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Activating transcription factor 6 regulated cell growth, migration and inhibiteds cell apoptosis and autophagy via MAPK pathway in cervical cancer
Journal of Reproductive Immunology 139 (2020) 103120
Contents lists available at ScienceDirect
Journal of Reproductive Immunology journal homepage: www.elsevier.com/locate/jri
Activating transcription factor 6 regulated cell growth, migration and inhibiteds cell apoptosis and autophagy via MAPK pathway in cervical cancer
T
Fang Liua,b, Li Changa,b, Jinliang Huc,d,* a
Department of Medical Laboratory, West China Second University Hospital, Sichuan University, Chengdu, Sichuan Province, China Key Laboratory of Obstetric & Gynecologic and Pediatric Diseases and Brith Defects of Ministry of Education, Chengdu, Sichuan Province, China c Institute of Health Policy & Hospital Management, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chinese Academy of Sciences Sichuan Translational Medicine Research Hospital, Chengdu, Sichuan Province, China d West China School of Public Health and West China Fourth Hospital, Sichuan University, Chengdu, Sichuan Province, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Activating transcription factor 6 MAPK pathway Cervical cancer ER stress
Background: Cervical cancer cell function is influence by ER. Therefore, in this study, ER stress senser-ATF6, was selected for detailed research in cervical cancer. Methods: ATF6 mRNA was assessed through RT-qPCR assays. Cell transfection was to regulate ATF6 and thereafter the differential ATF6 cancer cells were divided into two groups for further functional assays. Cell viabilities were analyzed by CCK-8 and migration by Scratch. RT-qPCR examined cell death biomarkers Caspas-3 and Bcl-2. 4-PBA was utilized to inhibit ER stress. After that, ATF6, viability, migration and apoptotic proteins were scrutinized after ER inhibition. Proteins signifying EMT, autophagy and MAPK signaling pathway were checked by western bolt. Last, we inactivated the MAPK signaling to investigate into the changes in cell functions. Results: ATF6 presented higher expression in cervical cancer cells. Inhibited ATF6 could reduce cell viabilities and migration but promote apoptosis through suppressing Bcl-2 and increasing caspase-3. ER stress antagonist witnessed a drop in ATF6 expression, cell viability, migration and Bcl-2 but a rise in caspase-3 activation, suggesting apoptosis increase. Cell autophagy was hindered in CC cells. Knockdown of ATF6 promoted autophagy and restrained EMT and MAPK signaling pathway. Suppressed ERK1/2 obstructed cell viabilities, migration, EMT and autophagy but promoted apoptosis. Conclusion: ATF6 might promote cell growth, migration, autophagy through ER stress and MAPK signaling in cervical cancer in vitro, indicating a potential regulatory gene in cervical cancer. However, in-depth researches are requested to enrich the knowledge of ATF6 in cervical cancer in vivo and in clinical in the future.
1. Introduction Cervical cancer (CC) is one of the most common malignant tumors in gynecology. Among all kinds of cancers worldwide, morbidity of cervical cancer ranks the third (Vu et al., 2018). Incidence rate and death rate of cervical cancer patients in developing countries are higher than those in developed countries (Denny et al., 2015). Thus, cervical cancer brings huge threat to females and requires extensive fundamental researches into the formation and development of CC in vitro and in vivo so as to advance the treatment potentials for CC. Gene therapy has become prospective, which helps to induce cancer cell apoptosis and inhibit cell viability, suggesting the possibility of
⁎
treatment in cancers. It was reported that gene RIZ1 and paclitaxel could promote the inhibitory effect in Siha CC cells than paclitaxel applied alone (Cheng et al., 2016). C5orf66-A/ miR-637/RING1 was likely to be the therapeutic target in CC cells (Rui et al., 2018). More studies into the roles of different genes in CC cells can enrich the knowledge of CC progression in cell level, to some extent promoting the discoveries of treatment in the future. ATF6 (activating transcription factor 6) is located in endoplasmic reticulum membrane (ER), which is a signaling molecule in ER unfolded protein response (Chen et al., 2002). It can be transferred to Golgi apparatus and conduct expressions of factors like XBP1(X-box binding protein 1) and GRP78 (glucose response protein 78) to
Corresponding author at: NO.32, 2nd western block of Yihuan road, Chengdu, Sichuan, China; Postcode: 610072 E-mail addresses: [email protected] (F. Liu), [email protected] (L. Chang), [email protected] (J. Hu).
https://doi.org/10.1016/j.jri.2020.103120 Received 29 January 2020; Received in revised form 27 February 2020; Accepted 11 March 2020 0165-0378/ © 2020 Published by Elsevier B.V.
Journal of Reproductive Immunology 139 (2020) 103120
F. Liu, et al.
in cervical cancer cells. Cell in log phase were selected and seeded into 6-well plate with 1 × 105 cells per well until cell confluences reached 35 %. siNC and siRNA of ATF6 were produced by GenePharma (Shanghai, China). Before transfection, opti-MEM medium (Thermo Fisher, USA) were added into plate. siRNA was diluted and added into 6-well plate with incubation for 48 h at 37℃. Lipofectamine 3000 (Invitrogen, USA) was picked as the reagent for transfection. when siNC and siATF6 was transfected, cells were cultured for another 24 h and RT-qPCR was performed to checked ATF6 expression later. Cells with siNC belonged to control group and cells with siATF6 were experimental groups.
maintain cell viabilities or induce apoptosis of cells (Wang et al., 2000). ATF6 was first discovered as binding protein of serum response factor, which could activate factors in regulating proliferation (Shen and Prywes, 2005). Researches regarding ATF6 in cervical cancer are mainly related to ER stress. It was previously demonstrated that Quercetin (Q) and Aconitine (A) could promote ER stress can cell death in cervical cancer, in which ATF6 was upregulated as a sensor of ER stress (Li et al., 2018).Similar findings were reported about ATF6 as an ER stress marker in cervical cancer cells (Kim et al., 2016; Lai and Wong, 2008). Nevertheless, functional role of ATF6 in cervical cancer has not been elucidated. This study focused on its role in in cervical cancer in vitro. Autophagy can be regulated by ER stress based on recent researches (Yorimitsu and Klionsky, 2007). All three signaling pathways in ER stress, IRE1-XBP1 pathway, PERK-eIF2α pathway and ATF6 pathway, can induce autophagy (Ogata et al., 2006). Based on previous researches, ER stress had close connection with development of cervical cancer (Tiansheng et al., 2020; Tan et al., 2018). MAPK signaling pathway has also been detected with development of cervical cancer. Normally, activation of factors in MAPK signaling pathway can help progression of cervical cancer (Sun et al., 2017a). P38, a subtribe of MAPK signaling pathway, has been found to have connection with ER stress in cervical cancer cells, implying the potential interplay between ATF6 and MAPK signaling pathway (Lin et al., 2018). Therefore, as a part in ER stress, ATF6 interaction with MAPK signaling pathway will also be investigated in this study.
