- Email: [email protected]
mTOR pathway
Author’s Accepted Manuscript hnRNP A1 promotes keratinocyte cell survival post UVB radiation through PI3K/Akt/mTOR pathway Jianguo Feng, Yi Liao, Xichao Xu, Qian Yi, Ling He, Liling Tang www.elsevier.com/locate/yexcr
PII: DOI: Reference:
S0014-4827(17)30644-4 https://doi.org/10.1016/j.yexcr.2017.12.002 YEXCR10841
To appear in: Experimental Cell Research Received date: 8 August 2017 Revised date: 29 November 2017 Accepted date: 6 December 2017 Cite this article as: Jianguo Feng, Yi Liao, Xichao Xu, Qian Yi, Ling He and Liling Tang, hnRNP A1 promotes keratinocyte cell survival post UVB radiation through PI3K/Akt/mTOR pathway, Experimental Cell Research, https://doi.org/10.1016/j.yexcr.2017.12.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
hnRNP A1 promotes keratinocyte cell survival post UVB radiation through PI3K/Akt/mTOR pathway Jianguo Feng
1,21
，Yi Liao
1,3,#
1
1,4
1
1,*
，Xichao Xu ，Qian Yi ，Ling He ，Liling Tang .
1
Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, China 2
Department of Anesthesiology, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan Province, China 3
Department of Cardiothoracic Surgery, Southwest Hospital, Third Military Medical University, Chongqing, China 4
Department of Physiology, College of preclinical medicine，Southwest Medical University, Luzhou, Sichuan Province, China
* Correspondence to: Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, China. Tel: 86-23-65102507; Fax: 86-23-65111901 E-mail: [email protected]
Abstract: hnRNP A1 acts as a critical splicing factor in regulating many alternative splicing events in various physiological and pathophysiological progressions. hnRNP A1 is capable of regulating UVB-induced hdm2 gene alternative splicing according to our previous study. However, the biological function and underlying molecular mechanism of hnRNP A1 in cell survival and cell cycle in response to UVB irradiation are still unclear. In this study, silencing hnRNP A1 expression by siRNA transfection led to decreased cell survival after UVB treatment, while promoting hnRNP A1 by lentiviruse vector resulted in increased cell survival. hnRNP A1 remarkably enhanced PI3K/Akt/mTOR signaling pathway by increasing phosphorylation of Akt, mTOR and P70S6 protein. Inhibition of PI3K/Akt signaling by LY294002 suppressed the expression of hnRNP A1. While mTOR signaling inhibitors, rapamycin and AZD8055, did not influence hnRNP A1 expression in HaCaT cells, suggesting that hnRNP A1 may be an upstream mediator of mTOR signaling. Furthermore, hnRNP A1 could alleviate UVB-provoked cell cycle arrest at G0/G1 phase and promoted cell cycle progression at G2/M phase. Our results indicate that hnRNP A1 promotes cell survival and cell cycle progression following UVB radiation.
1
Authors contributed equally in this study.
Keywords: Cell survival; Cell cycle; hnRNP A1; PI3K/Akt/mTOR signaling; UVB irradiation
Introduction Ultraviolet (UV) radiation of sunlight has been established as a prominent carcinogen in skin cancers [1, 2]. UV radiation causes DNA damage, cell cycle arrest, apoptosis and gene alternative splicing [3, 4]. The underlying molecular mechanisms in these cellular UV responses remain to be further elucidated. We have previously reported UVB radiation up-regulated the expression of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) [5], which is associated with tumor formation and progression[6, 7]. hnRNP A1 acts as a key factor regulating UVB-induced mdm2 gene alternative splicing to generate mdm2 B variant[5], which promotes mutant p53 accumulation and contributes to tumorigenesis[8]. hnRNP A1 also influences the metabolism and transport of certain mRNAs[9, 10]. However, the effects of hnRNP A1 on cell survial, cell cycle and relevant signaling pathway post UVB irradiation remain unknown. Akt, also known as Protein kinase B (PKB), is a downstream mediator of phosphatidylinositide 3 kinases (PI3Ks). PI3Ks, Akt and mammalian target of rapamycin (mTOR) constitute the core components of the PI3K/Akt/mTOR signaling cascade, regulating cell survival and proliferation [11]. UVB irradiation enhances PI3K/Akt/mTOR signaling in keratinocyte cells [12]. Therefore, in this study, we try to investigate the role of hnRNP A1 associated with PI3K/Akt/mTOR signaling. Our results demonstrated that hnRNP A1 increases HaCaT cell viability post UVB irradiation probably through activating PI3K/Akt/mTOR signaling. In addition, PI3K/Akt/mTOR signaling pathway was capable to regulate the progression of cell cycle[13], and UVB irradiation induced cell cycle arrest[14]. We also investigate whether hnRNP A1 is involved in cell cycle regulation in this study. Results showed that hnRNP A1 participated in cell cycle regulation by promoting cell cycle progression at G2/M phase.
Material and Methods Cell cluture Human embryonic kidney 293T cells (provided by Stem Cell Bank, Chinese Academy of Sciences.) and human keratinocyte HaCaT cells (a kind gift from Dr. Nihal Ahmad, University of Wisconsin–Madison) were maintained in RPMI 1640 medium (Hyclone) with 10% fetal bovine serum (Hyclone), 100 U/ml penicillin (Beyotime, China) and 100 μg/ml streptomycin (Beyotime, China) at 37℃ in a humidified atmosphere containing 5% carbon dioxide. UVB irradiation and Drug treatment UVB was generated by UVB lamps (SANKYO, Japan) and the intensity of UVB was measured by UV meter (UVP, USA). Cell culture medium was removed before UVB treatment and fresh medium was added to cell immediately after UVB irradiation. Cells were harvested at 1, 3 and 6 h post UVB irradiation. When performing drug treatment, 50 μM LY294002 was added into fresh medium directly after UVB exposure and incubated with cells for 6 h. Construction and transfection of plasmids and siRNA hnRNP A1 fragment was amplified from recombinant plasmid pcDNA3.1-hnRNP A1 prepared in our lab as previous reported[5], and then hnRNP A1 fragment conjugated with a flag tag coding DNA fragment at its 3’-terminal was cloned into pLenti-CMV-puro vector to form pLenti-CMV-hnRNP A1. The construction was confirmed by sequencing (Sangon Biotech, China). hnRNP A1 specific siRNA was purchased from Gene-Pharma (China) and the sequence was 5’-CAGCUGAGGAAGCUCUUCA-3’. Plasmids were transfected into 293T cells using Effectene Transfection Regent (Qiagen) and siRNA were transfected into HaCaT cells by X-tremeGENE Regent (Roche) according to manufacturer’s instructions. Immunoblotting Cells were lysed in RIPA buffer with PMFS (Beyotime, China). Protein concentration was determined by DC protein assay kit (Bio-Rad Laboratories, USA). Cytoplasmic and nuclear protein was extracted separately using Nuclear/Cytosol Fractionation Kit (Dualsystem Biotech, USA). Equal amounts of protein samples were subjected to SDS-PAGE (12%) and then transferred to PVDF membrane (PALL, USA). PVDF membrane was blocked in TBST with 5% skim milk at room temperature for 1 h. When performing the immunoblotting of phosphorylated proteins (p-Akt, p-mTOR and p-P70S6), the blocking buffer changed to TBST with 5% BSA.
