Long non‑coding RNA H19 protects H9c2 cells against hypoxia‑induced injury by activating the PI3K/AKT and ERK/p38 pathways

Introduction

Cardiovascular disease is one of the leading causes of death worldwide (1). During cardiac surgery, myocardial ischemia/reperfusion (I/R) injury often leads to adverse cardiovascular outcomes, including acute heart failure due to severe hypoxia (2). Multiple signaling pathways are involved in protective mechanisms against myocardial I/R injury, including the PI3K/AKT and the mitogen-activated protein kinase (MAPK) signaling pathways. The PI3K/AKT pathway is an important intracellular signaling pathway for the regulation of the cell cycle. Previous studies have suggested that the PI3K/AKT signaling pathway mediates a protection mechanism against myocardial I/R injury in rats (3,4). Inhibition of the cardiac p38-MAPK pathway has been reported to delay ischemic cell death and protect cardiac mitochondria from I/R injury (5,6).

Long non-coding RNAs (lncRNAs) are a class of non-coding RNA molecule with a length of >200 nucleotides. lncRNAs represent the most prevalent class of non-coding RNA, with the majority of the human genome transcribed into lncRNAs (7). lncRNAs can act as important regulators in a number of biological processes, including stem cell lineage differentiation (8), cancer development and metastasis (9,10) and angiogenesis (11,12). The lncRNA H19 (H19) is located on human chromosome 11 and expresses a 2.3 kb lncRNA transcribed from the maternal inherited allele (13). Increased expression of H19 was detected in rats following surgically induced myocardial I/R, suggesting that H19 may have a role in the protective mechanisms against I/R injury (14). Previous studies have also indicated that H19 exerts protective effects against hypoxia-induced injury in cardiomyocytes (15,16). Nevertheless, to date, the functional role of H19 in myocardial hypoxic injury has not been fully elucidated. The present study aimed to investigate the effects and the molecular mechanism of H19 in response to hypoxia-induced I/R injury using a myocardial cell model.

Materials and methods

Cell lines and culture

The rat cardiomyoblast cell line, H9c2, and 293T cells were purchased from BeNa Culture Collection. Cells were maintained in DMEM (Hyclone; GE Healthcare Life Sciences) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C and 5% CO2 in an incubator.

Design and construction of H19 expressing/silencing lentivirus

To construct an H19 expressing lentiviral vector, the H19 cDNA sequence (NR_002196.2) was synthesized by Sangon Biotech Co., Ltd. and cloned into the pLVX-Puro vector following the Lentivector User Manual (Takara Bio, Inc.). The following PCR reaction mixture was used: 25 µl 2× Super Pfx MasterMix (Beijing CoWin Biotech Co., Ltd.), 2.5 µl 10 µM forward primer, 2.5 µl 10 µM reverse primer, 2.0 µl DNA template, 18 µl ddH2O. The PCR was performed as follows: 98°C for 1 min, 30 cycles of 98°C for 5 sec, 58°C for 30 sec, and 72°C for 30 sec, followed by 72°C for 5 min. The pLVX-Puro vector was used as a negative control. To construct an H19 silencing lentiviral vector, the following oligonucleotides encoding a short hairpin RNA (shRNA) against H19 were designed and synthesized by Genscript and cloned into the pGreenPuro™ vector (Addgene, Inc.): Sense, 5′-GATCCTGAATATGCTGCACTTTACAACTCGAGTTGTAAAGTGCAGCATATTCATTTTTG-3′ and antisense, 5′-AATTCAAAAAATGAATATGCTGCACTTTACAACTCGAGTTGTAAAGTGCAGCATATTCAG-3′. The positive-sense and antisense strands harboring BamH and EcoR digestion sites annealed to form a double-stranded structure, which was then cloned into the pGreenPuro plasmid. The lentiviral vector containing a non-silencing sequence was used as a negative control. The sequence of constructs was confirmed by PCR and Sanger sequencing.

