DcR3表达对宫颈癌侵袭和转移的影响及机制研究
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摘要
目的研究诱饵因子3(decoy receptor 3,DcR3)在宫颈癌细胞株中的表达,并在体内体外探讨其对Caski细胞侵袭转移的影响以及作用机制。方法 Western-blot用于检测DcR3蛋白在He La、Caski宫颈癌细胞株及1株永生化人宫颈上皮细胞H8中的表达水平。胰酶消化呈对数生长期的Caski后铺置6孔板中,采用沉默慢病毒感染Caski,分为sh-DcR3组和sh-NC组,Western-blot检测沉默Caski细胞中DcR3基因的表达后,体外采用boyden实验检测细胞侵袭能力;transwell小室实验检测细胞转移能力;体内实验经尾静脉注射构建宫颈癌裸鼠转移瘤模型,观察DcR3对Caski细胞在裸鼠内转移能力的影响;Western-blot法检测STAT3以及下游蛋白MMP-7的表达情况。结果 Western-blot检测发现DcR3在宫颈癌细胞株Hela(7. 81±0. 11)、Caski(10. 96±0. 14)中的表达显著高于永生化人宫颈上皮细胞H8(1. 00±0. 02)(P <0. 05);沉默Caski细胞中DcR3的表达后,与sh-NC组相比,DcR3蛋白表达量明显降低(P <0. 05),细胞的侵袭和转移能力显著减弱(P <0. 05);尾静脉注射sh-DcR3的裸鼠肺转移灶的个数显著少于sh-NC细胞。STAT3以及下游蛋白MMP-7的表达量显著降低(P <0. 05)。结论 DcR3基因可能是通过调控STAT3/MMP-7信号通路,促进宫颈癌细胞Caski侵袭转移。
Objective To investigate the expression of decoy factor 3(DcR3) in cervical cancer cell lines,and to investigate its effect on the invasion and metastasis of Caski cells in vitro and in vivo. Methods Westernblot was used to detect the expression levels of DcR3 protein in HeLa,Caski cervical cancer cell line and one immortalized human cervical epithelial cell H8. After trypsin digestion,the Caski in the logarithmic growth phase was placed in a 6-well plate,and the Caski was infected with silencing lentivirus,which was then divided into the sh-DcR3 group and sh-NC group. The expression of DcR3 gene in Silk cells was detected by Western-blot. In vitro,the boyden assay was used to detect the cell invasion ability;the transwell chamber was used to detect the cell transfer ability;the in vivo experiment was performed to construct the cervical cancer xenograft metastasis model by tail vein injection,and the effect of DcR3 on the transfer ability of Caski cells in nude mice was observed; Western-blot method was used to detect the expression of STAT3 and the downstream protein MMP-7. Results Western-blot assay showed that the expressions of DcR3 in cervical cancer cell lines Hela(7. 81 ± 0. 11) and Caski(10. 96 ± 0. 14) were significantly higher than that of immortalized human cervical epithelial cells H8(1. 00 ± 0. 02)(P < 0. 05). After silencing the expression of DcR3 in Caski cells,the expression of DcR3 protein was significantly decreased compared with sh-NC group(P < 0. 05),and the invasion and metastasis ability of cells was significantly decreased(P < 0. 05);sh-DcR3 was injected into tail vein for inhibiting the expression and the number of lung metastases in nude mice was significantly less than that in sh-NC cells. The expression levels of STAT3 and downstream protein MMP-7 were significantly decreased(P < 0. 05). Conclusion The DcR3 gene may promote the invasion and metastasis of cervical cancer cells by regulating STAT3/MMP-7 signaling pathway.
