基于LC-MS/MS筛选宫颈病变和宫颈癌发生发展过程中的新型蛋白标志物
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摘要
目的筛选可用来指示宫颈癌从癌前病变到癌变的血浆蛋白标志物,并分析其潜在机制与功能。方法收集健康人(Control组),低度鳞状上皮内病变(LSIL)、高度鳞状上皮内病变(HSIL)、宫颈癌(CC)和治疗后患者血浆样本富集低丰度蛋白。LC-MS/MS检测样本的全多肽类型与序列,检测结果用Proteome Discoverer 2.2进行数据库检索,筛选差异蛋白类型与种类(要求Coverage≥20%,且Unique Peptides≥2蛋白质),LSIL、HSIL、CC组分别与Control组进行T检验比较,P≤0.05视为表达有显著差异的蛋白。通过观察差异蛋白在各组中的色谱图筛选出能提示宫颈病变及宫颈癌的潜在标志物及预后效能评估标记物,并通过靶向蛋白组学技术,在上述实验的基础上样本量对潜在蛋白标记物在各组的含量进行验证实验。进一步通过蛋白功能、功能富集及共表达分析明确差异蛋白的功能探讨其作为标志物的意义与病理机制。结果基于LC-MS/MS和蛋白组学分析发现LSIL、HSIL组相较于Control组分别存在9个异常表达蛋白,CC组相较于Control组存在5个差异蛋白。其中ORM2、HPR在LSIL组相对于Control组的表达存在一定差异,可能是指示宫颈癌癌前病变的潜在标志物;F9在LSIL、HSIL、CC组中相对于Control组,随着疾病的发展呈上升趋势,是一种潜在的指示宫颈病变程度的蛋白标记物;CFI、AFM蛋白在预后组和CC组差异明显,是一类潜在的评价疗效和预后的蛋白指标。各组中的差异蛋白在功能富集分析中发现与补体和凝血级联反应通路有关。结论 F9、CFI、AFM、HPR、ORM2五种新型蛋白可能是指示宫颈病变程度、疗效和预后的潜在标志物,联合分子标志物来评估宫颈病变的发生发展可以增强诊断的灵敏度和特异性,具有一定的临床应用前景。
Objective To screen potential plasma protein biomarkers for the progression of cervical precancerous lesions into cervical carcinoma and analyze their functions. Methods Plasma samples obtained from healthy control subjects, patients with low-grade squamous intraepithelial lesion(LSIL), high-grade squamous intraepithelial lesion(HSIL), cervical cancer(CC), and patients with CC after treatment were enriched for low-abundance proteins for liquid chromatography-tandem mass spectrometry(LC-MS/MS) analysis. The MS data of the samples were analyzed using Discoverer 2.2 software, and the differential proteins(peptide coverage≥20%, unique peptides≥2) were screened by comparison of LSIL, HSIL and CC groups against the control group followed by verification using target proteomics technology. Protein function enrichment and coexpression analyses were carried out to explore the role of the differentially expressed proteins as potential biomarkers and their pathological mechanisms. Results Compared with the control group, both LSIL group and HSIL group showed 9differential proteins; 5 differentially expressed proteins were identified in CC group. The proteins ORM2 and HPR showed obvious differential expressions in LSIL and HSIL groups compared with the control group, and could serve as potential biomarkers for the progression of cervical carcinoma. The expression of F9 increased consistently with the lesion progression from LSIL to HSIL and CC, suggesting its value as a potential biomarker for the progression of cervical cancer. CFI and AFM protein levels were obviously decreased in treated patients with CC compared with the patients before treatment, indicating their predictive value for the therapeutic efficacy. Protein function enrichment analysis showed that all these differentially expressed proteins were associated with the complement system and the coagulation cascades pathway. Conclusion We identified 5 new protein biomarkers(F9, CFI, AFM,HPR, and ORM2) for cervical precancerous lesions and for prognostic evaluation of CC, and combined detection of these biomarkers may help in the evaluation of the development and progression of CC and also in improving the diagnostic sensitivity and specificity of cervical lesions.
