EGFR敏感突变肺腺癌患者纵膈淋巴结转移相关基因的检测与分析

目的本研究采用转录组测序和生物信息学分析等方法,以期筛选出肺腺癌淋巴结转移相关基因。方法分别检测伴和不伴纵膈淋巴结转移的EGFR敏感突变肺腺癌临床标本转录组表达谱,筛选出差异表达基因(DEGs);对这些DEGs进行相关生物信息分析,探索这些基因在淋巴结转移进程中可能发挥的作用。q-PCR法验证高表达基因在PC9细胞株(EGFR敏感突变细胞株)中的表达情况。结果共检测了10例肺腺癌标本的转录组表达谱,通过差异基因表达分析筛选出169个DEGs,其中,68个在肺腺癌样本中表达上调、101个基因表达下调。q-PCR结果显示:高表达基因在PC-9细胞中,ZNF572、BRPF3、MMACHC等7个基因的表达量为中丰度,SMOC1、A1BG、BARX1等9个基因为低丰度,其余均为高丰度。结论 TFF3及DUSP6等多个差异表达基因可能涉及肺腺癌纵膈淋巴结转移进程。     

肿瘤抑制基因肝激酶B1在肺腺癌中的表达

目的:研究肝激酶B1(LKB1)在人肺腺癌组织中的表达。方法:收集肺腺癌组织标本(含癌旁组织、正常组织)共60例,采用实时荧光定量PCR法检测肺腺癌组织中LKB1的mRNA含量;Western blot法和免疫组化法检测肺癌组织中LKB1的表达情况。结果:实时荧光定量PCR检测显示LKB1的mRNA含量在正常对照组、癌旁组织和肺癌组织3组之间无显著差异;Western blot法检测和免疫组化结果显示肺腺癌组织中LKB1蛋白的表达量显著低于正常对照组和癌旁组织。结论:肺腺癌组织中LKB1的表达可能存在翻译后的调节。     

肺腺癌组织中CPNE1蛋白的表达及临床意义

目的探讨Copine 1(CPNE1)蛋白在肺腺癌组织中的表达及意义。方法采用免疫组化SP法检测CPNE1蛋白在186例肺腺癌组织及30例正常肺组织中的表达;采用RT-PCR技术检测肺腺癌组织及正常肺组织中CPNE1 mRNA的表达。结果在186例肺腺癌组织中68例CPNE1低表达(36.56%),118例呈CPNE1高表达(63.44%),CPNE1在正常组织中均呈低表达。相关性分析结果显示,CPNE1表达与肺腺癌分期、远处转移有关(P0.05),与患者年龄、性别、肿瘤大小等无关。RT-PCR结果显示,肺腺癌组织中CPNE1的表达量显著高于正常肺组织。结论 CPNE1蛋白在肺腺癌组织中的表达较正常肺组织高,且与淋巴结转移、临床分期有关。CPNE1蛋白表达与肺腺癌的发生、发展及预后密切相关,有望成为肺腺癌预后评估的重要指标。     

基于数据挖掘分析肺腺癌中ARHGEF16的表达及其临床意义

目的探讨Rho鸟嘌呤核苷酸交换因子16(Rho guanine nucleotide exchange factor 16, ARHGEF16)在肺腺癌中的表达及临床意义。方法使用Oncomine数据库挖掘ARHGEF16的信息,并对ARHGEF16在肺腺癌中的表达及临床意义进行分析,采用SPSS 22.0软件进行统计学分析,Graphpad Prism 7.0绘制患者生存曲线。结果与正常肺组织相比,肺腺癌组织中的ARHGEF16基因呈高表达(P0.001)。在肺腺癌组织中,EPHB3基因与ARHGEF16表达有相关性(r=0.933)。肺腺癌患者的总生存期与ARHGEF16基因高表达呈负相关(P=0.032)。结论肺腺癌组织中ARHGEF16的表达高于对应正常肺组织,且与患者预后呈负相关,提示ARHGEF16可能参与肺腺癌的发生、发展。     

