骨膜蛋白Periostin与大肠癌生物相关性的研究
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摘要
大肠癌(Colorectal cancer,CRC)是我国肿瘤发病率上升最快的肿瘤之一。复发和转移是导致大肠癌患者预后不良和死亡的主要原因，也是影响术后生存至关重要的因素，因此确定术后复发转移的危险因素非常重要。全球每年新增病例近600,000例，对人类健康和生命构成极大威胁。在美国，大肠癌的患病率居第三位，仅次于皮肤癌和肺癌。全球大肠癌的发病率和死亡率在不断上升，随着医疗技术的不断进步，大肠癌的术后5年生存率有了较大的提高，但在近十年大肠癌的术后5年生存率一直在50%~70%之间波动[1]。我国近年来大肠癌的发病率仅次于肺癌、胃癌以及肝癌，居第四位[2-5]。无淋巴结转移的大肠癌患者，术后可达90%的五年生存率[6]。发生淋巴结转移的大肠癌患者，术后只能达到65%的五年生存率[7,8]。显而易见，改善大肠癌的疗效和预后的关键就是早发现、早诊断及早治疗。早发现、早诊断及早治疗对提高患者生存质量、延长患者生命具有非常重要的意义。
医学科研工作者的努力目标是探寻早期诊断恶性肿瘤的有效的方法。目前学术界广泛关注的是分子标志物，因为其对恶性肿瘤的诊断、治疗以及预后都具有十分重要的作用。肿瘤标记物(tumor marker,TM)是指肿瘤组织和肿瘤细胞由于癌基因或抗癌基因和其他肿瘤相关基因及其产物异常表达所产生的抗原和生物活性物质。检测它们的存在或量变可以提示肿瘤的性质，还可以了解肿瘤的组织发生、细胞分化、细胞功能，以帮助肿瘤的诊断、分类、预后判断以及治疗指导。医学科研工作者们正在努力寻找具有高度灵敏度、高度特异性的肿瘤标记物来协助诊断结肠癌，这个研究具有非常重要的意义。
本研究通过采用二维色谱与质谱联用技术，对大肠癌及癌旁组织蛋白进行分析鉴定，结果发现有24个差异蛋白。在这24个差异蛋白质中，有15个上调蛋白，有9个下调蛋白。此结果与本实验室前期的试验结果一致[18]。实验鉴定出的这些差异蛋白很有可能成为大肠癌的高敏感性、高特异性的肿瘤标志物，但还需经进一步临床验证。本实验室将对这24个差异蛋白逐一进行临床验证，此次研究只对Periostin（POSTN）蛋白进行临床验证。
POSTN其分子量约90kDa，参与细胞的募集、黏附、迁移过程，是近些年来新发现的一种骨黏附分子。POSTN可有效促进成骨细胞的增殖、分化、黏附和伸展。POSTN最初是从小鼠的成骨细胞系中克隆得到。其编码蛋白与βig-h3、MBP-70、Algal-CAM、stablinⅠ和Ⅱ属于同一蛋白家族，被认为是间质细胞的标志物[19]。
POSTN可以由转化生长因子β1(TGF-β1)诱导而产生，这一发现在结直肠癌细胞中已经得到证实[20]。除了TGF-β1可以诱导产生POSTN外，还有一些潜在因子可以促进胰腺星形细胞分泌POSTN，如：血小板源性生长因子(PDGF-aa、PDGF-bb)、骨形态发生蛋白(BMP-2)以及成纤维细胞生长因子(FGF-B、FGF-A)[21]。血管紧张素II和成纤维细胞生长因子(FGF-1)可促进肺动脉平滑肌细胞表达POSTN[22]。除此之外，在对肺癌细胞株A549的研究中，发现在低氧情况下，转化生长因子A(TGF-A)和碱性成纤维细胞生长因子(bFGF)可以通过激活RTK/PI3K信号来增加POSTN的表达[23]。同时还有学者研究发现Twist、IL-3及IL-4都可以诱导POSTN的表达或分泌。
鼠类的POSTN定位于3号染色体，大约30kbp，含有811个氨基酸，分子量约为90.2kDa。鼠类的POSTN cDNA的长度为3187bp，包括一个733bp的3'非翻译区、一个18bp的5'非翻译区以及一个2436bp的开放读码框，其编码一个含811个氨基酸，分子量90.2kDa的蛋白质[24]。由于POSTN羧基末端存在不同的剪接，已有5种不同的POSTN亚型被分离出来。人POSTN定位于13号染色体，大约36kbp，含有836个氨基酸，分子量约为93.3kDa[25]。通过对骨肉瘤和人类胎盘cDNA文库研究发现POSTN有两个亚型，一个为人类骨肉瘤POSTN开放读码框编码的亚型，含836个氨基酸，分子量93.3kD；另一个为人类胎盘POSTN开放读码框编码的亚型，含779个氨基酸，分子量7kD。POSTN具有高度的保守性，人和小鼠的POSTN具有同源的氨基酸可达89.2%，与POSTN其他结构相比羧基端(C-末端)的保守性稍低，为85.5%。
在结构上，POSTN包括氨基端(N-末端)、C-末端、一个富含半胱氨酸区(EMI)以及四个同源性重复区。有一个典型的信号序列位于N-末端，提示其可能是一个分泌性的蛋白。POSTN是一个具有高度保守性的细胞外基质蛋白，其结构中的四个同源性重复区与昆虫蛋白fasciclin I(FAS I)具有同源性，这可能与细胞的黏附相关。EMI大约包括75个氨基酸，可能与蛋白的多聚化或蛋白-蛋白相互作用相关。近年来，研究发现，每个FAS I区域都含有一个C-羧化酶识别位点以及几个能羧化的谷氨酰胺残基,在FAS I区域还含有整合素结合位点，POSTN可与整合素相互作用，介导上皮细胞-间质细胞转化(EMT)，从而调节细胞的迁移和黏附[26]。C-末端具有亲水性，由于在转录水平上mRNA的选择性剪切，人类组织中POSTN被报道的同源异构体共有8种[27]，普遍认为POSTN同源异构体的表达方式与某些肿瘤的发生有关[28]。
许多正常组织表达POSTN蛋白：胃、结肠、肺、肾上腺、甲状腺、阴道、卵巢、睾丸、前列腺等[20]，而且在发育中的心脏瓣膜、发育中的牙齿、损伤后的骨骼肌、低氧刺激后的肺动脉平滑肌细胞中呈现高表达[29]。近年来，研究认为肿瘤组织与正常组织之间POSTN的表达具有明显的差异，因此POSTN被认为是肿瘤转移候选基因之一，与恶性肿瘤侵袭性和转移性相关。
为了探究POSTN在大肠癌中的表达情况以及POSTN在大肠癌中的功能，我们的主要研究结果如下：
第一部分应用二维色谱、质谱联用技术对大肠癌与癌旁组织的差异蛋白质组学研究
方法：
1、临床样本取材：65例Dukes C期腺癌患者，癌组织及癌旁组织标本。
2、样品总蛋白提取，蛋白浓度分析。
3、制备样品蛋白水解多肽混合物。
4、二维色谱与质谱联用分析，数据库检索。
结果：
1、癌组织：质谱数据采集、整理，获得846个蛋白的鉴定资料。
2、癌旁组织：质谱数据采集、整理，获得535个蛋白质的鉴定资料。
3、将癌组织与癌旁组织的蛋白质组进行比对分析，有显著差异蛋白24个，其中癌组织上调表达蛋白15个，下调表达蛋白9个。
小结：
1、成功应用二维色谱与质谱联用技术完成大肠癌组织及癌旁组织蛋白组数据鉴定。
2、通过数据鉴定、对比分析共得到癌组织与癌旁组织的差异蛋白24个，其中15个蛋白表达上调，9个蛋白表达下调。
第二部分大肠癌病人血清POSTN水平与临床病理参数之间的关系