To package lentiviruses, 293T packaging cells were plated in a 10 cm plate. At 70% confluency, the cells were co-transfected with 2.5 µg of the appropriate lentiviral vector (pLVX-Puro-H19 vector, pLVX-Puro vector, pGreenPuro™ shH19 vector or pGreenPuro™ non-silencing vector), 5 µg of psPAX and 5 µg of pMD2G (Addgene, Inc.) using a Lipofectamine® 3000 transfection kit (Invitrogen; Thermo Fisher Scientific, Inc.). The viral supernatants were harvested after 48 h and filtered with a 0.45 µm filter. The titer of the lentivirus was determined using a lentivirus titration kit (Applied Biological Materials), following the manufacturer's instructions.

Cell culture and infection

H9c2 cells were cultured in 6-well plates and divided into six groups: Normal control, model control, H19 expression, blank expression, H19 interference and blank interference groups. Cells in the H19 expression, blank expression, H19 interference and blank interference groups were infected with H19 overexpression, blank expression, H19-targeting shRNA and blank interference lentiviruses, respectively, at a multiplicity of infection of 1:100. Cells in the normal control group were then cultured under normoxia (21% O2, 5% CO2 and 74% N2). Cells in all other groups were incubated in an incubator containing 94% N2, 5% CO2 and 1% O2 to stimulate hypoxia injury. After 48 h, cells in each group were collected and subjected to subsequent analyses.

Apoptosis assay

At 48 h after transfection, apoptosis was determined using a FITC Annexin V Apoptosis Detection kit I (BD Biosciences). Briefly, cells were resuspended in 1X binding buffer, stained with 5 µl FITC Annexin V (BD Biosciences) and 10 µl of propidium iodide (PI; BD Biosciences) in the dark for 10 min. The fluorescence of cells was detected using a NovoCyte 2060R flow cytometer (ACEA Biosciences, Inc.) at 488 nm within 1 h and analyzed using NovoExpress software version 1.3.0 (ACEA Biosciences, Inc.).

Measurement of mitochondrial membrane potential

At 48 h after transfection, the mitochondrial membrane potential of cells was determined using a JC-1 mitochondrial membrane potential assay kit (Abcam). Briefly, cells were resuspended in 500 µl of 1X incubation buffer and stained with 1 µl JC-1 at 37°C, 5% CO2 for 10 min. Cells were collected by centrifugation at 420 × g at room temperature for 5 min and washed twice with 1X incubation buffer. The fluorescence of cells was analyzed using a NovoCyte 2060R flow cytometer (ACEA Biosciences, Inc.) at 488 nm within 1 h and analyzed using NovoExpress software version 1.3.0 (ACEA Biosciences, Inc.).

Cell cycle analysis

At 48 h after transfection, the cell cycle was analyzed using flow cytometry. Briefly, cells were fixed with 70% ethanol for 24 h at 4°C and stained with 100 µl of PI (30 ng/ml) for 30 min at 37°C in the dark. The percentage of the population in G1, S or G2/M phase was analyzed using a NovoCyte 2060R flow cytometer (ACEA Biosciences, Inc.) at 488 nm within 1 h and analyzed using NovoExpress software version 1.3.0 (ACEA Biosciences, Inc.).

Western blot analysis

Cells were collected at 48 h after transfection. Total protein was extracted using RIPA lysis buffer (Beijing CoWin Biotech Co., Ltd.) and quantified using bicinchoninic acid protein assay kit (Beijing CoWin Biotech Co., Ltd.). Protein extracts (60 µg) were separated by SDS-PAGE using 10% gels and transferred onto PVDF membranes. The membranes were blocked with 5% skim milk for 30 min at room temperature and incubated overnight at 4°C with the following antibodies: β-actin (1:2,000; cat. no. TA-09; OriGene Technologies, Inc.), AKT (1:2,000; cat. no. bs-0115R; BIOSS), ERK1/2 (1:1,000; cat. no. bs-0022R; BIOSS), p38 (1:2,000; cat. no. bs-0637R; BIOSS), PI3K (1:2,000; cat. no. ab191606; Abcam), phosphorylated (p-)AKT (1:1,000; cat. no. ab38449; Abcam), p-p38 (1:1,000; cat. no. ab47363; Abcam), p-ERK1/2 (1:1,000; cat. no. ab214362; Abcam) and p-PI3K (1:1,000; cat. no. ab127617; Abcam). The membranes were washed with PBS and incubated with horseradish peroxidase-conjugated goat anti-mouse (1:2,000; cat. no. ZB-2305; OriGene Technologies, Inc.) or anti-rabbit (1:2,000; cat. no. ZB-2305; OriGene Technologies, Inc.) secondary antibodies for 2 h at room temperature. Protein bands were visualized using an ECL Western blotting detection system (GE Healthcare). The relative expression of proteins was quantified using ImageJ software version 1.52e (National Institutes of Health) with β-actin used as the internal control.

