Mol. Cells 2019; 42(11): 794-803
Published online November 7, 2019
https://doi.org/10.14348/molcells.2019.0121
© The Korean Society for Molecular and Cellular Biology
Correspondence to : lmhk@mail.ustc.edu.cn
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/.
Ultraviolet light (UV)-induced cellular response has been studied by numerous investigators for many years. Long noncoding RNAs (lncRNAs) are emerging as new regulators of diverse cellular process; however, little is known about the role of lncRNAs in the cellular response to UV treatment. Here, we demonstrate that levels of lncRNA-HOTTIP significantly increases after UV stimulation and regulates the UV-mediated cellular response to UV through the coordinate activation of its neighboring gene Hoxa13 in GC-1 cells (spermatogonia germ cell line). UV-induced, G2/M-phase arrest and early apoptosis can be regulated by lncRNA-HOTTIP and Hoxa13. Furthermore, lncRNA-HOTTIP can up-regulate γ-H2AX and p53 expression via Hoxa13 in UV-irradiated GC-1 cells. In addition, p53 has the ability to regulate the expression of both lncRNA-HOTTIP and Hoxa13
Keywords DNA damage, HOTTIP, Hoxa13, spermatogenic cell, ultraviolet light
Solar ultraviolet light (UV) irradiation is the most prominent and ubiquitous physical carcinogen in our natural environment and UV-mediated DNA damage plays a critical role in the development of skin cancer (Armstrong and Kricker, 2001; de Gruijl, 1999; de Gruijl et al., 2001). DNA damage induced by UV irradiation initiates cellular recovery mechanisms, including the activation of signaling pathways that respond to DNA damage, cell cycle arrest and apoptosis (Gentile et al., 2003). UV irradiation can regulate testicular germ cell fate through DNA damage (Koberle et al., 2008). Recent studies have indicated that the mechanism for the cellular response to UV involves noncoding RNAs (Degueurce et al., 2016; Li et al., 2012; Williamson et al., 2017). UV stimulation promotes the transcription of the long noncoding RNA (lncRNA), ASCC3, which inhibits the expression of the ASCC3 protein encoded by the same gene, and plays important role in the cellular response induced by UV light (Williamson et al., 2017). Further, the expression of intermediate-size (70–500 nucleotides) ncRNAs have been shown to be altered post UV irradiation in
Throughout eukaryotic genomes, lncRNAs are pervasively transcribed and range in size from 200 bp to over 100 kb (Brosnan and Voinnet, 2009; Chen and Carmichael, 2010; Kapranov et al., 2007). Depending on their proximity to protein-coding genes, lncRNAs may be classified as antisense, intronic, bidirectional or intergenic lncRNAs (Rinn and Chang, 2012). Mounting evidence have recently suggested that lncRNAs are important molecules with roles in a in a diverse set of cellular processes, including growth, cell cycle, differentiation and apoptosis, tumor suppression and promotion, and the stress response (Liu et al., 2012; Tripathi et al., 2013; Zhou et al., 2007). Furthermore, lncRNAs can regulate the expression of neighboring protein-coding genes at the level of chromatin remodelling, and transcriptional and posttranscriptional processing (Mercer et al., 2009). Although lncRNAs have been investigated extensively, very little is known about the function of lncRNAs with respect to the testicular germ cell response to UV stimulation.
The lncRNA-HOTTIP (HOXA transcript at the distal tip) is spliced and polyadenylated transcript located at the 5′ tip of the Hoxa13 gene. The lncRNA-HOTTIP coordinates the activation of several HOXA genes through binding to and driving MLL1/WDR5 occupancy and H3K4 trimethylation of the HOXA gene promoter in human primary fibroblasts (Burgess, 2011; Wang et al., 2011). In addition, HOTTIP regulates human cartilage development and destruction by modulating integrin-α1 transcription via Hoxa13, and the HOTTIP transcript could be a potent predictive biomarker for osteoarthritis (Kim et al., 2013). In hepatocellular carcinoma patients, HOTTIP and Hoxa13 expression are associated with disease progression and can predict patient outcome (Quagliata et al., 2013). Furthermore, the expression levels of HOTTIP RNA are up-regulated in both osteoarthritis chondrocytes and hepatocellular carcinoma specimens (Kim et al., 2013; Quagliata et al., 2013), suggesting that HOTTIP is involved in multiple types of conditions in which the misregulation of cellular functioning occurrs. HOTTIP can be used as aprognostic biomarker for early-stages of human non–small-cell lung cancer and is correlated with a number of mRNAs and miRNAs signatures (Navarro et al., 2019). Moreover, HOTTIP enhances insulin secretion and regulates cell proliferation and the cell cycle by modifying the MEK/ERK cascade in islet-β cells (Xu et al., 2018). It is not clear; however, whether HOTTIP participates in the cellular response to UV-induced DNA damage in germ cells.