Primary antibodies were incubated with membranes at 4℃ overnight. After triple TBST wash, appropriate secondary antibodies in TBST buffer with 5% skim milk were added to membranes for incubation at room temperature for 1 h. Immunoblotting bands were detected via West Pico Super Signal chemiluminescent substrate (Pierce, USA). The integrated optical density of blotting bands was quantified using Image J software. hnRNP A1 and β-actin antibodies were purchased from Santa Cruz， as Akt，p-Akt，mTOR，p-mTOR and p-P70S6 antibodies from Cell Signaling Technology. HRP-conjugated goat anti-rabbit and anti-mouse secondary antibodies were purchased from Proteintech. Establishment of hnRNP A1 stable cell line hnRNP A1 overexpression stable HaCaT cell line was established through lentivirus infection and puromycin selection. GFP overexpression stable cell line was served as control group. The lentiviral plasmids plenti-CMV-hnRNP A1 or plenti-CMV-GFP, pLP/VSVG, and pCMV-dR8.2 dvpr were co-transfected into 293T cells using Effectene Transfection Regent (Qiagen, Germany). Lentivirus supernatant was collected at 48 h and 72 h after transfection. 72 h after infection by lentivirus supernatant, the stably transfected cells were selected by using 0.5 μg/ml puromycin. hnRNP A1 stable cell line was validated through immunofluorescence and immunoblotting. Immunofluorescence HaCaT cells were fixed by 4% paraformaldehyde at room temperature for 30 min, and 0.2% Triton X-100 in PBS was used to permeabilize cells for 10 min after triple PBS wash. Cells were incubated with 2% BSA in PBS at room temperature for 1 h, then hnRNP A1 (Santa Cruz, USA) or Flag (Proteintech, China) primary antibodies were added to cells for incubation at 4 ℃ overnight. Cells were incubated with appropriate Cy3-conjugated secondary antibody. Nucleus was dyed by DAPI. Images were taken using System Microscope Olympus BX53. Cell viability HaCaT cell viability was assessed using a Cell Counting Kit (CCK-8/WST-8) purchased from Beyotime (China). 3×105 cells/well were seeded into 12-well plates and cultured overnight. Transfected cells were then treated by UVB. At 6 h post UVB radiation, CCK-8 was added to cells for 1.5 h incubation, then measuring absorbance at 450 nm. Cell flow cytometry analysis 60%-70% confluent established stable cells in 6-well plate were starved for 24 hours in serum free medium, and then harvested at 6 h after UVB exposure. Cells were digested by trypsin and then suspended in cold 75% ethanol. After incubation with RNaseA (Sigma-Aldrich), cells were dyed by propidum iodide （ PI, Sigma-Aldrich） and 1-5×105 cells in each sample were taken for flow cytometry（BD
Biosciences）. GFP stable expression HaCaT cells were chosen as a control. Cell cycle was analyzed by Flowjo software.
Results UVB irradiation increased hnRNP A1 levels in HaCaT cells We analyzed the expression of hnRNP A1 after 25 mJ/cm2 (Figure 1 A) and 50 mJ/cm2 (Figure 1 C) UVB irradiation. Similar to earlier studies, we observed an increase of hnRNP A1 protein level in HaCaT cells at 1 h, 3 h and 6 h post UVB irradiation (Figure 1). Comparing to control group, hnRNP A1 expression increased by 27% at 1 h，45% at 3 h and 47% at 6 h after 25 mJ/cm2 UVB irradiation (Figure 1B). After 50 mJ/cm2 UVB treatment, hnRNP A1 levels increased by 20% at 1 h, 41% at 3 h and 44% at 6 h in HaCaT cells(Figure 1D). hnRNP A1 expression alteration showed a time-dependent manner of increasing at 1 h and 3 h after UVB exposure, though the expression of hnRNP A1 present further increase trend at 6 h, the change was not statistical significance comparing to 3 h timepoint. Additionally, no significant changes of hnRNP A1 elevation were found between 25 mJ/cm2 and 50 mJ/cm2 UVB treated HaCaT cells. Therefore, 50mJ/cm2 UVB was used in the subsequent experiments. hnRNP A1 shuttled from nucleus to cytoplasm in response to UVB radiation hnRNP A1 is a RNA binding protein and associates with pre-mRNAs in the nucleus. Our previous study indicated that hnRNP A1 bound to mdm2 pre-mRNA and promoted the generation of mdm2 variant, mdm2 B. Similar to other heterogeneous nuclear ribonucleoproteins, we have confirmed hnRNP A1 also mainly localized at nucleus in HaCaT cells by immunofluoresence (Figure 2A). Next, we investigated whether hnRNP A1 could shuttle between the nucleus and the cytoplasm in response to UVB. Immunofluoresence and immunoblotting were performed to detect the translocation of hnRNP A1 after UVB treatment. Results showed that hnRNP A1 shuttled from nucleus to cytoplasm in response to UVB radiation (Figure 2A). Isolated cytoplasmic and nuclear protein were further analyzed by immunoblotting, and the result showed hnRNP A1 protein level obviously increased in cytoplasmic upon UVB irradiation (Figure 2B). These data suggested an accumulation of hnRNP A1 in the cytoplasm after UVB irradiation. hnRNP A1 promote HaCaT cell survival after UVB treatment UVB irradiation leads to DNA damage, and further causes cell death. Given expression of hnRNP A1 was up-regulated in HaCaT cells after UVB exposure (Figure 1), we further investigated the role of hnRNP A1 in cell survival. Specific siRNA was used to silence the expression of hnRNP A1 in HaCaT cells. The effect of siRNA was verified by immunoblotting (Figure 3A). Cell viability was evaluated by CCK-8 assay. Results showed that cell viability was decreased by 10.6% in hnRNPA1 silent HaCaT cells compared to control cells (NC). The survival rate (ODUVB-6h/ODUVB-0h) in hnRNP A1 silencing group (42.3%) was lower than in NC
group (56.5%), suggesting that hnRNP A1 deficiency promotes cell death by UVB treatment(Figure 3B). hnRNP A1 stable expression HaCaT cell line was established by recombinant lentivirus infection and puromycin selection. The stable cell line was verified by immunofluoresence using flag tag staining (Figure 4A). HaCaT cell viability at 6 h after UVB exposure was examined by CCK-8 assay. Results showed that hnRNPA1 overexpression increased cell viability by 14.2% compared to GFP stable cells. The survival rate (ODUVB-6h/ODUVB-0h) in LV-hnRNP A1-Flag group (73.3%) was higher than in GFP group (55.7%), suggesting that hnRNP A1 overexpression protects cells from death caused by UVB exposure (Figure 4B). hnRNP A1 activated Akt/mTOR signaling Our finding supported that hnRNP A1 expression was important for HaCaT cell survival after UVB treatment. PI3K/Akt signaling was regarded as a crucial pathway of cell viability. However, whether hnRNP A1 involves into PI3K/Akt signaling remained unclear. To elucidate it, protein level of p-Akt was used to assess PI3K/Akt signaling activation in hnRNP A1 stable overexpressed HaCaT cells. The results indicated an activation of PI3K/Akt signaling induced by UVB exposure (Figure 5A). Furthermore, enhanced p-Akt expression was found in hnRNP A1 overexpression cells, and further increase of p-Akt level was determined after UVB treatment (Figure 5A). PI3K/Akt signaling is critical for protein translation. To determine whether PI3K/Akt signaling affect the expression of hnRNP A1, PI3K/Akt inhibitor LY294002 (50 μM) was used to block signaling pathway activity. Our results demonstrated LY294002 treatment impaired the expression of hnRNP A1 in both normal culture and UVB exposure HaCaT cells (Figure 5B). mTOR is a key downstream factor of PI3K/Akt signaling. p-mTOR level was used to evaluate mTOR signaling activation, as well as p-P70S6K1 (Ribosomal protein S6 kinase beta-1) which functions as part of mTOR signaling pathway. We further examine the influence of hnRNP A1 on mTOR signaling activation and results suggested that hnRNP A1 also significantly up-regulated p-mTOR and p-P70S6K1 levels in HaCaT cells (Figure 5C). To investigate the influence of mTOR signaling on expression of hnNP A1, two mTOR signaling inhibitors, Rapamycin and AZD8055, were used to treat HaCaT cells, and no significant change of hnRNP A1 was found (Figure 5D), suggesting inhibition of mTOR signaling activation may not affect hnRNP A1 expression. hnRNP A1 promoted G2/M cell cycle progression and inhibited UVB irradiation-provoked G1 cell cycle arrest UVB irradiation leads to a cell cycle arrest[15]. Whether hnRNP A1 involves in the cell cycle progression regulation is unknown. Therefore, we detected the effect of hnRNP A1 on HaCaT cell cycle using hnRNP A1 stable cell line by cell flow cytometry analysis. For non-UVB treated cells, higher level of hnRNP A1 protein decreased cells in S phase and promote cell cycle in G2/M phase (Figure 6, Normal culture group). In addition, UVB irradiation was capable of inducing G1 phase cell
cycle arrest in HaCaT cells, while elevated hnRNP A1 alleviated the UVB irradiation-provoked G1 cell cycle arrest and increased cells in G2/M phase (Figure 6, UVB treatment group).