Reverse transcription-quantitative PCR (RT-qPCR)

Cells were collected 48 h after transfection. Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA using SuperScript II (Invitrogen; Thermo Fisher Scientific, Inc.) at 42°C. qPCR was performed using UltraSYBR mixture (Takara Biotechnology Co., Ltd.) in an ABI 7500 System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The reactions conditions were as follows: 95°C for 10 min followed by 40 cycles of 95°C for 10 sec, 58°C for 30 sec and 72°C for 30 sec. The following primers were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.) and used in the PCR: H19 forward, 5′-GTGGGACACTGCCGTAGAA-3′ and reverse, 5′-CAGGAAAGGAGGAAGAAGAAAA-3′; and U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. Data was analyzed using the 2−ΔΔCq method (17). The relative expression of lncRNA H19 was calculated using U6 as the internal control.

Statistical analysis

All experiments were performed in triplicate. Data are presented as the mean ± SD and were analyzed using SPSS 19.0 software (IBM Corp.). Differences among groups were compared using one-way ANOVA followed by Tukey's post-hoc test. Ratios were compared using the χ2 tests. P<0.05 was considered to indicate a statistically significant difference.

Results

Hypoxia-induced apoptosis is alleviated by H19 overexpression and is aggravated by H19 interference

The expression of H19 in all groups was determined using RT-qPCR. As shown in Fig. 1, the model control, blank expression, blank interference and normal control groups had a similar level of H19 expression (all P>0.05). H19 expression was significantly increased in the H19 expression group (P=0.015) and significantly reduced in the H19 interference group (P=0.023) compared with the model control group. As shown in Fig. 2, the rate of apoptosis in the model group was significantly higher than in normal control group (P=0.021), indicative of hypoxia-induced myocardial cell injury. The rate of apoptosis was significantly reduced in the H19 expression group (P=0.016) and was significantly increased in the H19 interference group compared with the model group (P=0.022). The blank expression and blank interference groups had similar rates of apoptosis as the model group (both P>0.05).

Figure 1. Reverse transcription-quantitative PCR analysis of H19 expression in the different groups. Cells in the normal control group were cultured under normoxia. Cells in the model control and different infection groups (blank expression, H19 expression, H19 interference and blank interference) were collected after 48 h of culture in hypoxic conditions. The model control, blank expression, blank interference and normal control groups had a similar level of H19 expression (all P>0.05). H19 expression was significantly increased in the H19 expression group (P=0.015) and significantly reduced in the H19 interference group (P=0.023) compared with the model control group. Data were analyzed using the 2−ΔΔCq method. The relative expression of H19 was calculated using U6 as the internal control. All samples were measured in triplicate. *P<0.05 vs. model group. H19, long non-coding RNA H19.

Figure 2. Hypoxia-induced H9c2 apoptosis is aggravated by H19 knockdown and is alleviated by H19 overexpression. Cells in the normal control group were cultured under normoxia. Cells in the model control and different infection groups (H19 expression, blank expression, H19 interference and blank interference) were collected after 48 h of culture in hypoxic conditions. The level of apoptosis was determined using flow cytometry. All samples were measured in triplicate. *P<0.05, **P<0.01 vs. normal group; #P<0.05 vs. model group. H19, long non-coding RNA H19; PI, propidium iodide.

