【作者】 张菁；

【导师】 吴晓娟； 高鹏；

【作者基本信息】 山东大学 ， 临床病理学（专业学位）， 2020， 硕士

【摘要】 目的:肺癌是人类常见的恶性肿瘤之一,其中腺癌的发病率最高。尽管肺腺癌的治疗取得了较大进展,但肿瘤的复发转移使其死亡率位居恶性肿瘤之首,5年生存率不到19%。探寻新的能够预测肺腺癌侵袭转移的蛋白分子,有望为高侵袭性肺癌的治疗提供新靶点。序列相似性家族83成员B(FAM83B)是一种新型潜在癌基因,它可在多种人类腺癌中过表达,并促进肿瘤的增殖、侵袭、转移等恶性生物学行为,是各类肿瘤预后不良的标志之一,但其在肺腺癌中的表达水平及作用机制尚未完全阐明。方法:应用免疫组化二步法(PV8000),检测肺腺癌原发灶与转移灶中FAM83B的差异表达,分析FAM83B在原发性肺腺癌患者中的表达与临床病理特征的相关性以及EGFR野生型与常见EGFR突变之间的差异:数据库分析FAM83B表达与肺腺癌临床病理因素及预后的相关性。构建FAM83B过表达载体的慢病毒(GV492/FAM83B cDNA)和慢病毒干扰载体(GV248/siRNA),分别感染H1299和PC9/GR细胞株,应用qPCR、WB、Transwell小室、划痕实验及裸鼠成瘤等方法检测FAM83B过表达对肺癌细胞迁移及侵袭等生物学行为的影响:应用WB检测过表达FAM83B细胞系中MMP-9、TIMP-1、p-AKT/AKT的蛋白表达,初步探讨其促进肺腺癌侵袭转移的机制。结果:(1)在组织水平,免疫组化结果显示:FAM83B在原发灶中表达的阳性率为23.80%,在转移灶中的阳性率为68.80%,转移灶中表达的阳性率明显高于原发灶,差异具有统计学意义(P<0.0001)。此外,还分析了 FAM83B在EGFR WT组、19Del组和L858R组间的表达差异,结果发现:FAM83B在WT组的阳性率为53.12%,19Del组阳性率为37.93%,表达具有统计学意义(P<0.05);而在L858R组与19Del组间,以及WT组与L858R组间的表达差异无统计学意义(P>0.05)。(2)TCGA数据库分析显示:FAM83B在肺腺癌的表达高于周围正常组织、在淋巴结高分期组的肺腺癌中其表达高于无淋巴结受累组和淋巴结低分期组,与此相似,肺腺癌高分期组FAM83B的表达也高于低分期组。生存分析显示:FAM83B高表达组患者生存期明显低于低表达组,差异均具有统计学意义。(3)在细胞水平,构建FAM83B过表达及干扰慢病毒载体,分别建立FAM83B cDNA 过表达(FAM83B Over Expression,FAM83B OE)及靶向沉默FAM83B(silencing RNA,FAM83B siRNA)的肺腺癌细胞株,划痕实验观察到:H1299/FAM83B OE组的细胞迁移率约71.72±2.742%,而对照组的迁移率约28.56±4.930%,差异具有统计学意义(P<0.05);PC9/GR FAM83B siRNA 组细胞迁移率约48.59±2.849%、而对照组迁移率约77.50±2.660%,差异具有统计学意义(P<0.05)。Transwell小室结果显示:FAM83B OE组细胞穿过小室的数目为191.7±6.94个,而对照组为129.0±8.7,两组比较具有统计学意义(P<0.05);PC9/GR FAM83B siRNA组细胞穿过小室的数目为191.7±13.53个,而对照组为384.3±47.60个,两组比较具有统计学意义(P<0.01)。裸鼠成瘤实验显示:FAM83B OE组成瘤速度明显强于对照组(P<0.001),并有包膜侵犯。综上,FAM83B过表达可促使肺腺癌细胞株的侵袭及迁移能力增强。在蛋白分子水平发现,FAM83B OE组p-AKT/AKT的比值以及TIMP-1的表达水平均高于其对照组,差异具有统计学意义(P<0.05和P<0.01)。结论:(1)FAM83B是肺腺癌预后不良的标志之一,FAM83B高表达的肺腺癌对EGFR-TKIs治疗不敏感、或更易出现耐药。(2)FAM83B高表达可促进肺腺癌迁移及侵袭等恶性表型。(3)FAM83B过表达可促使p-AKT/AKT表达的比值升高以及TIMP-1的表达增加,因此推测,FAM83B可以活化PI3 K/AKT/NF-κB促进TIMP-1的表达,进而促进肺腺癌细胞的侵袭转移。更多还原