2.4. CCK-8 Cell proliferation can be evaluated through cell viability. Usually, higher cell viability represented higher ability in proliferation. Meanwhile, cancer cells were well known with their high capability in proliferation. Cells in log phase were collected and digested by 0.25 % trypsin to get cell suspension with 5 × 104 cell/ml and seeded into 96well plate. After incubation for 24 h, 20 μl CCK-8 was added at specific time point (24 h, 48 h and 72 h) and incubation for 4 h. In blank group, the well only contained medium and CCK-8 Kit without cells and in control groups, cells without siATF6 were added containing CCK-8 as well as medium. Then, optical density values (OD value) were detected at 450 nm wave length by microplate reader (Thermo Fisher, USA).
2. Methods 2.5. Scratch test
2.1. Cell culture
Scratch test was applied to measure migration ability of cells. Cells in log phase were gathered and seed into 6-well plate with 5 × 105 cells per well. After plate was full covered with cells, 20 μl pipette tip was used to scratch parallel lines vertically. Then cells were rinsed by PBS three times and added with serum free medium. After that, cells were incubated in full humidity incubator at 37℃, 5% CO2 and images were taken 24 h after.
Human cervical cancer cell lines (Hela, Caski and SiHa) and normal endometrium epithelial cell (ESC) were bought from ATCC (USA). Cells were cultured in DMEM complete medium (Thermo Fisher, USA) with 10 % fetal bovine serum, 100 μmol/l penicillin and 100 μmol/l streptomycin at 37℃, 5% CO2 saturated humidity incubator. Adhered cells were digested by 0.25 % trypsin to subculture. ESC cell line was the control group. Hela, Caski and SiHa cell lines are experimental groups. All the assays were performed in the affiliated lab of West China Second University Hospital, Chengdu city, Sichuan Province, China.
2.6. Western blot
2.2. RT-qPCR
Cells were lysed by RIPA Lysis and Extraction Buffer (Thermo Fisher, USA) on ice for 30 min. Then supernatant liquid was extracted and qualified by BCA Protein Assay Kit (Beyotime, Shanghai, China). Next, 50 μg proteins were separated by SDS-PAGE and transferred into PVDF membranes. Then QuickBlock™ Blocking Buffer was applied to block membranes and primary antibodies were used for incubation overnight at 4℃. Primary antibodies were shown as below: anti-LC3Ⅰ(1:1000; ab52628, Cambridge, UK), anti-LC3-Ⅱ (1:1000; ab48394), anti-p62 (1:1000; ab109012), anti-E-cadherin (1:1000; ab40772), antiSnail (1:1000; ab53519), anti-Vimentin (1:1000; ab92547), anti-pERK1/2 (1:1000; ab176640), anti-p38 (1:1000; ab170099) and GAPDH (1:2000; ab9485). After incubation with primary antibodies, membranes were rinsed by TBST buffer and incubated with secondary antibodies marked with HRP. Secondary antibodies were displayed: goat anti-rabbit IgG (1:900; ab6721) and goat anti-mouse IgG (1:900; ab150113). Thereafter, membranes were added with BeyoECL Moon (Beyotime, Shanghai, China) to be developed. Images of gray values were selected and qualified with GAPDH as the standard.
In order to dig out expressions of ATF6, Bcl-2 and caspase-3 in RNA level, RT-qPCR was adopted. Total RNAs were extracted from cells strictly according to Trizol reagent (Thermo Fisher, USA). Reverse transcription was performed through TaqMan™ MicroRNA Reverse Transcription Kit (Thermo Fisher, USA). Sequences of primers were designed by Primer Premier 6.0 (USA) and sequences were displayed as below, ATF6: forward, 5′-TGGAAGCAGCAAATGAGACG-3′, reverse, 5′-TGAGGAGGCTGGAGAAAGTG-3′, Bcl-2: sense, 5′−CCTCGCTGCAC AAATACTCC-3′, antisense, 5′-TGGAGAGAATGTTGGCGTCT-3′, caspase-3: forward, 5′-AAGGAGCAGCTTTGTGTGTG-3′, reverse, 5′-GGCA GGCCTGAATGATGAAG-3′, ERK1: forward, 5′- CCCGTTCCATCGCTAG TAGT-3′, reverse, 5′-AGCCTACAGACCAGACATCG-3′, ERK-2: forward, 5′- TGTACCCTCGCATGACTGTT-3′, reverse, 5′- TCCGTGGAACACAAC TCAGA-3′, p38: forward, 5′- AATCCTCACCATCCACAGCA-3′, reverse, 5′- 1GCTTAGAGTCCAGGCTTCCA-3′ and GAPDH: forward, 5′- ACCCA GAAGACTGTGGATGG-3′, reverse, 5′- TCAGCTCAGGGATGACCTTG-3′. ABI quantstudio 3 was used for this assay (4482603, Invitrogen, CA, US). Reaction conditions were showed. Pre-denaturation was at 95℃, 30 s. Denaturation was at 95℃, 5 s and extension was at 60℃, 30 s, 38 cycles. Relative expressions levels were qualified by 2−△△Ct methods.
2.7. Statistical analysis Data were displayed as mean ± SD and analyzed by SPSS 22.0 (IBM). Each experiment was repeated three times. Analyses of two groups were used t-test and groups more than two analyzed with one way ANOVA. P < 0.05 was considered to have statistical meaning.
2.3. Cell transfection Cell transfection was performed to acquire differential ATF6 levels 2
Journal of Reproductive Immunology 139 (2020) 103120
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Fig. 1. ATF6 expressed higher in cervical cancer and promoted proliferation and inhibited apoptosis. A. Expressions of ATF6 in normal cell line and cervical cancer cell lines were detected by RT-qPCR, P < 0.05. B. Cell viabilities in normal cell line and cervical cancer cell lines were measured by CCK-8, P < 0.05. C. Inhibited ATF6 expression was evaluated in Hela cell line by RT-qPCR, P < 0.05. D. cell viabilities were measured through CCK-8, P < 0.05. E. Migration of cell line was detected by scratch test, P < 0.05. F: Expressions of Bcl-2 and caspase-3 were validated by RT-qPCR, P < 0.05.
