Tristetraprolin通过NF-?B通路抑制肺腺癌细胞自噬
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摘要
目的研究RNA结合蛋白tristetraprolin在肺腺癌中的表达及抑制自噬作用的分子机制。方法瞬时转染tristetraprolin过表达质粒,分别在转染tristetraprolin 24、48及72 h后采用实时荧光定量PCR(RT-qPCR)和Western blot检测肺腺癌细胞中tristetraprolin表达及自噬相关分子Beclin1、LC3II/LCI及p62的表达变化。瞬时转染tristetraprolin过表达质粒及加入TNF-α,将肺腺癌细胞分为空载组、tristetraprolin组、空载+TNF-α组及tristetraprolin+TNF-α组,应用免疫荧光和Western blot检测NF-?B p65、c-rel、p50分子在细胞核中表达情况。共转染tristetraprolin过表达质粒及IκBα-mut质粒,将肺腺癌细胞分为tristetraprolin空载组、IκBα-mut空载组、tristetraprolin组、IκBα-mut组及tristetraprolin+IκBα-mut组,采用RT-qPCR和Western blot检测tristetraprolin表达及自噬相关基因表达变化。结果 Tristetraprolin在肺腺癌细胞中RNA及蛋白水平表达低(P<0.001)。过表达tristetraprolin后,自噬相关基因Beclin1、LC3-Ⅱ/LC3-Ⅰ在RNA及蛋白水平表达均较空载组降低(P<0.001)。同时,过表达tristetraprolin后,细胞核内的p65及c-rel蛋白表达较空载组及空载+TNF-α组减少(P<0.05),但p50表达无明显变化(P>0.05)。过表达tristetraprolin后,p65及c-rel核移位较空载组减少。共转染IκBα突变质粒及tristetraprolin过表达质粒后,NF-κB信号通路被阻断,自噬相关基因Beclin1、LC3-Ⅱ/LC3-Ⅰ在RNA及蛋白水平表达较tristetraprolin过表达组升高(P<0.05),NF-?B信号通路被阻断后tristetraprolin对自噬的抑制作用减弱。结论 Tristetraprolin在肺腺癌细胞中低表达,过表达tristetraprolin可能通过抑制NF-?B p65及c-rel核移位而抑制肺腺癌细胞自噬。
Objective To explore the expression of the RNA-binding protein tristetraprolin in lung adenocarcinoma cells and its molecular mechanism for inhibiting autophagy. Methods Quantitative real-time PCR and Western blotting were performed to detect the expression of autophagy-related genes(including Beclin1, LC3-II/LC3-I and SQSTM1/p62) in cultured lung adenocarcinoma cells at 24, 48 and 72 h after transient transfection with a tristetraprolin-overexpressing plasmid and the empty plasmid. The effects of transfection with the tristetraprolin-overexpressing plasmid and empty plasmids in the presence or absence of tumor necrosis factor-α(TNF-α) on the expressions of nuclear factor-κB(NF-κB) p65, c-rel, and p50 were examined in lung adenocarcinoma cells using immunofluorescence assay and Western blotting. The cells were also transfected with the IκBα-mut plasmid and the tristetraprolin-overexpressing plasmid, either alone or in combination, and the changes in the expressions of tristetraprolin and autophagy-related genes were detected using RT-qPCR and Western blotting. Results The expressions of tristetraprolin were significantly reduced at both the mRNA and protein levels in lung adenocarcinoma cells(P<0.001). Overexpression of tristetraprolin in the cells significantly lowered the expressions of autophagy-related genes Beclin1 and the ratio of LC3-II/LC3-I at the mRNA and protein levels(P<0.001), obviously lowered the expressions of NF-κB p65 and c-rel, and almost totally blocked the nuclear translocation of NF-κB p65 and c-rel(P<0.05); the expression of p50,however, did not undergo significant changes in response to tristetraprolin overexpression(P>0.05). The inhibitory effect of tristetraprolin overexpression on autophagy was abrogated by transfection of the cells with IκBα-mut plasmid, which blocked the NF-κB signaling pathway. Co-transfection of the cells with IκBα-mut also attenuated the inhibitory effect of tristetraprolin overexpression on Beclin1 and the LC3-II/LC3-I ratio at both the mRNA and protein levels(P<0.05). Conclusion The expression of tristetraprolin is low in lung adenocarcinoma cells. Tristetraprolin overexpression causes inhibition of autophagy in lung adenocarcinoma cells possibly by blocking NF-κB p65 and c-rel nuclear translocation.
