Oxidative stress plays a critical role in cardiovascular diseases. Salidroside, a glycoside from
Salidroside, an adaptogen purified from
miRNAs are a class of small endogenous noncoding RNAs that regulate gene expression by interacting with the 3′-UTR of the target mRNAs, thereby indirectly or directly causing their degradation [
In this study, we investigated changes in miR-103 expression in a cellular model of oxidative stress induced by H2O2. Then, we assessed the effects of miR-103 on cardiotoxicity in vitro. We demonstrated that miRNA-103 could regulate BNIP3 expression at the translational level. Finally, we revealed that the inhibition of BNIP3 by siRNA rescued cell viability and oxidative damage under a cellular oxidative stress state.
Human embryonic kidney (HEK293T) cells were obtained from the American Type Culture Collection. Human umbilical vein endothelial cells were conserved in our laboratory and routinely maintained under the culture conditions reported previously [
Total RNA was extracted from cultured cells using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. For the detection of miR-103 expression, stem-loop reverse transcription-polymerase chain reaction (RT-PCR) was performed using the TaqMan miRNA assay (Applied Biosystems, Foster City, CA, USA). The relative amounts of miR-103 were normalized to the expression of the U6 small RNA.
To measure the mRNA levels of BNIP3, 500 ng of total RNA was reverse transcribed using the Reverse Transcription Kit (Promega, Madison, WI, USA), and qPCR was performed using the TaKaRa SYBR Green PCR Kit (TAKARA, Dalian, China). The relative mRNA expression levels of the BNIP3 gene were normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primers for BNIP3 were designed using Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA, USA). The primer BNIP3 sequences were (forward) 5′-TGAGTCTGGACGGAGTAGCTC-3′ and (reverse) 5′-CCCTGTTGGTATCTTGTGGTGT-3′. The primer GAPDH sequences were (forward) 5′-AGCCTTCTCCATGGTGGTGAA-3′ and (reverse) 5′-ATCACCATCTTCCAGGAGCGA-3′. All qRT-PCR reactions were performed with the Applied Biosystems 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA) in triplicate.
Each group of cells was plated into 96-well plates at a density of 1 × 104 cells/well. The culture medium was removed after overnight incubation, and then the HUVECs were rinsed with phosphate buffered saline (PBS) and incubated with different treatments. After another 24 h of treatment, cell viability was assessed using the cell counting kit-8 (CCK-8) assay (Doujin Laboratories, Kumamoto, Japan). The absorbance of each well at 450 nm (OD = 450) was read on an ELISA plate reader (Tecan Group Ltd., Männedorf, Switzerland). Three independent experiments were performed in triplicate.
Apoptosis was measured by staining with annexin V and propidium iodide using the Annexin V-PE Apoptosis Detection Kit (Abnova, Taipei, Taiwan). Briefly, the cells were treated as indicated, trypsinized, and harvested by centrifugation. Then, the cells were incubated with annexin V and PI for 20 min at room temperature in the dark. The samples were analyzed by flow cytometry using a FACScan cytometer (BD Biosciences, Woburn, MA, USA) and FlowJo software (BD Biosciences).
HUVECs (105 cells) were seeded into each well of a 24-well plate. The next day, the cells were washed once with PBS and labeled at 37°C for 30 min in culture medium containing 10
To overexpress miR-103, a fragment encoding the pre-miR-103 sequence was amplified by PCR from human genomic DNA (Cwbiotech, Beijing, China) with the following primers: (forward) 5′-AAAGGATCCTACTGCCCTCGGCTTCTTTAC-3′ and (reverse) 5′-AAACTCGAGCAATGCCTTCATAGCCCTGTAC-3′. Then, the PCR product was cloned into the BamHI/XhoI sites of the pCDH-CMV-EF1-copGFP vector (SBI, Mountain View, CA, USA).