方法：
1、临床病理资料数据库的建立。
2、ELISA法血清测定POSTN。
结果：
1、大肠癌病人血清中POSTN水平显著升高。
2、大肠癌Dukes分期中，C期和D期的血清POSTN水平显著高于A期和B期(p<0.01)。
3、大肠癌TNM分期中，临床Ⅲ期和Ⅳ期的表达水平明显高于临床I期和临床Ⅱ期(p<0.01)。
4、伴淋巴结转移的大肠癌血清中POSTN水平显著高于无淋巴结转移的大肠癌(p=0.001)。
5、伴远处转移的大肠癌血浆中POSTN水平显著高于无远处转移的大肠癌(p=0.003)。
6、低分化型大肠癌血清中POSTN水平显著高于中分化、高分化大肠癌(p=0.003)。
小结：
大肠癌血清中POSTN水平与肿瘤的Dukes分期、TNM分期、淋巴结转移及远处转移及分化程度有关，而与病人性别、年龄无关(p>0.05)。
第三部分POSTN基因在大肠癌细胞系sw480中的表达及功能研究
方法：
1、POSTN蛋白在大肠癌细胞系sw480中的表达。
2、POSTN siRNA表达载体的构建和质粒扩增
结果：
1、POSTN蛋白定位于sw480细胞的细胞浆，为中等强度表达。
2、构建的POSTN siRNA慢病毒重组表达载体能够高效转染sw480细胞。
小结：
1、成功从大肠癌细胞株中筛选出POSTN高表达的SW480细胞株，为下一步实验打下基础。
2、本部分实验成功构建了能高效抑制POSTN基因的表达载体，酶切鉴定和测序证实质粒构建完全正确，即POSTN siRNA-1、POSTN siRNA-2。
3、成功利用脂质体介导法将重组质粒POSTN siRNA-1、POSTN siRNA-2转染到大肠癌细胞。
第四部分POSTN siRNA转染对人大肠癌细胞生物学行为的影响
方法：
1、POSTN siRNA转染对sw480细胞POSTN mRNA转录的影响。
2、POSTN siRNA转染对sw480细胞表达POSTN蛋白的影响。
3、POSTN RNAi对sw480细胞凋亡的影响。
4、POSTN siRNA转染后sw480细胞的亚细胞结构改变。
5、POSTN RNAi对sw480细胞增殖和细胞周期的影响。
结果：
1、转染siRNA-1质粒组，POSTN基因转录下降4.38倍，转染siRNA-2质粒组，POSTN基因转录下降6.2倍，与转染control质粒组相比，有明显统计学差异。
2、统计学分析结果显示，转染POSTN siRNA的细胞中POSTN蛋白表达明显低于转染control质粒组。
3、转染siRNA的sw480细胞凋亡指数明显高于转染control质粒组，两组比较具有统计学差异。
4、POSTN siRNA转染后sw480细胞的亚细胞结构发生改变。
5、RNAi质粒转染后转染组细胞存活明显低于正常培养的sw480细胞，也低于转染control质粒组，差异有统计学意义。
6、siRNA转染组与对照组的G1和G2期相比差异有统计学意义。
小结：
1.特异性siRNA可减低SW480细胞中POSTN的表达，POSTN mRNA以及POSTN蛋白的表达，其抑制效应较为明显。
2.干扰SW480细胞POSTN的表达能有效促进大肠癌细胞的凋亡。
3.干扰SW480细胞POSTN的表达能改变大肠癌细胞的亚结构。
4.干扰SW480细胞POSTN的表达能有效抑制大肠癌细胞的增殖。
5.干扰SW480细胞POSTN的表达能改变大肠癌细胞的细胞周期。
第五部分结论
Colorectal cancer (Colorectal cancer, CRC) is one of the fastest rising incidenceof cancer tumors in our country.Relapse and metastasis are the leading cause of poorprognosis and death of patients with colorectal cancer, but also a crucial factoraffecting survival, and therefore determine the risk factors for recurrence andmetastasis is very important.Nearly600,000new cases worldwide cases of humanhealth and pose a great threat to life each year.In the United States, the prevalence ofcolorectal cancer ranks third, behind skin cancer and lung cancer.The world ofcolorectal cancer incidence and mortality in sub-rise, with the continuousdevelopment of medical technology, five-year survival rate for colorectal cancer hasgreatly improved, but after nearly a decade of colorectal cancer5year survival ratehas been fluctuating between50%to70%[1].In recent years the incidence of colorectalcancer ranking fourth in China which after lung cancer, stomach cancer and livercancer[2-5]. Colorectal cancer patients without lymph node metastasis, survival rateafter five years, up to90%[6],the incidence of lymph node metastasis, survival rate ofonly65percent after five years[7,8].Therefore, the key to improving the efficacy andprognosis of colorectal cancer is early detection, early diagnosis and earlytreatment.Early detection, early diagnosis and early treatment to improve the qualityof life of patients, prolong the lives of patients with a very important significance.