肺腺癌基因谱分析及p53、KRAS的表达及意义

目的分析肺腺癌基因谱,探讨p53和KRAS在肺腺癌中的表达及其意义。方法采用二代测序技术检测29例肺腺癌石蜡样本中26个常见肿瘤相关热点基因;采用免疫组化法检测99例肺腺癌组织中p53蛋白和KRAS蛋白的表达,分析两者表达与肺腺癌临床病理特征的关系。结果 26个被检基因中发现6个基因发生突变,分别是EGFR(21/29)、p53(12/29)、KRAS(3/29)、ROS1(3/29)、ALK(1/29)和RET(1/29);EGFR突变多发生在19号(9/21例)和21号(10/21例)外显子上,2例发生在20号外显子上(其中1例在20和21号外显子上均有突变),1例发生在18号外显子上;p53突变位于6号(4/12)、4号(2/12)、5号(2/12)和8号(2/12)、7号(1/12)和10号(1/12)外显子上;KRAS突变均发生在2号(3/3)外显子上。29例中14例存在共突变,其中1例发生3种基因共突变(EGFR、KRAS、ROS1),13例发生双基因突变(10例EGFR和p53,1例EGFR和KRAS、1例EGFR和ROS1,1例p53和KRAS),12例发生单基因突变,3例未检出基因突变。免疫组化检测结果显示肺腺癌组织中p53阳性率为30.3%,KRAS阳性率为23.2%,两者表达无明显相关(r=0.054,P=0.594)。肺腺癌中p53表达与患者性别、年龄、病理亚型、肿瘤大小、有无淋巴结转移、TNM分期和分化程度均无关(P0.05),KRAS表达也与患者年龄、病理亚型、肿瘤大小、有无淋巴结转移、TNM分期和分化程度无关(P0.05),但KRAS在男性(36.4%)肺腺癌中的表达高于女性(12.7%)(P0.05)。结论肺腺癌组织中存在多种基因突变,并可发现共突变,EGFR突变多在19和21号外显子上,p53突变多集中在6、4、5和8号外显子上,KRAS突变多在2号外显子上,且KRAS突变更多见于男性患者。     

基因芯片检测人肺鳞癌和肺腺癌基因表达的异同

目的用基因芯片技术检测肺鳞癌和肺腺癌基因表达的异同。方法提取人肺鳞癌和肺腺癌组织及正常肺组织的RNA,分别用Cy5-dCTP或Cy3-dCTP标记,再与4096点基因芯片杂交,检测肺鳞癌和肺腺癌组织基因表达的异同。结果肺鳞癌和肺腺癌表达共同上调的基因17条,共同下调的基因19条;肺鳞癌表达显著高于肺腺癌的基因20条,显著低于肺腺癌的基因14条。结论多基因参与肺癌发病,基因芯片技术是肺癌基因表达检测的有效方法。     

丝氨酸蛋白酶抑制因子Kazal1型在人肺腺癌组织表达上调并促进肺腺癌细胞的增殖

目的探讨肺腺癌组织丝氨酸蛋白酶抑制因子Kazal 1型(SPINK1)表达与生存时间的关系及其对肺腺癌细胞生长的影响。方法用Real-time PCR法、免疫组织化学法检测285例肺腺癌组织和癌旁组织SPINK1的表达,并分析其表达水平与临床病理特征及预后的关系。利用SPINK1慢病毒表达载体及小干扰RNA(si RNA),处理人肺腺癌细胞系,光学显微镜下观察绿色荧光蛋白(GFP)表达情况,ELISA或Real-time PCR检测SPINK1表达水平,细胞计数和集落形成法检测肺腺癌细胞生长情况。结果肺腺癌组织SPINK1 mRNA和蛋白表达均上调(均P0. 0001),SPINK1高表达组患者总生存时间短于低表达组(50. 0个月vs 64. 5个月,P0. 001)。SPINK1过表达组,肺腺癌细胞上清液SPINK1蛋白高表达(P 0. 05),增殖与克隆形成能力均上调(P 0. 05);SPINK1敲减组,肺腺癌细胞SPINK1 mRNA表达下调,增殖能力下调(P0. 05)。结论肺腺癌组织SPINK1高表达提示患者预后不良; SPINK1促进人肺腺癌细胞的增殖。     