In the current study, we have elucidated that expression of lncRNA-HOTTIP and Hoxa13 in mouse tissues and cells, and HOTTIP-Hoxa13 expression is involved in the response to UV-mediated DNA damage in the spermatogonia germ cell line GC-1. Moreover, we have found that HOTTIP-Hoxa13 plays a major role in UV-induced cell cycle arrest and apoptosis via regulating γ-H2AX and p53 expression. Collectively, this study uncovers new insights into the function of HOTTIP-Hoxa13 in response to UV damage in spermatogenic cells.
C57BL6/J mice were originally acquired from the Laboratory Animal Center, Bengbu Medical College (Bengbu, China), and housed at room temperature (23 ± 2°C) under of 14 h light and 10 h dark. The mice had free access to water and food. This study received ethical approval from the Ethical Committee for Bengbu Medical College (approval No. 2016004).
The mouse Hoxa13 expression vector was constructed by cloning the mouse Hoxa13 cDNA into the pcDNA3.1(+) vector at the
NIH3T3, STO, GC-1, GC-2 and C18-4 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Life Technologies, USA) or DMEM/F12 (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies) and 1% penicillin and streptomycin (100 U/ml penicillin and 100 μg/ml streptomycin; Life Technologies) and incubated at 37°C and 5% CO2. Transfection of GC-1 cells were carried out with Lipofectamine 2000 reagent (Invitrogen, USA) following the manufacturer’s protocol and cells were transfected with 100 nM si-HOTTIP or si-NC. The DNA plasmids, si-HOTTIP and si-NC were diluted in Opti-MEM I reduced serum medium (Life Technologies).
For UV irradiation, the cultured medium of transfected cells was discarded and cells were rinsed with phosphate-buffered saline (PBS) (van der Wees et al., 2003). GC-1 cells were irradiated at 0, 5 or 10 J/m2 using UV Crosslinker with a peak emission at 254 nm (UVP CL-1000; Analytic Jena US LLC, USA). Immediately after UV treatment, the cells were cultured at 5% CO2 and 37°C in fresh medium for 24 h and then collected to study cellular responses to UV.
Total RNA was extracted from either cultured cells or mouse tissues with Trizol (Invitrogen) (Ma et al., 2013). RT-qPCR assays were used to analyze lncRNA-HOTTIP and Hoxa13 mRNA expression according to protocols outlined previously (Bustin et al., 2009). Briefly, RNA was reverse transcribed into cDNA by using the PrimeScript RT reagent kit (TaKaRa, Japan) and real-time PCR was executed on ABI Step One System (Applied Biosystems, USA) using the SYBR Premix Ex Taq II kit (TaKaRa) according to the manufacturer’s instructions. The relative expression levels of lncRNA-HOTTIP and Hoxa13 mRNA were normalized using endogenous β-actin mRNA. Primers for β-actin, HOTTIP, and Hoxa13 have been listed in Supplementary Table S2.
The proliferation of GC-1 cells was measured using a CCK-8 kit (Dojindo Laboratories, Japan) according the manufacturer’s protocols. After 24 h of transfection with si-NC/si-HOTTIP and another 24 h after UV treatment, the GC-1 cells were cultured in fresh medium with 10% CCK-8 reagent for 2 h. Subsequently, absorbance at 450 nm for GC-1 cells were measured using a Cytation 3 imaging reader (BioTek, USA).
Cell apoptosis and cell cycle were analyzed using a FACScalibur Flow Cytometer (BD Biosciences, USA). Data analysis was carried out using WinMDI software. To assess cell apoptosis, cells were collected 48-h post treatment, stained with the Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology, China) and analyzed by FACS. To assess cell cycle, cells were collected 48-h post treatment, washed with PBS and fixed overnight in 70% ethanol. Cells were then harvested, washed with PBS, incubated at 37°C for 10 minutes with PBS, 100 μg/ml RNase A (Sigma-Aldrich, USA) and 25 μg/ml propidium iodide (PI) (Sigma-Aldrich), and analyzed by FACS.
GC-1 cells were lysed in RIPA buffer (Millipore, USA) with 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml CompleteTM EDTA free protease inhibitor cocktail (Roche, USA) and phosphatase inhibitors (5 mM sodium orthovanadate). Protein lysates were loaded on to SDS-PAGE gels, transferred to nitrocellulose membranes (Amersham Biosciences, Germany), immunoblotted with antibodies and visualized using enhanced chemiluminescence substrate (Thermo Fisher Scientific, USA). Protein levels of p53, p21 and γ-H2AX were normalized to GAPDH. The primary antibodies in this study were the following: anti-GAPDH (Cell Signaling Technology, USA), anti-p53 (Santa Cruz, USA), anti-γ-H2AX (Abcam, USA) and anti-p21 (Santa Cruz).