Medical researchers effort aims to explore effective methods for early diagnosisof malignant tumors.The current academic attention molecular marker for thediagnosis of malignant tumors since the treatment, and prognosis have a veryimportant role.Tumor marker (tumor marker, TM) is the tumor tissue and tumor cells due to the anti-cancer genes or oncogenes and other cancer-related genes and theirproducts and abnormal antigen expression of bioactive substances produced.Detecttheir presence or quantitative nature of the tumor can be prompted, you can also learnabout the tumor tissue, cell differentiation, cell function, to help diagnose tumors,classification, prognosis and treatment guidelines.Medical researchers are trying tofind a highly sensitive, highly specific tumor markers to help diagnose colon cancer,this study has very important significance.In this study, through the application oftwo-dimensional chromatography coupled with mass spectrometry techniques,analyzed and identified differences in colorectal cancer tissues and adjacent tissuesexpressed protein profiling, identification and found there are24differentproteins.The identification of the24differentially expressed proteins, the differentialprotein upregulation has15, there are differences in protein downregulation9. This isconsistent with the preliminary laboratory test results[18].These differences incolorectal cancer tumor protein is expected to be highly sensitive, highly specificmarkers, but need further clinical validation. The lab will be this one by24differentproteins for clinical validation of all, this study only Periostin (POSTN) protein forclinical validation.
First, the general characteristics POSTN
POSTN having a molecular weight of about90kDa, primarily bone precursorcells and their secreted by the cells into the involved cell recruitment, adhesion,migration process is a bone adhesion molecules newly discovered in recentyears.POSTN can effectively promote osteoblast proliferation, differentiation,adhesion and spreading, adhesion and aggregation and promote the role of periostealprogenitor cells.POSTN originally cloned from a mouse osteoblastic cell lines.Encoding a protein βig-h3, MBP-70, Algal-CAM, stablin Ⅰ Ⅱ belong to the sameprotein family and is considered a marker of mesenchymal cells[19].
POSTN may consist of transforming growth factor-β1(TGF-β1) induced toproduce this discovery in colon cancer cells has been confirmed[20].In addition to TGF-β1can induce POSTN, there are some potential factors can promote thesecretion of pancreatic stellate cells POSTN, such as: platelet-derived growth factor(PDGF-aa, PDGF-bb), bone morphogenetic protein (BMP-2) and fibroblast growthfactor (FGF-B, FGF-A)[21].Angiotensin II, and fibroblast growth factor (FGF-1) canpromote the expression of pulmonary artery smooth muscle cells POSTN[22].Inaddition, in the study of lung cancer cell line A549, we found that under hypoxicconditions, transforming growth factor A (TGF-A) and basic fibroblast growth factor(bFGF) signals by activating RTK/PI3K the increased expression POSTN[23].Alongresearchers found Twist, IL-3and IL-4can induce the expression or secretionPOSTN.
Rodent POSTN located in chromosome3, about30kbp, containing811aminoacids, molecular weight of about90.2kDa.Rodent POSTN cDNA length is3187bp,comprising a733bp3'untranslated region, a18bp5' untranslated region and a2436bp open reading frame which encodes a811amino acids and902protein [24].Due tothe presence of the carboxyl terminus of alternative splicing POSTN, five differentsubtypes are separated POSTN.People POSTN located in chromosome13, about36kbp, containing836amino acids, molecular weight of about93.3kDa[25].By humanosteosarcoma cDNA library and found that placental POSTN two subtypes, thehuman osteosarcoma POSTN an open reading frame encoding the subtypescontaining836amino acids and a molecular weight of93.3kD;. Another for humanplacental POSTN the open reading frame encoding isoforms containing779aminoacids and7kD.POSTN highly conserved, homologous human and mouse aminoPOSTN up89.2%.Compared with other structures POSTN carboxy terminal(C-terminal) conserved lower,85.5%.
