摘要
背景和目的
肺癌是严重威胁人类健康和生命的恶性肿瘤，其发病率将在相当长时期内呈现显著上升趋势，已成为引起世界上癌症死亡率最主要原因~1。高迁移率族蛋白B1(high mobility group box B 1，HMGB1)，是一种存在于真核生物细胞内的非组蛋白染色体结合蛋白，参与多种生物学过程包括诸如基因转录、DNA修复等功能。HMGB 1过表达有抑制细胞凋亡，诱导细胞分化、细胞迁移、细胞增殖等作用~2。最近研究表明它不但是一种转录因子和促生长因子，而且是一种重要的炎性细胞因子，并与肿瘤的发生、浸润、转移等生物学行为关系密切，有很大的临床价值~3。HMGB1在肺癌中的研究处于起步和探索阶段。国内外并无其他关于HMGB1在肺癌中全方位系统性研究的文献报道。本研究在人肺鳞癌细胞株YTMLC-9、人肺腺癌细胞株A549、人大细胞肺癌细胞株L9981及人小细胞癌细胞株NCI-H446中筛选高表达HMGB1的细胞株，作为人肺癌细胞HMGB1基因研究的理想材料，为下一步进行的RNAi实验研究的细胞模型的选择奠定了基础；设计针对HMGB1为靶点的siRNA，观察通过RNA干扰抑制HMGB1基因表达后，高表达HMGB1的人肺癌细胞株L9981增殖和侵袭能力的改变，体外验证HMGB1基因做为治疗靶点的可行性；为临床开发应用和探索肿瘤治疗的新途径提供实验和理论基础；应用RNA干扰联合基因芯片技术检测HMGB1表达下调后肺癌细胞基因表达谱的差异情况，筛选与HMGB1相互作用的基因及可能的信号通路，探讨HMGB1基因在肺癌中的作用机制。
方法
1、常规培养人肺鳞癌细胞株YTMLC-9、人肺腺癌细胞株A549、人大细胞肺癌细胞株L9981及人小细胞癌细胞株NCI-H446，采用实时荧光定量PCR和Westernblot方法对人肺癌细胞HMGB1基因在4株细胞中的mRNA和蛋白水平的表达进行检测，筛选出高表达HMGB1基因的人肺癌细胞株。
2、根据siRNA设计原则，设计合成靶向HMGB1基因的siRNA，采用阳离子脂质体试剂瞬时转染已经筛选出的高表达HMGB1的人大细胞肺癌细胞株L9981，利用实时荧光定量PCR和Western blot检测RNA干扰后HMGB1基因的沉默效果，评价siRNA设计的合理性及RNA干扰抑制HMGB1表达的有效性；确定RNA干扰抑制HMGB1表达确实有效后，培养人大细胞肺癌细胞株L9981，分为3组进行RNA干扰研究，分别为转染靶向HMGB1基因的siRNA组，转染无关序列组和空白对照组，于RNA干扰后48小时，用细胞活力计数仪测定3组细胞活力；分别在RNA干扰后24、48、72和96小时应用MTT实验检测3组细胞生存状态，计算生长抑制率并绘制生长曲线；应用boyden chamber法检测3组侵袭能力的差异。
3、采用阳离子脂质体试剂向人肺癌细胞L9981转染靶向HMGB1基因的siRNA，下调HMGB1的表达。应用Affymetrix HU133 plus 2基因芯片检测人肺癌细胞L9981 HMGB1基因表达下调后，全基因表达谱的变化，并应用GCOS(Gene-ChipOperation Software)软件分析筛查数据，对相关基因和信号通路进行综合分析。
结果：
1、人肺鳞癌细胞株YTMLC-9、人肺腺癌细胞株A549、人大细胞肺癌细胞株L9981及人小细胞癌细胞株NCI-H446中均有HMGB1基因表达，其中人大细胞肺癌细胞株L9981表达水平最高，mRNA是表达量最低的人肺腺癌细胞株A549mRNA表达量的10．3倍，人大细胞肺癌细胞株L9981与其它3种人肺癌细胞株比较差异有统计学意义(P＜0．01)
2、靶向HMGB1的siRNA成功抑制人大细胞肺癌细胞株L9981中HMGB1基因及蛋白表达(P=0．000)，细胞活力检测仪测定RNA干扰48小时后，空白对照组和阴性对照组细胞活力显著大于siRNA转染组；MTT测定siRNA转染组细胞生长抑制率显著大于空白对照组和阴性对照组；boyden chamber小室细胞侵袭实验结果siRNA转染组穿膜细胞数明显减少，与空白对照组和阴性对照组比较，差异有显著性(P＜0．05)。
3、RNA干扰抑制人大细胞肺癌细胞株L9981HMGB1基因表达后，GCOS软件分析基因芯片分析其表达谱的变化。结果发现1433个探针表达明显改变，对应有明确名称的基因为879个，其中上调的基因有295个，下调的有584个；EST序列216个，其中上调96个，下调120个。差异基因共涉及131个信号通路，主要为肺癌、钙信号通路、细胞通讯、细胞因子受体相互作用、ErbB信号通路、细胞基质黏附、细胞迁移、细胞信号转导、Jak-STAT信号通路、MAPK信号通路、p53信号通路、细胞周期、细胞凋亡以及Wnt信号通路等相关通路。经过Milano网站分析，其中678个基因有报道。在这678个基因中，与肺癌(检索词lung cancer)有相关文献报道的有209个，与癌症(检索词cancer)有相关报道的有526个，与侵袭(检索词invasion)有相关报道的有170个，或转移(检索词metastasis)有相关报道的有194个。
结论：
1、本研究在国内外首次报道HMGB1在人肺鳞癌细胞株、腺癌细胞株、大细胞癌细胞株及小细胞癌细胞株等4株肺癌细胞中的表达水平，筛选出HMGB1高表达的人肺癌细胞株L9981，HMGB1低表达的人肺癌细胞株A549，人大细胞肺癌L9981细胞株为HMGB1高表达细胞株，可作为进行肺癌细胞中研究HMGB1的实验材料。
2、成功设计了靶向HMGB1的siRNA，应用RNA干扰技术在国内外首次将靶向HMGB1的siRNA转染至筛选出的高表达HMGB1的肺癌细胞株中，并成功的高效地下调人肺癌细胞L9981HMGB1基因的表达，成功构建了转染HMGB1的siRNA的肺癌细胞株和转染无关序列的肺癌细胞株。人肺癌细胞L9981HMGB1基因下调后，细胞增值和侵袭能力显著被抑制，表明HMGB1可作为人肺癌基因治疗潜在的靶点。
3、本研究在国内外首先应用表达谱芯片研究人肺癌细胞L9981HMGB1基因下调后，其细胞基因表达谱变化情况，确定表达差异基因879个，主要涉及肺癌、钙信号通路、细胞通讯、细胞因子受体相互作用、ErbB信号通路、细胞基质黏附、细胞迁移、细胞信号转导、Jak-STAT信号通路、MAPK信号通路、p53信号通路、细胞周期、细胞凋亡以及Wnt信号通路等方面。经过Milano网站分析，其中678个基因有报道。在这678个基因中，与肺癌(检索词lung cancer)、侵袭和转移(检索词metastasis)报道较多的基因中。筛选出与肺癌、侵袭和转移相关报道较多的基因进行分析，其中上调的基因功能主要有血管形成、细胞通讯、抗凋亡、细胞增殖正调控和细胞迁移正调控等；在下调的基因中，主要功能与信号转导、细胞之间信号联系、细胞粘附、细胞与基质黏附、细胞周期调控、DNA修复、DNA复制、细胞迁移、细胞分化、细胞生长、细胞增殖和钙离子通道有关。表明HMGB1基因表达受很多相关基因及信号通路的调控，与肺癌的侵袭、转移及肺癌形成等生物学过程密切相关，其分子机制有待进一步深入探讨。
Objective:
Lung cancer is a type of malignant tumor which theats human health and life.Itsmorbidity will increase dramatically in a long period.Lung cancer is the leadingcause of cancer all over the world.HMGBl(high mobility group box B 1)is anon-histone chromsone binding protein locates in the cells.It takes part in manybiological processions including gene transcription and DNA repair.HMGBloverexpression can results in cell apoptosis,cell diffrenial,cell metastisis and cellprolifration.Recent studys indicates it is not only a transciption factor but also aimportant factor that relate to tumor development,infiltration and metastasis.Thestudy of HMGB1 in lung cancer is in the beginning step around the world.There arenot literatures about systemic reseach of HMGB 1 in lung cancer.The main purpose ofthis study is detect the HMGB1 expression of 4 lung cancer cell lines to select themost suitable cell line to do the work next step.Then to suppress HMGB1 geneexpression by RNA interference in lung cancer cell,to research the invasion andmigration behavior of lung cancer ceil after HMGB l gene silence.Then we further tostudy the molecular mechanisms of the invasion of lung cancer cell by genechip.
Methods:
1.Four lung cancer cell lines were cultured by normal method,Western blot andreal-time quantitative PCR were used to verify the expression level of HMGB1.Select the cell line which HMGB 1 over-expressed.
2.siRNA targeting HMGB1 were designed and synthesized.The siRNA wastransiently transfected into L9981 cell line which was selected that HMGB1over-epressed via cationic liposome Lipofectamine 2000,Western Blot andreal-time quantitative PCR were used to verify the interference efficiency of HMGB 1protein and mRNA expression levels.After HMGB1 was down-regulated in lungcancer cell line L9981 by RNAi,Determination and analysis of cell viability forL9981 cell line with HMGB1 gene silencing;.the ability of cell invasion was measured by Boyden chamber,
3.The alteration of gene expression profiles after HMGB1 gene silencing wasinvestigated through Affymetrix HU133 plus 2 gene chip,and bioinformatics wasused to analysize data.
Results:
1.HMGB1 expressed in all 4 lung cancer cell lines,The cell line L9981 is themost highly expressed cell line(P＜0.01).
2.HMGB1 gene mRNA and protein expression levels of lung cancer cell lineL9981 were both dramatically decreased by RNA interference(P＜0.01).AfterHMGB1 was down-regulated in lung cancer cell line L9981 by RNAi,both cellinvasion and migration ability were inhibited significantly(P＜0.01).
3.Microarray assay revealed that expression of 1433 probes were altered inresponse to HMGB1 gene silencing,including 879 genes and 216 ESTs.Among 879genes there are 295 up-regulated genes and 584 down-regulated genes.These genesare involved in cell cycle,apoptosis,cell signal transduction,cell adhesion,cellmigration and aging.
Conclusion:
1.All 4 lung cancer cell lines expree HMGB1 gene.As the HMGB1overexpression cell line,L9981 is an ideal material for follow-up research.
2.RNAi can efficiently down-regulate the HMGB 1 gene in lung cancer cell lineL9981,The ability of growth and invasion of lung cancer cell line L9981 can beinhibited by HMGB1 silencing.HMGB1 can be regarded as a target for gene therapyof lung cancer.
4.HMGB1 is an important gene to regulate cells′adhesion and invasion.Afterthe silence of HMGB1 gene,many genes had a significantly differential expression.These genes are involved in cell cycle,apoptosis,cell signal transduction,celladhesion,cell migration and aging.However,the present study still can not completely reveal the HMGB1 signal pathways and molecular mechanism;morestudies are needed to carry out.
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