Experiments were repeated at least three times and performed in triplicate. Data have been shown as mean ± SEM. Means for groups were analyzed using the Student’s
According to the UCSC Genome Browser (
We next explored how lncRNA-HOTTIP regulates proliferation post UV irradiation in GC-1 cells. The effects of stimulation with 0, 5, 10 J/m2 UV on cell-cycle progression was determined using FACS analysis. These experiments revealed that stimulation with 5 J/m2 and 10 J/m2 UV can inhibit GC-1 from exiting G2/M phase relative to those that were not irradiated with UV (Figs. 2A and 2B). Knockdown of HOTTIP expression partially reversed the effects of UV, reducing the level of G2/M phase arrest in GC-1 cells stimulated with either 5 J/m2 or 10 J/m2 UV (Figs. 2A and 2B). Furthermore, stimulation with either 5 J/m2 or 10 J/m2 UV causes a significant increase in the proportion of cells that undergo early apoptosis compared with compared with those not subjected to UV treatment (Figs. 2C and 2D). Knockdown of HOTTIP partially reversed the occurrence of early apoptosis in GC-1 cells irradiated with either 5 J/m2 or 10 J/m2 UV (Figs. 2C and 2D).
To determine whether levels of the cell cycle and apoptosis-related proteins, p53 and p21, and the UV-induced, DNA-damage-repair protein, γ-H2AX, were affected, relative expression of the proteins were assessed by western blot. As shown in Figure 2E, irradiation with 5 J/m2 and 10 J/m2 UV promoted expression of both p53 and p21, proving a mechanistic explanation of why GC-1 cells accumulated in G2/M phase and underwent early apoptosis post UV exposure. Stimulation with 5 J/m2 and 10 J/m2 UV also enhanced γ-H2AX expression, which suggests that DNA damage occurs in UV-irradiated GC-1 cells (Fig. 2E). Knockdown of HOTTIP can reverse the effects stimulation with 5 J/m2 and 10 J/m2 UV with respect to p53, p21 and γ-H2AX levels (Fig. 2E). Furthermore, lncRNA-HOTTIP can promote γ-H2AX transcription in UV-induced GC-1 cells with DNA damage (Supplementary Fig. S2). These results suggest that HOTTIP may have the ability to inhibit UV-induced cell-cycle arrest, apoptosis and DNA damage repair. Induction of G2/M cell cycle arrest by lncRNA-HOTTIP indicates that HOTTIP acts as a pro-apoptotic activator and then causes DNA damage in UV-stimulated GC-1 cells.
Previous studies have reported that Hoxa13 is involved in cell proliferation, the cell cycle and apoptosis both
The mutual regulation of p53 and lncRNA has already been shown to vital roles in physiological and pathological processes such as development and cancer (Joo et al., 2019; Voce et al., 2019). In this research, we revealed that expression of lncRNA-HOTTIP and Hoxa13 was lower in p53 −/− mouse testis, ovaries and lung cells compared with the same type of cells from p53 +/+ mice (Fig. 4A). Furthermore, overexpression of p53 in GC-1 cells significantly increased the expression level of both lncRNA-HOTTIP and Hoxa13 (Fig. 4B), while knockdown of p53 in GC-1 cells significantly decreased the expression of lncRNA-HOTTIP and Hoxa13 (Fig. 4C). These data identified a p53-HOTTIP-hoxa13 signaling pathway that participates in the cellular response to UV. Further, expression of lncRNA-HOTTIP and Hoxa13 was higher in p53 −/− mouse liver and heart cells compared with liver and heart cells from a p53 +/+ mouse (Fig. 4A), these findings suggest that p53 regulates HOTTIP expression in a tissue-specific manner. In the future, further study will be conducted both
The majority of the animal genomes encode a large number of noncoding RNAs, which can be broadly divided into two groups comprised of small ncRNAs (18–200 bp) and long ncRNAs (200 bp to > 100 kb) (Bohacek and Mansuy, 2015; Prasanth and Spector, 2007). In addition to well-studied small ncRNAs (e.g., microRNAs, piwi-interacting RNAs and small interfering RNAs), lncRNAs have recently been identified as important molecules that are responsible for the regulation of diverse cellular processes including dosage compensation and genomic imprinting (Militti et al., 2014; Park et al., 2017). Furthermore, the coordinated expression of lncRNAs with proximal or distal genes plays an important role in physiological and pathological processes in lower and higher organisms (Carmona et al., 2018; Li et al., 2017; Tan et al., 2019; Zhao et al., 2018). For example, the expression of the β-catenin-coordinated lncRNA, MALAT1, and downstream signaling factors, affects the function of hepatocyte growth factors and promotes differentiation of bone marrow mesenchymal stem cells into hepatocytes (Tan et al., 2019). The lncRNA, Jpx, regulates expression of the lncRNA, Xist, in a dose-dependent manner using both trans and cis methods, which were confirmed using transgenic mouse models (Carmona et al., 2018). The lncRNA, MACC1-AS1, is an antisense version of the transcriptional regulator, MACC1, which regulates the mesenchymal to epithelial transition (MET). MACC1 expression increases under conditions of metabolic stimulation and facilitates metabolic plasticity through enhancing MACC1 expression via mRNA stabilization (Zhao et al., 2018). The signaling pathway including lncRNA linc1281, miRNA let-7 and DNA methylation promote the acute extinction of naive pluripotency, which is required for the rapid transition from the pre-implantation epiblast stage to gastrulation in rodents (Li et al., 2017). In this research, we demonstrated that lncRNA-HOTTIP is coordinated with expression of Hoxa13 both