Structurally, POSTN include amino terminal (N-terminus), C-terminus, acysteine-rich region (EM I) and four homologous repeat region. There is a typicalN-terminal signal sequence is located, suggesting that it might be a secretory protein.POSTN is a highly conserved extracellular matrix proteins, the structure of the repeat region of homology with the four insect protein fasciclin I (FAS I) having homology,which may be associated with cell adhesion. EMI includes about75amino acids, maybe of the protein or multimeric protein-associated protein interactions. In recentyears, studies have found that each region contains a FAS I C-carboxylase and severalrecognition sites can be carboxylated glutamine residue, further comprising the FAS Iarea integrin binding sites, POSTN with integrin interactions mediated epithelial-mesenchymal transition (EMT), thereby regulating cell migration and adhesion[26]. Ahydrophilic C-terminus, the alternative splicing of the mRNA transcription level, thehuman tissue POSTN isoforms reported a total of8[27]is generally considered theexpression of isoforms POSTN and the occurrence of certain tumors[28].
Many POSTN protein expression in normal tissues: stomach, colon, lung,adrenal gland, thyroid, vagina, ovaries, testes, prostate, etc.[20],and in the developingheart valves, the developing teeth, skeletal muscle injury after hypoxia afterpulmonary artery smooth muscle cells showed high expression[29].In recent years,studies suggest that the expression of POSTN has obvious differences between tumortissue and normal tissue, thus POSTN tumor metastasis is considered one of thecandidate genes associated with tumor invasion and metastasis.
To explore the expression in colorectal cancer cases POSTN and POSTNfunction in colorectal cancer, and our main findings are as follows:
Part I: Application of two-dimensional chromatography, mass spectrometrytechnique differences beside colorectal cancer tissue and proteomics research
Method:
1, drawn from clinical samples:65cases of patients with Dukes Cadenocarcinoma, adjacent tissues and cancer tissues.
2, total protein samples were extracted protein concentration analysis.
3, the sample preparation proteolytic peptide mixtures.
4, the two-dimensional chromatography and mass spectrometry analysis, thedatabase retrieval.
Results:
1, carcinoma: MS data collection, collation, obtain identification information846protein.
2, adjacent tissues: MS data collection, collation, identification data obtained535proteins.
3, the cancer tissue and adjacent tissues were compared proteome analysis, thereare significant differences in protein24, which regulate the expression of protein incancer tissue15, down-regulated protein9.
4Summary:
1, two-dimensional chromatography and mass spectrometry with the successfulapplication of technology to complete colorectal cancer and adjacent tissuesproteomic data identification.
2, through the identification data, comparative analysis of tumor tissues wereobtained and adjacent tissues of24different proteins, including15proteins wereup-regulated and9down-regulated protein expression.
Part II:Plasma levels of colorectal cancer Periostin relationship withclinicopathological parameters between patients
Method:Established clinical and pathological data of the database.Measured byELISAplasma POSTN.
Results:
1, POSTN plasma of patients with colorectal cancer were significantlyincreased.Dukes staging of colorectal cancer, plasma POSTN levels C and D of theperiod was significantly higher than theAand B stages (p <0.01).
2, TNM staging of colorectal cancer, clinical expression level Ⅲ and Ⅳ wassignificantly higher than clinical and clinical Phase I Phase Ⅱ (p <0.01).
3, POSTN levels in plasma of colorectal cancer with lymph node metastasis wassignificantly higher than those without lymph node metastasis of colorectal cancer (p =0.001).
4, With distant metastases in colorectal cancer POSTN plasma levels weresignificantly higher than those without distant metastasis of colorectal cancer (p=0.003).
5, Poorly differentiated type of colorectal cancer in plasma levels weresignificantly higher than in POSTN differentiation, poorly differentiated withcolorectal cancer (p=0.003).
Summary:POSTN levels in plasma of colorectal cancer and tumor Dukes staging, TNM stage,lymph node metastasis and distant metastasis and differentiation, but not with patientgender and age (p>0.05).
Part III: POSTN gene expression and function of the colon cancer cell linesw480
Method:
1, POSTN protein expression in colorectal cancer cell lines sw480.
2,Build POSTN siRNAexpression vector and plasmid amplification.
Results:
1, POSTN sw480protein localized in the cytoplasm of cells, expressed as amoderate intensity.
2, POSTN siRNA lentiviral construct the recombinant expression vectors canefficiently transfected sw480cells.
Summary:
1, successfully screened from colon cancer cell lines SW480POSTN highexpression cell lines, lay the foundation for further experiments.
2, this part of the experiment successfully constructed efficiently inhibited gene expression vector POSTN, restriction enzyme digestion and sequencing confirmedplasmid entirely correct, that POSTN siRNA-1, postn siRNA-2.
3, the successful use of liposome-mediated recombinant plasmid POSTNsiRNA-1, POSTN siRNA-2transfected into colon cancer cells.
Part IV: POSTN siRNA transfection on the biological behavior of humancolon cancer cells
Method:
1, POSTN siRNAtransfection on sw480cells POSTN mRNAtranscription.
2, POSTN siRNAtransfection protein expression of POSTN of sw480cells.
3, POSTN RNAi on apoptosis sw480's.
4, POSTN siRNAtransfected cells sw480structural changes in subcellular.
5, POSTN RNAi effects on sw480cell proliferation and cell cycle.
Results:
1, transfected siRNA-1plasmid, POSTN gene transcription decreased4.38timestransfected siRNA-2plasmid group, POSTN gene transcription decreased6.2times,compared with the control plasmid transfected group, a significant statisticaldifference.
2, statistical analysis showed POSTN siRNA transfected cells, POSTN proteinexpression was significantly lower than the control plasmid transfected group.
3, transfection of siRNA sw480apoptotic index was significantly higher than thecontrol plasmid transfected group, a significant difference between the two groups.
4, POSTN siRNAtransfected cells sw480structural changes in subcellular.
5, Sw480POSTN RNAi effects on cell proliferation and cell cycle.