肺腺癌中SETD7的表达及其临床意义

目的 探讨肺腺癌中SETD7的表达及其临床意义。方法 采用qRT-PCR技术检测22例肺腺癌组织和15例癌旁组织中SETD7 mRNA的表达水平；采用免疫组化GTVision法检测162例肺腺癌和57例癌旁组织中SETD7蛋白表达，分析其表达水平与肺腺癌临床病理特征及预后的关系。结果 与癌旁组织相比，肺腺癌组织中SETD7 mRNA表达水平明显升高(t=5.212,P<0.000 1);同时，SETD7在肺腺癌组织中的蛋白表达水平亦升高，其高表达率(58.6%,95/162)高于癌旁组织(0,0/57)(P<0.001)。SETD7蛋白表达与肺腺癌临床分期(c-TNM分期)相关(P<0.05),与患者性别、年龄、吸烟、饮酒、p-TNM分期、淋巴结转移、复发、EGFR和KRAS突变以及PD-L1表达水平均无关(P>0.05)。Kaplan-Meier生存分析：SETD7蛋白高表达组患者生存率明显低于SETD7低表达组(P<0.000 1)。Cox单因素及多因素回归分析显示，SETD7蛋白高表达是影响肺腺癌患者预后的独立危险因素(P<0.001)。结论 ...     

肺腺癌中HMGB1的表达及意义

目的 探讨肺腺癌组织中HMGB1表达与上皮-间质转化(epithelial-mesenchymal transformation,EMT)的关系.方法 采用qRT-PCR法检测20例肺腺癌及20例癌旁正常肺组织中HMGB1 mRNA的表达;应用免疫组化法检测90例肺腺癌及30例癌旁正常肺组织中EMT表型蛋白(E-cad...     

lncRNA TTN-AS1通过miR-519d-3p/MMP2调控肺腺癌A549细胞的活力和侵袭

目的:观察肺腺癌组织中长链非编码RNA(lncRNA)TTN反义RNA 1(TTN-AS1)的表达情况,以及沉默TTN-AS1表达对肺腺癌A549细胞活力和侵袭的影响。方法:RT-qPCR法检测32例肺腺癌和癌旁正常组织中TTN-AS1、微小RNA-519d-3p(miR-519d-3p)和基质金属蛋白酶2(MMP2)的mRNA表达水平。将未转染的A549细胞设为空白组,转染si-NC的为si-NC对照组,转染沉默TTN-AS1表达的siRNA为si-lncRNA组(n=5),采用CCK8和Transwell方法检测沉默TTN-AS1表达对A549细胞活力和侵袭的影响。使用双萤光素酶报告基因实验、RNA免疫沉淀实验、RT-qPCR和Western blot测定TTN-AS1对miR-519d-3p和miR-519d-3p对MMP2的靶向调控作用。结果:32例肺腺癌组织中TTN-AS1的表达水平显著高于对应癌旁正常组织(P0.05)。沉默A549细胞中TTN-AS1的表达可抑制细胞的活力和侵袭。TTN-AS1可通过海绵吸附的方式负调控miR-519d-3p的表达;MMP2是miR-519d-3p的靶基因,受其负调控。过表达MMP2可部分逆转沉默TTN-AS1和过表达miR-519d-3p对A549细胞侵袭的抑制作用。结论:肺腺癌组织中lncRNA TTN-AS1呈现高表达情况,它可能通过miR-519d-3p/MMP2调控肺腺癌A549细胞的活力和侵袭能力。     

微小RNA-140-3p靶向细胞分裂周期相关蛋白8抑制肺腺癌细胞侵袭转移的生物信息学分析及验证

目的 在生物信息学基础上探讨微小RNA(miR)-140-3p靶向细胞分裂周期相关蛋白8(CDCA8)抑制肺腺癌细胞的侵袭和转移.方法 通过GEO数据库中的GEO2R分析肺腺癌芯片数据中差异表达的miRNA.TargetScanHuman7.2和 miRWalk 数据库查找 miR-140-3p 的靶基因.Cytosc...     

