Mol. Cells 2019; 42(5): 397-405
Published online April 15, 2019
https://doi.org/10.14348/molcells.2018.0180
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *lengjiyan424@sina.com
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
The regulatory role of long noncoding RNA (lncRNA) growth arrest-specific transcript 5 (
Keywords H9c2 cell, heart failure, hypoxia, lncRNA
Heart failure is a complex clinical syndrome caused by systolic and diastolic dysfunction, resulting in a mismatch between demand and supply of oxygenated blood (Vucicevic et al., 2018). It represents a debilitating disorder, affecting approximately 26 million people worldwide, and leading to more than 1 million hospitalizations in United States and Europe (Ambrosy et al., 2014). What’s worse, heart failure is associated with high morbidity and mortality as itself increases the risk of stroke (Kim and Kim, 2018). Recently, cardiac function can be significantly improved by medical treatment and instrument therapies. However, heart failure remains the major cause of death worldwide (Ziaeian and Fonarow, 2016) due to the current management is limited in improving symptoms and preventing disease progression. This phenomenon calls for a better understanding of heart failure, which will be helpful for improving the development of novel treatment strategies.
Noncoding RNAs (ncRNAs) are a class of RNAs without protein-coding capacity. ncRNAs were initially considered as “junk DNAs”. But recent decades, researchers found that approximately 98% of the human genome are ncRNAs (Mattick, 2001), and they have regulatory functions that effectively feedback into a larger communication network (Adams et al., 2017). Long ncRNAs (lncRNAs) and microRNAs (miRNAs) are two main groups of ncRNAs that have gained widespread attention recently. It is believed that lncRNAs and miRNAs are key regulators in modulation of cell proliferation, cell-cycle progression, differentiation, apoptosis, migration,
LncRNA growth arrest specific transcript 5 (
Rat embryonic ventricular cardiomyocyte H9c2 (CRL-1446, ATCC, USA) was routinely cultured in DMEM (Sigma-Aldrich, USA) containing 10% fetal bovine serum (Gibco, USA). The cells were maintained at an atmosphere with 95% air and 5% CO2 at 37°C.
To make hypoxic injury, H9c2 cells were incubated in a hypoxic incubator containing 94% N2, 5% CO2, and 1% O2. The cells incubated in normoxic condition (with 21% O2) were used as control.
shRNA specific for lncRNA
The untransfected and transfected cells were seeded in 96-well plates with a density of 5 × 103 cells/well for adhesion. The cells were subjected to hypoxia for 6 h, after which the plates were placed in the normoxic condition for 48 h. The culture medium of each well was removed, the cells were washed twice with phosphate buffer saline (PBS), and 10 μl CCK-8 solution (Dojindo Molecular Technologies, USA) was added. The plates were incubated at 37°C for 1 h, thereafter the absorbance was measured by a Microplate Reader (Bio-Rad, USA) at 450 nm.
The FITC-annexin V/PI detection kit (Biosea Biotechnology, China) was utilized in this study for testing cell apoptosis. The untransfected and transfected cells were seeded in 6-well plates with a density of 5 × 105 cells/well, and subjected to hypoxia for 6 h. Subsequently, cells were collected by trypsin-EDTA solution (Sigma-Aldrich), and resuspended in 200 μl Binding Buffer. The cells were then stained by 10 μl FITC-annexin V and 5 μl PI for 30 min at room temperature in the dark. At least 1 × 105 cells per sample were analyzed by the cytometry (Beckman Coulter, USA). Apoptotic cells (FITC-annexin V-positive and PI-negative) were analyzed by using FlowJo software (Treestar, USA).
For testing the endogenous association between lncRNA
The 3′UTR fragment of
After transfection and hypoxia exposure, the cells in 24-well plates were washed twice with ice-cold PBS. Total RNA in cell was extracted by using the Trizol reagent (Life Technologies Corporation, USA). The purity and concentration of RNA in the extracts was tested by UV spectrophotometry. To test the expression of lncRNA
After transfection and hypoxia exposure, the cells in 24-well plates were washed twice with ice-cold PBS. Total protein in cell was extracted by using RIPA lysis buffer (Santa Cruz Biotechnology, USA). The proteins were separated by SDS-PAGE and were transferred onto PVDF membranes (Millipore, MA). After blocking for 1 h at room temperature with 5% nonfat milk in Tris buffered saline-0.01% Tween 20 (Santa Cruz Biotechnology), the membranes were incubated with primary antibodies at 4°C overnight for detection of p53 (ab26), CyclinD1 (ab16663), CDK4 (ab199728), Bax (ab32503), Bcl-2 (ab32124), pro-caspase-3 (ab32150), cleaved-caspase-3 (ab2302), PI3K (ab191606), p-PI3K (ab182651), AKT (ab8805), p-AKT (ab38449), MEK (ab32091), p-MEK (ab96379), ERK (ab32537), p-ERK (ab131438), β-actin (ab8227, Abcam, USA) and TP53INP1 (orb163035, Biorbyt, USA). After three washes with Tris buffered saline-0.01% Tween 20, the membranes were incubated with goat anti-mouse IgG (ab6785, Abcam) or goat anti-rabbit IgG (ab6721, Abcam) for 1 h at room temperature. The signals were developed by using the chemiluminescence detection kit (Pierce, USA), and the intensity was quantified by using Image Lab™ Software (Bio-Rad).