Discussion We previously showed that UVB irradiation increased the expression of hnRNP A1, which contributed to the regulation of mdm2 alternative splicing process[5]. However, the effects of hnRNP A1 on cellular UV responses such as cell survival and cell cycle arrest are still unclear. In this study, we first examined the expression of hnRNP A1 at 1 h, 3 h and 6 h post 25 and 50 mJ/cm2 UVB exposure, and found that hnRNP A1 level increased gradually at 1 h and 3 h, but not further increased at 6 h (Figure 1A and B). One possible explanation could be that negative factors such as p53 and ROS inhibited the further increase of hnRNP A1 expression at 6 h after UVB treatment. pcDNA6/p53 plasmid was transient transfected into H1299 cells, a p53 negative cell line. The results showed p53 decreased hnRNP A1 level in normal culture condition and inhibited the UVB-induced elevated hnRNP A1 expression (Supplement 1). In addition, ROS level was regarded as a negative factor influencing the expression of hnRNP A1 [5], and probably became another reason for none significant change of hnRNP A1 expression between 3 h and 6 h after UVB treatment. Besides, we demonstrated that PI3K/Akt signaling pathway was capable of regulating the expression of hnRNP A1 (Figure 5B). LY294002, a PI3K/Akt signaling specific inhibitor, dramatically suppressed hnRNP A1 expression and inhibited the increase of hnRNP A1 expression induced by UVB irradiation (Figure 5B). Enhanced activation of PI3K/Akt/mTOR signaling pathway also presented in hnRNP A1 stable overexpressed HaCaT cells (Figure 5A), and the interplay between PI3K/Akt signaling and hnRNP A1 probably was an explanation for hnRNP A1 level contributing to cell survival after UVB treatment (Figure 3 and 4). However, mTOR specific inhibitors rapamycin and AZD8055 had no effect on the expression of hnRNP A1, suggesting that hnRNP A1 might be an upstream factor of mTOR signaling pathway. Moreover, hnRNP A1 shuttled from nucleus to cytoplasm after UVB treatment (Figure 2), and whether this phenomenon contributed to hnRNP A1-mediated PI3K/Akt/mTOR signaling activation is still unknown. Both phosphorylation and O-GlcNAcylation were sufficient to cause translocation of hnRNP A1 from nucleus to cytoplasm [16, 17], and which type of modification on hnRNP A1 predominantly regulated nuclear export activity induced by UVB irradiation also need further study. UVB irradiation causes DNA damage to cells in the form of pyrimidine dimers and 6-4 photoproducts [18, 19], which are repaired by nucleotide excision repair (NER). Global genomic repair will restore the rest of the genome [20]. However, incomplete repair and damaged bases might be misinterpreted during replication [18]. In this study, data indicated that overexpression of hnRNP A1 promote cell cycle progression at G2/M phase of HaCaT cells post UVB exposure (Figure 6B), which
probably lead to accumulation of cancer predisposing mutations, making hnRNP A1 a risk factor for skin cancers. UVB irradiation lead to cell cycle arrest at G0/G1 phase in primary human fibroblasts [15], and UVB has been proved to manifest a similar effect on HaCaT cells in our research (Figure 6). Furthermore, elevated hnRNP A1 in HaCaT cells significantly alleviated G0/G1 phase cell cycle arrest (Figure 6B), suggesting the association of hnRNP A1 with G0/G1 phase cell cycle regulation. hnRNP A1 also promoted cell cycle progression at G2/M phase in normal culture condition (Figure 6). Yu and colleagues [21] found that hnRNP A1 was highly expressed in the G2/M cell cycle phase of oral squamous cancer cells. A research from Sui [22] showed that DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylated hnRNP A1 during the G2 and M phases, critical for capping of the newly replicated telomeres, which maybe one potential explanation for hnRNP A1 promoting cell cycle progression at G2/M phase. In summary, our results indicated that (1) UVB induced hnRNP A1 shuttled from nucleus to cytoplasm in HaCaT cells; (2) hnRNP A1 promoted HaCaT cell survival post UVB irradiation; (3) hnRNP A1 enhanced PI3K/Akt/mTOR signaling pathway, and probably was a key upstream factor of mTOR signaling; (4) hnRNP A1 alleviated the UVB irradiation-provoked G1 cell cycle arrest, and was capable of promoting cell cycle progression at G2/M phase. Acknowledgements This work was supported by the National Natural Science Foundation of China [No.31670952]; Scientific Research Project of Sichuan Provincial Health and Family Planning Commission [No.16PJ547].
Conflict of Interest The authors of this article declared they have no conflicts of interest.