Many studies have focused on the functions of lncRNAs with respect to cellular proliferation, cell cycle and apoptosis and have revealed both positive and negative effects of lncRNAs on the cellular functioning (Lei et al., 2018; Li et al., 2018b; Wang et al., 2018a; Zeng et al., 2017; Zhang et al., 2018). Low expression of lncRNA NBAT-1 is associated with the occurrence of lung cancer and overexpression of lncRNA NBAT-1 suppress cellular proliferation, the cell cycle and stimulate apoptosis in A549 cells (Lei et al., 2018). LncRNA AF113014 restrains proliferation of the hepatocllular carcinoma cell line both
UV is a vital external stimulus that can regulate cellular activities in various biological systems through the modification of multiple signaling pathways (Ascer et al., 2015; Farrell et al., 2018; Kaneko et al., 2015; Matsunuma et al., 2016; Siegenthaler et al., 2018). UV irradiation facilitates the phosphorylation of histone acetyltransferases and affects CRL4 ubiquitylation and, subsequently, inhibits cellular proliferation (Matsunuma et al., 2016). Over-activation of CDC42 is induced by UV stimulation and results in G2/M phase arrest and suppresses proliferation and survival in Hela cells (Ascer et al., 2015). The deficiency of chromatin remodeling protein Brahma in keratinocytes enhances UV-induced carcinogenesis through promoting the cell cycle progression and promotes the accumulation of DNA mutations (Farrell et al., 2018). BV-2 microglial cells treated with lipopolysaccharide can resist UV-stimulated apoptosis via cell cycle arrest in G1 through the upregulation of expression of p21 and GADD45α (Kaneko et al., 2015). The transcription factor nuclear factor erythroid 2-related factor 3 (Nrf3) activates UV-induced apoptosis in keratinocytes through the inhibition of cell-cell and cell-matrix adhesion (Siegenthaler et al., 2018). This study has revealed that UV treatment significantly promotes lncRNA-HOTTIP expression, which coordinates the activation of Hoxa13, a neighboring gene, in GC-1 cells. In addition, UV-induced G2/M phase arrest and early apoptosis are regulated by HOTTIP and Hoxa13. Signaling through lncRNA-HOTTIP-Hoxa13 regulates the expression of p53, p21 and γ-H2AX in UV irradiated GC-1 cells. Taken together, these results have shown that a novel lncRNA-related signaling cascade participates in regulating the response to UV irradiation in reproductive cells.
A growing number of studies have indicated that lncRNA-HOTTIP contributes to a variety of human conditions and are key candidates as biomarkers of disease (Li et al., 2018a; Su et al., 2019; Sun and Liu, 2018; Wang et al., 2018b; Zhuang et al., 2019). Upregulation of lncRNA-HOTTIP in acute myeloid leukemia-M5 patients significantly activated cellular proliferation and cell cycle progression through the modification miR-608-DDA1 (Zhuang et al., 2019). In renal carcinoma cells, increased expression of lncRNA-HOTTIP promotes cellular proliferation, migration and invasion and inhibits cellular autophagy via the PI3K-Akt-Atg13 signaling pathway (Su et al., 2019). In addition, the lncRNA-HOTTIP levels were decreased in an ischemic stroke mouse model, while overexpression of lncRNA-HOTTIP weakened oxygen-glucose-deprivation-induced neuronal injury. The lncRNA affected glycolytic metabolism by sequestering miR-143, which directly targeted HK-2 expression (Wang et al., 2018b). Expression of lncRNA-HOTTIP was remarkably increased in diabetic retinopathy rat and mouse models, which aggravate diabetic retinal microangiopathy via p38-MAPK signaling (Sun and Liu, 2018). The reduced level of expression of lncRNA-HOTTIP in preeclampsia patients has been associated with maternal blood pressure and urinary protein levels. Overexpression of lncRNA-HOTTIP, however has been shown to promote trophoblast cellular proliferation and cell cycle progression through suppressing Rho family GTPase 3 (Li et al., 2018a). This research demonstrated that the expression of lncRNA-HOTTIP and Hoxa13 was significantly decreased in the testis of p53 −/− versus wild type mouse models. Overexpression of p53 in GC-1 cells significantly promoted the expression of lncRNA-HOTTIP and Hoxa13
This research showed a novel lncRNA-HOTTIP-Hoxa13 signaling pathway in the regulation of the UV-induced GC-1 cellular response. UV irradiation causes spermatogonia germ cell line, GC-1, cells to arrest in the G2/M phase and undergo early apoptosis. This occurs, at least partiall0y, through the synergistic co-expression of HOTTIP and Hoxa13, along with increased expression of downstream signaling factors including p53, p21 and γ-H2AX (Fig. 5). Further analysis of the roles of lncRNA-HOTTIP
This work was supported by the following grants: National Natural Science Foundation of China (81801516), Key project of Natural Science Foundation of Anhui Provincial Department of Education (P.R. China) (KJ2018A0992).