All experiments were repeated three times in triplicate. All results were presented as mean ± SD. Statistical analyses were done in the SPSS 19.0 software (SPSS Inc., USA). Difference between groups was analyzed by Student
To start with, H9c2 cells were subjected to hypoxic condition for 0–24 h. We observed that the viability of H9c2 cells was significantly decreased in hypoxia group as compared to the normoxia control group (
To explore the functional effects of lncRNA
Next, we measured the expression changes of
In order to validate the abovementioned hypothesis, an inhibitor specific for
Finally, by using the TargetScan online database,
The hypothesis of the present study was that silence of lncRNA
A growing number of lncRNAs have been linked to various kinds of cardiovascular diseases. For instance, increased expression of lncRNA Kcna2 Antisense RNA (
Recent studies indicate that lncRNAs can interact with miRNAs and these interactions play significant roles in the determination of cell fate (Cao et al., 2017; Duval et al., 2017). Herein, we focused on the regulation between lncRNA
PI3K/AKT and MEK/ERK signaling pathways are known to play key regulatory roles in numerous cellular functions, including proliferation, cell-cycle progression, apoptosis, differentiation and neoplastic transformation (Bader et al., 2005; Chang et al., 2003; Peyssonnaux and Eychene, 2001). The importance of these two signaling in heart failure has been widely revealed. For instance, Chinese medicine Qishenkeli could prevent cardiomyocytes apoptosis through modulation of PI3K/AKT signaling pathway in animal and H9c2 cell model of heart failure (Chang et al., 2017). Advanced glycation end products caused cardiac hypertrophy via the MEK/ERK pathway (Ko et al., 2013). Under hypoxic condition, p-AKT is decreased, while the total level of AKT is unchanged (Hirai et al., 2003). Besides, AKT is a downstream effector of PI3K, and PI3K is importance for the activation of MEK and ERK (Schmidt et al., 2004). In the present study, we found that hypoxia exposure for 6 h significantly decreased p-PI3K and p-AKT levels, while have no impacts on the levels of p-MEK and p-ERK. More importantly, silence of lncRNA
To sum up, we demonstrated that silence of lncRNA
Mol. Cells 2019; 42(5): 397-405
Published online May 31, 2019 https://doi.org/10.14348/molcells.2018.0180
Copyright © The Korean Society for Molecular and Cellular Biology.
Jian Du, Si-Tong Yang, Jia Liu, Ke-Xin Zhang, and Ji-Yan Leng*
Department of Cadre Ward, The First Hospital of Jilin University, Changchun, Jilin 130021, China
Correspondence to:*lengjiyan424@sina.com
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
The regulatory role of long noncoding RNA (lncRNA) growth arrest-specific transcript 5 (
Keywords: H9c2 cell, heart failure, hypoxia, lncRNA
Heart failure is a complex clinical syndrome caused by systolic and diastolic dysfunction, resulting in a mismatch between demand and supply of oxygenated blood (Vucicevic et al., 2018). It represents a debilitating disorder, affecting approximately 26 million people worldwide, and leading to more than 1 million hospitalizations in United States and Europe (Ambrosy et al., 2014). What’s worse, heart failure is associated with high morbidity and mortality as itself increases the risk of stroke (Kim and Kim, 2018). Recently, cardiac function can be significantly improved by medical treatment and instrument therapies. However, heart failure remains the major cause of death worldwide (Ziaeian and Fonarow, 2016) due to the current management is limited in improving symptoms and preventing disease progression. This phenomenon calls for a better understanding of heart failure, which will be helpful for improving the development of novel treatment strategies.
Noncoding RNAs (ncRNAs) are a class of RNAs without protein-coding capacity. ncRNAs were initially considered as “junk DNAs”. But recent decades, researchers found that approximately 98% of the human genome are ncRNAs (Mattick, 2001), and they have regulatory functions that effectively feedback into a larger communication network (Adams et al., 2017). Long ncRNAs (lncRNAs) and microRNAs (miRNAs) are two main groups of ncRNAs that have gained widespread attention recently. It is believed that lncRNAs and miRNAs are key regulators in modulation of cell proliferation, cell-cycle progression, differentiation, apoptosis, migration,
LncRNA growth arrest specific transcript 5 (
Rat embryonic ventricular cardiomyocyte H9c2 (CRL-1446, ATCC, USA) was routinely cultured in DMEM (Sigma-Aldrich, USA) containing 10% fetal bovine serum (Gibco, USA). The cells were maintained at an atmosphere with 95% air and 5% CO2 at 37°C.