Hypoxia-induced G1 phase cell cycle arrest is attenuated by H19 overexpression and is aggravated by H19 interference

The cell cycle distribution was analyzed using flow cytometry. As shown in Fig. 3, the proportion of H9c2 cells in the G1 phase in the model group was significantly higher compared with the normal control group (P=0.020) and the percentage of S phase cells was significantly lower (P=0.043), suggesting that hypoxia induced a G1 phase cell cycle arrest. The H19 expression and normal control groups had a similar proportion of H9c2 cells in G1 phase (P>0.05). The H19 interference group had a significantly higher G1 population compared with the normal control group (P=0.012). When compared with the model group, the H19 expression group had a significantly lower G1 and a higher S phase population (P=0.018 and 0.031, respectively), whereas the H19 interference group had a significantly higher G1 population and a smaller S phase population (P=0.029 and 0.045, respectively). The G1 and S phase populations in the blank expression and blank interference groups were similar to the model group (all P>0.05).

Figure 3. Flow cytometry analysis of the cell cycle distribution of H9c2 cells. Cells in the normal control group were cultured under normoxia. Cells in the model control and different infection groups (H19 expression, blank expression, H19 interference and blank interference) were collected after 48 h of culture in hypoxic conditions. The percentage of cells in the G1 phase was quantified. All samples were measured in triplicate. *P<0.05 vs. normal group; #P<0.05 vs. model group. PE, phycoerythrin; RMS, root mean square; Freq, frequency; CV, coefficient of variation; H19, long non-coding RNA H19.

Hypoxia-induced mitochondrial membrane depolarization is reduced by H19 overexpression, but is enhanced by H19 interference

Under hypoxic treatment, the depolarization rate of the mitochondrial membrane potential in the model group was increased compared with the normal control group (P=0.033; Fig. 4). The mitochondrial depolarization rate was significantly reduced in the H19 expression group (P=0.036) and was increased in the H19 interference group (P=0.012) compared with the model group. By contrast, the blank expression and blank interference groups had similar depolarization rates as the model group (both P>0.05).

Figure 4. Hypoxia-induced mitochondrial membrane depolarization is reduced by H19 overexpression and is enhanced by H19 interference. Cells in normal control group were cultured under normoxia. Cells in the model control and different infection groups (H19 expression, blank expression, H19 interference and blank interference) were collected after 48 h of culture in hypoxic conditions. The mitochondrial depolarization rate was determined using flow cytometry. All samples were measured in triplicate. *P<0.05, **P<0.01 vs. normal group; #P<0.05 vs. model group. H19, long non-coding RNA H19; FL, fluorescence.

PI3K/AKT and ERK/p38 pathways are activated by H19 overexpression, but are inhibited by H19 interference

The underlying mechanism for the H19-asssociated regulation of apoptosis, the cell cycle and mitochondrial membrane potential was analyzed by western blotting. The results showed that the levels of p-PI3K, p-AKT and p-p38 in the model group were higher compared with the normal control group (all P>0.05). p-ERK1/2 expression was significantly lower in the model group compared with the normal control (P<0.05). The overexpression of H19 increased the levels of p-PI3K, p-ERK1/2 and p-p38 compared with the model group (all P<0.05). p-AKT level in the H19 overexpression group was similar as compared to the model group (P>0.05). The effects of H19 interference were the opposite to H19 overexpression in regulating the expression of p-p38 and p-PIK3 (all P<0.05, Fig. 5). H19 interference also significantly reduced the p-AKT expression as compared to model group (P<0.05). H19 interference and model groups had similar p-ERK1/2 expression (P>0.05). These findings indicated that the overexpression of H19 may attenuate hypoxia-induced cell injury by regulating the PI3K/AKT and ERK/p38 pathways. Nevertheless, it is worth noting that there are some variation among the model and blank expression/interference groups, indicating that some other pathways may also be involved in the H19 regulatory process.

Figure 5. PI3K/AKT and ERK/p38 pathways were regulated by H19. Cells in the normal control group were cultured under normoxia. Cells in the model control and different infection groups (H19 expression, blank expression, H19 interference and blank interference) were collected after 48 h of culture in hypoxic conditions. The expression levels of p-PI3K, p-AKT, p-ERK1/2, p-p38, PI3K, AKT, ERK1/2 and p38 were determined using western blotting with β-actin as the internal control. All samples were measured in triplicate. *P<0.05 vs. normal group; #P<0.05 vs. model group. H19, long non-coding RNA H19; p-, phosphorylated.

