KRAS基因突变与分化型甲状腺癌~(131)I放疗耐受性及预后的关系
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摘要
目的:探究KRAS基因突变与分化型甲状腺癌(differentiated thyroid carcinoma,DTC)~(131)I放疗疗效和预后的相关性,并阐明其可能的机制。方法:收集经~(131)I放射治疗DTC临床组织样本,聚合酶链反应-单链构象分析法(single strand conformationpolymorphism analysis of polymerase chain reaction products,PCR-SSCP)检测KRAS的遗传突变;采用qPCR和Wb检测p21蛋白的表达水平;亚致死剂量的~(131)I放射治疗DTC细胞系,采用CCK-8、流式细胞术(FCM)、Transwell实验检测细胞活力的变化,并通过动物模型验证。结果:~(131)I放射治疗耐受DTC患者的KRAS基因突变增加(P<0.01),KRAS基因突变导致p21蛋白表达下调(P<0.05),且与DTC临床分期及预后较差相关(P<0.05,P<0.01)。体内外实验证明,亚致死剂量的~(131)I放射治疗导致DTC细胞KRAS基因的突变率增加、p21蛋白的表达水平降低,导致DTC细胞产生~(131)I放射耐受,而超表达KRAS基因显著提高p21的表达,抑制肿瘤增长及转移。结论:KRAS基因突变与DTC临床分期及~(131)I放射耐受相关,亚致死剂量~(131)I放射治疗DTC促进KRAS基因突变产生放射耐受,而超表达KRAS基因能够提高DTC对~(131)I放射治疗的敏感性。
Objective: To investigate the correlation between KRAS gene mutation and differentiated thyroid carcinoma(DTC) treatment effect and prognosis, and to explore the mechanism. Methods: Clinical tissue samples from DTC patients undergoing ~(131) I Radiotherapy were collected. Then single strand conformation polymorphism analysis of polymerase chain reaction products(PCRC-SSCP)was used to detect KRAS mutation rate in thyroid cancer patients of different TNM stages; p21 protein expression level was detected by real-time quantitative polymerase chain reaction(qPCR) and western blotting. DTC cells were treated by sub-lethal dose of ~(131) I Radiotherapy, and then CCK-8 assay, transwell assay and flow cytometry(FCM) were used to evaluate the changes of cells viability. Animal models were then constructed for verification. Results: The results showed that KRAS gene mutants were increased in ~(131) I-resistant DTC patients; KRAS gene mutation suppressed p21 protein expression and was associated with clinical stage and poor prognosis. In vivo and in vitro experiments proved that sub-lethal dose of ~(131) I increased KRAS gene mutation rate, suppressed p21 expression level, and caused ~(131) I radiotherapy resistance. Reversely, over-expression of KRAS gene could significantly increase p21 expression, and inhibit tumor proliferation and metastasis. Conclusion: KRAS gene mutations were associated with DTC TNM stages and ~(131) I resistance in DTC patients. Sub-lethal dose of ~(131) I treatment could improve ~(131) I resistance in DTC cells line, inversely, over-expressed KRAS gene could increase the sensitivity to ~(131) I radiotherapy in DTC patients.
Objective: To investigate the correlation between KRAS gene mutation and differentiated thyroid carcinoma(DTC) treatment effect and prognosis, and to explore the mechanism. Methods: Clinical tissue samples from DTC patients undergoing ~(131) I Radiotherapy were collected. Then single strand conformation polymorphism analysis of polymerase chain reaction products(PCRC-SSCP)was used to detect KRAS mutation rate in thyroid cancer patients of different TNM stages; p21 protein expression level was detected by real-time quantitative polymerase chain reaction(qPCR) and western blotting. DTC cells were treated by sub-lethal dose of ~(131) I Radiotherapy, and then CCK-8 assay, transwell assay and flow cytometry(FCM) were used to evaluate the changes of cells viability. Animal models were then constructed for verification. Results: The results showed that KRAS gene mutants were increased in ~(131) I-resistant DTC patients; KRAS gene mutation suppressed p21 protein expression and was associated with clinical stage and poor prognosis. In vivo and in vitro experiments proved that sub-lethal dose of ~(131) I increased KRAS gene mutation rate, suppressed p21 expression level, and caused ~(131) I radiotherapy resistance. Reversely, over-expression of KRAS gene could significantly increase p21 expression, and inhibit tumor proliferation and metastasis. Conclusion: KRAS gene mutations were associated with DTC TNM stages and ~(131) I resistance in DTC patients. Sub-lethal dose of ~(131) I treatment could improve ~(131) I resistance in DTC cells line, inversely, over-expressed KRAS gene could increase the sensitivity to ~(131) I radiotherapy in DTC patients.