Objective To screen potential plasma protein biomarkers for the progression of cervical precancerous lesions into cervical carcinoma and analyze their functions. Methods Plasma samples obtained from healthy control subjects, patients with low-grade squamous intraepithelial lesion(LSIL), high-grade squamous intraepithelial lesion(HSIL), cervical cancer(CC), and patients with CC after treatment were enriched for low-abundance proteins for liquid chromatography-tandem mass spectrometry(LC-MS/MS) analysis. The MS data of the samples were analyzed using Discoverer 2.2 software, and the differential proteins(peptide coverage≥20%, unique peptides≥2) were screened by comparison of LSIL, HSIL and CC groups against the control group followed by verification using target proteomics technology. Protein function enrichment and coexpression analyses were carried out to explore the role of the differentially expressed proteins as potential biomarkers and their pathological mechanisms. Results Compared with the control group, both LSIL group and HSIL group showed 9differential proteins; 5 differentially expressed proteins were identified in CC group. The proteins ORM2 and HPR showed obvious differential expressions in LSIL and HSIL groups compared with the control group, and could serve as potential biomarkers for the progression of cervical carcinoma. The expression of F9 increased consistently with the lesion progression from LSIL to HSIL and CC, suggesting its value as a potential biomarker for the progression of cervical cancer. CFI and AFM protein levels were obviously decreased in treated patients with CC compared with the patients before treatment, indicating their predictive value for the therapeutic efficacy. Protein function enrichment analysis showed that all these differentially expressed proteins were associated with the complement system and the coagulation cascades pathway. Conclusion We identified 5 new protein biomarkers(F9, CFI, AFM,HPR, and ORM2) for cervical precancerous lesions and for prognostic evaluation of CC, and combined detection of these biomarkers may help in the evaluation of the development and progression of CC and also in improving the diagnostic sensitivity and specificity of cervical lesions.
引文
[1]Ferlay J,Soerjomataram I,Ervik M.FGLOBOCAN 2012 v1.0,Cancer incidence and mortality worldwide:IARC cancer base No.11[Internet].Lyon,France:International agency for research on cancer[OL].2013.Available from:http://globocan.iarc.fr,accessed on 15/5/2015.
[2]Chen W,Zheng R,Baade PD,et al.Cancer statistics in China,2015[J].CACancer J Clin,2016,66(2):115-32.
[3]王玉东,孙璐璐.妊娠期宫颈癌筛查[J].中国实用妇科与产科杂志,2016,32(5):421-5.
[4]陈晓琴,王晓银,罗晓菊,等.妊娠期妇女对宫颈癌筛查认知行为的调查研究[J].中国计划生育和妇产科,2018,28(2):67-71.
[5]Stoler MH,Vichnin MD,Ferenczy A,et al.The accuracy of colposcopic biopsy:analyses from the placebo arm of the Gardasil clinical trials[J].Int J Cancer,2011,128(6):1354-62.
[6]Wentzensen N,Fetterman B,Castle PE,et al.p16/Ki-67 dual stain cytology for detection of cervical precancer in HPV-Positive women[J].J Natl Cancer Inst,2015,107(12):257.
[7]He XK,Luo XP,Mao LZ,et al.An optoelectronic cervical cancer screening system for screening cervical cancer:comparison with cervical cytology[J].J SouthMed Univ,2010,30(10):2304-6.
[8]Li XC,Shang JB,Wu XM,et al.MRI findings of uterine cervical cancer and value of MRI in preoperative staging[J].J South Med Univ,2007,27(3):352-4.
[9]Guo X,Hao Y,Kamilijiang M,et al.Potential predictive plasma biomarkers for cervical cancer by 2D-DIGE proteomics and ingenuity pathway analysis[J].Tumour Biol,2015,36(3):1711-20.
[10]Lokamani I,Looi ML,Ali SA,et al.Gelsolin and ceruloplasmin as potential predictive biomarkers for cervical cancer by 2D-DIGEproteomics analysis[J].Pathol Oncol Res,2014,20(1):119-29.
[11]Boichenko AP,Govorukhina N,Klip HG,et al.A panel of regulated proteins in serum from patients with cervical intraepithelial neoplasia and cervical cancer[J].J Proteome Res,2014,13(11):4995-5007.
[12]Liang Z,Hong Z,Li DJ,et al.Comparative proteomic study of cervical cancer with different radiotherapy sensitivity[J].ChineseGerman J Clinic Oncol,2009,8(4):219-24.
[13]Qing S,TulakeW,Ru MF,et al.Proteomic identification of potential biomarkers for cervical squamous cell carcinoma and human papillomavirus infection[J].Tumour Biol,2017,39(4):1010428317697547.
[14]谢松喜,李伟雄,黄玉娟,等.脑脊液蛋白质质谱在非小细胞肺癌脑转移诊断中的应用[J].南方医科大学学报,2010,30(3):498-501.
[15]蔡思娜,刘国炳,郭晓红,等.应用蛋白芯片-飞行质谱技术筛选宫颈癌血清差异蛋白质[J].南方医科大学学报,2009,29(1):32-5.
