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The inhibition of miR-101a-3p alleviates H/R injury in H9C2 cells by regulating the JAK2/STAT3 pathway

Hypoxia/reoxygenation (H/R) is used as an in vivo model of ischemia/reperfusion injury, and myocardial ischemia can lead to heart disease. Therefore, it is necessary to prevent myocardial H/R injury to avoid the risk of heart disease. The aim of the present study was to investigate whether inhibiting microRNA (miR)-101a-3p attenuated H9C2 cell H/R injury, apoptosis mechanisms and key target proteins. Cell viability and apoptosis were determined by Cell Counting Kit-8 assays and flow cytometry using a cell apoptosis kit, respectively. The contents of creatine kinase (CK) and lactate dehydrogenase (LDH) were detected using colorimetric assays. Dual luciferase assays were carried out to determine if miR-101a-3p inhibited Janus kinase (JAK)2. Western blot analysis and reverse transcription-quantitative PCR were used to determine proteins levels and mRNAs expression. It was found that the inhibition of miR-101a-3p increased the growth of H9C2 cells and decreased H9C2 cell apoptosis during H/R injury. The inhibition of miR-101a-3p reduced the amounts of CK and LDH in H/R model H9C2 cells. The inhibition of miR-101a-3p lowered the levels of Bax, interleukin-6 and tumor necrosis factor-α, but raised the levels of phosphorylated (p)-STAT3 and p-JAK2 in H9C2 cells subjected to H/R injury treatment. miR-101a-3p mimic was found to inhibit H9C2 cell viability, raise p-JAK2 level and slightly increase p-STAT3 during H/R injury. AG490 induced H9C2 cell apoptosis, and decreased the levels of p-JAK2 and p-STAT3 during H/R injury. The data indicated that inhibiting miR-101a-3p reduced H/R damage in H9C2 cells and decreased apoptosis via Bax/Bcl-2 signaling during H/R injury. In addition, it was suggested that the inhibition of miR-101a-3p decreased H/R injury in H9C2 cell by regulating the JAK2/STAT3 signaling pathway.


Introduction
In vivo, ischemia/reperfusion (i/r) injury refers to organ damage produced by the restoration of blood flow after ischemia. Myocardial i/r injury is a common phenomenon that occurs after ischemic heart diseases such as myocardial infarction (MI), or is the result of a reduction in blood flow and oxygen supply insufficiency (1). MI is a cause of morbidity in cardiovascular diseases (2,3). limiting or reducing the damage following i/r has received extensive research attention. In vitro, an hypoxia/reoxygenation (H/r) injury model simulates in vivo i/r injury (2).
The differentiation, proliferation and migration of cells is critical in the early stages of cell healing, and apoptosis affects the elimination of inflammatory factors during cell healing (9). a number of studies have investigated cell apoptosis via the Bax/Bcl-2 signaling pathway (10)(11)(12). in addition, elevated interleukin-6 (il-6) levels may cause tissue damage and inflammation (13). Studies have demonstrated that tumor necrosis factor-α (TnF-α) is connected with inflammation and injury (14)(15)(16)(17). STaT3, a member of the STaT family of transcription factor, regulates cell proliferation, cellular transformation, metastasis and immune responses, whereas Janus kinase (JaK)2 participates in the immune system and other signaling transductions (18,19). Previous studies have demonstrated that inactivation of the JaK/STaT3 signaling pathway relieved cell injury, suggesting that activation of the JaK/STaT3 signaling pathway was correlated with cell injury (20,21).
in the present study, H9c2 cells (rat myocardial cells) were subjected to H/r treatment and established as an i/r injury model in vitro. The aim of the study was to investigate the effect and mechanism of the inhibition of mir-101a-3p on H/r injury. cell apoptosis in cardiomyocytes, and critical regulation of target factors by inhibition of mir-101a-3p and H/r injury were also studied.