3. Results
viabilities of cells in tumor cell line (Fig. 2B). As for migration, compared to negative control group, ER stress inhibitor group showed that migration of Hela cell line was suppressed (Fig. 3B). Apoptosis of cells were also measured, compared with control group, 4-PBA group could reduce expression of Bcl-2 and increase expression of caspase-3 (Fig. 2D, E).
3.1. ATF6 was up-regulated in cervical cancer cells and promoted proliferation with inhibiting apoptosis Expressions of ATF6 were evaluated in cervical cancer cell lines (Hela, CaSki and SiHa) and normal cervical epithelial cell line ESC. Results showed that expressions of ATF6 were higher in cancer cell lines than normal cell line, especially in Hela cell line (Fig. 1A). Cell viabilities were also detected, which showed that cell viabilities were also higher in cancer cell lines rather than normal cell line (Fig. 1B). Therefore, ATF6 was inhibited to measure the expression of ATF6 in Hela cell line. After ATF6 was suppressed, expression of ATF6 was lower than negative control groups as well as cell viabilities ((Fig. 1C, D). Next, migration was detected, suggesting that inhibited ATF6 could decrease migration in Hela cell line (Fig. 1E). RNAs related to apoptosis were also validated, which showed that expression of Bcl-2 was significantly decreased while expression of caspase-3 was promoted (Fig. 1F, G).
3.3. ATF6 suppressed autophagy and promoted EMT via MAPK signaling pathway Expressions of proteins in autophagy were measured. In normal cell line, expressions of LC3-Ⅰ and p62 were higher in cervical cancer cell lines while expression of LC3-Ⅱ was lower (Fig. 3A). In cancer cell line selected, knockdown of ATF6 could up regulate expression of LC3-Ⅱ compared to control group (Fig. 3B). Expressions of LC3-Ⅰ and p62 in inhibited ATF6 groups were reduced. In former experiments, migration was inhibited by repressed ATF6. Thus, proteins related to EMT were evaluated, compared with negative control groups, expression of Ecadherin was higher in inhibited ATF6 and expressions of Vimentin and Snail were lower in transfected ATF6 groups (Fig. 3C). After that, proteins in MAPK signaling pathway were validated, which showed that phosphorylated ERK1/2 and p38 were up-regulated in cervical cancer cell lines (Fig. 3D). Furthermore, in suppressed ATF6 groups in cervical cancer cell line, p-ERK1/2 and p38 were decreased (Fig. 3E).
3.2. ATF6 regulated proliferation, migration and apoptosis of cervical cancer cells via ER stress In order to figure out further mechanism of ATF6, 4-PBA was applied to inhibit ER stress. RT-qPCR was used to detect expressions of ATF6 in Hela cell line. Results displayed that cells with 4-PBA could inhibit expression of ATF6 (Fig. 2A). Meanwhile, cell viabilities of Hela cell line also demonstrated that cells with 4-PBA could reduce 3
Journal of Reproductive Immunology 139 (2020) 103120
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Fig. 2. ATF6 regulated proliferation, migration and apoptosis of cervical cancer cells via ER stress. 4-PBA was used to suppress ER stress. A. ATF6 expressions in negative control and 4-PBA group were measured by RT-qPCR, P < 0.05. B. CCK-8 was used to evaluate cell viabilities, P < 0.05. C. Migration was tested through scratch test, P < 0.05. D. Bcl-2 and caspase-3 expressions were evaluated by RT-qPCR, P < 0.05.
Fig. 3. ATF6 suppressed autophagy and promoted EMT via MAPK signaling pathway. A. Proteins in autophagy in normal cell line and cervical cancer cell lines were validated by western blot. B. Proteins in autophagy in NC and siATF6 group were measured by western blot. C. Proteins in EMT were detected by western blot. D. Proteins in MAPK signaling pathway were evaluated through western blot. E. Proteins of MAPK signaling pathway group were measured in NC and siATF6 using western blot.
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Journal of Reproductive Immunology 139 (2020) 103120
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Fig. 4. Suppressed MAPK signaling pathway inhibited proliferation, apoptosis, migration, EMT and autophagy. SCH773984 was inhibitor of ERK1/2 A. Expression of ERK1/2 was detected by western blot. B. Cell viabilities were measured through CCK-8, P < 0.05. C. Scratch test was used for evaluating migration of cells in NC group and SCH772984 group, P < 0.05. D. RT-qPCR was applied for validating expressions of Bcl-2 and caspase-3, P < 0.05. E, F. Proteins in EMT and autophagy were measured through western blot.
in CC cells, showing that ATF6 downregulation could improve apoptosis in Hela cell line. 4-phenylbutyric acid (4-PBA) is a small molecular weight fatty acid, which could suppress ER stress (Zhu et al., 2014). Next, we used 4-PBA for suppressing ER stress in cells, which decreased ATF6 expression in CC cells. Meanwhile, we found lower cell viabilities and migration in cells with 4-PBA groups. Expression of Bcl-2 was suppressed and caspase-3 was up-regulated, signifying the promoted apoptosis in ER inhibited CC cells. Therefore, we decided that ATF6 might regulate progression of cervical cancer cells through ER stress. Physiological functions of autophagy contain four parts, cleaning injured cells to maintain stability of inner environment, regulating innate immunity and adaptive immunity, inducing cell death and extending life of cells (Liu and Levine, 2015). Autophagy could induce cell apoptosis in early stage of cancers (Huang et al., 2014). LC3-II is resultant protein after ubiquitination of LC3-I, which marked autophagy condition (Zhou et al., 2018).P62 was reported to be an autophagy adaptor in cancers (Moscat et al., 2016). In this study, we observed that after ATF6 was down-regulated, expressions of LC3-Ⅰ and p62 decreased and LC3-Ⅱ was promoted, suggesting the severity of autophagy increased with the inhibition of ATF6. Epithelial to mesenchymal transition (EMT) is a process that epithelial cells transformed into mesenchymal cells (Thiery and Lim, 2013). E-cadherin mainly existed in epithelial cell (Tiwari et al., 2012). Snail could bind E-cadherin to suppress expression of E-cadherin (Cade et al., 2010). Vimentin could promote EMT in cancer. Therefore, we measured E-cadherin and vimentin and found that inhibited ATF6 could increase expression of Ecadherin and reduce expressions of Snail and Vimentin, thus hindered EMT process. Mitogen-activated protein kinase (MAPK) signaling pathway
3.4. Effects on proliferation, apoptosis, migration, EMT and autophagy by suppressed MAPK signaling pathway In former figure, it was indicated that ATF6 regulated cell progressions via MAPK signaling pathway. Thus, functions of MAPK signaling pathway were measured. SCH772984 was a kind of inhibitor which could suppress ERK1/2 proteins in MAPK signaling pathway (Fig. 4A). After expression was inhibited in SCH772984 group, cell viabilities were shown, which revealed that SCH772984 could down regulate cell viabilities in tumor cell line (Fig. 4B). In scratch test, SCH772984 could decrease migration in cervical tumor cell line (Fig. 4C). As for apoptosis, Bcl-2 was repressed in SCH772984 group while caspase-3 was promoted (Fig. 4D, E). In EMT, E-cadherin expression was higher in suppressed ERK1/2 group but Vimentin expression was reduced as well as Sanil expression (Fig. 4F). Expressions of LC3-Ⅰ and p62 in autophagy were lower in SCH772984 groups with promoted LC3-Ⅱ compared to the negative group (Fig. 4G).