Objective To explore the expression of the RNA-binding protein tristetraprolin in lung adenocarcinoma cells and its molecular mechanism for inhibiting autophagy. Methods Quantitative real-time PCR and Western blotting were performed to detect the expression of autophagy-related genes(including Beclin1, LC3-II/LC3-I and SQSTM1/p62) in cultured lung adenocarcinoma cells at 24, 48 and 72 h after transient transfection with a tristetraprolin-overexpressing plasmid and the empty plasmid. The effects of transfection with the tristetraprolin-overexpressing plasmid and empty plasmids in the presence or absence of tumor necrosis factor-α(TNF-α) on the expressions of nuclear factor-κB(NF-κB) p65, c-rel, and p50 were examined in lung adenocarcinoma cells using immunofluorescence assay and Western blotting. The cells were also transfected with the IκBα-mut plasmid and the tristetraprolin-overexpressing plasmid, either alone or in combination, and the changes in the expressions of tristetraprolin and autophagy-related genes were detected using RT-qPCR and Western blotting. Results The expressions of tristetraprolin were significantly reduced at both the mRNA and protein levels in lung adenocarcinoma cells(P<0.001). Overexpression of tristetraprolin in the cells significantly lowered the expressions of autophagy-related genes Beclin1 and the ratio of LC3-II/LC3-I at the mRNA and protein levels(P<0.001), obviously lowered the expressions of NF-κB p65 and c-rel, and almost totally blocked the nuclear translocation of NF-κB p65 and c-rel(P<0.05); the expression of p50,however, did not undergo significant changes in response to tristetraprolin overexpression(P>0.05). The inhibitory effect of tristetraprolin overexpression on autophagy was abrogated by transfection of the cells with IκBα-mut plasmid, which blocked the NF-κB signaling pathway. Co-transfection of the cells with IκBα-mut also attenuated the inhibitory effect of tristetraprolin overexpression on Beclin1 and the LC3-II/LC3-I ratio at both the mRNA and protein levels(P<0.05). Conclusion The expression of tristetraprolin is low in lung adenocarcinoma cells. Tristetraprolin overexpression causes inhibition of autophagy in lung adenocarcinoma cells possibly by blocking NF-κB p65 and c-rel nuclear translocation.
引文
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[18]Hitti E, Bakheet T, Al-Souhibani N, et al. Systematic analysis of AURich element expression in cancer reveals common functional clusters regulated by key RNA-Binding proteins[J]. Cancer Res,2016, 76(14):4068-80.
[19]Mohammad F, Antonio LA, John LC. CREB targets define the gene expression signature of malignancies having reduced levels of the tumor suppressor tristetraprolin[J]. PLoS One, 2014, 9(12):e115517-26.
[20]Rebecca VW, Amaravadi RK. Emerging strategies to effectively target autophagy in cancer[J]. Oncogene, 2016, 35(1):1-11.
[21]Liu GB, Pei F, Yang FQ, et al. Role of autophagy and apoptosis in non-small-cell lung cancer[J]. Int J Mol Sci, 2017, 18(2):E367-75.
[22]Karsli UG, Jy G, Price S, et al. Autophagy is required for glucose homeostasis and lung tumor maintenance[J]. Cancer Discov, 2014, 4(8):914-27.
[23]Lee SJ, Kim HP, Jin Y, et al. Beclin1 deficiency is associat-ed with increased hypoxia-induced angiogenesis[J]. Autophagy, 2011, 7(8):829-39.
[24]Wang XF, Du ZY, Li LY, et al. Beclin1 and p62 expression in nonsmall cell lung cancer:relation with malignant behaviors and clinical outcome[J]. Int J Clin Exp Pathol, 2015, 8(9):10644-52.
[25]Wu SF, Su J, Qian H, et al. SLC27A4 regulate ATG4B activity and control reactions to chemotherapeutics-induced autophagy in human lung cancer cells[J]. Tumour Biol, 2016, 37(5):6943-52.
[26]Xu L, Ning H, Gu L, et al. Tristetraprolin induces cell cycle arrest in breast tumor cells through targeting AP-1/c-Jun and NF-kappa B pathway[J]. Oncotarget, 2015, 6(39):41679-91.
[27]Salminen A, Hyttinen JM, Kauppinen A, et al. Context-dependent regulation of autophagy by IKK-NF-κB signaling:impact on the aging process[J]. Int J Cell Biol, 2012, 20(18):849541-53.
[28]Pankiv S, Clausen TH, Lamark T, et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy[J]. J Biol Chem, 2007, 282(33):24131-45.
[29]Seibenhener ML, Geetha T, Wooten MW. Sequestosome 1/p62-more than just a scaffold[J]. FEBS Lett, 2007, 581(2):175-9.
[2] Guo J, Wang H, Jiang SY, et al. The cross-talk between tristetraprolin and cytokines in cancer[J]. Anticanc Agents Med Chem, 2017, 17(11):1477-86.
[3] Gu L, Ning H, Qian XE, et al. Suppression of IL-12 production by tristetraprolin through blocking NF-kappa B nuclear translocation[J]. J Immunol, 2013, 191(7):3922-30.
[4] Schichl YM, Resch U, Martin R. Tristetraprolin impairs NF-kappaB/p65 nuclear translocation[J]. J Biol Chem, 2009, 284(43):29571-81.
[5] Liang J, Lei TH, Song YT, et al. RNA-destabilizing factor tristetraprolin negatively regulates NF-kappa B signaling[J]. Biol Chem, 2009, 284(43):29383-90.
[6] Park JM, Lee TH, Kang TH. Roles of tristetraprolin in tumorigenesis[J]. Int J Mol Sci, 2018, 19(11):3384-95.