Reference: [1] [2] [3]
[4] [5] [6]
F.R. de Gruijl, Skin cancer and solar UV radiation, Eur J Cancer 35 (1999) 2003-2009. B.K. Armstrong, A. Kricker, The epidemiology of UV induced skin cancer, J Photochem Photobiol B 63 (2001) 8-18. M. Gentile, L. Latonen, M. Laiho, Cell cycle arrest and apoptosis provoked by UV radiation-induced DNA damage are transcriptionally highly divergent responses, Nucleic Acids Res 31 (2003) 4779-4790. C.N. Sprung, J. Li, D. Hovan, M.J. McKay, H.B. Forrester, Alternative transcript initiation and splicing as a response to DNA damage, PLoS One 6 (2011) e25758. J. Feng, L. Li, L. Tong, L. Tang, S. Wu, The Involvement of Splicing Factor hnRNP A1 in UVB-induced Alternative Splicing of hdm2, Photochem Photobiol 92 (2016) 318-324. Z.J. Zhou, Z. Dai, S.L. Zhou, X.T. Fu, Y.M. Zhao, Y.H. Shi, J. Zhou, J. Fan, Overexpression of HnRNP A1 promotes tumor invasion through regulating CD44v6 and
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14] [15]
[16] [17]
[18]
[19] [20] [21]
indicates poor prognosis for hepatocellular carcinoma, Int J Cancer 132 (2013) 1080-1089. S. Bonomi, A. di Matteo, E. Buratti, D.S. Cabianca, F.E. Baralle, C. Ghigna, G. Biamonti, HnRNP A1 controls a splicing regulatory circuit promoting mesenchymal-to-epithelial transition, Nucleic Acids Res 41 (2013) 8665-8679. T. Zheng, J. Wang, Y. Zhao, C. Zhang, M. Lin, X. Wang, H. Yu, L. Liu, Z. Feng, W. Hu, Spliced MDM2 isoforms promote mutant p53 accumulation and gain-of-function in tumorigenesis, Nat Commun 4 (2013) 2996. L.K. Zerbe, I. Pino, R. Pio, P.F. Cosper, L.D. Dwyer-Nield, A.M. Meyer, J.D. Port, L.M. Montuenga, A.M. Malkinson, Relative amounts of antagonistic splicing factors, hnRNP A1 and ASF/SF2, change during neoplastic lung growth: implications for pre-mRNA processing, Mol Carcinog 41 (2004) 187-196. R. Roy, D. Durie, H. Li, B.Q. Liu, J.M. Skehel, F. Mauri, L.V. Cuorvo, M. Barbareschi, L. Guo, M. Holcik, M.J. Seckl, O.E. Pardo, hnRNPA1 couples nuclear export and translation of specific mRNAs downstream of FGF-2/S6K2 signalling, Nucleic Acids Res 42 (2014) 12483-12497. J.S. Yu, W. Cui, Proliferation, survival and metabolism: the role of PI3K/AKT/mTOR signalling in pluripotency and cell fate determination, Development 143 (2016) 3050-3060. B.B. Bridgeman, P. Wang, B. Ye, J.C. Pelling, O.V. Volpert, X. Tong, Inhibition of mTOR by apigenin in UVB-irradiated keratinocytes: A new implication of skin cancer prevention, Cell Signal 28 (2016) 460-468. L. Vadlakonda, M. Pasupuleti, R. Pallu, Role of PI3K-AKT-mTOR and Wnt Signaling Pathways in Transition of G1-S Phase of Cell Cycle in Cancer Cells, Front Oncol 3 (2013) 85. T.R. Berton, A. Pavone, S.M. Fischer, Ultraviolet-B irradiation alters the cell cycle machinery in murine epidermis in vivo, J Invest Dermatol 117 (2001) 1171-1178. T.G. Ortolan, C.F. Menck, UVB-induced cell death signaling is associated with G1-S progression and transcription inhibition in primary human fibroblasts, PLoS One 8 (2013) e76936. S. Roth, I. Khalaila, The effect of O-GlcNAcylation on hnRNP A1 translocation and interaction with transportin1, Exp Cell Res 350 (2017) 210-217. E. Allemand, S. Guil, M. Myers, J. Moscat, J.F. Caceres, A.R. Krainer, Regulation of heterogenous nuclear ribonucleoprotein A1 transport by phosphorylation in cells stressed by osmotic shock, Proc Natl Acad Sci U S A 102 (2005) 3605-3610. F.R. de Gruijl, H.J. van Kranen, L.H. Mullenders, UV-induced DNA damage, repair, mutations and oncogenic pathways in skin cancer, J Photochem Photobiol B 63 (2001) 19-27. J.L. Ravanat, T. Douki, J. Cadet, Direct and indirect effects of UV radiation on DNA and its components, J Photochem Photobiol B 63 (2001) 88-102. P.C. Hanawalt, Subpathways of nucleotide excision repair and their regulation, Oncogene 21 (2002) 8949-8956. C. Yu, J. Guo, Y. Liu, J. Jia, R. Jia, M. Fan, Oral squamous cancer cell exploits hnRNP A1 to regulate cell cycle and proliferation, J Cell Physiol 230 (2015) 2252-2261.
[22]
J. Sui, Y.F. Lin, K. Xu, K.J. Lee, D. Wang, B.P. Chen, DNA-PKcs phosphorylates hnRNP-A1 to facilitate the RPA-to-POT1 switch and telomere capping after replication, Nucleic Acids Res 43 (2015) 5971-5983.
Figure 1. UVB irradiation increased hnRNP A1 expression in HaCaT cells within time dependent. A. ＆ C. The levels of hnRNP A1 in HaCaT cells at 1 h, 3 h and 6 h post 25 mJ/cm2（A）and 50 mJ/cm2 （C） radiation. B. ＆ D. hnRNPA1 expressions showed in A and C were quantified by Image J software. Data presents three sets of independent experiments. *P<0.05 vs control group,#P<0.05 vs UVB(1h) group.
Figure 2. UVB radiation induced translocation of hnRNPA1 in HaCaT cells. A. Subcelluar location of hnRNPA1(Red) in HaCaT cells at 6 h after UVB treatment, and DAPI was used to label nuclei (400X). B. Cytoplasm hnRNPA1 was increased and nucleus hnRNPA1 decreased at 6 h after UVB treatment by western blot.
Figure 3. Knockdown hnRNP A1 in HaCaT cells decreased cell survival after UVB treatment. A. The expression of hnRNP A1 was detected by western blot at 24 h after specific siRNA or NC transfection in HaCaT cells. B. Transfected HaCaT cells by siRNA or NC were subjected to CCK-8 assay at 6 h after UVB exposure or without UVB irradiation. The data represents three sets of independent experiments and were shown as the means ± SD. *P < 0.05.
Figure 4. Overexpression hnRNP A1 in HaCaT cells increased cell survival after UVB treatment. A. hnRNP A1 stable cell line was established by lentivirus infection, verified by Flag tag staining(Cy3), and GFP stable cell line was used as control (200X). B. Stable HaCaT cells (hnRNP A1 or GFP) were subjected to CCK-8 assay at 6 h after UVB exposure or without UVB irradiation. The data represents three sets of independent experiments and were shown as the means ± SD. *P < 0.05.
Figure 5. hnRNP A1 enhanced the activation of PI3K/Akt/mTOR signaling pathway. A. The phosphorylation levels of Akt were examined by western blot in hnRNP A1 or GFP stable cell line with or without UVB irradiation. Relative expression of p-Akt was quantitatively analyzed by Image J software. Results are mean±SD from three independent experiments. *P<0.05 vs LV-GFP without UVB irradiation, # P<0.05 vs LV-GFP with UVB irradiation. B. The expression of hnRNP A1 was detected by western blot at 6 h after UVB exposure in HaCaT cells treated by 50 μM LY294002. Results are mean±SD from three independent experiments. *P<0.05 vs group 1 (without UVB irradiation nor LY294002 treatment), # P<0.05 vs group 3(with only UVB irradiation without LY294002 treatment). C. The phosphorylation levels of mTOR and P70S6 were detected by western blot in hnRNP A1 or GFP stable cells with or without UVB irradiation. Relative expression of p-mTOR was quantitatively analyzed by Image J software. The data represents three sets of independent experiments and were shown as the means ± SD. *P<0.05 vs LV-GFP without UVB irradiation, # P<0.05 vs LV-GFP with UVB irradiation. D. Western blot analysis of hnRNP A1 expression in HaCaT cells after treated with various concentration of Rapamycin and AZD8055 for 24 h.
Figure 6. hnRNP A1 promoted cell cycle post UVB irradiation. A. Stable cells (hnRNP A1 or GFP) were starved for 24 h and then harvested at 6 h after UVB irradiation, subsequently dyed by propidum iodide and subjected to flow cytometry. Cell cycle was analyzed by flowjo software. B. The population percentages at G0/G1, S and G2/M phases are shown as mean±SD. P* < 0.05.