Summary:
1, the interference can be reduced expression POSTN POSTN mRNA expressionin colon cancer cells and POSTN protein.
2, can inhibit the overexpression POSTN apoptosis in colon carcinoma cells.
3, the interference POSTN colon cancer cells change expression substructure.
4, the overexpression POSTN can promote the proliferation of colon cancer cells.
5, After transfection of RNAi plasmid transfected cells survived significantlylower than the normal cells cultured sw480,also lower than the control plasmidtransfected group,the difference was statistically significant.
6,The difference was statistical significant compared to G1and G2phasessiRNAtransfection group and control group.
Part V: Conclusion:
医学科研工作者的努力目标是探寻早期诊断恶性肿瘤的有效的方法。目前学术界广泛关注的是分子标志物，因为其对恶性肿瘤的诊断、治疗以及预后都具有十分重要的作用。肿瘤标记物(tumor marker,TM)是指肿瘤组织和肿瘤细胞由于癌基因或抗癌基因和其他肿瘤相关基因及其产物异常表达所产生的抗原和生物活性物质。检测它们的存在或量变可以提示肿瘤的性质，还可以了解肿瘤的组织发生、细胞分化、细胞功能，以帮助肿瘤的诊断、分类、预后判断以及治疗指导。医学科研工作者们正在努力寻找具有高度灵敏度、高度特异性的肿瘤标记物来协助诊断结肠癌，这个研究具有非常重要的意义。
本研究通过采用二维色谱与质谱联用技术，对大肠癌及癌旁组织蛋白进行分析鉴定，结果发现有24个差异蛋白。在这24个差异蛋白质中，有15个上调蛋白，有9个下调蛋白。此结果与本实验室前期的试验结果一致[18]。实验鉴定出的这些差异蛋白很有可能成为大肠癌的高敏感性、高特异性的肿瘤标志物，但还需经进一步临床验证。本实验室将对这24个差异蛋白逐一进行临床验证，此次研究只对Periostin（POSTN）蛋白进行临床验证。
POSTN其分子量约90kDa，参与细胞的募集、黏附、迁移过程，是近些年来新发现的一种骨黏附分子。POSTN可有效促进成骨细胞的增殖、分化、黏附和伸展。POSTN最初是从小鼠的成骨细胞系中克隆得到。其编码蛋白与βig-h3、MBP-70、Algal-CAM、stablinⅠ和Ⅱ属于同一蛋白家族，被认为是间质细胞的标志物[19]。
POSTN可以由转化生长因子β1(TGF-β1)诱导而产生，这一发现在结直肠癌细胞中已经得到证实[20]。除了TGF-β1可以诱导产生POSTN外，还有一些潜在因子可以促进胰腺星形细胞分泌POSTN，如：血小板源性生长因子(PDGF-aa、PDGF-bb)、骨形态发生蛋白(BMP-2)以及成纤维细胞生长因子(FGF-B、FGF-A)[21]。血管紧张素II和成纤维细胞生长因子(FGF-1)可促进肺动脉平滑肌细胞表达POSTN[22]。除此之外，在对肺癌细胞株A549的研究中，发现在低氧情况下，转化生长因子A(TGF-A)和碱性成纤维细胞生长因子(bFGF)可以通过激活RTK/PI3K信号来增加POSTN的表达[23]。同时还有学者研究发现Twist、IL-3及IL-4都可以诱导POSTN的表达或分泌。
鼠类的POSTN定位于3号染色体，大约30kbp，含有811个氨基酸，分子量约为90.2kDa。鼠类的POSTN cDNA的长度为3187bp，包括一个733bp的3'非翻译区、一个18bp的5'非翻译区以及一个2436bp的开放读码框，其编码一个含811个氨基酸，分子量90.2kDa的蛋白质[24]。由于POSTN羧基末端存在不同的剪接，已有5种不同的POSTN亚型被分离出来。人POSTN定位于13号染色体，大约36kbp，含有836个氨基酸，分子量约为93.3kDa[25]。通过对骨肉瘤和人类胎盘cDNA文库研究发现POSTN有两个亚型，一个为人类骨肉瘤POSTN开放读码框编码的亚型，含836个氨基酸，分子量93.3kD；另一个为人类胎盘POSTN开放读码框编码的亚型，含779个氨基酸，分子量7kD。POSTN具有高度的保守性，人和小鼠的POSTN具有同源的氨基酸可达89.2%，与POSTN其他结构相比羧基端(C-末端)的保守性稍低，为85.5%。
在结构上，POSTN包括氨基端(N-末端)、C-末端、一个富含半胱氨酸区(EMI)以及四个同源性重复区。有一个典型的信号序列位于N-末端，提示其可能是一个分泌性的蛋白。POSTN是一个具有高度保守性的细胞外基质蛋白，其结构中的四个同源性重复区与昆虫蛋白fasciclin I(FAS I)具有同源性，这可能与细胞的黏附相关。EMI大约包括75个氨基酸，可能与蛋白的多聚化或蛋白-蛋白相互作用相关。近年来，研究发现，每个FAS I区域都含有一个C-羧化酶识别位点以及几个能羧化的谷氨酰胺残基,在FAS I区域还含有整合素结合位点，POSTN可与整合素相互作用，介导上皮细胞-间质细胞转化(EMT)，从而调节细胞的迁移和黏附[26]。C-末端具有亲水性，由于在转录水平上mRNA的选择性剪切，人类组织中POSTN被报道的同源异构体共有8种[27]，普遍认为POSTN同源异构体的表达方式与某些肿瘤的发生有关[28]。
许多正常组织表达POSTN蛋白：胃、结肠、肺、肾上腺、甲状腺、阴道、卵巢、睾丸、前列腺等[20]，而且在发育中的心脏瓣膜、发育中的牙齿、损伤后的骨骼肌、低氧刺激后的肺动脉平滑肌细胞中呈现高表达[29]。近年来，研究认为肿瘤组织与正常组织之间POSTN的表达具有明显的差异，因此POSTN被认为是肿瘤转移候选基因之一，与恶性肿瘤侵袭性和转移性相关。
为了探究POSTN在大肠癌中的表达情况以及POSTN在大肠癌中的功能，我们的主要研究结果如下：
第一部分应用二维色谱、质谱联用技术对大肠癌与癌旁组织的差异蛋白质组学研究
方法：
1、临床样本取材：65例Dukes C期腺癌患者，癌组织及癌旁组织标本。
2、样品总蛋白提取，蛋白浓度分析。
3、制备样品蛋白水解多肽混合物。
4、二维色谱与质谱联用分析，数据库检索。
结果：
1、癌组织：质谱数据采集、整理，获得846个蛋白的鉴定资料。
2、癌旁组织：质谱数据采集、整理，获得535个蛋白质的鉴定资料。
3、将癌组织与癌旁组织的蛋白质组进行比对分析，有显著差异蛋白24个，其中癌组织上调表达蛋白15个，下调表达蛋白9个。
小结：