miRNA-181a在人肺腺癌细胞中的表达及对细胞功能的影响

目的:研究微小RNA-181a(mi RNA-181a)在不同人肺腺癌细胞中的表达及其转染人肺腺癌耐药细胞A549/DDP后对其细胞功能的影响及机制。方法:利用实时荧光定量PCR方法检测mi RNA-181a在人正常肺上皮细胞系BEAS-2B、人肺腺癌细胞系A549、人肺腺癌耐药细胞系A549/DDP中的表达;利用p Genesil-mi RNA-181a真核表达质粒转染A549/DDP细胞,同时设置未转染组和空载组;分别采用实时荧光定量PCR、MTT法、流式细胞术、Transwell实验以及Western blot法检测mi RNA-181a转染前后的表达情况、对A549/DDP细胞活力、细胞周期、细胞侵袭能力和顺铂(DDP)作用下的细胞生长抑制率、细胞凋亡率的影响以及对A549/DDP细胞中mi RNA-181a的靶基因bcl-2和p53蛋白表达的影响。结果:mi RNA-181a在A549和A549/DDP中的表达量显著低于BEAS-2B(P0.05),且在A549/DDP中表达量最低;mi RNA-181a转染A549/DDP细胞后其表达显著升高(P0.05)且能够抑制A549/DDP细胞活力、细胞周期和细胞侵袭能力(P0.05),同时升高DDP作用下A549/DDP细胞的生长抑制率和细胞凋亡率(P0.05);mi RNA-181a转染A549/DDP细胞后抑制Bcl-2蛋白的表达而促进P53蛋白的表达(P0.05)。结论:mi RNA-181a可能参与了肺腺癌的发生发展,mi RNA-181a可作为人肺腺癌治疗的新靶点。     

Metastasis suppressor 1 (MTSS1) expression is associated with reduced in-vivo metastasis and enhanced patient survival in lung adenocarcinoma

Metastasis suppressor 1 (MTSS1) has been shown to be a metastasis suppressor in a number of cancers. However, its role in lung adenocarcinoma is largely unknown. To evaluate the significance of MTSS1 expression on lung adenocarcinoma metastatic properties, the gain or loss of MTSS1 in in vivo and in vitro experiments were employed. Using an in vivo orthotopic mouse xenograft model mimicking human disease progression, stable overexpression of MTSS1 in lung adenocarcinoma cells resulted in a significant decrease in metastatic burden. Stable overexpression of MTSS1 in NCI-H1299 decreased in vitro lung adenocarcinoma invasion and migration while knockdown of MTSS1 in A549 resulted in a significant increase in cell invasion and migration. Using The Cancer Genome Atlas dataset of over 500 patient lung adenocarcinoma specimens, we demonstrated a 20% increase in 5-year survival associated with preserved intratumoral MTSS expression. MTSS1 expression in lung adenocarcinoma is associated with decreased metastatic burden, as assessed by an in vivo orthotopic model, and correlates with a 20% survival advantage at 5 years following diagnosis. In vitro data suggests MTSS1 regulates lung adenocarcinoma through augmentation of cell invasion and migration.     

甘氨双唑钠协同顺铂有效促进肺腺癌细胞中p21表达*

 目的:探讨甘氨双唑钠（CMNa）协同顺铂对肺腺癌细胞中p21表达的影响及其分子机制。方法:使用NCBI公共数据库分析顺铂对人肺腺癌细胞作用的mRNA芯片数据,寻找差异基因;对人肺腺癌A549细胞系用CMNa和顺铂处理,用real-time PCR技术验证A549细胞系差异基因的表达;用RT-PCR及染色质免疫沉淀技术进一步检测CMNa对p21及其上游分子p53表达的影响。结果:通过参考公共数据库中顺铂处理A549细胞系的mRNA芯片数据,对若干差异表达基因进行实验验证,发现p21受顺铂影响最为显著;CMNa联合顺铂处理可以有效促进A549细胞系p21的表达,但对A549细胞系单纯进行CMNa处理,p21无明显变化。此外,通过染色质免疫沉淀检测发现,CMNa联合顺铂处理可增加上游分子p53的表达,从而引起p21的上调。结论:CMNa可以协同顺铂上调p53表达,从而促进人肺腺癌细胞p21的表达,这提示CMNa对肺腺癌细胞的放疗增敏作用可能还有新的作用机制。       

miR-183 promotes radioresistance of lung adenocarcinoma H1299 cells via epithelial-mesenchymal transition