PII: DOI: Reference:
S0014-4827(17)30644-4 https://doi.org/10.1016/j.yexcr.2017.12.002 YEXCR10841
To appear in: Experimental Cell Research Received date: 8 August 2017 Revised date: 29 November 2017 Accepted date: 6 December 2017 Cite this article as: Jianguo Feng, Yi Liao, Xichao Xu, Qian Yi, Ling He and Liling Tang, hnRNP A1 promotes keratinocyte cell survival post UVB radiation through PI3K/Akt/mTOR pathway, Experimental Cell Research, https://doi.org/10.1016/j.yexcr.2017.12.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
hnRNP A1 promotes keratinocyte cell survival post UVB radiation through PI3K/Akt/mTOR pathway Jianguo Feng
1,21
，Yi Liao
1,3,#
1
1,4
1
1,*
，Xichao Xu ，Qian Yi ，Ling He ，Liling Tang .
1
Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, China 2
Department of Anesthesiology, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan Province, China 3
Department of Cardiothoracic Surgery, Southwest Hospital, Third Military Medical University, Chongqing, China 4
Department of Physiology, College of preclinical medicine，Southwest Medical University, Luzhou, Sichuan Province, China
* Correspondence to: Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, China. Tel: 86-23-65102507; Fax: 86-23-65111901 E-mail: [email protected]
Abstract: hnRNP A1 acts as a critical splicing factor in regulating many alternative splicing events in various physiological and pathophysiological progressions. hnRNP A1 is capable of regulating UVB-induced hdm2 gene alternative splicing according to our previous study. However, the biological function and underlying molecular mechanism of hnRNP A1 in cell survival and cell cycle in response to UVB irradiation are still unclear. In this study, silencing hnRNP A1 expression by siRNA transfection led to decreased cell survival after UVB treatment, while promoting hnRNP A1 by lentiviruse vector resulted in increased cell survival. hnRNP A1 remarkably enhanced PI3K/Akt/mTOR signaling pathway by increasing phosphorylation of Akt, mTOR and P70S6 protein. Inhibition of PI3K/Akt signaling by LY294002 suppressed the expression of hnRNP A1. While mTOR signaling inhibitors, rapamycin and AZD8055, did not influence hnRNP A1 expression in HaCaT cells, suggesting that hnRNP A1 may be an upstream mediator of mTOR signaling. Furthermore, hnRNP A1 could alleviate UVB-provoked cell cycle arrest at G0/G1 phase and promoted cell cycle progression at G2/M phase. Our results indicate that hnRNP A1 promotes cell survival and cell cycle progression following UVB radiation.
1
Authors contributed equally in this study.
Keywords: Cell survival; Cell cycle; hnRNP A1; PI3K/Akt/mTOR signaling; UVB irradiation
Introduction Ultraviolet (UV) radiation of sunlight has been established as a prominent carcinogen in skin cancers [1, 2]. UV radiation causes DNA damage, cell cycle arrest, apoptosis and gene alternative splicing [3, 4]. The underlying molecular mechanisms in these cellular UV responses remain to be further elucidated. We have previously reported UVB radiation up-regulated the expression of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) [5], which is associated with tumor formation and progression[6, 7]. hnRNP A1 acts as a key factor regulating UVB-induced mdm2 gene alternative splicing to generate mdm2 B variant[5], which promotes mutant p53 accumulation and contributes to tumorigenesis[8]. hnRNP A1 also influences the metabolism and transport of certain mRNAs[9, 10]. However, the effects of hnRNP A1 on cell survial, cell cycle and relevant signaling pathway post UVB irradiation remain unknown. Akt, also known as Protein kinase B (PKB), is a downstream mediator of phosphatidylinositide 3 kinases (PI3Ks). PI3Ks, Akt and mammalian target of rapamycin (mTOR) constitute the core components of the PI3K/Akt/mTOR signaling cascade, regulating cell survival and proliferation [11]. UVB irradiation enhances PI3K/Akt/mTOR signaling in keratinocyte cells [12]. Therefore, in this study, we try to investigate the role of hnRNP A1 associated with PI3K/Akt/mTOR signaling. Our results demonstrated that hnRNP A1 increases HaCaT cell viability post UVB irradiation probably through activating PI3K/Akt/mTOR signaling. In addition, PI3K/Akt/mTOR signaling pathway was capable to regulate the progression of cell cycle[13], and UVB irradiation induced cell cycle arrest[14]. We also investigate whether hnRNP A1 is involved in cell cycle regulation in this study. Results showed that hnRNP A1 participated in cell cycle regulation by promoting cell cycle progression at G2/M phase.
Material and Methods Cell cluture Human embryonic kidney 293T cells (provided by Stem Cell Bank, Chinese Academy of Sciences.) and human keratinocyte HaCaT cells (a kind gift from Dr. Nihal Ahmad, University of Wisconsin–Madison) were maintained in RPMI 1640 medium (Hyclone) with 10% fetal bovine serum (Hyclone), 100 U/ml penicillin (Beyotime, China) and 100 μg/ml streptomycin (Beyotime, China) at 37℃ in a humidified atmosphere containing 5% carbon dioxide. UVB irradiation and Drug treatment UVB was generated by UVB lamps (SANKYO, Japan) and the intensity of UVB was measured by UV meter (UVP, USA). Cell culture medium was removed before UVB treatment and fresh medium was added to cell immediately after UVB irradiation. Cells were harvested at 1, 3 and 6 h post UVB irradiation. When performing drug treatment, 50 μM LY294002 was added into fresh medium directly after UVB exposure and incubated with cells for 6 h. Construction and transfection of plasmids and siRNA hnRNP A1 fragment was amplified from recombinant plasmid pcDNA3.1-hnRNP A1 prepared in our lab as previous reported[5], and then hnRNP A1 fragment conjugated with a flag tag coding DNA fragment at its 3’-terminal was cloned into pLenti-CMV-puro vector to form pLenti-CMV-hnRNP A1. The construction was confirmed by sequencing (Sangon Biotech, China). hnRNP A1 specific siRNA was purchased from Gene-Pharma (China) and the sequence was 5’-CAGCUGAGGAAGCUCUUCA-3’. Plasmids were transfected into 293T cells using Effectene Transfection Regent (Qiagen) and siRNA were transfected into HaCaT cells by X-tremeGENE Regent (Roche) according to manufacturer’s instructions. Immunoblotting Cells were lysed in RIPA buffer with PMFS (Beyotime, China). Protein concentration was determined by DC protein assay kit (Bio-Rad Laboratories, USA). Cytoplasmic and nuclear protein was extracted separately using Nuclear/Cytosol Fractionation Kit (Dualsystem Biotech, USA). Equal amounts of protein samples were subjected to SDS-PAGE (12%) and then transferred to PVDF membrane (PALL, USA). PVDF membrane was blocked in TBST with 5% skim milk at room temperature for 1 h. When performing the immunoblotting of phosphorylated proteins (p-Akt, p-mTOR and p-P70S6), the blocking buffer changed to TBST with 5% BSA.