4. Discussion In previous study, ATF6 could conduct activation of GRP78, which could inhibit activation of caspase-3 and maintain stability of ER and inner environment to improve survival of cancer cells (Dong et al., 2004; Shen et al., 2002). Therefore, in this research, we found that ATF6 was expressed higher in CC cells and suppressed ATF6 could reduce cell viabilities and migration in Hela, indicating that inhibited ATF6 could reduce proliferation and ignite migration of CC cells. RTqPCR showed that pro-apoptotic protein Bcl-2 was down-regulated and apoptotic biomarker caspase-3 was promoted when ATF6 was silenced 5
Journal of Reproductive Immunology 139 (2020) 103120
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contains extracellular signal-regulated kinase (ERK1/2), Jun N-lerminal kinase (JNK), p38,MAPK and ERK5 (Sun et al., 2015). ERK1/2 is considered to have connections with proliferation, transformation and differentiation of cells and activation of ERK1/2 could suppress apoptosis in cells and P38 is related to apoptosis, inflammations, ER stress, etc (Sun et al., 2017b; Song et al., 2017). The findings in this research showed that p-ERK1/2 and p38 were higher in cancer cells and knockdown of ATF6 could down regulate p-ERK1/2 and p38 in cervical cancer cells. Furthermore, SCH772984 was used to suppress expression of ERK1/2, which inhibited cell viability and migration in cervical cancer cell line. Bcl-2 was decreased with SCH772984 added and caspase-3 was up-regulated. E-cadherin in EMT was higher in SCH772984 and Vimentin and Snail were lower. LC3-Ⅰ and p62 related to autophagy were lower in SCH772984 group while LC3-Ⅱ was up-regulated. Therefore, knockdown of ATF6 could help silence MAPK signaling and inactivated MAPK signaling pathway suppressed cell viability and promoted cell apoptosis and autophagy.
Huang, Y.H., et al., 2014. A role of autophagy in PTP4A3-driven cancer progression. Autophagy 10 (10), 1787–1800. Kim, B., et al., 2016. Curcumin induces ER stress-mediated apoptosis through selective generation of reactive oxygen species in cervical cancer cells. Mol. Carcinog. 55 (5), 918–928. Lai, W.L., Wong, N.S., 2008. The PERK/eIF2 alpha signaling pathway of Unfolded Protein Response is essential for N-(4-hydroxyphenyl)retinamide (4HPR)-induced cytotoxicity in cancer cells. Exp. Cell Res. 314 (8), 1667–1682. Li, X.M., et al., 2018. Quercetin and aconitine synergistically induces the human cervical carcinoma HeLa cell apoptosis via endoplasmic reticulum (ER) stress pathway. PLoS One 13 (1), e0191062. Lin, C.L., et al., 2018. Protodioscin induces apoptosis through ROS-mediated endoplasmic reticulum stress via the JNK/p38 activation pathways in human cervical cancer cells. Cell. Physiol. Biochem. 46 (1), 322–334. Liu, Y., Levine, B., 2015. Autosis and autophagic cell death: the dark side of autophagy. Cell Death Differ. 22 (3), 367–376. Moscat, J., Karin, M., Diaz-Meco, M.T., 2016. p62 in Cancer: signaling adaptor beyond autophagy. Cell 167 (3), 606–609. Ogata, M., et al., 2006. Autophagy is activated for cell survival after endoplasmic reticulum stress. Mol. Cell. Biol. 26 (24), 9220–9231. Rui, X., et al., 2018. Long non-coding RNA C5orf66-AS1 promotes cell proliferation in cervical cancer by targeting miR-637/RING1 axis. Cell Death Dis. 9 (12), 1175. Shen, J., Prywes, R., 2005. ER stress signaling by regulated proteolysis of ATF6. Methods 35 (4), 382–389. Shen, J., et al., 2002. ER stress regulation of ATF6 localization by dissociation of BiP/ GRP78 binding and unmasking of Golgi localization signals. Dev. Cell 3 (1), 99–111. Song, X.F., et al., 2017. ZEB1 promotes prostate cancer proliferation and invasion through ERK1/2 signaling pathway. Eur. Rev. Med. Pharmacol. Sci. 21 (18), 4032–4038. Sun, Y., et al., 2015. Signaling pathway of MAPK/ERK in cell proliferation, differentiation, migration, senescence and apoptosis. J. Recept. Signal Transduct. Res. 35 (6), 600–604. Sun, Q., et al., 2017a. ER-alpha36 mediates estrogen-stimulated MAPK/ERK activation and regulates migration, invasion, proliferation in cervical cancer cells. Biochem. Biophys. Res. Commun. 487 (3), 625–632. Sun, X., et al., 2017b. Cigarette smoke extract induces epithelial-mesenchymal transition of human bladder cancer T24 cells through activation of ERK1/2 pathway. Biomed. Pharmacother. 86, 457–465. Tan, B., et al., 2018. Astragaloside attenuates the progression of prostate cancer cells through endoplasmic reticulum stress pathways. Oncol. Lett. 16 (3), 3901–3906. Thiery, J.P., Lim, C.T., 2013. Tumor dissemination: an EMT affair. Cancer Cell 23 (3), 272–273. Tiansheng, G., et al., 2020. lncRNA metastasis-associated lung adenocarcinoma transcript 1 promotes proliferation and invasion of non-small cell lung Cancer cells via downregulating miR-202 expression. Cell J. 22 (3), 375–385. Tiwari, N., et al., 2012. EMT as the ultimate survival mechanism of cancer cells. Semin. Cancer Biol. 22 (3), 194–207. Vu, M., et al., 2018. Cervical cancer worldwide. Curr. Probl. Cancer 42 (5), 457–465. Wang, Y., et al., 2000. Activation of ATF6 and an ATF6 DNA binding site by the endoplasmic reticulum stress response. J. Biol. Chem. 275 (35), 27013–27020. Yorimitsu, T., Klionsky, D.J., 2007. Endoplasmic reticulum stress: a new pathway to induce autophagy. Autophagy 3 (2), 160–162. Zhou, L., et al., 2018. Glucose deprivation promotes apoptotic response to S1 by enhancing autophagy in human cervical cancer cells. Cancer Manag. Res. 10, 6195–6204. Zhu, M., et al., 2014. 4-phenylbutyric acid attenuates endoplasmic reticulum stressmediated pancreatic beta-cell apoptosis in rats with streptozotocin-induced diabetes. Endocrine 47 (1), 129–137.