引文
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[22]MISALE S,YAEGER R,HOBOR S,et al.Emergence of KRASmutations and acquired resistance to anti-EGFR therapy in colorectal cancer[J].Nature,2012,486(7404):532-536.DOI:10.1038/nature11156.
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[24]LEE W Y,CHEN P C,WU W S,et al.Panobinostat sensitizes KRAS-mutant non-small-cell lung cancer to gefitinib by targeting TAZ[J].Int J Cancer,2017,141(9):1921-1931.DOI:10.1002/ijc.30888.
[25]NAKATANI K,YAMAOKA T,OHBA M,et al.KRAS and EGFRamplifications mediate resistance to rociletinib and osimertinib in acquired afatinib-resistant NSCLC harboring exon 19 deletion/T790M in EGFR[J].Mol Cancer Ther,2019,18(1):112-126.DOI:10.1158/1535-7163.Mct-18-0591.
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[27]LEVY R,GRAFI-COHEN M,KRAIEM Z,et al.Galectin-3 promotes chronic activation of K-Ras and differentiation block in malignant thyroid carcinomas[J].Mol Cancer Ther,2010,9(8):2208-2219.DOI:10.1158/1535-7163.Mct-10-0262.
[28]WAGNER P L,PERNER S,RICKMAN D S,et al.In situ evidence of KRAS amplification and association with increased p21 levels in non-small cell lung carcinoma[J].Am J Clin Pathol,2009,132(4):500-505.DOI:10.1309/ajcpf10zunsolifg.
[29]BANYS-PALUCHOWSKI M,FEHM T,JANNI W,et al.Elevated serum RAS p21 is an independent prognostic factor in metastatic breast cancer[J/OL].BMC Cancer,2018,18(1):541[2018-09-13].https://bmccancer.biomedcentral.com/articles/10.1186/s12885-018-4282-0.DOI:10.1186/s12885-018-4282-0.
[30]EL-DEIRY W S.p21(WAF1)mediates cell-cycle inhibition,relevant to cancer suppression and therapy[J].Cancer Res,2016,76(18):5189-5191.DOI:10.1158/0008-5472.Can-16-2055.
[31]ZHANG X,LIU J,ZANG D,et al.Upregulation of miR-572 transcriptionally suppresses SOCS1 and p21 and contributes to human ovarian cancer progression[J].Oncotarget,2015,6(17):15180-15193.DOI:10.18632/oncotarget.3737.
[32]VALESKY E M,HRGOVIC I,DOLL M,et al.Dimethylfumarate effectively inhibits lymphangiogenesis via p21 induction and G1cell cycle arrest[J].Exp Dermatol,2016,25(3):200-205.DOI:10.1111/exd.12907.
[2]MIZOKAMI D,KOSUDA S,SHIOTANI A,et al.Impact of131ISPECT/CT on the management of differentiated thyroid carcinoma outpatients with radioablation[J].Nihon Jibiinkoka Gakkai Kaiho,2014,117(5):673-680.DOI:10.3950/jibiinkoka.117.673.
[3]CHMIELIK E,RUSINEK D,OCZKO-WOJCIECHOWSKA M,et al.Heterogeneity of thyroid cancer[J].Pathobiology,2018,85(1/2):117-129.DOI:10.1159/000486422.
[4]TSUCHIDA N,RYDER T,OHTSUBO E.Nucleotide sequence of the oncogene encoding the p21 transforming protein of Kirsten murine sarcoma virus[J].Science,1982,217(4563):937-939.
[5]SMEBY J,SVEEN A,MEROK M A,et al.CMS-dependent prognostic impact of KRAS and BRAFV600E mutations in primary colorectal cancer[J].Ann Oncol,2018,29(5):1227-1234.DOI:10.1093/annonc/mdy085.