[16]Wang W,Jia HL,Huang JM,et al.Identification of biomarkers for lymph node metastasis in early-stage cervical cancer by tissue-based proteomics[J].Br J Cancer,2014,110(7):1748-58.
[17]Matsubara J,Honda K,Ono M,et al.Identification of adipophilin as a potential plasma biomarker for colorectal cancer using label-free quantitative mass spectrometry and protein microarray[J].Cancer Epidemiol Biomarkers Prev,2011,20(10):2195-203.
[18]王睿,王岚,黎明新,等.血清PARP-1蛋白高表达在胃癌中的临床意义[J].中国癌症杂志,2015,35(12):972-7.
[19]Lin YW,Lai HC,Lin CY,et al.Plasma proteomic profiling for detecting and differentiating in situ and invasive carcinomas of the uterine cervix[J].Int J Gynecol Cancer,2006,16(3):1216-24.
[20]Xia T,Zheng ZG,Gao Y,et al.Application of SELDI-TOF serum proteome profiling in cervical squamous cell carcinoma[J].Ai Zheng,2008,27(3):279-82.
[21]Looi ML,Karsani SA,Rahman MA,et al.Plasma proteome analysis of cervical intraepithelial neoplasia and cervical squamous cell carcinoma[J].J Biosci,2009,34(6):917-25.
[22]Dae HJ,Hyoung KK,Abd-Ei BP,et al.Plasma proteomic analysis of patients with squamous cell carcinoma of the uterine cervix[J].Gynecol Oncol,2008,19(3):173-80.
[23]Ceciliani F,Pocacqua V.The acute phase protein alpha 1-acid glycoprotein:a model for altered glycosylation during diseases[J].Curr Protein Pept Sci,2007,8(1):91-108.
[24]Jo M,Kim JH,Song GJ,et al.Astrocytic orosomucoid-2 modulates microglial activation and neuroinflammation[J].J Neurosci,2017,37(11):2878-94.
[25]Baraniuk JN,Casado B,Maibach H,et al.A chronic fatigue syndrome-related proteome in human cerebrospinal fluid[J].BMCNeurol,2005,5(1):22.
[26]Mestriner FL,Spiller F,Laure HJ,et al.Acute-phase protein alpha-1-acid glycoprotein mediates neutrophil migration failure in sepsis by a nitric oxide-dependent mechanism[J].Proc Natl Acad Sci USA,2007,104(49):19595-600.
[27]Sai K,Kurose K,Koizumi T,et al.Distal promoter regions are responsible for differential regulation of human orosomucoid-1 and-2 gene expression and acute phase responses[J].Biol Pharm Bull,2014,37(1):164-8.
[28]Sun YH,Cui L,Chen J,et al.Analysis of relationships between prethrombotic states and cervical cancer[J].Asian Pac J Cancer Prev,2015,16(14):6163-6.
[29]Piva M,Horowitz GM,Sharpe-Timms KL.Interleukin-6 differentially stimulates haptoglobin production by peritoneal and endometriotic cells in vitro:a model for endometrial-peritoneal interaction in endometriosis[J].J Clin Endocrinol Metab,2001,86(6):2553-61.
[30]Khosa F,Otero HJ,Prevedello LM,et al.Imaging presentation of venous thrombosis in patients with cancer[J].AJRAm J Roentgenol,2010,194(4):1099-108.
[31]Maldonado PA,Pimentel VC,Negrini LA,et al.Role of the purinergic system in patients with cervical intraepithelial neoplasia and uterine cancer[J].Biomed Pharmacother,2012,66(1):6-11.
[32]Dieplinger H,Ankerst DP,Burges AA,et al.Afamin and apolipoprotein A-IV:novel protein markers for ovarian cancer[J].Cancer Epidemiol Biomarkers Prev,2009,18(4):1127-33.
[33]Kratzer I,Bernhart E,Wintersperger A,et al.Afamin is synthesized by cerebrovascular endothelial cells and mediates alpha-tocopherol transport across an in vitro model of the blood-brain barrier[J].JNeurochem,2009,108(3):707-18.
[34]Riihila P,Nissinen L,Farshchian M,et al.Complement factor Ipromotes progression of cutaneous squamous cell carcinoma[J].JInvest Dermatol,2014,134(2):S18.
[35]Humphries JM,Penno MA,Weiland FA,et al.Identification and validation of novel candidate protein biomarkers for the detection of human gastric cancer[J].Biochim Biophys Acta,2014,1844(5,SI):1051-8.
[36]Choi JW,Liu H,Shin DH,et al.Proteomic and cytokine plasma biomarkers for predicting progression from colorectal adenoma to carcinoma in human patients[J].Proteomic,2013,13(15):2361-74.