5. Conclusion This study has evaluated the regulatory role of ATF6 in cervical cancer cells, which elucidated that ATF6 might the cell proliferation and migration, inhibited cell autophagy and apoptosis in CC through ER stress and MAPK signaling. Our observations might offer basic research experience for future animal researches and clinical treatments related to cervical cancer. Declaration of Competing Interest Nothing to be disclosed by authors. References Cade, T.J., et al., 2010. Progestogen treatment options for early endometrial cancer. Bjog 117 (7), 879–884. Chen, X., Shen, J., Prywes, R., 2002. The luminal domain of ATF6 senses endoplasmic reticulum (ER) stress and causes translocation of ATF6 from the ER to the Golgi. J. Biol. Chem. 277 (15), 13045–13052. Cheng, H.Y., et al., 2016. Synergism between RIZ1 gene therapy and paclitaxel in SiHa cervical cancer cells. Cancer Gene Ther. 23 (11), 392–395. Denny, L., et al., 2015. Cervical cancer. In: third edition. In: Gelband, H. (Ed.), Cancer: Disease Control Priorities 3 The International Bank for Reconstruction and Development / The World Bank (c) 2015 International Bank for Reconstruction and Development The World Bank: Washington (DC). Dong, D., et al., 2004. Spontaneous and controllable activation of suicide gene expression driven by the stress-inducible grp78 promoter resulting in eradication of sizable human tumors. Hum. Gene Ther. 15 (6), 553–561.
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Contents lists available at ScienceDirect
Journal of Reproductive Immunology journal homepage: www.elsevier.com/locate/jri
Activating transcription factor 6 regulated cell growth, migration and inhibiteds cell apoptosis and autophagy via MAPK pathway in cervical cancer
T
Fang Liua,b, Li Changa,b, Jinliang Huc,d,* a
Department of Medical Laboratory, West China Second University Hospital, Sichuan University, Chengdu, Sichuan Province, China Key Laboratory of Obstetric & Gynecologic and Pediatric Diseases and Brith Defects of Ministry of Education, Chengdu, Sichuan Province, China c Institute of Health Policy & Hospital Management, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chinese Academy of Sciences Sichuan Translational Medicine Research Hospital, Chengdu, Sichuan Province, China d West China School of Public Health and West China Fourth Hospital, Sichuan University, Chengdu, Sichuan Province, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Activating transcription factor 6 MAPK pathway Cervical cancer ER stress
Background: Cervical cancer cell function is influence by ER. Therefore, in this study, ER stress senser-ATF6, was selected for detailed research in cervical cancer. Methods: ATF6 mRNA was assessed through RT-qPCR assays. Cell transfection was to regulate ATF6 and thereafter the differential ATF6 cancer cells were divided into two groups for further functional assays. Cell viabilities were analyzed by CCK-8 and migration by Scratch. RT-qPCR examined cell death biomarkers Caspas-3 and Bcl-2. 4-PBA was utilized to inhibit ER stress. After that, ATF6, viability, migration and apoptotic proteins were scrutinized after ER inhibition. Proteins signifying EMT, autophagy and MAPK signaling pathway were checked by western bolt. Last, we inactivated the MAPK signaling to investigate into the changes in cell functions. Results: ATF6 presented higher expression in cervical cancer cells. Inhibited ATF6 could reduce cell viabilities and migration but promote apoptosis through suppressing Bcl-2 and increasing caspase-3. ER stress antagonist witnessed a drop in ATF6 expression, cell viability, migration and Bcl-2 but a rise in caspase-3 activation, suggesting apoptosis increase. Cell autophagy was hindered in CC cells. Knockdown of ATF6 promoted autophagy and restrained EMT and MAPK signaling pathway. Suppressed ERK1/2 obstructed cell viabilities, migration, EMT and autophagy but promoted apoptosis. Conclusion: ATF6 might promote cell growth, migration, autophagy through ER stress and MAPK signaling in cervical cancer in vitro, indicating a potential regulatory gene in cervical cancer. However, in-depth researches are requested to enrich the knowledge of ATF6 in cervical cancer in vivo and in clinical in the future.
1. Introduction Cervical cancer (CC) is one of the most common malignant tumors in gynecology. Among all kinds of cancers worldwide, morbidity of cervical cancer ranks the third (Vu et al., 2018). Incidence rate and death rate of cervical cancer patients in developing countries are higher than those in developed countries (Denny et al., 2015). Thus, cervical cancer brings huge threat to females and requires extensive fundamental researches into the formation and development of CC in vitro and in vivo so as to advance the treatment potentials for CC. Gene therapy has become prospective, which helps to induce cancer cell apoptosis and inhibit cell viability, suggesting the possibility of
⁎
treatment in cancers. It was reported that gene RIZ1 and paclitaxel could promote the inhibitory effect in Siha CC cells than paclitaxel applied alone (Cheng et al., 2016). C5orf66-A/ miR-637/RING1 was likely to be the therapeutic target in CC cells (Rui et al., 2018). More studies into the roles of different genes in CC cells can enrich the knowledge of CC progression in cell level, to some extent promoting the discoveries of treatment in the future. ATF6 (activating transcription factor 6) is located in endoplasmic reticulum membrane (ER), which is a signaling molecule in ER unfolded protein response (Chen et al., 2002). It can be transferred to Golgi apparatus and conduct expressions of factors like XBP1(X-box binding protein 1) and GRP78 (glucose response protein 78) to
Corresponding author at: NO.32, 2nd western block of Yihuan road, Chengdu, Sichuan, China; Postcode: 610072 E-mail addresses: [email protected] (F. Liu), [email protected] (L. Chang), [email protected] (J. Hu).
https://doi.org/10.1016/j.jri.2020.103120 Received 29 January 2020; Received in revised form 27 February 2020; Accepted 11 March 2020 0165-0378/ © 2020 Published by Elsevier B.V.