【Abstract】 Objective:Lung cancer is one of the most common malignancies,among which adenocarcinoma has the highest morbidity.Although the treatment of lung adenocarcinoma has made great progress,the recurrence and metastasis of the tumor makes its mortality in the first place of among most common malignant tumors,with the 5-year survival rate lower than 19%.Exploring new protein molecules that can predict the invasion and metastasis of lung adenocarcinoma is expected to provide a new target for the treatment of high-invasive lung cancer.FAM83B,a family 83 member with sequence similarity,is a new potential oncogene.It can be overexpressed in a variety of human adenocarcinomas and promote malignant biological behaviors such as proliferation,invasion,metastasis,etc.It is one of the markers of poor prognosis of various tumors,but its expression level and mechanism of action in lung adenocarcinoma have not been clear.Method:The differences of FAM83B expression in primary and metastatic tumors were detected by immunohistochemistry(PV8000),then the relationship between and FAM83B expression with clinical pathological characteristics,and differences between EGFR wild type and common EGFR mutations were analyzed.The correlation between FAM83B expression and clinicopathological factors and prognosis in lung adenocarcinoma was analyzed using the databases.Construct stable H1299 and PC9/GR cell lines respectively using the lentivirus overexpression vector(GV492/FAM83B cDNA)and lentivirus interference vector(GV248/shRNA),The effects of FAM83B on the migration and invasion of lung cancer cells were detected by WB,transwell assay,scratch test and tumor implantations experiment.The expression of MMP-9,TIMP-1,p-Akt/Akt in the overexpressed FAM83B cell line was detected by WB,and the possible mechanism of promoting the invasion and metastasis of lung adenocarcinoma was discussed.Result:(1)At the tissue level,our immunohistochemical results showed that the positive rate of FAM83B was 23.80%in primary group and 68.80%in metastatic group,the different expression of FAM83B in the metastasis group and the primary group was statistically significant(P<0.0001).In addition,the differences in expression of FAM83B between EGFR WT group,19Del group and L858R group were also analyzed,the results showed that the positive rate of FAM83B was 53.12%in WT group and 37.93%in 19del group,the difference was statistically significant(P<0.05),but there was no significant difference between L858R group and 19del group,nor do there any difference between WT group and L858R group(P>0.05)(2)At TCGA database,FAM83B was highly expressed in patients with lung adenocarcinoma than that in surrounding normal tissues and in lung adenocarcinoma,the expression level of FAM83B was higher in patients with high lymph node stage than that in the group without lymph node involvement and the group with low lymph node stage,similarly,the higher the stage of lung adenocarcinoma,the higher its expression rate;survival analysis,that survival rate of patients in high expression group was significantly lower than low expression group.(3)At the cell level,Construct the lentivirus vector for over-expression and interference of FAM83B gene,then establish lung adenocarcinoma cell line stably expressing FAM83B(FAM83B Over Expression,OE)and silencing FAM83B(silencing RNA,FAM83B siRNA),scratch test observe that the cell migration rate of H1299/FAM83B OE group was about 71.72±2.742%,while that of the control group was about 28.56±4.930%,the difference was statistically significant(P<0.05);the cell migration rate of PC9/GR FAM83B siRNA group was about 48.59±2.849%,while that of the control group was about 77.50±2.660%,the difference was statistically significant(P<0.05).Transwell assay observe that there were 191.7±6.94 cells in FAM83B OE group and 129.0±8.7 in control group,which were statistically significant(P<0.05);191.7±13.53 cells in PC9/GR FAM83B siRNA group and 384.3±47.60 cells in control group,which were also statistically significant(P<0.01).The tumor formation experiments in nude mice showed that the growth rate of tumor in FAM83B OE group was significantly stronger than that in the control group(P<0.001),and OE group had capsule invasion.In conclusion,the overexpression of FAM83B can promote the invasion and migration of lung adenocarcinoma cell lines.At the protein molecular level,the ratio of p-AKT/AKT and the expression level of TIMP-1 in FAM83B OE group were higher than those in the control group,the difference was statistically significant(P<0.05,P<0.01).Conclusions:(1)FAM83B is one of the markets of poor prognosis in lung adenocarcinoma,and the lung adenocarcinoma with high expression of fam83b is not sensitive to EGFR-TKIs,or more susceptible.(2)High expression of FAM83B can promote migration,invasion and other malignant phenotypes.(3)Overexpression of FAM83B can increase the ratio of p-AKT/AKT and the expression level of TIMP-1.Therefore,it is speculated that FAM83B can increase in the expression of TIMP-1 by activating PI3K/AKT/NF-κB,thus promoting the invasion and metastasis of lung adenocarcinoma cells.更多还原