Mol. Cells 2019; 42(11): 794-803
Published online November 30, 2019 https://doi.org/10.14348/molcells.2019.0121
Copyright © The Korean Society for Molecular and Cellular Biology.
Meng Liang* and Ke Hu
Department of Biotechnology, School of Life Science, Bengbu Medical College, Bengbu 233030, China
Correspondence to:lmhk@mail.ustc.edu.cn
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/.
Ultraviolet light (UV)-induced cellular response has been studied by numerous investigators for many years. Long noncoding RNAs (lncRNAs) are emerging as new regulators of diverse cellular process; however, little is known about the role of lncRNAs in the cellular response to UV treatment. Here, we demonstrate that levels of lncRNA-HOTTIP significantly increases after UV stimulation and regulates the UV-mediated cellular response to UV through the coordinate activation of its neighboring gene Hoxa13 in GC-1 cells (spermatogonia germ cell line). UV-induced, G2/M-phase arrest and early apoptosis can be regulated by lncRNA-HOTTIP and Hoxa13. Furthermore, lncRNA-HOTTIP can up-regulate γ-H2AX and p53 expression via Hoxa13 in UV-irradiated GC-1 cells. In addition, p53 has the ability to regulate the expression of both lncRNA-HOTTIP and Hoxa13
Keywords: DNA damage, HOTTIP, Hoxa13, spermatogenic cell, ultraviolet light
Solar ultraviolet light (UV) irradiation is the most prominent and ubiquitous physical carcinogen in our natural environment and UV-mediated DNA damage plays a critical role in the development of skin cancer (Armstrong and Kricker, 2001; de Gruijl, 1999; de Gruijl et al., 2001). DNA damage induced by UV irradiation initiates cellular recovery mechanisms, including the activation of signaling pathways that respond to DNA damage, cell cycle arrest and apoptosis (Gentile et al., 2003). UV irradiation can regulate testicular germ cell fate through DNA damage (Koberle et al., 2008). Recent studies have indicated that the mechanism for the cellular response to UV involves noncoding RNAs (Degueurce et al., 2016; Li et al., 2012; Williamson et al., 2017). UV stimulation promotes the transcription of the long noncoding RNA (lncRNA), ASCC3, which inhibits the expression of the ASCC3 protein encoded by the same gene, and plays important role in the cellular response induced by UV light (Williamson et al., 2017). Further, the expression of intermediate-size (70–500 nucleotides) ncRNAs have been shown to be altered post UV irradiation in
Throughout eukaryotic genomes, lncRNAs are pervasively transcribed and range in size from 200 bp to over 100 kb (Brosnan and Voinnet, 2009; Chen and Carmichael, 2010; Kapranov et al., 2007). Depending on their proximity to protein-coding genes, lncRNAs may be classified as antisense, intronic, bidirectional or intergenic lncRNAs (Rinn and Chang, 2012). Mounting evidence have recently suggested that lncRNAs are important molecules with roles in a in a diverse set of cellular processes, including growth, cell cycle, differentiation and apoptosis, tumor suppression and promotion, and the stress response (Liu et al., 2012; Tripathi et al., 2013; Zhou et al., 2007). Furthermore, lncRNAs can regulate the expression of neighboring protein-coding genes at the level of chromatin remodelling, and transcriptional and posttranscriptional processing (Mercer et al., 2009). Although lncRNAs have been investigated extensively, very little is known about the function of lncRNAs with respect to the testicular germ cell response to UV stimulation.
The lncRNA-HOTTIP (HOXA transcript at the distal tip) is spliced and polyadenylated transcript located at the 5′ tip of the Hoxa13 gene. The lncRNA-HOTTIP coordinates the activation of several HOXA genes through binding to and driving MLL1/WDR5 occupancy and H3K4 trimethylation of the HOXA gene promoter in human primary fibroblasts (Burgess, 2011; Wang et al., 2011). In addition, HOTTIP regulates human cartilage development and destruction by modulating integrin-α1 transcription via Hoxa13, and the HOTTIP transcript could be a potent predictive biomarker for osteoarthritis (Kim et al., 2013). In hepatocellular carcinoma patients, HOTTIP and Hoxa13 expression are associated with disease progression and can predict patient outcome (Quagliata et al., 2013). Furthermore, the expression levels of HOTTIP RNA are up-regulated in both osteoarthritis chondrocytes and hepatocellular carcinoma specimens (Kim et al., 2013; Quagliata et al., 2013), suggesting that HOTTIP is involved in multiple types of conditions in which the misregulation of cellular functioning occurrs. HOTTIP can be used as aprognostic biomarker for early-stages of human non–small-cell lung cancer and is correlated with a number of mRNAs and miRNAs signatures (Navarro et al., 2019). Moreover, HOTTIP enhances insulin secretion and regulates cell proliferation and the cell cycle by modifying the MEK/ERK cascade in islet-β cells (Xu et al., 2018). It is not clear; however, whether HOTTIP participates in the cellular response to UV-induced DNA damage in germ cells.