Materials and methods
Cell culture and H/R models. The rat embryonic cardiomyoblast cell line H9c2 was purchased from the american Type culture collection. H9c2 cells were cultured in dMeM (Thermo Fisher Scientific, Inc.) at 37˚C in an incubator (Thermo Fisher Scientific, inc.) with 5% co 2 . dMeM contained high-glucose basic DMEM (Invitrogen; Thermo Fisher Scientific, Inc.), 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% 10,000 u/ml penicilin-10,000 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.). Subculture was conducted when the cells reached ~90% confluence in the culture flask.
Cell Counting Kit-8 (CCK-8) assay. cell viability was detected by ccK-8 (Medchemexpress, llc). The cells were plated in 96-well plates at 1.5x10 3 cells/well for 24 h. Following transfection and H/r treatment, ccK-8 solution was added into 96-well plates and diluted in phosphate buffered saline (Gibco; Thermo Fisher Scientific, Inc.) at 9:1, and then incubated in a 37˚C, 5% CO 2 atmosphere for 1 h. Then, the od value at a wavelength of 450 nm was measured by Multiskan™ Fc (Thermo Fisher Scientific, Inc.).
Colorimetric assays. The supernatants (centrifugation at 10,000 x g for 30 min at 4˚C) from the control group, H/r group, ic + H/r group and i + H/r group were collected into centrifuge tubes. creatine kinase (cK) was detected using a cK test kit (cat. no. Bc1140; Beijing Solarbio Science & Technology co., ltd.) and lactate dehydrogenase (ldH) was analyzed using an ldH test kit (cat. no. Te0159; Beijing leagene Biotech co. ltd.), according to the manufacturers' protocols.
Flow cytometry. cell apoptosis was determined via flow cytometry. The cells were suspended in PBS after having been treated for 48 h. Then, 1 µl annexin V/FiTc 5X, 150 µl annexin-binding buffer and 2.5 µl propidium iodide (Pi) in a Dead Cell Apoptosis kit (Thermo Fisher Scientific, Inc.) were added to 300 µl cell suspension for conducting flow cytometry at room temperature for 15-20 min in the dark. The fluorescence was detected by a flow cytometer (BD Biosciences) and the cell apoptosis was calculated using Bd FacSuite software (version 1.0; Bd Biosciences). apoptosis was calculated as the sum of early apoptosis and late apoptosis.

Effects of miR-101a-3p on the H/R-induced H9C2 cell injury.
M significantly increased miR-101a-3p expression, and I inhibited mir-101a-3p expression in H9c2 cells (Fig. 1a). although the expression of mir-101a-3p was higher in H9c2 cells that had been subjected to H/r injury, its expression was inhibited by i (Fig. 1B).
Effects of I on viability, and the contents of CK and LDH in H/R model H9C2 cells. cK has been reported to be linked to diastolic dysfunction in rats (23), and cardiomyocyte damage can be assessed by detecting the content of ldH (24). The expression levels of cK and ldH increased when the cells were subjected to H/r injury, and such expression was decreased by i transfection during H/r (Fig. 1c and d). The data also demonstrated that H/r decreased H9c2 cell viability, whereas i improved cell viability following H/r (Fig. 1e).

Effects of I on H/R-induced H9C2 cell apoptosis.
The effects of i and aG490 on H9c2 cell apoptosis were determined via flow cytometry using a cell apoptosis kit with Annexin V-FITC and Pi. it was determined that the apoptosis rate of H/r group was significantly higher than that of control group, while the apoptosis rate of i + H/R group was significantly lower than that of H/r group, suggesting that H/r induced H9c2 cell apoptosis, and that i can decrease H9c2 cell apoptosis during H/r injury (P<0.01; Fig. 1F and G).  Bcl-2, Bax, il-6 and TnF-α were detected via rT-qPcr and western blot analyses. compared with the H/r group, the i + H/r group had a lower level of Bax but a higher level of Bcl-2 expression (Fig. 2a, B, e and F), suggesting that i inhibited the Bax/Bcl-2 signaling pathway during cell H/r injury. The i + H/R group exhibited significantly lower expression levels of il-6 and TnF-α in comparison with the H/r group (Fig. 2c-F). of note, the expression levels of Bcl-2, Bax, il-6 and TnF-α in the H/r group were similar to those in the ic + H/R group, and the significant differences between Figure 3. effects of i and aG490 on the expression levels of JaK2, p-JaK2, STaT3, and p-STaT3 in H9c2 cells during H/r injury. (a) psi-cHecK-2 containing the JaK2 3'uTr, and M or i were used to transfect cells, and a dual luciferase assay kit was used to detect luciferase activity. (B) TargetScan7.2 was used to predict target genes of mir-101a-3p. (c) Protein levels of JaK2, p-JaK2, STaT3 and p-STaT3 were detected via western blot analysis. ratios of (d) p-JaK2/JaK2 and (e) total JaK2 were used to assess the activation of JaK2. (F) p-STaT3/STaT3 and (G) total STaT3 were used to assess the activation of STAT3. Data are presented as the mean ± standard deviation and were analyzed by ANOVA with Tukey-Kramer multiple comparison. * P<0.05, ** P<0.01 vs. Blank; # P<0.05, ## P<0.01; ^P <0.05, ^^P <0.01. mir, microrna; JaK, Janus kinase; H/r, hypoxia/reoxygenation; p-, phosphorylated; uTr, untranslated region; WT, wild-type; MuT, mutant; i, mir-101a-3p inhibitor; ic, mir-101a-3p inhibitor control; M, mir-101a-3p mimic; Mc, mir-101a-3p mimic control. groups in the RT-qPCR assays were similar to those identified via western blot analysis.