[6]TELECHEA-FERNANDEZ M,RODRIGUEZ-FERNANDEZ L,GARCIA C,et al.New localization and function of calpain-2 in nucleoli of colorectal cancer cells in ribosomal biogenesis:effect of KRAS status[J].Oncotarget,2018,9(10):9100-9113.DOI:10.18632/oncotarget.23888.
[7]BISHEHSARI F,ZHANG L,BARLASS U,et al.KRAS mutation and epithelial-macrophage interplay in pancreatic neoplastic transformation[J].Int J Cancer,2018,143(8):1994-2007.DOI:10.1002/ijc.31592.
[8]刘蕾,魏素菊.KRAS突变的非小细胞肺癌的研究进展[J].中国肺癌杂志,2018,21(5):419-424.DOI:10.3779/j.issn.1009-3419.2018.05.11.
[9]EGELI U,TEZCAN G,CECENER G,et al.miR-216b targets FG-FR1 and confers sensitivity to radiotherapy in pancreatic ductal adenocarcinoma patients without EGFR or KRAS mutation[J].Pancreas,2016,45(9):1294-1302.DOI:10.1097/mpa.0000000000000640.
[10]WANG M,HAN J,MARCAR L,et al.Radiation resistance in KRAS-mutated lung cancer is enabled by stem-like properties mediated by an osteopontin-EGFR pathway[J].Cancer Res,2017,77(8):2018-2028.DOI:10.1158/0008-5472.can-16-0808.
[11]DUMAN B B,KARA O I,UGUZ A,et al.Evaluation of PTEN,PI3K,MTOR,and KRAS expression and their clinical and prognostic relevance to differentiated thyroid carcinoma[J].Contemp Oncol(Pozn),2014,18(4):234-240.DOI:10.5114/wo.2014.43803.
[12]VOLANTE M,RAPA I,GANDHI M,et al.RAS mutations are the predominant molecular alteration in poorly differentiated thyroid carcinomas and bear prognostic impact[J].J Clin Endocrinol Metab,2009,94(12):4735-4741.DOI:10.1210/jc.2009-1233.
[13]MENG Z,LOU S,TAN J,et al.Nuclear factor-kappa B inhibition can enhance apoptosis of differentiated thyroid cancer cells induced by131I[J/OL].PLoS One,2012,7(3):e33597[2018-09-11].https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033597.DOI:10.1371/journal.pone.0033597.
[14]PEDROZA-TORRES A,CAMPOS-PARRA A D,MILLAN-CATA-LAN O,et al.MicroRNA-125 modulates radioresistance through targeting p21 in cervical cancer[J].Oncol Rep,2018,39(3):1532-1540.DOI:10.3892/or.2018.6219.
[15]STOJANOVIC-RUNDIC S,JANKOVIC R,MICEV M,et al.p21does,but p53 does not predict pathological response to preoperative chemoradiotherapy in locally advanced rectal cancer[J].J BUON,2017,22(6):1463-1470.
[16]RANDLE R W,BUSHMAN N M,ORNE J,et al.Papillary thyroid cancer:the good and bad of the"good cancer"[J].Thyroid,2017,27(7):902-907.DOI:10.1089/thy.2016.0632.
[17]BIKA,A,VAN NOSTRAND D,JENSEN K,et al.Metformin attenuates131I-induced decrease in peripheral blood cells in patients with differentiated thyroid cancer[J].Thyroid,2016,26(2):280-286.DOI:10.1089/thy.2015.0413.
[18]LAI H,WANG Y,DUAN F,et al.Krukovine suppresses KRAS-mutated lung cancer cell growth and proliferation by inhibiting the RAF-ERK pathway and inactivating AKT pathway[J/OL].Front Pharmacol,2018,9:958[2018-09-11].http://inpst.net/feed-items/krukovine-suppresses-kras-mutated-lung-cancer-cell-growth-andproliferation-by-inhibiting-the-raf-erk-pathway-and-inactivatingakt-pathway/.DOI:10.3389/fphar.2018.00958.