[7] Al-Haj A, L, Blackshear PJ, et al. Regulation of p21/CIP1/WAF-1mediated cell-cyclearrest by RNase L and tristetraprolin, and involvement of AU-rich elements[J]. Nucleic Acids Res, 2012, 40(16):7739-52.
[8] Marderosian M, Sharma A, Funk AP, et al. Ttristetraprolin regulates cyclin D1 and c-Myc mRNA stability in response to rapamycin in an Akt-dependent manner via p38 MAPK signaling[J]. Oncogene,2006, 25(47):6277-90.
[9] Lee HH, Lee SR, Leem SH. Tristetraprolin regulates prostate cancer cell growth throughsuppression of E2F1[J]. Microbiol Biotechnol,2014, 24(2):287-94.
[10]Lee HH, Vo MT, Kim HJ, et al. Stability of the LATS2 tumor suppressor gene is regulated by tristetraprolin[J]. J Biol Chem,2010, 285(23):17329-37.
[11]Wang H, Ding NN, Guo J, et al. Dysregulation of TTP and HuR plays an important role in cancers[J]. Tumour Biol, 2016, 37(11):14451-61.
[12]Hai DD, Koch A, Allister A, et al. Treatment with MAPKAP2(Mk2)inhibitor and DNA methylation inhibitor, 5-aza dC, synergistically triggers apoptosis in hepatocellular carcinoma(HCC)via tristetraprolin(TTP)[J]. Cell Signal, 2016, 28(12):1872-80.
[13]Bourcier C, Griseri P, Grépin R, et al. Constitutive ERK activity induces downregulation of tristetraprolin, a major protein controlling interleukin8/CXCL8 mRNA stability in melanoma cells[J]. Am J Physiol Cell Physiol, 2011, 301(3):C609-18.
[14]Suswam EA, Shacka JJ, Walker K, et al. Mutant tristetraprolin:a potent inhibitor of malignant glioma cell growth[J]. Neuro Oncol,2013, 113(2):195-205.
[15]Tilija PN, Park PH. Adiponectin inhibits inflammatory cytokines production by Beclin-1 phosphorylation and Bcl-2 mRNA destabilization:role for autophagy induction[J]. Br J Pharmaco,2018, 175(7):1066-84.
[16]María VG, Albana G, Johanna M. Expression of the mRNA stability regulator Tristetraprolin is required for lactation maintenance in the mouse mammary gland[J]. Oncotarget, 2018, 9(9):8278-89.
[17]Dong F, Li C, Xu L. The RNA binding protein tristetraprolin downregulates autophagy in lung adenocarcinoma cells[J]. Exp Cell Res,2018, 367(1):89-96.
[18]Hitti E, Bakheet T, Al-Souhibani N, et al. Systematic analysis of AURich element expression in cancer reveals common functional clusters regulated by key RNA-Binding proteins[J]. Cancer Res,2016, 76(14):4068-80.
[19]Mohammad F, Antonio LA, John LC. CREB targets define the gene expression signature of malignancies having reduced levels of the tumor suppressor tristetraprolin[J]. PLoS One, 2014, 9(12):e115517-26.
[20]Rebecca VW, Amaravadi RK. Emerging strategies to effectively target autophagy in cancer[J]. Oncogene, 2016, 35(1):1-11.
[21]Liu GB, Pei F, Yang FQ, et al. Role of autophagy and apoptosis in non-small-cell lung cancer[J]. Int J Mol Sci, 2017, 18(2):E367-75.
[22]Karsli UG, Jy G, Price S, et al. Autophagy is required for glucose homeostasis and lung tumor maintenance[J]. Cancer Discov, 2014, 4(8):914-27.
[23]Lee SJ, Kim HP, Jin Y, et al. Beclin1 deficiency is associat-ed with increased hypoxia-induced angiogenesis[J]. Autophagy, 2011, 7(8):829-39.
[24]Wang XF, Du ZY, Li LY, et al. Beclin1 and p62 expression in nonsmall cell lung cancer:relation with malignant behaviors and clinical outcome[J]. Int J Clin Exp Pathol, 2015, 8(9):10644-52.
[25]Wu SF, Su J, Qian H, et al. SLC27A4 regulate ATG4B activity and control reactions to chemotherapeutics-induced autophagy in human lung cancer cells[J]. Tumour Biol, 2016, 37(5):6943-52.
[26]Xu L, Ning H, Gu L, et al. Tristetraprolin induces cell cycle arrest in breast tumor cells through targeting AP-1/c-Jun and NF-kappa B pathway[J]. Oncotarget, 2015, 6(39):41679-91.
[27]Salminen A, Hyttinen JM, Kauppinen A, et al. Context-dependent regulation of autophagy by IKK-NF-κB signaling:impact on the aging process[J]. Int J Cell Biol, 2012, 20(18):849541-53.
[28]Pankiv S, Clausen TH, Lamark T, et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy[J]. J Biol Chem, 2007, 282(33):24131-45.
[29]Seibenhener ML, Geetha T, Wooten MW. Sequestosome 1/p62-more than just a scaffold[J]. FEBS Lett, 2007, 581(2):175-9.