Objective To investigate the expression of decoy factor 3(DcR3) in cervical cancer cell lines,and to investigate its effect on the invasion and metastasis of Caski cells in vitro and in vivo. Methods Westernblot was used to detect the expression levels of DcR3 protein in HeLa,Caski cervical cancer cell line and one immortalized human cervical epithelial cell H8. After trypsin digestion,the Caski in the logarithmic growth phase was placed in a 6-well plate,and the Caski was infected with silencing lentivirus,which was then divided into the sh-DcR3 group and sh-NC group. The expression of DcR3 gene in Silk cells was detected by Western-blot. In vitro,the boyden assay was used to detect the cell invasion ability;the transwell chamber was used to detect the cell transfer ability;the in vivo experiment was performed to construct the cervical cancer xenograft metastasis model by tail vein injection,and the effect of DcR3 on the transfer ability of Caski cells in nude mice was observed; Western-blot method was used to detect the expression of STAT3 and the downstream protein MMP-7. Results Western-blot assay showed that the expressions of DcR3 in cervical cancer cell lines Hela(7. 81 ± 0. 11) and Caski(10. 96 ± 0. 14) were significantly higher than that of immortalized human cervical epithelial cells H8(1. 00 ± 0. 02)(P < 0. 05). After silencing the expression of DcR3 in Caski cells,the expression of DcR3 protein was significantly decreased compared with sh-NC group(P < 0. 05),and the invasion and metastasis ability of cells was significantly decreased(P < 0. 05);sh-DcR3 was injected into tail vein for inhibiting the expression and the number of lung metastases in nude mice was significantly less than that in sh-NC cells. The expression levels of STAT3 and downstream protein MMP-7 were significantly decreased(P < 0. 05). Conclusion The DcR3 gene may promote the invasion and metastasis of cervical cancer cells by regulating STAT3/MMP-7 signaling pathway.
引文
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[15]GE H,LIANG C,LI Z,et al. DcR3 induces proliferation,migration,invasion,and EMT in gastric cancer cells via the PI3K/AKT/GSK-3β/β-catenin signaling pathway[J]. Onco Targets Ther,2018,11:4177-4187.
[16]ZHANG H,CHENX,LI D,et al. DcR3 promotes hepatoma cell migration by downregulating E-cadherin expression[J]. Oncol Rep,2017,38(1):377-383.
[17]伊日贵,徐晓艳,李时荣.肿瘤侵袭转移机制研究进展[J].中华实用诊断与治疗杂志,2014,28(10):937-939.
[18]牛艳珍,陈显久,王军. STAT3通路与肿瘤关系的研究进展[J].医学研究杂志,2018,47(03):9-11,16.
[19]NAKAMURA H,TAGUCHI A,KAWANA K,et al. STAT3 activity regulates sensitivity to tumor necrosis factor-related apoptosisinducing ligand-induced apoptosis in cervical cancer cells[J]. Int J Oncol,2016,49(5):2155-2162.
[20]FURTEK S L,BACKOS D S,MATHESON C J. Strategies and Approaches of Targeting STAT3 for Cancer Treatment[J]. ACS ChemBiol,2016,11(2):308-318.
[21]KANG D Y,SP N,KIM D H,et al. Salidroside inhibits migration,invasion and angiogenesis of MDAMB 231 TNBC cells by regulating EGFR/Jak2/STAT3 signaling via MMP2[J]. Int J Oncol,2018,53(2):877-885.
[22]ZHANG G, MIYAKE M, LAWTON A, et al. Matrix metalloproteinase-10 promotes tumor progression through regulation of angiogenic and apoptotic pathways in cervical tumors[J]. BMC cancer,2014,14(1):300-310.
[23]ZHOU D,TIAN Y,SUN L,et al. Matrix Metalloproteinase-7 Is a Urinary Biomarker and Pathogenic Mediator of Kidney Fibrosis[J].J Am Soc Nephrol,2017,28(2):598-611.
[24]ZHU L,ZHENG X,DU Y,et al. Matrix metalloproteinase-7 may serve as a novel biomarker for cervical cancer[J]. Onco Targets Ther,2018,11(1):4207-4220.
[2]BOBDEY S,SATHWARA J,JAIN A. Burden of cervical cancer and role of screening in India[J]. Indian J Med Paediatr Oncol,2016,37(4):278-285.
[3]SUNEJA G,BROWN D,CHANG A,et al. American Brachytherapy Society:Brachytherapy treatment recommendationsfor locally advanced cervix cancer for low-income and middle-income countries[J].Brachytherapy,2017,16(1):85-94.
[4]MEZEI A K,ARMSTRONG H L,PEDERSEN H N,et al. Costeffectiveness of cervical cancer screening methods in low-and middle-income countries:A systematic review[J]. Int J Cancer,2017,141(3):437-446.