To make hypoxic injury, H9c2 cells were incubated in a hypoxic incubator containing 94% N2, 5% CO2, and 1% O2. The cells incubated in normoxic condition (with 21% O2) were used as control.
shRNA specific for lncRNA
The untransfected and transfected cells were seeded in 96-well plates with a density of 5 × 103 cells/well for adhesion. The cells were subjected to hypoxia for 6 h, after which the plates were placed in the normoxic condition for 48 h. The culture medium of each well was removed, the cells were washed twice with phosphate buffer saline (PBS), and 10 μl CCK-8 solution (Dojindo Molecular Technologies, USA) was added. The plates were incubated at 37°C for 1 h, thereafter the absorbance was measured by a Microplate Reader (Bio-Rad, USA) at 450 nm.
The FITC-annexin V/PI detection kit (Biosea Biotechnology, China) was utilized in this study for testing cell apoptosis. The untransfected and transfected cells were seeded in 6-well plates with a density of 5 × 105 cells/well, and subjected to hypoxia for 6 h. Subsequently, cells were collected by trypsin-EDTA solution (Sigma-Aldrich), and resuspended in 200 μl Binding Buffer. The cells were then stained by 10 μl FITC-annexin V and 5 μl PI for 30 min at room temperature in the dark. At least 1 × 105 cells per sample were analyzed by the cytometry (Beckman Coulter, USA). Apoptotic cells (FITC-annexin V-positive and PI-negative) were analyzed by using FlowJo software (Treestar, USA).
For testing the endogenous association between lncRNA
The 3′UTR fragment of
After transfection and hypoxia exposure, the cells in 24-well plates were washed twice with ice-cold PBS. Total RNA in cell was extracted by using the Trizol reagent (Life Technologies Corporation, USA). The purity and concentration of RNA in the extracts was tested by UV spectrophotometry. To test the expression of lncRNA
After transfection and hypoxia exposure, the cells in 24-well plates were washed twice with ice-cold PBS. Total protein in cell was extracted by using RIPA lysis buffer (Santa Cruz Biotechnology, USA). The proteins were separated by SDS-PAGE and were transferred onto PVDF membranes (Millipore, MA). After blocking for 1 h at room temperature with 5% nonfat milk in Tris buffered saline-0.01% Tween 20 (Santa Cruz Biotechnology), the membranes were incubated with primary antibodies at 4°C overnight for detection of p53 (ab26), CyclinD1 (ab16663), CDK4 (ab199728), Bax (ab32503), Bcl-2 (ab32124), pro-caspase-3 (ab32150), cleaved-caspase-3 (ab2302), PI3K (ab191606), p-PI3K (ab182651), AKT (ab8805), p-AKT (ab38449), MEK (ab32091), p-MEK (ab96379), ERK (ab32537), p-ERK (ab131438), β-actin (ab8227, Abcam, USA) and TP53INP1 (orb163035, Biorbyt, USA). After three washes with Tris buffered saline-0.01% Tween 20, the membranes were incubated with goat anti-mouse IgG (ab6785, Abcam) or goat anti-rabbit IgG (ab6721, Abcam) for 1 h at room temperature. The signals were developed by using the chemiluminescence detection kit (Pierce, USA), and the intensity was quantified by using Image Lab™ Software (Bio-Rad).
All experiments were repeated three times in triplicate. All results were presented as mean ± SD. Statistical analyses were done in the SPSS 19.0 software (SPSS Inc., USA). Difference between groups was analyzed by Student
To start with, H9c2 cells were subjected to hypoxic condition for 0–24 h. We observed that the viability of H9c2 cells was significantly decreased in hypoxia group as compared to the normoxia control group (
To explore the functional effects of lncRNA
Next, we measured the expression changes of
In order to validate the abovementioned hypothesis, an inhibitor specific for
Finally, by using the TargetScan online database,
The hypothesis of the present study was that silence of lncRNA
A growing number of lncRNAs have been linked to various kinds of cardiovascular diseases. For instance, increased expression of lncRNA Kcna2 Antisense RNA (
Recent studies indicate that lncRNAs can interact with miRNAs and these interactions play significant roles in the determination of cell fate (Cao et al., 2017; Duval et al., 2017). Herein, we focused on the regulation between lncRNA
PI3K/AKT and MEK/ERK signaling pathways are known to play key regulatory roles in numerous cellular functions, including proliferation, cell-cycle progression, apoptosis, differentiation and neoplastic transformation (Bader et al., 2005; Chang et al., 2003; Peyssonnaux and Eychene, 2001). The importance of these two signaling in heart failure has been widely revealed. For instance, Chinese medicine Qishenkeli could prevent cardiomyocytes apoptosis through modulation of PI3K/AKT signaling pathway in animal and H9c2 cell model of heart failure (Chang et al., 2017). Advanced glycation end products caused cardiac hypertrophy via the MEK/ERK pathway (Ko et al., 2013). Under hypoxic condition, p-AKT is decreased, while the total level of AKT is unchanged (Hirai et al., 2003). Besides, AKT is a downstream effector of PI3K, and PI3K is importance for the activation of MEK and ERK (Schmidt et al., 2004). In the present study, we found that hypoxia exposure for 6 h significantly decreased p-PI3K and p-AKT levels, while have no impacts on the levels of p-MEK and p-ERK. More importantly, silence of lncRNA
To sum up, we demonstrated that silence of lncRNA
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