In the current study, we have elucidated that expression of lncRNA-HOTTIP and Hoxa13 in mouse tissues and cells, and HOTTIP-Hoxa13 expression is involved in the response to UV-mediated DNA damage in the spermatogonia germ cell line GC-1. Moreover, we have found that HOTTIP-Hoxa13 plays a major role in UV-induced cell cycle arrest and apoptosis via regulating γ-H2AX and p53 expression. Collectively, this study uncovers new insights into the function of HOTTIP-Hoxa13 in response to UV damage in spermatogenic cells.
C57BL6/J mice were originally acquired from the Laboratory Animal Center, Bengbu Medical College (Bengbu, China), and housed at room temperature (23 ± 2°C) under of 14 h light and 10 h dark. The mice had free access to water and food. This study received ethical approval from the Ethical Committee for Bengbu Medical College (approval No. 2016004).
The mouse Hoxa13 expression vector was constructed by cloning the mouse Hoxa13 cDNA into the pcDNA3.1(+) vector at the
NIH3T3, STO, GC-1, GC-2 and C18-4 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Life Technologies, USA) or DMEM/F12 (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies) and 1% penicillin and streptomycin (100 U/ml penicillin and 100 μg/ml streptomycin; Life Technologies) and incubated at 37°C and 5% CO2. Transfection of GC-1 cells were carried out with Lipofectamine 2000 reagent (Invitrogen, USA) following the manufacturer’s protocol and cells were transfected with 100 nM si-HOTTIP or si-NC. The DNA plasmids, si-HOTTIP and si-NC were diluted in Opti-MEM I reduced serum medium (Life Technologies).
For UV irradiation, the cultured medium of transfected cells was discarded and cells were rinsed with phosphate-buffered saline (PBS) (van der Wees et al., 2003). GC-1 cells were irradiated at 0, 5 or 10 J/m2 using UV Crosslinker with a peak emission at 254 nm (UVP CL-1000; Analytic Jena US LLC, USA). Immediately after UV treatment, the cells were cultured at 5% CO2 and 37°C in fresh medium for 24 h and then collected to study cellular responses to UV.
Total RNA was extracted from either cultured cells or mouse tissues with Trizol (Invitrogen) (Ma et al., 2013). RT-qPCR assays were used to analyze lncRNA-HOTTIP and Hoxa13 mRNA expression according to protocols outlined previously (Bustin et al., 2009). Briefly, RNA was reverse transcribed into cDNA by using the PrimeScript RT reagent kit (TaKaRa, Japan) and real-time PCR was executed on ABI Step One System (Applied Biosystems, USA) using the SYBR Premix Ex Taq II kit (TaKaRa) according to the manufacturer’s instructions. The relative expression levels of lncRNA-HOTTIP and Hoxa13 mRNA were normalized using endogenous β-actin mRNA. Primers for β-actin, HOTTIP, and Hoxa13 have been listed in Supplementary Table S2.
The proliferation of GC-1 cells was measured using a CCK-8 kit (Dojindo Laboratories, Japan) according the manufacturer’s protocols. After 24 h of transfection with si-NC/si-HOTTIP and another 24 h after UV treatment, the GC-1 cells were cultured in fresh medium with 10% CCK-8 reagent for 2 h. Subsequently, absorbance at 450 nm for GC-1 cells were measured using a Cytation 3 imaging reader (BioTek, USA).
Cell apoptosis and cell cycle were analyzed using a FACScalibur Flow Cytometer (BD Biosciences, USA). Data analysis was carried out using WinMDI software. To assess cell apoptosis, cells were collected 48-h post treatment, stained with the Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology, China) and analyzed by FACS. To assess cell cycle, cells were collected 48-h post treatment, washed with PBS and fixed overnight in 70% ethanol. Cells were then harvested, washed with PBS, incubated at 37°C for 10 minutes with PBS, 100 μg/ml RNase A (Sigma-Aldrich, USA) and 25 μg/ml propidium iodide (PI) (Sigma-Aldrich), and analyzed by FACS.
GC-1 cells were lysed in RIPA buffer (Millipore, USA) with 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml CompleteTM EDTA free protease inhibitor cocktail (Roche, USA) and phosphatase inhibitors (5 mM sodium orthovanadate). Protein lysates were loaded on to SDS-PAGE gels, transferred to nitrocellulose membranes (Amersham Biosciences, Germany), immunoblotted with antibodies and visualized using enhanced chemiluminescence substrate (Thermo Fisher Scientific, USA). Protein levels of p53, p21 and γ-H2AX were normalized to GAPDH. The primary antibodies in this study were the following: anti-GAPDH (Cell Signaling Technology, USA), anti-p53 (Santa Cruz, USA), anti-γ-H2AX (Abcam, USA) and anti-p21 (Santa Cruz).