Primary antibodies were incubated with membranes at 4℃ overnight. After triple TBST wash, appropriate secondary antibodies in TBST buffer with 5% skim milk were added to membranes for incubation at room temperature for 1 h. Immunoblotting bands were detected via West Pico Super Signal chemiluminescent substrate (Pierce, USA). The integrated optical density of blotting bands was quantified using Image J software. hnRNP A1 and β-actin antibodies were purchased from Santa Cruz， as Akt，p-Akt，mTOR，p-mTOR and p-P70S6 antibodies from Cell Signaling Technology. HRP-conjugated goat anti-rabbit and anti-mouse secondary antibodies were purchased from Proteintech. Establishment of hnRNP A1 stable cell line hnRNP A1 overexpression stable HaCaT cell line was established through lentivirus infection and puromycin selection. GFP overexpression stable cell line was served as control group. The lentiviral plasmids plenti-CMV-hnRNP A1 or plenti-CMV-GFP, pLP/VSVG, and pCMV-dR8.2 dvpr were co-transfected into 293T cells using Effectene Transfection Regent (Qiagen, Germany). Lentivirus supernatant was collected at 48 h and 72 h after transfection. 72 h after infection by lentivirus supernatant, the stably transfected cells were selected by using 0.5 μg/ml puromycin. hnRNP A1 stable cell line was validated through immunofluorescence and immunoblotting. Immunofluorescence HaCaT cells were fixed by 4% paraformaldehyde at room temperature for 30 min, and 0.2% Triton X-100 in PBS was used to permeabilize cells for 10 min after triple PBS wash. Cells were incubated with 2% BSA in PBS at room temperature for 1 h, then hnRNP A1 (Santa Cruz, USA) or Flag (Proteintech, China) primary antibodies were added to cells for incubation at 4 ℃ overnight. Cells were incubated with appropriate Cy3-conjugated secondary antibody. Nucleus was dyed by DAPI. Images were taken using System Microscope Olympus BX53. Cell viability HaCaT cell viability was assessed using a Cell Counting Kit (CCK-8/WST-8) purchased from Beyotime (China). 3×105 cells/well were seeded into 12-well plates and cultured overnight. Transfected cells were then treated by UVB. At 6 h post UVB radiation, CCK-8 was added to cells for 1.5 h incubation, then measuring absorbance at 450 nm. Cell flow cytometry analysis 60%-70% confluent established stable cells in 6-well plate were starved for 24 hours in serum free medium, and then harvested at 6 h after UVB exposure. Cells were digested by trypsin and then suspended in cold 75% ethanol. After incubation with RNaseA (Sigma-Aldrich), cells were dyed by propidum iodide （ PI, Sigma-Aldrich） and 1-5×105 cells in each sample were taken for flow cytometry（BD
Biosciences）. GFP stable expression HaCaT cells were chosen as a control. Cell cycle was analyzed by Flowjo software.
Results UVB irradiation increased hnRNP A1 levels in HaCaT cells We analyzed the expression of hnRNP A1 after 25 mJ/cm2 (Figure 1 A) and 50 mJ/cm2 (Figure 1 C) UVB irradiation. Similar to earlier studies, we observed an increase of hnRNP A1 protein level in HaCaT cells at 1 h, 3 h and 6 h post UVB irradiation (Figure 1). Comparing to control group, hnRNP A1 expression increased by 27% at 1 h，45% at 3 h and 47% at 6 h after 25 mJ/cm2 UVB irradiation (Figure 1B). After 50 mJ/cm2 UVB treatment, hnRNP A1 levels increased by 20% at 1 h, 41% at 3 h and 44% at 6 h in HaCaT cells(Figure 1D). hnRNP A1 expression alteration showed a time-dependent manner of increasing at 1 h and 3 h after UVB exposure, though the expression of hnRNP A1 present further increase trend at 6 h, the change was not statistical significance comparing to 3 h timepoint. Additionally, no significant changes of hnRNP A1 elevation were found between 25 mJ/cm2 and 50 mJ/cm2 UVB treated HaCaT cells. Therefore, 50mJ/cm2 UVB was used in the subsequent experiments. hnRNP A1 shuttled from nucleus to cytoplasm in response to UVB radiation hnRNP A1 is a RNA binding protein and associates with pre-mRNAs in the nucleus. Our previous study indicated that hnRNP A1 bound to mdm2 pre-mRNA and promoted the generation of mdm2 variant, mdm2 B. Similar to other heterogeneous nuclear ribonucleoproteins, we have confirmed hnRNP A1 also mainly localized at nucleus in HaCaT cells by immunofluoresence (Figure 2A). Next, we investigated whether hnRNP A1 could shuttle between the nucleus and the cytoplasm in response to UVB. Immunofluoresence and immunoblotting were performed to detect the translocation of hnRNP A1 after UVB treatment. Results showed that hnRNP A1 shuttled from nucleus to cytoplasm in response to UVB radiation (Figure 2A). Isolated cytoplasmic and nuclear protein were further analyzed by immunoblotting, and the result showed hnRNP A1 protein level obviously increased in cytoplasmic upon UVB irradiation (Figure 2B). These data suggested an accumulation of hnRNP A1 in the cytoplasm after UVB irradiation. hnRNP A1 promote HaCaT cell survival after UVB treatment UVB irradiation leads to DNA damage, and further causes cell death. Given expression of hnRNP A1 was up-regulated in HaCaT cells after UVB exposure (Figure 1), we further investigated the role of hnRNP A1 in cell survival. Specific siRNA was used to silence the expression of hnRNP A1 in HaCaT cells. The effect of siRNA was verified by immunoblotting (Figure 3A). Cell viability was evaluated by CCK-8 assay. Results showed that cell viability was decreased by 10.6% in hnRNPA1 silent HaCaT cells compared to control cells (NC). The survival rate (ODUVB-6h/ODUVB-0h) in hnRNP A1 silencing group (42.3%) was lower than in NC
group (56.5%), suggesting that hnRNP A1 deficiency promotes cell death by UVB treatment(Figure 3B). hnRNP A1 stable expression HaCaT cell line was established by recombinant lentivirus infection and puromycin selection. The stable cell line was verified by immunofluoresence using flag tag staining (Figure 4A). HaCaT cell viability at 6 h after UVB exposure was examined by CCK-8 assay. Results showed that hnRNPA1 overexpression increased cell viability by 14.2% compared to GFP stable cells. The survival rate (ODUVB-6h/ODUVB-0h) in LV-hnRNP A1-Flag group (73.3%) was higher than in GFP group (55.7%), suggesting that hnRNP A1 overexpression protects cells from death caused by UVB exposure (Figure 4B). hnRNP A1 activated Akt/mTOR signaling Our finding supported that hnRNP A1 expression was important for HaCaT cell survival after UVB treatment. PI3K/Akt signaling was regarded as a crucial pathway of cell viability. However, whether hnRNP A1 involves into PI3K/Akt signaling remained unclear. To elucidate it, protein level of p-Akt was used to assess PI3K/Akt signaling activation in hnRNP A1 stable overexpressed HaCaT cells. The results indicated an activation of PI3K/Akt signaling induced by UVB exposure (Figure 5A). Furthermore, enhanced p-Akt expression was found in hnRNP A1 overexpression cells, and further increase of p-Akt level was determined after UVB treatment (Figure 5A). PI3K/Akt signaling is critical for protein translation. To determine whether PI3K/Akt signaling affect the expression of hnRNP A1, PI3K/Akt inhibitor LY294002 (50 μM) was used to block signaling pathway activity. Our results demonstrated LY294002 treatment impaired the expression of hnRNP A1 in both normal culture and UVB exposure HaCaT cells (Figure 5B). mTOR is a key downstream factor of PI3K/Akt signaling. p-mTOR level was used to evaluate mTOR signaling activation, as well as p-P70S6K1 (Ribosomal protein S6 kinase beta-1) which functions as part of mTOR signaling pathway. We further examine the influence of hnRNP A1 on mTOR signaling activation and results suggested that hnRNP A1 also significantly up-regulated p-mTOR and p-P70S6K1 levels in HaCaT cells (Figure 5C). To investigate the influence of mTOR signaling on expression of hnNP A1, two mTOR signaling inhibitors, Rapamycin and AZD8055, were used to treat HaCaT cells, and no significant change of hnRNP A1 was found (Figure 5D), suggesting inhibition of mTOR signaling activation may not affect hnRNP A1 expression. hnRNP A1 promoted G2/M cell cycle progression and inhibited UVB irradiation-provoked G1 cell cycle arrest UVB irradiation leads to a cell cycle arrest[15]. Whether hnRNP A1 involves in the cell cycle progression regulation is unknown. Therefore, we detected the effect of hnRNP A1 on HaCaT cell cycle using hnRNP A1 stable cell line by cell flow cytometry analysis. For non-UVB treated cells, higher level of hnRNP A1 protein decreased cells in S phase and promote cell cycle in G2/M phase (Figure 6, Normal culture group). In addition, UVB irradiation was capable of inducing G1 phase cell
cycle arrest in HaCaT cells, while elevated hnRNP A1 alleviated the UVB irradiation-provoked G1 cell cycle arrest and increased cells in G2/M phase (Figure 6, UVB treatment group).