Effects of I, M and AG490 on the expression levels of JAK2, p-JAK2, STAT3, and p-STAT3 in H/R-treated H9C2 cells.
aG490 can inhibit the activation of STaT3 and JaK2 (25). A dual luciferase assay identified that I promoted luciferase activity, which was inhibited by M, supporting the hypothesis that i promoted JaK2 expression and M was able to inhibit expression (Fig. 3a,B). The H/r, ic + H/r and Mc + H/r groups exhibited lower levels of JaK2 compared with control group (Fig. 3c-e). M inhibited the expression of JaK2, which was found to be upregulated by i in the H/r model. aG490 + M + H/r group was found to have the lowest expression levels of JaK2. in addition, M inhibited the phosphorylation of JAK2, which was significantly promoted by i in comparison (Fig. 3c-e). it was also found that i increased the level of STaT3 in the H/r group, while M decreased the level of STaT3 in the H/r group, and that the aG490 + M + H/r group had the lowest levels of STaT3 ( Fig. 3F and G).
Effects of AG490 on H/R-induced H9C2 cell apoptosis. H/r treatment significantly induced apoptosis of H9C2 cells, while i inhibited the promoting effect of H/r on apoptosis of H9c2 cells (Fig. 4); AG490, a JAK2-specific inhibitor (26), was found to induce H9c2 cell apoptosis during H/r injury (Fig. 4); the results also showed that the apoptosis rate of aG490 + i + H/r group were higher than that of i + H/r group (Fig. 4), indicating that aG490 reversed the inhibitory effect of i on H/r-induced cell apoptosis.
Effects of M on H9C2 cell viability. The M + H/r group exhibited significantly reduced cell viability compared with the Mc + H/r group (Fig. 5), suggesting that mir-101a-3p upregulation aggravated H/r-induced damage in H9c2 cells. in addition, the aG490 + M + H/r group exhibited the lowest cell viability (Fig. 5), indicating that aG490 enhanced the inhibitory effect of M on H/r-induced cell viability.

Discussion
The results of the present study demonstrated that i can improve H9c2 cell viability and decrease cell apoptosis during cell H/r injury. as damage of cardiomyocytes gradually  intensifies, LDH and CK will be increasingly released from cardiomyocytes (27,28). in the H9c2 cells, H/r caused cardiomyocyte damage, which was attenuated by i.
The Bcl-2 family contains Bcl-2 and Bax, and downregulating the level of Bax and upregulating the level of Bcl-2 can reduce cell apoptosis (29). The Bax/Bcl-2 ratio is regarded as a main factor in determining cell apoptosis, and the majority of studies investigate apoptosis via the Bax/Bcl signaling pathway (30)(31)(32)(33). The present study demonstrated that i attenuated H/r-induced apoptosis by decreasing Bax and increasing Bcl-2. IL-6, a pro-inflammatory factor, is rapidly produced in response to tissue injury (34,35). a reduction of il-6 has beneficial functions in tissues and prevents destructive effects (36), as it can reduce inflammation and prevent further injury (37). Injury causes significant immune dysfunction, and TNF-α is a potent proinflammatory cytokine (38,39). It was revealed in the present study that i could reduce immune dysfunction caused by H/R by decreasing expression of the inflammatory cytokines il-6 and TnF-α.
Previous studies have demonstrated that the downregulation of p-JaK2 and p-STaT3 can increase cell apoptosis and decrease cell viability (40)(41)(42). it has also been reported that proapoptotic factors such as Bax are upregulated in H/r injury-treated cells (3,43). The present study revealed that M could not only inhibit p-JaK2 and p-STaT3, but also inhibit cell viability during H/r, whereas i promoted JaK2 and STaT3 during H/r and increased cell viability. in order to investigate whether the JaK2/STaT3 signaling pathway contributed to the effects of i on H/r damage, aG490 (which inhibits JaK2 and STaT3 during H/r) was used to investigate cell viability during H/r. notably, aG490 inhibited cell viability during H/r, and aG490 inhibited the p-JaK2/iaK2 and p-STaT3/STaT3 ratios in M-transfected and H/r-treated cells. However, i promoted cell viability, and the p-JaK2/JaK and p-STaT3/STaT3 ratios during H/r. Therefore, it was hypothesized that the levels of JaK2, p-JaK2 and p-STaT3 were important indicators to regulate the effects of mir-101a-3p on H9c2 cells during H/r. However, the present study did not investigate in depth whether the levels of these molecules were of clear importance for H9c2 cells during H/r. in future studies, the effects of the levels of JaK2, p-JaK2 and p-STaT3 on H9c2 cells during H/r should be studied via quantitative analysis. it has been demonstrated that the activation of JaK2 results in phosphorylation of downstream STaT3 signaling pathways (44). However, the role of STaT3 in i-treated cardiomyocytes during H/r, and the association between JaK2 activation and STaT activation during H/r remains unclear and should be determined in future studies.
in conclusion, the present study indicated that downregulation of mir-101a-3p prevented not only H/r-induced damage to cardiomyocytes by decreasing inflammatory responses, but also reduced cell apoptosis during H/r injury via the Bax/Bcl-2 signaling pathway. The present study provided evidence that the JaK2/STaT3 signaling pathway served an important role in decreasing H/r injury in cardiomyocytes following inhibition of mir-101a-3p.