Journal of Reproductive Immunology 139 (2020) 103120
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in cervical cancer cells. Cell in log phase were selected and seeded into 6-well plate with 1 × 105 cells per well until cell confluences reached 35 %. siNC and siRNA of ATF6 were produced by GenePharma (Shanghai, China). Before transfection, opti-MEM medium (Thermo Fisher, USA) were added into plate. siRNA was diluted and added into 6-well plate with incubation for 48 h at 37℃. Lipofectamine 3000 (Invitrogen, USA) was picked as the reagent for transfection. when siNC and siATF6 was transfected, cells were cultured for another 24 h and RT-qPCR was performed to checked ATF6 expression later. Cells with siNC belonged to control group and cells with siATF6 were experimental groups.
maintain cell viabilities or induce apoptosis of cells (Wang et al., 2000). ATF6 was first discovered as binding protein of serum response factor, which could activate factors in regulating proliferation (Shen and Prywes, 2005). Researches regarding ATF6 in cervical cancer are mainly related to ER stress. It was previously demonstrated that Quercetin (Q) and Aconitine (A) could promote ER stress can cell death in cervical cancer, in which ATF6 was upregulated as a sensor of ER stress (Li et al., 2018).Similar findings were reported about ATF6 as an ER stress marker in cervical cancer cells (Kim et al., 2016; Lai and Wong, 2008). Nevertheless, functional role of ATF6 in cervical cancer has not been elucidated. This study focused on its role in in cervical cancer in vitro. Autophagy can be regulated by ER stress based on recent researches (Yorimitsu and Klionsky, 2007). All three signaling pathways in ER stress, IRE1-XBP1 pathway, PERK-eIF2α pathway and ATF6 pathway, can induce autophagy (Ogata et al., 2006). Based on previous researches, ER stress had close connection with development of cervical cancer (Tiansheng et al., 2020; Tan et al., 2018). MAPK signaling pathway has also been detected with development of cervical cancer. Normally, activation of factors in MAPK signaling pathway can help progression of cervical cancer (Sun et al., 2017a). P38, a subtribe of MAPK signaling pathway, has been found to have connection with ER stress in cervical cancer cells, implying the potential interplay between ATF6 and MAPK signaling pathway (Lin et al., 2018). Therefore, as a part in ER stress, ATF6 interaction with MAPK signaling pathway will also be investigated in this study.
2.4. CCK-8 Cell proliferation can be evaluated through cell viability. Usually, higher cell viability represented higher ability in proliferation. Meanwhile, cancer cells were well known with their high capability in proliferation. Cells in log phase were collected and digested by 0.25 % trypsin to get cell suspension with 5 × 104 cell/ml and seeded into 96well plate. After incubation for 24 h, 20 μl CCK-8 was added at specific time point (24 h, 48 h and 72 h) and incubation for 4 h. In blank group, the well only contained medium and CCK-8 Kit without cells and in control groups, cells without siATF6 were added containing CCK-8 as well as medium. Then, optical density values (OD value) were detected at 450 nm wave length by microplate reader (Thermo Fisher, USA).
2. Methods 2.5. Scratch test
2.1. Cell culture
Scratch test was applied to measure migration ability of cells. Cells in log phase were gathered and seed into 6-well plate with 5 × 105 cells per well. After plate was full covered with cells, 20 μl pipette tip was used to scratch parallel lines vertically. Then cells were rinsed by PBS three times and added with serum free medium. After that, cells were incubated in full humidity incubator at 37℃, 5% CO2 and images were taken 24 h after.
Human cervical cancer cell lines (Hela, Caski and SiHa) and normal endometrium epithelial cell (ESC) were bought from ATCC (USA). Cells were cultured in DMEM complete medium (Thermo Fisher, USA) with 10 % fetal bovine serum, 100 μmol/l penicillin and 100 μmol/l streptomycin at 37℃, 5% CO2 saturated humidity incubator. Adhered cells were digested by 0.25 % trypsin to subculture. ESC cell line was the control group. Hela, Caski and SiHa cell lines are experimental groups. All the assays were performed in the affiliated lab of West China Second University Hospital, Chengdu city, Sichuan Province, China.
2.6. Western blot
2.2. RT-qPCR
Cells were lysed by RIPA Lysis and Extraction Buffer (Thermo Fisher, USA) on ice for 30 min. Then supernatant liquid was extracted and qualified by BCA Protein Assay Kit (Beyotime, Shanghai, China). Next, 50 μg proteins were separated by SDS-PAGE and transferred into PVDF membranes. Then QuickBlock™ Blocking Buffer was applied to block membranes and primary antibodies were used for incubation overnight at 4℃. Primary antibodies were shown as below: anti-LC3Ⅰ(1:1000; ab52628, Cambridge, UK), anti-LC3-Ⅱ (1:1000; ab48394), anti-p62 (1:1000; ab109012), anti-E-cadherin (1:1000; ab40772), antiSnail (1:1000; ab53519), anti-Vimentin (1:1000; ab92547), anti-pERK1/2 (1:1000; ab176640), anti-p38 (1:1000; ab170099) and GAPDH (1:2000; ab9485). After incubation with primary antibodies, membranes were rinsed by TBST buffer and incubated with secondary antibodies marked with HRP. Secondary antibodies were displayed: goat anti-rabbit IgG (1:900; ab6721) and goat anti-mouse IgG (1:900; ab150113). Thereafter, membranes were added with BeyoECL Moon (Beyotime, Shanghai, China) to be developed. Images of gray values were selected and qualified with GAPDH as the standard.