Discussion We previously showed that UVB irradiation increased the expression of hnRNP A1, which contributed to the regulation of mdm2 alternative splicing process[5]. However, the effects of hnRNP A1 on cellular UV responses such as cell survival and cell cycle arrest are still unclear. In this study, we first examined the expression of hnRNP A1 at 1 h, 3 h and 6 h post 25 and 50 mJ/cm2 UVB exposure, and found that hnRNP A1 level increased gradually at 1 h and 3 h, but not further increased at 6 h (Figure 1A and B). One possible explanation could be that negative factors such as p53 and ROS inhibited the further increase of hnRNP A1 expression at 6 h after UVB treatment. pcDNA6/p53 plasmid was transient transfected into H1299 cells, a p53 negative cell line. The results showed p53 decreased hnRNP A1 level in normal culture condition and inhibited the UVB-induced elevated hnRNP A1 expression (Supplement 1). In addition, ROS level was regarded as a negative factor influencing the expression of hnRNP A1 [5], and probably became another reason for none significant change of hnRNP A1 expression between 3 h and 6 h after UVB treatment. Besides, we demonstrated that PI3K/Akt signaling pathway was capable of regulating the expression of hnRNP A1 (Figure 5B). LY294002, a PI3K/Akt signaling specific inhibitor, dramatically suppressed hnRNP A1 expression and inhibited the increase of hnRNP A1 expression induced by UVB irradiation (Figure 5B). Enhanced activation of PI3K/Akt/mTOR signaling pathway also presented in hnRNP A1 stable overexpressed HaCaT cells (Figure 5A), and the interplay between PI3K/Akt signaling and hnRNP A1 probably was an explanation for hnRNP A1 level contributing to cell survival after UVB treatment (Figure 3 and 4). However, mTOR specific inhibitors rapamycin and AZD8055 had no effect on the expression of hnRNP A1, suggesting that hnRNP A1 might be an upstream factor of mTOR signaling pathway. Moreover, hnRNP A1 shuttled from nucleus to cytoplasm after UVB treatment (Figure 2), and whether this phenomenon contributed to hnRNP A1-mediated PI3K/Akt/mTOR signaling activation is still unknown. Both phosphorylation and O-GlcNAcylation were sufficient to cause translocation of hnRNP A1 from nucleus to cytoplasm [16, 17], and which type of modification on hnRNP A1 predominantly regulated nuclear export activity induced by UVB irradiation also need further study. UVB irradiation causes DNA damage to cells in the form of pyrimidine dimers and 6-4 photoproducts [18, 19], which are repaired by nucleotide excision repair (NER). Global genomic repair will restore the rest of the genome [20]. However, incomplete repair and damaged bases might be misinterpreted during replication [18]. In this study, data indicated that overexpression of hnRNP A1 promote cell cycle progression at G2/M phase of HaCaT cells post UVB exposure (Figure 6B), which
probably lead to accumulation of cancer predisposing mutations, making hnRNP A1 a risk factor for skin cancers. UVB irradiation lead to cell cycle arrest at G0/G1 phase in primary human fibroblasts [15], and UVB has been proved to manifest a similar effect on HaCaT cells in our research (Figure 6). Furthermore, elevated hnRNP A1 in HaCaT cells significantly alleviated G0/G1 phase cell cycle arrest (Figure 6B), suggesting the association of hnRNP A1 with G0/G1 phase cell cycle regulation. hnRNP A1 also promoted cell cycle progression at G2/M phase in normal culture condition (Figure 6). Yu and colleagues [21] found that hnRNP A1 was highly expressed in the G2/M cell cycle phase of oral squamous cancer cells. A research from Sui [22] showed that DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylated hnRNP A1 during the G2 and M phases, critical for capping of the newly replicated telomeres, which maybe one potential explanation for hnRNP A1 promoting cell cycle progression at G2/M phase. In summary, our results indicated that (1) UVB induced hnRNP A1 shuttled from nucleus to cytoplasm in HaCaT cells; (2) hnRNP A1 promoted HaCaT cell survival post UVB irradiation; (3) hnRNP A1 enhanced PI3K/Akt/mTOR signaling pathway, and probably was a key upstream factor of mTOR signaling; (4) hnRNP A1 alleviated the UVB irradiation-provoked G1 cell cycle arrest, and was capable of promoting cell cycle progression at G2/M phase. Acknowledgements This work was supported by the National Natural Science Foundation of China [No.31670952]; Scientific Research Project of Sichuan Provincial Health and Family Planning Commission [No.16PJ547].
Conflict of Interest The authors of this article declared they have no conflicts of interest.