Experiments were repeated at least three times and performed in triplicate. Data have been shown as mean ± SEM. Means for groups were analyzed using the Student’s
According to the UCSC Genome Browser (
We next explored how lncRNA-HOTTIP regulates proliferation post UV irradiation in GC-1 cells. The effects of stimulation with 0, 5, 10 J/m2 UV on cell-cycle progression was determined using FACS analysis. These experiments revealed that stimulation with 5 J/m2 and 10 J/m2 UV can inhibit GC-1 from exiting G2/M phase relative to those that were not irradiated with UV (Figs. 2A and 2B). Knockdown of HOTTIP expression partially reversed the effects of UV, reducing the level of G2/M phase arrest in GC-1 cells stimulated with either 5 J/m2 or 10 J/m2 UV (Figs. 2A and 2B). Furthermore, stimulation with either 5 J/m2 or 10 J/m2 UV causes a significant increase in the proportion of cells that undergo early apoptosis compared with compared with those not subjected to UV treatment (Figs. 2C and 2D). Knockdown of HOTTIP partially reversed the occurrence of early apoptosis in GC-1 cells irradiated with either 5 J/m2 or 10 J/m2 UV (Figs. 2C and 2D).
To determine whether levels of the cell cycle and apoptosis-related proteins, p53 and p21, and the UV-induced, DNA-damage-repair protein, γ-H2AX, were affected, relative expression of the proteins were assessed by western blot. As shown in Figure 2E, irradiation with 5 J/m2 and 10 J/m2 UV promoted expression of both p53 and p21, proving a mechanistic explanation of why GC-1 cells accumulated in G2/M phase and underwent early apoptosis post UV exposure. Stimulation with 5 J/m2 and 10 J/m2 UV also enhanced γ-H2AX expression, which suggests that DNA damage occurs in UV-irradiated GC-1 cells (Fig. 2E). Knockdown of HOTTIP can reverse the effects stimulation with 5 J/m2 and 10 J/m2 UV with respect to p53, p21 and γ-H2AX levels (Fig. 2E). Furthermore, lncRNA-HOTTIP can promote γ-H2AX transcription in UV-induced GC-1 cells with DNA damage (Supplementary Fig. S2). These results suggest that HOTTIP may have the ability to inhibit UV-induced cell-cycle arrest, apoptosis and DNA damage repair. Induction of G2/M cell cycle arrest by lncRNA-HOTTIP indicates that HOTTIP acts as a pro-apoptotic activator and then causes DNA damage in UV-stimulated GC-1 cells.
Previous studies have reported that Hoxa13 is involved in cell proliferation, the cell cycle and apoptosis both
The mutual regulation of p53 and lncRNA has already been shown to vital roles in physiological and pathological processes such as development and cancer (Joo et al., 2019; Voce et al., 2019). In this research, we revealed that expression of lncRNA-HOTTIP and Hoxa13 was lower in p53 −/− mouse testis, ovaries and lung cells compared with the same type of cells from p53 +/+ mice (Fig. 4A). Furthermore, overexpression of p53 in GC-1 cells significantly increased the expression level of both lncRNA-HOTTIP and Hoxa13 (Fig. 4B), while knockdown of p53 in GC-1 cells significantly decreased the expression of lncRNA-HOTTIP and Hoxa13 (Fig. 4C). These data identified a p53-HOTTIP-hoxa13 signaling pathway that participates in the cellular response to UV. Further, expression of lncRNA-HOTTIP and Hoxa13 was higher in p53 −/− mouse liver and heart cells compared with liver and heart cells from a p53 +/+ mouse (Fig. 4A), these findings suggest that p53 regulates HOTTIP expression in a tissue-specific manner. In the future, further study will be conducted both
The majority of the animal genomes encode a large number of noncoding RNAs, which can be broadly divided into two groups comprised of small ncRNAs (18–200 bp) and long ncRNAs (200 bp to > 100 kb) (Bohacek and Mansuy, 2015; Prasanth and Spector, 2007). In addition to well-studied small ncRNAs (e.g., microRNAs, piwi-interacting RNAs and small interfering RNAs), lncRNAs have recently been identified as important molecules that are responsible for the regulation of diverse cellular processes including dosage compensation and genomic imprinting (Militti et al., 2014; Park et al., 2017). Furthermore, the coordinated expression of lncRNAs with proximal or distal genes plays an important role in physiological and pathological processes in lower and higher organisms (Carmona et al., 2018; Li et al., 2017; Tan et al., 2019; Zhao et al., 2018). For example, the expression of the β-catenin-coordinated lncRNA, MALAT1, and downstream signaling factors, affects the function of hepatocyte growth factors and promotes differentiation of bone marrow mesenchymal stem cells into hepatocytes (Tan et al., 2019). The lncRNA, Jpx, regulates expression of the lncRNA, Xist, in a dose-dependent manner using both trans and cis methods, which were confirmed using transgenic mouse models (Carmona et al., 2018). The lncRNA, MACC1-AS1, is an antisense version of the transcriptional regulator, MACC1, which regulates the mesenchymal to epithelial transition (MET). MACC1 expression increases under conditions of metabolic stimulation and facilitates metabolic plasticity through enhancing MACC1 expression via mRNA stabilization (Zhao et al., 2018). The signaling pathway including lncRNA linc1281, miRNA let-7 and DNA methylation promote the acute extinction of naive pluripotency, which is required for the rapid transition from the pre-implantation epiblast stage to gastrulation in rodents (Li et al., 2017). In this research, we demonstrated that lncRNA-HOTTIP is coordinated with expression of Hoxa13 both