[19]SIMANSHU D K,NISSLEY D V,MCCORMICK F.RAS proteins and their regulators in human disease[J].Cell,2017,170(1):17-33.DOI:10.1016/j.cell.2017.06.009.
[20]WOJAS-KRAWCZYK K,KALINKA-WARZOCHA E,RESZKAK,et al.Analysis of KRAS,NRAS,BRAF,and PIK3CA mutations could predict metastases in colorectal cancer:A preliminary study[J].Adv Clin Exp Med,2018,epub ahead of print[2018-09-11].www.advances.umed.wroc.pl/ahead-of-print/76162.pdf.DOI:10.17219/acem/76162.
[21]XIANG C,ZHANG M L,ZHAO Q Z,et al.LncRNA-SLC6A9-5:2:a potent sensitizer in131I-resistant papillary thyroid carcinoma with PARP-1 induction[J].Oncotarget,2017,8(14):22954-22967.DOI:10.18632/oncotarget.14578.
[22]MISALE S,YAEGER R,HOBOR S,et al.Emergence of KRASmutations and acquired resistance to anti-EGFR therapy in colorectal cancer[J].Nature,2012,486(7404):532-536.DOI:10.1038/nature11156.
[23]HSU H C,THIAM T K,LU Y J,et al.Mutations of KRAS/NRAS/BRAF predict cetuximab resistance in metastatic colorectal cancer patients[J].Oncotarget,2016,7(16):22257-22270.DOI:10.18632/oncotarget.8076.
[24]LEE W Y,CHEN P C,WU W S,et al.Panobinostat sensitizes KRAS-mutant non-small-cell lung cancer to gefitinib by targeting TAZ[J].Int J Cancer,2017,141(9):1921-1931.DOI:10.1002/ijc.30888.
[25]NAKATANI K,YAMAOKA T,OHBA M,et al.KRAS and EGFRamplifications mediate resistance to rociletinib and osimertinib in acquired afatinib-resistant NSCLC harboring exon 19 deletion/T790M in EGFR[J].Mol Cancer Ther,2019,18(1):112-126.DOI:10.1158/1535-7163.Mct-18-0591.
[26]VOLANTE M,RAPA I,GANDHI M,et al.RAS mutations are the predominant molecular alteration in poorly differentiated thyroid carcinomas and bear prognostic impact[J].J Clin Endocrinol Metab,2009,94(12):4735-4741.DOI:10.1210/jc.2009-1233.
[27]LEVY R,GRAFI-COHEN M,KRAIEM Z,et al.Galectin-3 promotes chronic activation of K-Ras and differentiation block in malignant thyroid carcinomas[J].Mol Cancer Ther,2010,9(8):2208-2219.DOI:10.1158/1535-7163.Mct-10-0262.
[28]WAGNER P L,PERNER S,RICKMAN D S,et al.In situ evidence of KRAS amplification and association with increased p21 levels in non-small cell lung carcinoma[J].Am J Clin Pathol,2009,132(4):500-505.DOI:10.1309/ajcpf10zunsolifg.
[29]BANYS-PALUCHOWSKI M,FEHM T,JANNI W,et al.Elevated serum RAS p21 is an independent prognostic factor in metastatic breast cancer[J/OL].BMC Cancer,2018,18(1):541[2018-09-13].https://bmccancer.biomedcentral.com/articles/10.1186/s12885-018-4282-0.DOI:10.1186/s12885-018-4282-0.
[30]EL-DEIRY W S.p21(WAF1)mediates cell-cycle inhibition,relevant to cancer suppression and therapy[J].Cancer Res,2016,76(18):5189-5191.DOI:10.1158/0008-5472.Can-16-2055.
[31]ZHANG X,LIU J,ZANG D,et al.Upregulation of miR-572 transcriptionally suppresses SOCS1 and p21 and contributes to human ovarian cancer progression[J].Oncotarget,2015,6(17):15180-15193.DOI:10.18632/oncotarget.3737.
[32]VALESKY E M,HRGOVIC I,DOLL M,et al.Dimethylfumarate effectively inhibits lymphangiogenesis via p21 induction and G1cell cycle arrest[J].Exp Dermatol,2016,25(3):200-205.DOI:10.1111/exd.12907.