In order to dig out expressions of ATF6, Bcl-2 and caspase-3 in RNA level, RT-qPCR was adopted. Total RNAs were extracted from cells strictly according to Trizol reagent (Thermo Fisher, USA). Reverse transcription was performed through TaqMan™ MicroRNA Reverse Transcription Kit (Thermo Fisher, USA). Sequences of primers were designed by Primer Premier 6.0 (USA) and sequences were displayed as below, ATF6: forward, 5′-TGGAAGCAGCAAATGAGACG-3′, reverse, 5′-TGAGGAGGCTGGAGAAAGTG-3′, Bcl-2: sense, 5′−CCTCGCTGCAC AAATACTCC-3′, antisense, 5′-TGGAGAGAATGTTGGCGTCT-3′, caspase-3: forward, 5′-AAGGAGCAGCTTTGTGTGTG-3′, reverse, 5′-GGCA GGCCTGAATGATGAAG-3′, ERK1: forward, 5′- CCCGTTCCATCGCTAG TAGT-3′, reverse, 5′-AGCCTACAGACCAGACATCG-3′, ERK-2: forward, 5′- TGTACCCTCGCATGACTGTT-3′, reverse, 5′- TCCGTGGAACACAAC TCAGA-3′, p38: forward, 5′- AATCCTCACCATCCACAGCA-3′, reverse, 5′- 1GCTTAGAGTCCAGGCTTCCA-3′ and GAPDH: forward, 5′- ACCCA GAAGACTGTGGATGG-3′, reverse, 5′- TCAGCTCAGGGATGACCTTG-3′. ABI quantstudio 3 was used for this assay (4482603, Invitrogen, CA, US). Reaction conditions were showed. Pre-denaturation was at 95℃, 30 s. Denaturation was at 95℃, 5 s and extension was at 60℃, 30 s, 38 cycles. Relative expressions levels were qualified by 2−△△Ct methods.
2.7. Statistical analysis Data were displayed as mean ± SD and analyzed by SPSS 22.0 (IBM). Each experiment was repeated three times. Analyses of two groups were used t-test and groups more than two analyzed with one way ANOVA. P < 0.05 was considered to have statistical meaning.
2.3. Cell transfection Cell transfection was performed to acquire differential ATF6 levels 2
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Fig. 1. ATF6 expressed higher in cervical cancer and promoted proliferation and inhibited apoptosis. A. Expressions of ATF6 in normal cell line and cervical cancer cell lines were detected by RT-qPCR, P < 0.05. B. Cell viabilities in normal cell line and cervical cancer cell lines were measured by CCK-8, P < 0.05. C. Inhibited ATF6 expression was evaluated in Hela cell line by RT-qPCR, P < 0.05. D. cell viabilities were measured through CCK-8, P < 0.05. E. Migration of cell line was detected by scratch test, P < 0.05. F: Expressions of Bcl-2 and caspase-3 were validated by RT-qPCR, P < 0.05.
3. Results
viabilities of cells in tumor cell line (Fig. 2B). As for migration, compared to negative control group, ER stress inhibitor group showed that migration of Hela cell line was suppressed (Fig. 3B). Apoptosis of cells were also measured, compared with control group, 4-PBA group could reduce expression of Bcl-2 and increase expression of caspase-3 (Fig. 2D, E).
3.1. ATF6 was up-regulated in cervical cancer cells and promoted proliferation with inhibiting apoptosis Expressions of ATF6 were evaluated in cervical cancer cell lines (Hela, CaSki and SiHa) and normal cervical epithelial cell line ESC. Results showed that expressions of ATF6 were higher in cancer cell lines than normal cell line, especially in Hela cell line (Fig. 1A). Cell viabilities were also detected, which showed that cell viabilities were also higher in cancer cell lines rather than normal cell line (Fig. 1B). Therefore, ATF6 was inhibited to measure the expression of ATF6 in Hela cell line. After ATF6 was suppressed, expression of ATF6 was lower than negative control groups as well as cell viabilities ((Fig. 1C, D). Next, migration was detected, suggesting that inhibited ATF6 could decrease migration in Hela cell line (Fig. 1E). RNAs related to apoptosis were also validated, which showed that expression of Bcl-2 was significantly decreased while expression of caspase-3 was promoted (Fig. 1F, G).
3.3. ATF6 suppressed autophagy and promoted EMT via MAPK signaling pathway Expressions of proteins in autophagy were measured. In normal cell line, expressions of LC3-Ⅰ and p62 were higher in cervical cancer cell lines while expression of LC3-Ⅱ was lower (Fig. 3A). In cancer cell line selected, knockdown of ATF6 could up regulate expression of LC3-Ⅱ compared to control group (Fig. 3B). Expressions of LC3-Ⅰ and p62 in inhibited ATF6 groups were reduced. In former experiments, migration was inhibited by repressed ATF6. Thus, proteins related to EMT were evaluated, compared with negative control groups, expression of Ecadherin was higher in inhibited ATF6 and expressions of Vimentin and Snail were lower in transfected ATF6 groups (Fig. 3C). After that, proteins in MAPK signaling pathway were validated, which showed that phosphorylated ERK1/2 and p38 were up-regulated in cervical cancer cell lines (Fig. 3D). Furthermore, in suppressed ATF6 groups in cervical cancer cell line, p-ERK1/2 and p38 were decreased (Fig. 3E).
3.2. ATF6 regulated proliferation, migration and apoptosis of cervical cancer cells via ER stress In order to figure out further mechanism of ATF6, 4-PBA was applied to inhibit ER stress. RT-qPCR was used to detect expressions of ATF6 in Hela cell line. Results displayed that cells with 4-PBA could inhibit expression of ATF6 (Fig. 2A). Meanwhile, cell viabilities of Hela cell line also demonstrated that cells with 4-PBA could reduce 3
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Fig. 2. ATF6 regulated proliferation, migration and apoptosis of cervical cancer cells via ER stress. 4-PBA was used to suppress ER stress. A. ATF6 expressions in negative control and 4-PBA group were measured by RT-qPCR, P < 0.05. B. CCK-8 was used to evaluate cell viabilities, P < 0.05. C. Migration was tested through scratch test, P < 0.05. D. Bcl-2 and caspase-3 expressions were evaluated by RT-qPCR, P < 0.05.
Fig. 3. ATF6 suppressed autophagy and promoted EMT via MAPK signaling pathway. A. Proteins in autophagy in normal cell line and cervical cancer cell lines were validated by western blot. B. Proteins in autophagy in NC and siATF6 group were measured by western blot. C. Proteins in EMT were detected by western blot. D. Proteins in MAPK signaling pathway were evaluated through western blot. E. Proteins of MAPK signaling pathway group were measured in NC and siATF6 using western blot.