Many studies have focused on the functions of lncRNAs with respect to cellular proliferation, cell cycle and apoptosis and have revealed both positive and negative effects of lncRNAs on the cellular functioning (Lei et al., 2018; Li et al., 2018b; Wang et al., 2018a; Zeng et al., 2017; Zhang et al., 2018). Low expression of lncRNA NBAT-1 is associated with the occurrence of lung cancer and overexpression of lncRNA NBAT-1 suppress cellular proliferation, the cell cycle and stimulate apoptosis in A549 cells (Lei et al., 2018). LncRNA AF113014 restrains proliferation of the hepatocllular carcinoma cell line both
UV is a vital external stimulus that can regulate cellular activities in various biological systems through the modification of multiple signaling pathways (Ascer et al., 2015; Farrell et al., 2018; Kaneko et al., 2015; Matsunuma et al., 2016; Siegenthaler et al., 2018). UV irradiation facilitates the phosphorylation of histone acetyltransferases and affects CRL4 ubiquitylation and, subsequently, inhibits cellular proliferation (Matsunuma et al., 2016). Over-activation of CDC42 is induced by UV stimulation and results in G2/M phase arrest and suppresses proliferation and survival in Hela cells (Ascer et al., 2015). The deficiency of chromatin remodeling protein Brahma in keratinocytes enhances UV-induced carcinogenesis through promoting the cell cycle progression and promotes the accumulation of DNA mutations (Farrell et al., 2018). BV-2 microglial cells treated with lipopolysaccharide can resist UV-stimulated apoptosis via cell cycle arrest in G1 through the upregulation of expression of p21 and GADD45α (Kaneko et al., 2015). The transcription factor nuclear factor erythroid 2-related factor 3 (Nrf3) activates UV-induced apoptosis in keratinocytes through the inhibition of cell-cell and cell-matrix adhesion (Siegenthaler et al., 2018). This study has revealed that UV treatment significantly promotes lncRNA-HOTTIP expression, which coordinates the activation of Hoxa13, a neighboring gene, in GC-1 cells. In addition, UV-induced G2/M phase arrest and early apoptosis are regulated by HOTTIP and Hoxa13. Signaling through lncRNA-HOTTIP-Hoxa13 regulates the expression of p53, p21 and γ-H2AX in UV irradiated GC-1 cells. Taken together, these results have shown that a novel lncRNA-related signaling cascade participates in regulating the response to UV irradiation in reproductive cells.
A growing number of studies have indicated that lncRNA-HOTTIP contributes to a variety of human conditions and are key candidates as biomarkers of disease (Li et al., 2018a; Su et al., 2019; Sun and Liu, 2018; Wang et al., 2018b; Zhuang et al., 2019). Upregulation of lncRNA-HOTTIP in acute myeloid leukemia-M5 patients significantly activated cellular proliferation and cell cycle progression through the modification miR-608-DDA1 (Zhuang et al., 2019). In renal carcinoma cells, increased expression of lncRNA-HOTTIP promotes cellular proliferation, migration and invasion and inhibits cellular autophagy via the PI3K-Akt-Atg13 signaling pathway (Su et al., 2019). In addition, the lncRNA-HOTTIP levels were decreased in an ischemic stroke mouse model, while overexpression of lncRNA-HOTTIP weakened oxygen-glucose-deprivation-induced neuronal injury. The lncRNA affected glycolytic metabolism by sequestering miR-143, which directly targeted HK-2 expression (Wang et al., 2018b). Expression of lncRNA-HOTTIP was remarkably increased in diabetic retinopathy rat and mouse models, which aggravate diabetic retinal microangiopathy via p38-MAPK signaling (Sun and Liu, 2018). The reduced level of expression of lncRNA-HOTTIP in preeclampsia patients has been associated with maternal blood pressure and urinary protein levels. Overexpression of lncRNA-HOTTIP, however has been shown to promote trophoblast cellular proliferation and cell cycle progression through suppressing Rho family GTPase 3 (Li et al., 2018a). This research demonstrated that the expression of lncRNA-HOTTIP and Hoxa13 was significantly decreased in the testis of p53 −/− versus wild type mouse models. Overexpression of p53 in GC-1 cells significantly promoted the expression of lncRNA-HOTTIP and Hoxa13
This research showed a novel lncRNA-HOTTIP-Hoxa13 signaling pathway in the regulation of the UV-induced GC-1 cellular response. UV irradiation causes spermatogonia germ cell line, GC-1, cells to arrest in the G2/M phase and undergo early apoptosis. This occurs, at least partiall0y, through the synergistic co-expression of HOTTIP and Hoxa13, along with increased expression of downstream signaling factors including p53, p21 and γ-H2AX (Fig. 5). Further analysis of the roles of lncRNA-HOTTIP
This work was supported by the following grants: National Natural Science Foundation of China (81801516), Key project of Natural Science Foundation of Anhui Provincial Department of Education (P.R. China) (KJ2018A0992).
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