Lung adenocarcinomas are usually sensitive to radiation therapy, but some develop resistance. Radiation resistance can lead to poor patient prognosis. Studies have shown that lung adenocarcinoma cells (H1299 cells) can develop radioresistance through epithelial-mesenchymal transition (EMT), and this process is regulated by miRNAs. However, it is unclear which miRNAs are involved in the process of EMT. In our present study, we found that miR-183 expression was increased in a radioresistant lung adenocarcinoma cell line (H1299R cells). We then explored the regulatory mechanism of miR-183 and found that it may be involved in the regulation of zinc finger E-box-binding homeobox 1 (ZEB1) expression and mediate EMT in lung adenocarcinoma cells. qPCR results showed that miR-183, ZEB1, and vimentin were highly expressed in H1299R cells, whereas no difference was observed in E-cadherin expression. Western blot results showed that ZEB1 and vimentin were highly expressed in H1299R cells, while E-cadherin expression was decreased. When miR-183 expression was inhibited in H1299R cells, radiation resistance, proliferation, and cell migration were decreased. The expression of ZEB1 and vimentin in H1299R cells was decreased, while the expression of E-cadherin was increased. Moreover, miR-183 overexpression in H1299 cells enhanced radiation resistance, proliferative capacity, and cell migration ability. The expression of ZEB1 and vimentin in H1299 cells was increased, while that of E-cadherin was decreased. In conclusion, miR-183 may promote EMT and radioresistance in H1299 cells, and targeting the miR-183-ZEB1 signaling pathway may be a promising approach for lung cancer treatment.     

Heat shock factor 5 correlated with immune infiltration serves as a prognostic biomarker in lung adenocarcinoma

Lung adenocarcinoma (LUAD) is the predominant subtype of lung cancer with a relatively poor prognosis. The dramatic improvements of new immunotherapy strategies have shown promising results in lung cancer patients. This study aimed to elucidate the functions of immune-associated genes in LUAD prognosis and pathogenesis by analyzing public databases. We obtained expression profiles of LUAD patients from The Cancer Genome Atlas (TCGA) database and applied the ESTIMATE algorithm to calculate immune scores and stromal scores. A series of microenvironment-related genes with prognostic value was then identified. Of note, heat shock factor 5 (HSF5) was found to be decreased in LUAD patients and positively correlated with overall survival, which was further confirmed in the Gene Expression Omnibus (GEO) database. Moreover, Gene Ontology (GO) analysis based on the correlated genes of HSF5 demonstrated that HSF5 expression was significantly associated with the immune response and inflammatory activities. Based on the Tumor IMmune Estimation Resource (TIMER) and Gene Expression Profiling Interactive Analysis (GEPIA) datasets, HSF5 expression showed strong correlations with various immune cell infiltration and diverse immune marker sets. These findings suggest that HSF5 can be used as a promising biomarker for determining prognosis and immune infiltration in LUAD patients.     

CircRNA_23113抑制肺腺癌细胞的增殖和迁移

目的探究circRNA₂3113在肺腺癌中的表达及对肺腺癌细胞增殖和迁移的影响。方法通过高通量测序技术测序5例临床肺腺癌、癌旁组织标本,筛选出差异性表达的circRNA₂3113。实时荧光定量PCR检测肺腺癌组织、血清和细胞中circRNA₂3113的表达;构建circRNA₂3113的过表达质粒,用CCK-8法、克隆形成实验、细胞划痕和Transwell小室法分析其对细胞增殖和迁移的影响;用Western blot检测β-catenin、cyclin D1以及c-myc蛋白水平的表达。结果 CircRNA₂3113在肺腺癌患者组织、血清和细胞中显著低表达（P<0.05）;过表达circRNA₂3113后抑制A549和H1299细胞增殖和迁移（P<0.05）;CircRNA₂3113抑制β-catenin、cyclin D1以及c-myc表达（P<0.05）。结论 CircRNA₂3113在肺腺...     

CDCA7 promotes lung adenocarcinoma proliferation via regulating the cell cycle

CDCA7 is overexpressed in several malignant cancers and is predicted by bioinformatics to be a candidate oncogene in lung adenocarcinoma (LUAD). However, the clinical and biological function of CDCA7 in LUAD has never been investigated. In this study, we used quantitative real-time RT-PCR and immunohistochemistry to determine the expression level and clinical significance of CDCA7. As a result, CDCA7 was significantly overexpressed in LUAD compared to adjacent normal tissues. Furthermore, overexpression of CDCA7 was positively associated with more advanced clinical features. Silencing CDCA7 inhibited cell proliferation in LUAD through G1 phase arrest and induction of apoptosis. In conclusion, CDCA7 can be used as a potential therapeutic target for new biomarkers and LUAD.