1、成功应用二维色谱与质谱联用技术完成大肠癌组织及癌旁组织蛋白组数据鉴定。
2、通过数据鉴定、对比分析共得到癌组织与癌旁组织的差异蛋白24个，其中15个蛋白表达上调，9个蛋白表达下调。
第二部分大肠癌病人血清POSTN水平与临床病理参数之间的关系
方法：
1、临床病理资料数据库的建立。
2、ELISA法血清测定POSTN。
结果：
1、大肠癌病人血清中POSTN水平显著升高。
2、大肠癌Dukes分期中，C期和D期的血清POSTN水平显著高于A期和B期(p<0.01)。
3、大肠癌TNM分期中，临床Ⅲ期和Ⅳ期的表达水平明显高于临床I期和临床Ⅱ期(p<0.01)。
4、伴淋巴结转移的大肠癌血清中POSTN水平显著高于无淋巴结转移的大肠癌(p=0.001)。
5、伴远处转移的大肠癌血浆中POSTN水平显著高于无远处转移的大肠癌(p=0.003)。
6、低分化型大肠癌血清中POSTN水平显著高于中分化、高分化大肠癌(p=0.003)。
小结：
大肠癌血清中POSTN水平与肿瘤的Dukes分期、TNM分期、淋巴结转移及远处转移及分化程度有关，而与病人性别、年龄无关(p>0.05)。
第三部分POSTN基因在大肠癌细胞系sw480中的表达及功能研究
方法：
1、POSTN蛋白在大肠癌细胞系sw480中的表达。
2、POSTN siRNA表达载体的构建和质粒扩增
结果：
1、POSTN蛋白定位于sw480细胞的细胞浆，为中等强度表达。
2、构建的POSTN siRNA慢病毒重组表达载体能够高效转染sw480细胞。
小结：
1、成功从大肠癌细胞株中筛选出POSTN高表达的SW480细胞株，为下一步实验打下基础。
2、本部分实验成功构建了能高效抑制POSTN基因的表达载体，酶切鉴定和测序证实质粒构建完全正确，即POSTN siRNA-1、POSTN siRNA-2。
3、成功利用脂质体介导法将重组质粒POSTN siRNA-1、POSTN siRNA-2转染到大肠癌细胞。
第四部分POSTN siRNA转染对人大肠癌细胞生物学行为的影响
方法：
1、POSTN siRNA转染对sw480细胞POSTN mRNA转录的影响。
2、POSTN siRNA转染对sw480细胞表达POSTN蛋白的影响。
3、POSTN RNAi对sw480细胞凋亡的影响。
4、POSTN siRNA转染后sw480细胞的亚细胞结构改变。
5、POSTN RNAi对sw480细胞增殖和细胞周期的影响。
结果：
1、转染siRNA-1质粒组，POSTN基因转录下降4.38倍，转染siRNA-2质粒组，POSTN基因转录下降6.2倍，与转染control质粒组相比，有明显统计学差异。
2、统计学分析结果显示，转染POSTN siRNA的细胞中POSTN蛋白表达明显低于转染control质粒组。
3、转染siRNA的sw480细胞凋亡指数明显高于转染control质粒组，两组比较具有统计学差异。
4、POSTN siRNA转染后sw480细胞的亚细胞结构发生改变。
5、RNAi质粒转染后转染组细胞存活明显低于正常培养的sw480细胞，也低于转染control质粒组，差异有统计学意义。
6、siRNA转染组与对照组的G1和G2期相比差异有统计学意义。
小结：
1.特异性siRNA可减低SW480细胞中POSTN的表达，POSTN mRNA以及POSTN蛋白的表达，其抑制效应较为明显。
2.干扰SW480细胞POSTN的表达能有效促进大肠癌细胞的凋亡。
3.干扰SW480细胞POSTN的表达能改变大肠癌细胞的亚结构。
4.干扰SW480细胞POSTN的表达能有效抑制大肠癌细胞的增殖。
5.干扰SW480细胞POSTN的表达能改变大肠癌细胞的细胞周期。
第五部分结论
Colorectal cancer (Colorectal cancer, CRC) is one of the fastest rising incidenceof cancer tumors in our country.Relapse and metastasis are the leading cause of poorprognosis and death of patients with colorectal cancer, but also a crucial factoraffecting survival, and therefore determine the risk factors for recurrence andmetastasis is very important.Nearly600,000new cases worldwide cases of humanhealth and pose a great threat to life each year.In the United States, the prevalence ofcolorectal cancer ranks third, behind skin cancer and lung cancer.The world ofcolorectal cancer incidence and mortality in sub-rise, with the continuousdevelopment of medical technology, five-year survival rate for colorectal cancer hasgreatly improved, but after nearly a decade of colorectal cancer5year survival ratehas been fluctuating between50%to70%[1].In recent years the incidence of colorectalcancer ranking fourth in China which after lung cancer, stomach cancer and livercancer[2-5]. Colorectal cancer patients without lymph node metastasis, survival rateafter five years, up to90%[6],the incidence of lymph node metastasis, survival rate ofonly65percent after five years[7,8].Therefore, the key to improving the efficacy andprognosis of colorectal cancer is early detection, early diagnosis and earlytreatment.Early detection, early diagnosis and early treatment to improve the qualityof life of patients, prolong the lives of patients with a very important significance.