Reference: [1] [2] [3]
[4] [5] [6]
F.R. de Gruijl, Skin cancer and solar UV radiation, Eur J Cancer 35 (1999) 2003-2009. B.K. Armstrong, A. Kricker, The epidemiology of UV induced skin cancer, J Photochem Photobiol B 63 (2001) 8-18. M. Gentile, L. Latonen, M. Laiho, Cell cycle arrest and apoptosis provoked by UV radiation-induced DNA damage are transcriptionally highly divergent responses, Nucleic Acids Res 31 (2003) 4779-4790. C.N. Sprung, J. Li, D. Hovan, M.J. McKay, H.B. Forrester, Alternative transcript initiation and splicing as a response to DNA damage, PLoS One 6 (2011) e25758. J. Feng, L. Li, L. Tong, L. Tang, S. Wu, The Involvement of Splicing Factor hnRNP A1 in UVB-induced Alternative Splicing of hdm2, Photochem Photobiol 92 (2016) 318-324. Z.J. Zhou, Z. Dai, S.L. Zhou, X.T. Fu, Y.M. Zhao, Y.H. Shi, J. Zhou, J. Fan, Overexpression of HnRNP A1 promotes tumor invasion through regulating CD44v6 and
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14] [15]
[16] [17]
[18]
[19] [20] [21]
indicates poor prognosis for hepatocellular carcinoma, Int J Cancer 132 (2013) 1080-1089. S. Bonomi, A. di Matteo, E. Buratti, D.S. Cabianca, F.E. Baralle, C. Ghigna, G. Biamonti, HnRNP A1 controls a splicing regulatory circuit promoting mesenchymal-to-epithelial transition, Nucleic Acids Res 41 (2013) 8665-8679. T. Zheng, J. Wang, Y. Zhao, C. Zhang, M. Lin, X. Wang, H. Yu, L. Liu, Z. Feng, W. Hu, Spliced MDM2 isoforms promote mutant p53 accumulation and gain-of-function in tumorigenesis, Nat Commun 4 (2013) 2996. L.K. Zerbe, I. Pino, R. Pio, P.F. Cosper, L.D. Dwyer-Nield, A.M. Meyer, J.D. Port, L.M. Montuenga, A.M. Malkinson, Relative amounts of antagonistic splicing factors, hnRNP A1 and ASF/SF2, change during neoplastic lung growth: implications for pre-mRNA processing, Mol Carcinog 41 (2004) 187-196. R. Roy, D. Durie, H. Li, B.Q. Liu, J.M. Skehel, F. Mauri, L.V. Cuorvo, M. Barbareschi, L. Guo, M. Holcik, M.J. Seckl, O.E. Pardo, hnRNPA1 couples nuclear export and translation of specific mRNAs downstream of FGF-2/S6K2 signalling, Nucleic Acids Res 42 (2014) 12483-12497. J.S. Yu, W. Cui, Proliferation, survival and metabolism: the role of PI3K/AKT/mTOR signalling in pluripotency and cell fate determination, Development 143 (2016) 3050-3060. B.B. Bridgeman, P. Wang, B. Ye, J.C. Pelling, O.V. Volpert, X. Tong, Inhibition of mTOR by apigenin in UVB-irradiated keratinocytes: A new implication of skin cancer prevention, Cell Signal 28 (2016) 460-468. L. Vadlakonda, M. Pasupuleti, R. Pallu, Role of PI3K-AKT-mTOR and Wnt Signaling Pathways in Transition of G1-S Phase of Cell Cycle in Cancer Cells, Front Oncol 3 (2013) 85. T.R. Berton, A. Pavone, S.M. Fischer, Ultraviolet-B irradiation alters the cell cycle machinery in murine epidermis in vivo, J Invest Dermatol 117 (2001) 1171-1178. T.G. Ortolan, C.F. Menck, UVB-induced cell death signaling is associated with G1-S progression and transcription inhibition in primary human fibroblasts, PLoS One 8 (2013) e76936. S. Roth, I. Khalaila, The effect of O-GlcNAcylation on hnRNP A1 translocation and interaction with transportin1, Exp Cell Res 350 (2017) 210-217. E. Allemand, S. Guil, M. Myers, J. Moscat, J.F. Caceres, A.R. Krainer, Regulation of heterogenous nuclear ribonucleoprotein A1 transport by phosphorylation in cells stressed by osmotic shock, Proc Natl Acad Sci U S A 102 (2005) 3605-3610. F.R. de Gruijl, H.J. van Kranen, L.H. Mullenders, UV-induced DNA damage, repair, mutations and oncogenic pathways in skin cancer, J Photochem Photobiol B 63 (2001) 19-27. J.L. Ravanat, T. Douki, J. Cadet, Direct and indirect effects of UV radiation on DNA and its components, J Photochem Photobiol B 63 (2001) 88-102. P.C. Hanawalt, Subpathways of nucleotide excision repair and their regulation, Oncogene 21 (2002) 8949-8956. C. Yu, J. Guo, Y. Liu, J. Jia, R. Jia, M. Fan, Oral squamous cancer cell exploits hnRNP A1 to regulate cell cycle and proliferation, J Cell Physiol 230 (2015) 2252-2261.
[22]
J. Sui, Y.F. Lin, K. Xu, K.J. Lee, D. Wang, B.P. Chen, DNA-PKcs phosphorylates hnRNP-A1 to facilitate the RPA-to-POT1 switch and telomere capping after replication, Nucleic Acids Res 43 (2015) 5971-5983.
Figure 1. UVB irradiation increased hnRNP A1 expression in HaCaT cells within time dependent. A. ＆ C. The levels of hnRNP A1 in HaCaT cells at 1 h, 3 h and 6 h post 25 mJ/cm2（A）and 50 mJ/cm2 （C） radiation. B. ＆ D. hnRNPA1 expressions showed in A and C were quantified by Image J software. Data presents three sets of independent experiments. *P<0.05 vs control group,#P<0.05 vs UVB(1h) group.
Figure 2. UVB radiation induced translocation of hnRNPA1 in HaCaT cells. A. Subcelluar location of hnRNPA1(Red) in HaCaT cells at 6 h after UVB treatment, and DAPI was used to label nuclei (400X). B. Cytoplasm hnRNPA1 was increased and nucleus hnRNPA1 decreased at 6 h after UVB treatment by western blot.
Figure 3. Knockdown hnRNP A1 in HaCaT cells decreased cell survival after UVB treatment. A. The expression of hnRNP A1 was detected by western blot at 24 h after specific siRNA or NC transfection in HaCaT cells. B. Transfected HaCaT cells by siRNA or NC were subjected to CCK-8 assay at 6 h after UVB exposure or without UVB irradiation. The data represents three sets of independent experiments and were shown as the means ± SD. *P < 0.05.
Figure 4. Overexpression hnRNP A1 in HaCaT cells increased cell survival after UVB treatment. A. hnRNP A1 stable cell line was established by lentivirus infection, verified by Flag tag staining(Cy3), and GFP stable cell line was used as control (200X). B. Stable HaCaT cells (hnRNP A1 or GFP) were subjected to CCK-8 assay at 6 h after UVB exposure or without UVB irradiation. The data represents three sets of independent experiments and were shown as the means ± SD. *P < 0.05.
Figure 5. hnRNP A1 enhanced the activation of PI3K/Akt/mTOR signaling pathway. A. The phosphorylation levels of Akt were examined by western blot in hnRNP A1 or GFP stable cell line with or without UVB irradiation. Relative expression of p-Akt was quantitatively analyzed by Image J software. Results are mean±SD from three independent experiments. *P<0.05 vs LV-GFP without UVB irradiation, # P<0.05 vs LV-GFP with UVB irradiation. B. The expression of hnRNP A1 was detected by western blot at 6 h after UVB exposure in HaCaT cells treated by 50 μM LY294002. Results are mean±SD from three independent experiments. *P<0.05 vs group 1 (without UVB irradiation nor LY294002 treatment), # P<0.05 vs group 3(with only UVB irradiation without LY294002 treatment). C. The phosphorylation levels of mTOR and P70S6 were detected by western blot in hnRNP A1 or GFP stable cells with or without UVB irradiation. Relative expression of p-mTOR was quantitatively analyzed by Image J software. The data represents three sets of independent experiments and were shown as the means ± SD. *P<0.05 vs LV-GFP without UVB irradiation, # P<0.05 vs LV-GFP with UVB irradiation. D. Western blot analysis of hnRNP A1 expression in HaCaT cells after treated with various concentration of Rapamycin and AZD8055 for 24 h.
Figure 6. hnRNP A1 promoted cell cycle post UVB irradiation. A. Stable cells (hnRNP A1 or GFP) were starved for 24 h and then harvested at 6 h after UVB irradiation, subsequently dyed by propidum iodide and subjected to flow cytometry. Cell cycle was analyzed by flowjo software. B. The population percentages at G0/G1, S and G2/M phases are shown as mean±SD. P* < 0.05.