To construct the BNIP3 3′-UTR plasmid, a wild-type 3′-UTR fragment of human BNIP3 mRNA containing the putative miR-103 binding sequence was amplified by RT-PCR with the following primers: (forward) 5′-AAACTCGAGTGAAGAACTGGAGTCTGACTTGGTT-3′ and (reverse) 5′-AAAGCGGCCGCCCATTTCCAGTTTTTTAAAGTAGAC-3′. The PCR product was cloned into the XhoI/NotI sites of the psiCHECK-2 vector (Promega, Madison, WI, USA). A mutant of the single miR-103 binding site (5′-AATGCTGC-3′ to 5′-GGGCCTCG-3′) in the 3′-UTR of BNIP3 was generated using the Site-Directed Mutagenesis Kit (Stratagene, San Diego, CA, USA). All plasmids were validated by DNA sequencing.
miRNA mimics, the miRNA inhibitor and the negative control miRNA oligonucleotides for hsa-miR-103, were synthesized by GenePharma (Shanghai, China). A siRNA sequence targeting the human BNIP3 gene was designed and synthesized by GenePharma. The effective siRNA sequence was 5′-GGGCAUAUUCUCUGCAGAAdTdT-3′ (sense). The scrambled siRNA 5′-UUCUCCGAACGUGUCACGUdTdT-3′ (sense) was included as a negative control. HUVECs were transfected with negative control (control) or BNIP3 siRNA at a final concentration of 20 nM using the X-treme GENE siRNA Transfection Reagent (Roche, Indianapolis, IN, USA) according to the manufacturer’s instructions. The expression levels of BNIP3 were examined by Western blot analysis 48 h after transfection.
For production of viral particles, lentivirus-mediated miR-103 or the control vector from the pPACKH1 Lentivector Packaging Kit (SBI, Mountain View, CA, USA) was cotransfected into HEK293T cells with the X-tremeGENE siRNA Transfection Reagent. The HUVECs were transduced with miR-103 or the control vector, and the expression of miR-103 was determined by qRT-PCR 72 h after transfection.
For the luciferase reporter assays, HUVECs were seeded into 96-well plates. The cells in each well were transfected with the luciferase reporter plasmid containing either the BNIP3 wild type 3′-UTR (WT) or BNIP3 mutant type 3′-UTR (MUT) sequence and miR-103 mimics or the mimic control using the X-tremeGENE siRNA Transfection Reagent. Luciferase activity was measured 24 h after transfection using a Luciferase Assay Kit (Promega, Madison, WI, USA) following the manufacturer’s protocol. The results represent three independent experiments, each performed in triplicate.
Western blot analysis was performed according to our standard protocol as previously described [
Data were expressed as the mean ± standard deviation (SD). The Student’s
A previous report showed that miR-103 played an important role in angiogenesis in vascular endothelial cells [
Salidroside mediated the expression of miR-103 in HUVECs induced by H2O2. (a) The expression of miR-103 was detected by qRT-PCR after H2O2 treatment at the indicated time points. (b) The expression of miR-103 was determined in HUVECs treated with the indicated concentration of H2O2 for 6 h. (c) HUVECs were treated with H2O2 alone or in combination with salidroside. The relative expression of miR-103 was detected by qRT-PCR. (d) qRT-PCR was used to assess the relative expression of miR-103 in HUVECs treated with or without salidroside. Data are shown as the mean ± SD from three independent experiments.
Our previous study indicated that salidroside protected HUVECs from the cytotoxicity and oxidative stress induced by H2O2 [
To assess the biological function of miR-103, HUVECs were stably transduced with miR-103 by lentiviral infection. Empty vector-transfected cells were used as controls. Successful overexpression of miR-103 was confirmed by qRT-PCR (Figure
Effect of miR-103 on HUVECs. (a) qRT-PCR was performed to verify the expression of miR-103 in HUVECs transfected with miR-103. (b) Effect of miR-103 on HUVEC viability induced by H2O2 was measured with the CCK-8 assay. (c) An apoptosis assay was performed to assess the apoptosis levels of HUVECs treated as indicated. (d) Effects of miR-103 on the intracellular formation of ROS triggered by preincubation exposure of HUVECs to H2O2 assessed by DCF assays.
The above findings indicated that miR-103 had a strong protective effect on HUVECs. Next, we searched for potential target genes of miR-103 that might contribute to its function. We performed in silico studies to identify potential gene targets of miR-103 using the bioinformatics algorithms TargetScan (
Validation of BNIP3 as a miR-103 target gene. (a) The 3′-UTR of BNIP3 and mutant 3′-UTR sequences that abolished binding. (b) Luciferase activity was assessed in HUVECs transfected with BNIP3 3′-UTR-WT or BNIP3 3′-UTR-MUT and the mimic control or miR-103. (c) Luciferase activity was assessed in HUVECs transfected with BNIP3 3′-UTR-WT or BNIP3 3′-UTR-MUT and the inhibitor control or miR-103 inhibitor. (d) BNIP3 mRNA levels analyzed by qRT-PCR. (e) Western blot analysis of the endogenous expression of BNIP3 upon forced expression of miR-103. (f) The protein expression of BNIP3 in HUVECs transfected with the miR-103 inhibitor or inhibitor control was determined by western blotting.