Medical researchers effort aims to explore effective methods for early diagnosisof malignant tumors.The current academic attention molecular marker for thediagnosis of malignant tumors since the treatment, and prognosis have a veryimportant role.Tumor marker (tumor marker, TM) is the tumor tissue and tumor cells due to the anti-cancer genes or oncogenes and other cancer-related genes and theirproducts and abnormal antigen expression of bioactive substances produced.Detecttheir presence or quantitative nature of the tumor can be prompted, you can also learnabout the tumor tissue, cell differentiation, cell function, to help diagnose tumors,classification, prognosis and treatment guidelines.Medical researchers are trying tofind a highly sensitive, highly specific tumor markers to help diagnose colon cancer,this study has very important significance.In this study, through the application oftwo-dimensional chromatography coupled with mass spectrometry techniques,analyzed and identified differences in colorectal cancer tissues and adjacent tissuesexpressed protein profiling, identification and found there are24differentproteins.The identification of the24differentially expressed proteins, the differentialprotein upregulation has15, there are differences in protein downregulation9. This isconsistent with the preliminary laboratory test results[18].These differences incolorectal cancer tumor protein is expected to be highly sensitive, highly specificmarkers, but need further clinical validation. The lab will be this one by24differentproteins for clinical validation of all, this study only Periostin (POSTN) protein forclinical validation.
First, the general characteristics POSTN
POSTN having a molecular weight of about90kDa, primarily bone precursorcells and their secreted by the cells into the involved cell recruitment, adhesion,migration process is a bone adhesion molecules newly discovered in recentyears.POSTN can effectively promote osteoblast proliferation, differentiation,adhesion and spreading, adhesion and aggregation and promote the role of periostealprogenitor cells.POSTN originally cloned from a mouse osteoblastic cell lines.Encoding a protein βig-h3, MBP-70, Algal-CAM, stablin Ⅰ Ⅱ belong to the sameprotein family and is considered a marker of mesenchymal cells[19].
POSTN may consist of transforming growth factor-β1(TGF-β1) induced toproduce this discovery in colon cancer cells has been confirmed[20].In addition to TGF-β1can induce POSTN, there are some potential factors can promote thesecretion of pancreatic stellate cells POSTN, such as: platelet-derived growth factor(PDGF-aa, PDGF-bb), bone morphogenetic protein (BMP-2) and fibroblast growthfactor (FGF-B, FGF-A)[21].Angiotensin II, and fibroblast growth factor (FGF-1) canpromote the expression of pulmonary artery smooth muscle cells POSTN[22].Inaddition, in the study of lung cancer cell line A549, we found that under hypoxicconditions, transforming growth factor A (TGF-A) and basic fibroblast growth factor(bFGF) signals by activating RTK/PI3K the increased expression POSTN[23].Alongresearchers found Twist, IL-3and IL-4can induce the expression or secretionPOSTN.
Rodent POSTN located in chromosome3, about30kbp, containing811aminoacids, molecular weight of about90.2kDa.Rodent POSTN cDNA length is3187bp,comprising a733bp3'untranslated region, a18bp5' untranslated region and a2436bp open reading frame which encodes a811amino acids and902protein [24].Due tothe presence of the carboxyl terminus of alternative splicing POSTN, five differentsubtypes are separated POSTN.People POSTN located in chromosome13, about36kbp, containing836amino acids, molecular weight of about93.3kDa[25].By humanosteosarcoma cDNA library and found that placental POSTN two subtypes, thehuman osteosarcoma POSTN an open reading frame encoding the subtypescontaining836amino acids and a molecular weight of93.3kD;. Another for humanplacental POSTN the open reading frame encoding isoforms containing779aminoacids and7kD.POSTN highly conserved, homologous human and mouse aminoPOSTN up89.2%.Compared with other structures POSTN carboxy terminal(C-terminal) conserved lower,85.5%.
Structurally, POSTN include amino terminal (N-terminus), C-terminus, acysteine-rich region (EM I) and four homologous repeat region. There is a typicalN-terminal signal sequence is located, suggesting that it might be a secretory protein.POSTN is a highly conserved extracellular matrix proteins, the structure of the repeat region of homology with the four insect protein fasciclin I (FAS I) having homology,which may be associated with cell adhesion. EMI includes about75amino acids, maybe of the protein or multimeric protein-associated protein interactions. In recentyears, studies have found that each region contains a FAS I C-carboxylase and severalrecognition sites can be carboxylated glutamine residue, further comprising the FAS Iarea integrin binding sites, POSTN with integrin interactions mediated epithelial-mesenchymal transition (EMT), thereby regulating cell migration and adhesion[26]. Ahydrophilic C-terminus, the alternative splicing of the mRNA transcription level, thehuman tissue POSTN isoforms reported a total of8[27]is generally considered theexpression of isoforms POSTN and the occurrence of certain tumors[28].