[5]CHAFFER C L. A perspective on cancer cell metastasis[J].Science,2011,331(6024):1559-1564.
[6]XIAO J B,LI X L,LIU L,et al. The association of semaphorin 5A with lymph node metastasis and adverse prognosis in cervical cancer[J].Cancer Cell Int,2018,18(2):80-87.
[7]HAGAN M,ZHOU M,ASHRAF M,et al. Long noncoding RNAs and their roles in skeletal muscle fate determination[J]. Null,2017,1(1):14-21.
[8]ZHAO T,XU Y,REN S,et al. The siRNA silencing of DcR3 expression induces Fas ligand-mediated apoptosis in Hep G2 cells[J]. Exp Ther Med,2018,15(5):4370-4378.
[9]梁冬雨,侯彦强,娄晓丽.沉默诱骗受体3的表达对肝癌细胞生物学特性的影响[J].世界华人消化杂志,2017,25(03):234-240.
[10]HSIEH S L. Decoy receptor 3:an endogenous immunomodulator in cancer growth and inflammatory reactions[J]. J Biomed Sci,2017,24(1):39.
[11]王凤杰,王科坤,陈显兵,等.宫颈癌组织中DCR3表达与外周血T细胞亚群的相关性研究[J].免疫学杂志,2015,31(03):246-249.
[12]PITTI R M,MARSTERS S A,LAWRENCE D A,et al. Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer[J]. Nature,1998,396(6712):699-703.
[13]LIU W,RAMAGOPAL U,CHENG H,et al. Crystal Structure of the Complex of Human Fas L and Its Decoy Receptor DcR3[J].Structure,2016,24(11):2016-2023.
[14]SIAKAVELLAS S I. Tumor Necrosis Factor-like Cytokine TL1A and Its Receptors DR3 and DcR3:Important New Factors in Mucosal Homeostasis and Inflammation[J]. Inflamm Bowel Dis,2015,21(10):2441-2452.
[15]GE H,LIANG C,LI Z,et al. DcR3 induces proliferation,migration,invasion,and EMT in gastric cancer cells via the PI3K/AKT/GSK-3β/β-catenin signaling pathway[J]. Onco Targets Ther,2018,11:4177-4187.
[16]ZHANG H,CHENX,LI D,et al. DcR3 promotes hepatoma cell migration by downregulating E-cadherin expression[J]. Oncol Rep,2017,38(1):377-383.
[17]伊日贵,徐晓艳,李时荣.肿瘤侵袭转移机制研究进展[J].中华实用诊断与治疗杂志,2014,28(10):937-939.
[18]牛艳珍,陈显久,王军. STAT3通路与肿瘤关系的研究进展[J].医学研究杂志,2018,47(03):9-11,16.
[19]NAKAMURA H,TAGUCHI A,KAWANA K,et al. STAT3 activity regulates sensitivity to tumor necrosis factor-related apoptosisinducing ligand-induced apoptosis in cervical cancer cells[J]. Int J Oncol,2016,49(5):2155-2162.
[20]FURTEK S L,BACKOS D S,MATHESON C J. Strategies and Approaches of Targeting STAT3 for Cancer Treatment[J]. ACS ChemBiol,2016,11(2):308-318.
[21]KANG D Y,SP N,KIM D H,et al. Salidroside inhibits migration,invasion and angiogenesis of MDAMB 231 TNBC cells by regulating EGFR/Jak2/STAT3 signaling via MMP2[J]. Int J Oncol,2018,53(2):877-885.
[22]ZHANG G, MIYAKE M, LAWTON A, et al. Matrix metalloproteinase-10 promotes tumor progression through regulation of angiogenic and apoptotic pathways in cervical tumors[J]. BMC cancer,2014,14(1):300-310.
[23]ZHOU D,TIAN Y,SUN L,et al. Matrix Metalloproteinase-7 Is a Urinary Biomarker and Pathogenic Mediator of Kidney Fibrosis[J].J Am Soc Nephrol,2017,28(2):598-611.
[24]ZHU L,ZHENG X,DU Y,et al. Matrix metalloproteinase-7 may serve as a novel biomarker for cervical cancer[J]. Onco Targets Ther,2018,11(1):4207-4220.