Next, we determined whether miR-103 directly targeted the 3′-UTR of BNIP3, thereby potentially contributing to its biological functions. Luciferase reporter plasmids of BNIP3 carrying the 3′-UTR with the potential miR-103 binding site or the miR-103 mutant binding site were cotransfected into HUVECs with either miR-103 or the miRNA negative control. Transfection with miR-103 significantly reduced the luciferase activity compared with the negative control (Figure
Studies have shown that BNIP3 is a proapoptotic protein that is involved in the pathogenesis of cardiovascular disease through the regulation of mitochondrial functions [
H2O2 upregulated the expression of BNIP3 in HUVECs. (a) BNIP3 protein expression in HUVECs cultured in 200
Although H2O2 downregulated the expression of miR-103 and upregulated the expression of BNIP3, whether the downregulation of BNIP3 might play a role in miR-103-induced HUVEC viability and oxidative stress was not clear. Therefore, we inhibited BNIP3 expression in HUVECs treated with H2O2 using siRNA. BNIP3 protein levels were estimated by Western blotting after transfection of the HUVECs with BNIP3 siRNA 1, siRNA 2, siRNA 3, or the negative control for 48 h. BNIP3 siRNA 3-treated HUVECs showed a significant reduction in BNIP3 compared to the control cells (Figure
Cell viability and DCF analysis of HUVECs exposed to H2O2 with or without BNIP3 knockdown. (a) Western blot analysis showing levels of BNIP3 when HUVECs were transfected with different BNIP3 siRNA constructs or a negative control (NC) siRNA. (b) The CCK-8 assay was performed to evaluate the cell viability of HUVECs treated as indicated. (c) An apoptosis assay was used to assess the apoptosis levels of HUVECs treated as indicated. (d) Intracellular formation of ROS was measured in HUVECs treated as indicated. (e) BNIP3 expression was determined in HUVECs transfected with siRNA and treated with or without H2O2.
Oxidative stress is a major stimulus in the pathogenesis of cardiovascular diseases, including atherosclerosis, hypertension, myocardial infarction, and heart failure, via different molecular pathways [
Emerging evidence has implicated miRNAs in the regulation of a wide variety of biological processes, such as oxidative stress and apoptosis. However, although miRNAs have emerged as critical players in the cytotoxicity induced by H2O2, whether the beneficial effects of salidroside were related to miRNAs was unknown. Our results shed some light on the novel role of miR-103 in the oxidative stress of HUVECs.
In the present study, we found that H2O2 stimulation decreased the expression of miR-103 in a time- and concentration-dependent manner and that salidroside significantly attenuated these changes. However, the expression of miR-103 was not obviously altered in HUVECs. Then, we assessed the cardioprotection function of miR-103 in HUVECs under oxidative stress. Using the CCK-8 assay and flow cytometry, we demonstrated the attenuation of the loss in cell viability and ROS production induced by H2O2 in HUVECs stably overexpressing miR-103, implying that miR-103 may mimic many features of salidroside in HUVECs treated with H2O2. Consistent with this notion, experimental studies have revealed that miR-103 affects stroke-induced brain damage and neurological deficits by regulating NCX1 expression [
To determine the targets of miR-103 action, in silico algorithms were performed to identify BNIP3 as a potential target of miR-103. BNIP3 is a proapoptotic BH3-only protein that is primarily localized to the mitochondria [
In this report, we demonstrated that miR-103 was upregulated following pretreatment with salidroside in an H2O2-induced oxidative stress model of HUVECs, thereby dramatically decreasing the level of BNIP3 mRNA and protein expression by directly binding to the 3′-UTR of BNIP3. Thus, modulation of miR-103 levels may provide a new strategic therapeutic target for oxidative stress-associated disorders. However, the molecular mechanism by which miR-103 protects cultured HUVECs against oxidative stress needs to be further elucidated.
The authors declare that they have no competing interests.
The authors thank Xuchao Zhu (Tongji University, Shanghai, China) for experimental design and language editing. The study was funded by the Pudong New Area leading academic discipline project.