[37]RutkowskiMJ,SughrueME,KaneAJ,et al.The complement cascade as a mediator of tissue growth and regeneration[J].Inflamm Res,2010,59(11):897-905.
[38]Rutkowski MJ,Sughrue ME,Kane AJ,et al.Cancer and the complement cascade[J].Mol Cancer Res,2010,8(11):1453-65.
[39]unnikkala S,Hakulinen J,Jarva H,et al.Secretion of soluble complement inhibitors factor H and factor H-like protein(FHL-1)by ovarian tumour cells[J].Br J Cancer,2002,87(10):1119-27.
[40]Ajona D,Castano Z,Garayoa M,et al.Expression of complement factor H by lung cancer cells:Effects on the activation of the alternative pathway of complement[J].Cancer Res,2004,64(17):6310-8.
[2]Chen W,Zheng R,Baade PD,et al.Cancer statistics in China,2015[J].CACancer J Clin,2016,66(2):115-32.
[3]王玉东,孙璐璐.妊娠期宫颈癌筛查[J].中国实用妇科与产科杂志,2016,32(5):421-5.
[4]陈晓琴,王晓银,罗晓菊,等.妊娠期妇女对宫颈癌筛查认知行为的调查研究[J].中国计划生育和妇产科,2018,28(2):67-71.
[5]Stoler MH,Vichnin MD,Ferenczy A,et al.The accuracy of colposcopic biopsy:analyses from the placebo arm of the Gardasil clinical trials[J].Int J Cancer,2011,128(6):1354-62.
[6]Wentzensen N,Fetterman B,Castle PE,et al.p16/Ki-67 dual stain cytology for detection of cervical precancer in HPV-Positive women[J].J Natl Cancer Inst,2015,107(12):257.
[7]He XK,Luo XP,Mao LZ,et al.An optoelectronic cervical cancer screening system for screening cervical cancer:comparison with cervical cytology[J].J SouthMed Univ,2010,30(10):2304-6.
[8]Li XC,Shang JB,Wu XM,et al.MRI findings of uterine cervical cancer and value of MRI in preoperative staging[J].J South Med Univ,2007,27(3):352-4.
[9]Guo X,Hao Y,Kamilijiang M,et al.Potential predictive plasma biomarkers for cervical cancer by 2D-DIGE proteomics and ingenuity pathway analysis[J].Tumour Biol,2015,36(3):1711-20.
[10]Lokamani I,Looi ML,Ali SA,et al.Gelsolin and ceruloplasmin as potential predictive biomarkers for cervical cancer by 2D-DIGEproteomics analysis[J].Pathol Oncol Res,2014,20(1):119-29.
[11]Boichenko AP,Govorukhina N,Klip HG,et al.A panel of regulated proteins in serum from patients with cervical intraepithelial neoplasia and cervical cancer[J].J Proteome Res,2014,13(11):4995-5007.
[12]Liang Z,Hong Z,Li DJ,et al.Comparative proteomic study of cervical cancer with different radiotherapy sensitivity[J].ChineseGerman J Clinic Oncol,2009,8(4):219-24.
[13]Qing S,TulakeW,Ru MF,et al.Proteomic identification of potential biomarkers for cervical squamous cell carcinoma and human papillomavirus infection[J].Tumour Biol,2017,39(4):1010428317697547.
[14]谢松喜,李伟雄,黄玉娟,等.脑脊液蛋白质质谱在非小细胞肺癌脑转移诊断中的应用[J].南方医科大学学报,2010,30(3):498-501.
[15]蔡思娜,刘国炳,郭晓红,等.应用蛋白芯片-飞行质谱技术筛选宫颈癌血清差异蛋白质[J].南方医科大学学报,2009,29(1):32-5.
[16]Wang W,Jia HL,Huang JM,et al.Identification of biomarkers for lymph node metastasis in early-stage cervical cancer by tissue-based proteomics[J].Br J Cancer,2014,110(7):1748-58.
[17]Matsubara J,Honda K,Ono M,et al.Identification of adipophilin as a potential plasma biomarker for colorectal cancer using label-free quantitative mass spectrometry and protein microarray[J].Cancer Epidemiol Biomarkers Prev,2011,20(10):2195-203.
[18]王睿,王岚,黎明新,等.血清PARP-1蛋白高表达在胃癌中的临床意义[J].中国癌症杂志,2015,35(12):972-7.
[19]Lin YW,Lai HC,Lin CY,et al.Plasma proteomic profiling for detecting and differentiating in situ and invasive carcinomas of the uterine cervix[J].Int J Gynecol Cancer,2006,16(3):1216-24.
