes of miR-82. Furthermore, we performed the luciferase reporter assay. As shown in Figure 2C, miR-82 mimic caused a significant decrease in fluorescence of CDKN1A, PA2G4, GIPC3, ITGA5, and FHL3 wild-type reporter vectors 64%, 46%, 40%, 53%, and 31%, respectively. Combined with the results of Figure 2B, we speculated that PA2G4 and FHL3 may be the direct targets of miR-82. Next, we constructed mutant vectors of PA2G4 and FHL3, respectively, for confirmation. As shown in Figure 2D, miR-82 mimic did not cause significant changes in fluorescence of PA2G4 and FHL3 mutant reporter vectors. These results indicated that PA2G4 and FHL3 are the direct target genes of miR-82.
In the analysis of the correlation between miR-82 and PA2G4 or FHL3 in clinical patients, there was a high negative correlation between miR-82 and PA2G4, which was consistent with the results of target genes validation. However, miR-82 was positively correlated with FHL3 (Figure 2E). The final conclusion may need to be validated in more clinical samples.
Taken together, we discovered a novel miR-82 in a clinical trail and confirmed its expression. Our results provide the first evidence that the novel miR-82 was not only expressed in human, but also in rats. miR-82 was upregulated in ischemic AC16 cells and its downregulation had beneficial effect on ischemic AC16 cells. PA2G4 and FHL3 are the direct target genes of miR-82. miR-82 may be a druggable target for myocardial ischemia.

A C K N O W L E D G M E N T S
We thank Pro. Jianjun Zhang (Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College) for providing HEK293 cells. This work was supported by the National Major Scientific and Technological Special Project for "Significant New Drugs Development" (2017ZX09301059).

C O N F L I C T O F I N T E R E S T
The authors declare no conflict of interest.