Many POSTN protein expression in normal tissues: stomach, colon, lung,adrenal gland, thyroid, vagina, ovaries, testes, prostate, etc.[20],and in the developingheart valves, the developing teeth, skeletal muscle injury after hypoxia afterpulmonary artery smooth muscle cells showed high expression[29].In recent years,studies suggest that the expression of POSTN has obvious differences between tumortissue and normal tissue, thus POSTN tumor metastasis is considered one of thecandidate genes associated with tumor invasion and metastasis.
To explore the expression in colorectal cancer cases POSTN and POSTNfunction in colorectal cancer, and our main findings are as follows:
Part I: Application of two-dimensional chromatography, mass spectrometrytechnique differences beside colorectal cancer tissue and proteomics research
Method:
1, drawn from clinical samples:65cases of patients with Dukes Cadenocarcinoma, adjacent tissues and cancer tissues.
2, total protein samples were extracted protein concentration analysis.
3, the sample preparation proteolytic peptide mixtures.
4, the two-dimensional chromatography and mass spectrometry analysis, thedatabase retrieval.
Results:
1, carcinoma: MS data collection, collation, obtain identification information846protein.
2, adjacent tissues: MS data collection, collation, identification data obtained535proteins.
3, the cancer tissue and adjacent tissues were compared proteome analysis, thereare significant differences in protein24, which regulate the expression of protein incancer tissue15, down-regulated protein9.
4Summary:
1, two-dimensional chromatography and mass spectrometry with the successfulapplication of technology to complete colorectal cancer and adjacent tissuesproteomic data identification.
2, through the identification data, comparative analysis of tumor tissues wereobtained and adjacent tissues of24different proteins, including15proteins wereup-regulated and9down-regulated protein expression.
Part II:Plasma levels of colorectal cancer Periostin relationship withclinicopathological parameters between patients
Method:Established clinical and pathological data of the database.Measured byELISAplasma POSTN.
Results:
1, POSTN plasma of patients with colorectal cancer were significantlyincreased.Dukes staging of colorectal cancer, plasma POSTN levels C and D of theperiod was significantly higher than theAand B stages (p <0.01).
2, TNM staging of colorectal cancer, clinical expression level Ⅲ and Ⅳ wassignificantly higher than clinical and clinical Phase I Phase Ⅱ (p <0.01).
3, POSTN levels in plasma of colorectal cancer with lymph node metastasis wassignificantly higher than those without lymph node metastasis of colorectal cancer (p =0.001).
4, With distant metastases in colorectal cancer POSTN plasma levels weresignificantly higher than those without distant metastasis of colorectal cancer (p=0.003).
5, Poorly differentiated type of colorectal cancer in plasma levels weresignificantly higher than in POSTN differentiation, poorly differentiated withcolorectal cancer (p=0.003).
Summary:POSTN levels in plasma of colorectal cancer and tumor Dukes staging, TNM stage,lymph node metastasis and distant metastasis and differentiation, but not with patientgender and age (p>0.05).
Part III: POSTN gene expression and function of the colon cancer cell linesw480
Method:
1, POSTN protein expression in colorectal cancer cell lines sw480.
2,Build POSTN siRNAexpression vector and plasmid amplification.
Results:
1, POSTN sw480protein localized in the cytoplasm of cells, expressed as amoderate intensity.
2, POSTN siRNA lentiviral construct the recombinant expression vectors canefficiently transfected sw480cells.
Summary:
1, successfully screened from colon cancer cell lines SW480POSTN highexpression cell lines, lay the foundation for further experiments.
2, this part of the experiment successfully constructed efficiently inhibited gene expression vector POSTN, restriction enzyme digestion and sequencing confirmedplasmid entirely correct, that POSTN siRNA-1, postn siRNA-2.
3, the successful use of liposome-mediated recombinant plasmid POSTNsiRNA-1, POSTN siRNA-2transfected into colon cancer cells.
Part IV: POSTN siRNA transfection on the biological behavior of humancolon cancer cells
Method:
1, POSTN siRNAtransfection on sw480cells POSTN mRNAtranscription.
2, POSTN siRNAtransfection protein expression of POSTN of sw480cells.
3, POSTN RNAi on apoptosis sw480's.
4, POSTN siRNAtransfected cells sw480structural changes in subcellular.
5, POSTN RNAi effects on sw480cell proliferation and cell cycle.
Results:
1, transfected siRNA-1plasmid, POSTN gene transcription decreased4.38timestransfected siRNA-2plasmid group, POSTN gene transcription decreased6.2times,compared with the control plasmid transfected group, a significant statisticaldifference.
2, statistical analysis showed POSTN siRNA transfected cells, POSTN proteinexpression was significantly lower than the control plasmid transfected group.
3, transfection of siRNA sw480apoptotic index was significantly higher than thecontrol plasmid transfected group, a significant difference between the two groups.
4, POSTN siRNAtransfected cells sw480structural changes in subcellular.
5, Sw480POSTN RNAi effects on cell proliferation and cell cycle.
Summary:
1, the interference can be reduced expression POSTN POSTN mRNA expressionin colon cancer cells and POSTN protein.
2, can inhibit the overexpression POSTN apoptosis in colon carcinoma cells.
3, the interference POSTN colon cancer cells change expression substructure.
4, the overexpression POSTN can promote the proliferation of colon cancer cells.
5, After transfection of RNAi plasmid transfected cells survived significantlylower than the normal cells cultured sw480,also lower than the control plasmidtransfected group,the difference was statistically significant.
6,The difference was statistical significant compared to G1and G2phasessiRNAtransfection group and control group.
Part V: Conclusion:
引文
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