[20]Xia T,Zheng ZG,Gao Y,et al.Application of SELDI-TOF serum proteome profiling in cervical squamous cell carcinoma[J].Ai Zheng,2008,27(3):279-82.
[21]Looi ML,Karsani SA,Rahman MA,et al.Plasma proteome analysis of cervical intraepithelial neoplasia and cervical squamous cell carcinoma[J].J Biosci,2009,34(6):917-25.
[22]Dae HJ,Hyoung KK,Abd-Ei BP,et al.Plasma proteomic analysis of patients with squamous cell carcinoma of the uterine cervix[J].Gynecol Oncol,2008,19(3):173-80.
[23]Ceciliani F,Pocacqua V.The acute phase protein alpha 1-acid glycoprotein:a model for altered glycosylation during diseases[J].Curr Protein Pept Sci,2007,8(1):91-108.
[24]Jo M,Kim JH,Song GJ,et al.Astrocytic orosomucoid-2 modulates microglial activation and neuroinflammation[J].J Neurosci,2017,37(11):2878-94.
[25]Baraniuk JN,Casado B,Maibach H,et al.A chronic fatigue syndrome-related proteome in human cerebrospinal fluid[J].BMCNeurol,2005,5(1):22.
[26]Mestriner FL,Spiller F,Laure HJ,et al.Acute-phase protein alpha-1-acid glycoprotein mediates neutrophil migration failure in sepsis by a nitric oxide-dependent mechanism[J].Proc Natl Acad Sci USA,2007,104(49):19595-600.
[27]Sai K,Kurose K,Koizumi T,et al.Distal promoter regions are responsible for differential regulation of human orosomucoid-1 and-2 gene expression and acute phase responses[J].Biol Pharm Bull,2014,37(1):164-8.
[28]Sun YH,Cui L,Chen J,et al.Analysis of relationships between prethrombotic states and cervical cancer[J].Asian Pac J Cancer Prev,2015,16(14):6163-6.
[29]Piva M,Horowitz GM,Sharpe-Timms KL.Interleukin-6 differentially stimulates haptoglobin production by peritoneal and endometriotic cells in vitro:a model for endometrial-peritoneal interaction in endometriosis[J].J Clin Endocrinol Metab,2001,86(6):2553-61.
[30]Khosa F,Otero HJ,Prevedello LM,et al.Imaging presentation of venous thrombosis in patients with cancer[J].AJRAm J Roentgenol,2010,194(4):1099-108.
[31]Maldonado PA,Pimentel VC,Negrini LA,et al.Role of the purinergic system in patients with cervical intraepithelial neoplasia and uterine cancer[J].Biomed Pharmacother,2012,66(1):6-11.
[32]Dieplinger H,Ankerst DP,Burges AA,et al.Afamin and apolipoprotein A-IV:novel protein markers for ovarian cancer[J].Cancer Epidemiol Biomarkers Prev,2009,18(4):1127-33.
[33]Kratzer I,Bernhart E,Wintersperger A,et al.Afamin is synthesized by cerebrovascular endothelial cells and mediates alpha-tocopherol transport across an in vitro model of the blood-brain barrier[J].JNeurochem,2009,108(3):707-18.
[34]Riihila P,Nissinen L,Farshchian M,et al.Complement factor Ipromotes progression of cutaneous squamous cell carcinoma[J].JInvest Dermatol,2014,134(2):S18.
[35]Humphries JM,Penno MA,Weiland FA,et al.Identification and validation of novel candidate protein biomarkers for the detection of human gastric cancer[J].Biochim Biophys Acta,2014,1844(5,SI):1051-8.
[36]Choi JW,Liu H,Shin DH,et al.Proteomic and cytokine plasma biomarkers for predicting progression from colorectal adenoma to carcinoma in human patients[J].Proteomic,2013,13(15):2361-74.
[37]RutkowskiMJ,SughrueME,KaneAJ,et al.The complement cascade as a mediator of tissue growth and regeneration[J].Inflamm Res,2010,59(11):897-905.
[38]Rutkowski MJ,Sughrue ME,Kane AJ,et al.Cancer and the complement cascade[J].Mol Cancer Res,2010,8(11):1453-65.
[39]unnikkala S,Hakulinen J,Jarva H,et al.Secretion of soluble complement inhibitors factor H and factor H-like protein(FHL-1)by ovarian tumour cells[J].Br J Cancer,2002,87(10):1119-27.
[40]Ajona D,Castano Z,Garayoa M,et al.Expression of complement factor H by lung cancer cells:Effects on the activation of the alternative pathway of complement[J].Cancer Res,2004,64(17):6310-8.
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