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Fig. 4. Suppressed MAPK signaling pathway inhibited proliferation, apoptosis, migration, EMT and autophagy. SCH773984 was inhibitor of ERK1/2 A. Expression of ERK1/2 was detected by western blot. B. Cell viabilities were measured through CCK-8, P < 0.05. C. Scratch test was used for evaluating migration of cells in NC group and SCH772984 group, P < 0.05. D. RT-qPCR was applied for validating expressions of Bcl-2 and caspase-3, P < 0.05. E, F. Proteins in EMT and autophagy were measured through western blot.
in CC cells, showing that ATF6 downregulation could improve apoptosis in Hela cell line. 4-phenylbutyric acid (4-PBA) is a small molecular weight fatty acid, which could suppress ER stress (Zhu et al., 2014). Next, we used 4-PBA for suppressing ER stress in cells, which decreased ATF6 expression in CC cells. Meanwhile, we found lower cell viabilities and migration in cells with 4-PBA groups. Expression of Bcl-2 was suppressed and caspase-3 was up-regulated, signifying the promoted apoptosis in ER inhibited CC cells. Therefore, we decided that ATF6 might regulate progression of cervical cancer cells through ER stress. Physiological functions of autophagy contain four parts, cleaning injured cells to maintain stability of inner environment, regulating innate immunity and adaptive immunity, inducing cell death and extending life of cells (Liu and Levine, 2015). Autophagy could induce cell apoptosis in early stage of cancers (Huang et al., 2014). LC3-II is resultant protein after ubiquitination of LC3-I, which marked autophagy condition (Zhou et al., 2018).P62 was reported to be an autophagy adaptor in cancers (Moscat et al., 2016). In this study, we observed that after ATF6 was down-regulated, expressions of LC3-Ⅰ and p62 decreased and LC3-Ⅱ was promoted, suggesting the severity of autophagy increased with the inhibition of ATF6. Epithelial to mesenchymal transition (EMT) is a process that epithelial cells transformed into mesenchymal cells (Thiery and Lim, 2013). E-cadherin mainly existed in epithelial cell (Tiwari et al., 2012). Snail could bind E-cadherin to suppress expression of E-cadherin (Cade et al., 2010). Vimentin could promote EMT in cancer. Therefore, we measured E-cadherin and vimentin and found that inhibited ATF6 could increase expression of Ecadherin and reduce expressions of Snail and Vimentin, thus hindered EMT process. Mitogen-activated protein kinase (MAPK) signaling pathway
3.4. Effects on proliferation, apoptosis, migration, EMT and autophagy by suppressed MAPK signaling pathway In former figure, it was indicated that ATF6 regulated cell progressions via MAPK signaling pathway. Thus, functions of MAPK signaling pathway were measured. SCH772984 was a kind of inhibitor which could suppress ERK1/2 proteins in MAPK signaling pathway (Fig. 4A). After expression was inhibited in SCH772984 group, cell viabilities were shown, which revealed that SCH772984 could down regulate cell viabilities in tumor cell line (Fig. 4B). In scratch test, SCH772984 could decrease migration in cervical tumor cell line (Fig. 4C). As for apoptosis, Bcl-2 was repressed in SCH772984 group while caspase-3 was promoted (Fig. 4D, E). In EMT, E-cadherin expression was higher in suppressed ERK1/2 group but Vimentin expression was reduced as well as Sanil expression (Fig. 4F). Expressions of LC3-Ⅰ and p62 in autophagy were lower in SCH772984 groups with promoted LC3-Ⅱ compared to the negative group (Fig. 4G).
4. Discussion In previous study, ATF6 could conduct activation of GRP78, which could inhibit activation of caspase-3 and maintain stability of ER and inner environment to improve survival of cancer cells (Dong et al., 2004; Shen et al., 2002). Therefore, in this research, we found that ATF6 was expressed higher in CC cells and suppressed ATF6 could reduce cell viabilities and migration in Hela, indicating that inhibited ATF6 could reduce proliferation and ignite migration of CC cells. RTqPCR showed that pro-apoptotic protein Bcl-2 was down-regulated and apoptotic biomarker caspase-3 was promoted when ATF6 was silenced 5
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contains extracellular signal-regulated kinase (ERK1/2), Jun N-lerminal kinase (JNK), p38,MAPK and ERK5 (Sun et al., 2015). ERK1/2 is considered to have connections with proliferation, transformation and differentiation of cells and activation of ERK1/2 could suppress apoptosis in cells and P38 is related to apoptosis, inflammations, ER stress, etc (Sun et al., 2017b; Song et al., 2017). The findings in this research showed that p-ERK1/2 and p38 were higher in cancer cells and knockdown of ATF6 could down regulate p-ERK1/2 and p38 in cervical cancer cells. Furthermore, SCH772984 was used to suppress expression of ERK1/2, which inhibited cell viability and migration in cervical cancer cell line. Bcl-2 was decreased with SCH772984 added and caspase-3 was up-regulated. E-cadherin in EMT was higher in SCH772984 and Vimentin and Snail were lower. LC3-Ⅰ and p62 related to autophagy were lower in SCH772984 group while LC3-Ⅱ was up-regulated. Therefore, knockdown of ATF6 could help silence MAPK signaling and inactivated MAPK signaling pathway suppressed cell viability and promoted cell apoptosis and autophagy.
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5. Conclusion This study has evaluated the regulatory role of ATF6 in cervical cancer cells, which elucidated that ATF6 might the cell proliferation and migration, inhibited cell autophagy and apoptosis in CC through ER stress and MAPK signaling. Our observations might offer basic research experience for future animal researches and clinical treatments related to cervical cancer. Declaration of Competing Interest Nothing to be disclosed by authors. References Cade, T.J., et al., 2010. Progestogen treatment options for early endometrial cancer. Bjog 117 (7), 879–884. Chen, X., Shen, J., Prywes, R., 2002. The luminal domain of ATF6 senses endoplasmic reticulum (ER) stress and causes translocation of ATF6 from the ER to the Golgi. J. Biol. Chem. 277 (15), 13045–13052. Cheng, H.Y., et al., 2016. Synergism between RIZ1 gene therapy and paclitaxel in SiHa cervical cancer cells. Cancer Gene Ther. 23 (11), 392–395. Denny, L., et al., 2015. Cervical cancer. In: third edition. In: Gelband, H. (Ed.), Cancer: Disease Control Priorities 3 The International Bank for Reconstruction and Development / The World Bank (c) 2015 International Bank for Reconstruction and Development The World Bank: Washington (DC). Dong, D., et al., 2004. Spontaneous and controllable activation of suicide gene expression driven by the stress-inducible grp78 promoter resulting in eradication of sizable human tumors. Hum. Gene Ther. 15 (6), 553–561.
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