Discussion

Hypoxia is commonly used to induce a myocardial cell injury model (18). In the present study, H9c2 cells were exposed to hypoxic conditions to induce cell injury. As a result, these cells exhibited an increased level of apoptosis, G1 cell cycle arrest and depolarization of the mitochondrial membrane potential. The role of H19 has been previously studied in several cancers; however, its role remains controversial. For instance, H19 overexpression has been reported to enhance carcinogenesis and metastasis in gastric cancer (19). Contrary to this, it was found that H19 does not affect the proliferation or cell cycle distribution of breast cancer cells (20). There are few studies reporting the effects of H19 in hypoxia-injured myocardial cells (16). In the present study, H19 expression was found to be increased in hypoxia-treated cells, suggesting the possible involvement of H19 in protection against hypoxia-induced cell injury. H9c2 cells were transfected with H19 expressing or silencing lentiviruses to investigate the role of H19 in hypoxia-injured cells. It was found that H19 overexpression alleviated hypoxia-induced injury mediated apoptosis, cell cycle arrest and mitochondrial membrane potential depolarization; however, these processes were aggravated by H19 knockdown. These results suggested that H19 attenuated hypoxia-induced myocardial cell injury, which is consistent with previous studies (15,16). The findings of this previous report (16) and the present study are consistent with each other in regard to the molecular mechanism of H19 in protecting against myocardial I/R injury. Nevertheless, it is worth noting that the two studies have different experimental designs. Specifically, the present study examined the role of H19 regulation on the cell cycle and mitochondrial changes in cells in addition to cell apoptosis, which more directly reveals the cellular/intracellular responses during the process of myocardial I/R injury, whereas the reference only examined the cell behavior, including cell viability, migration and apoptosis.

The PI3K/AKT signal pathway is an important pathway for protecting myocardial cells against hypoxia-induced injury (21,22). Xiao et al (23) reported that hydrogen sulfide protects myocardial cells against hypoxia-induced injury via mTOR activation. Chen et al (24) reported that lipoxin A4-induced heme oxygenase-1 protects cardiomyocytes against hypoxia/reoxygenation injury via p38 MAPK activation and the nuclear factor erythroid 2-related/antioxidant responsive element complex. IT has been previously reported that AKT activation reduces myocardial cell apoptosis by upregulating the expression of Bcl-2 (25). The relationship between the Bcl-2 family proteins and mitochondria-mediated apoptosis pathway is well-established (26). Thus, it is speculated that H19 may regulate mitochondrial apoptosis by activating the PI3K/AKT/mTOR pathway. In the present study the effects of H19 on mitochondrial membrane potential were assessed, as was the activation of the PI3K/AKT/mTOR signaling pathway and MAPK activation, in order to elucidate the mechanisms underlying the possible protective effects of H19 against hypoxia-induced cell injury. It was demonstrated that the overexpression of H19 stabilized the mitochondrial membrane potential and upregulated the PI3K/AKT/mTOR pathway in hypoxia-treated H9c2 cells, while H19 interference had the opposite effect, indicating that H19 alleviated hypoxia-induced cell injury by reducing the mitochondrial apoptosis pathway and activating the PI3K/AKT and MAPK pathways.

In conclusion, the present study demonstrated that the overexpression of H19 decreased hypoxia-induced cell injury by increasing cell viability and decreasing apoptosis, whereas knockdown of H19 had the opposite effects. Furthermore, it was found that the overexpression of H19 may protect H9c2 cells from hypoxia-induced injury by activating the PI3K/AKT/mTOR and MAPK pathways. The present study may provide new insights into the prevention and treatment of acute myocardial infarction. Nevertheless, the present study was limited by the use of only one cell line. Future experiments should be conducted with other types of cardiomyocytes (for example, AC16 human cardiomyocyte and HCFB human cardiac fibroblasts) to verify the findings of the present study. Additionally, in-depth mechanistic studies should be performed to further elucidate the protective effects of H19. Findings from future studies should also be verified in animal models.

Acknowledgements

Not applicable.

Funding

No funding was received.

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Authors' contributions

LiY performed the majority of experiments and drafted the paper. LeY, JZ, ZZ and CL helped with experiments. BZ and XH analyzed the data and drafted part of the paper. GX and YT conceived the study, supervised the experiments and edited the manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References