摘要
目的：
环指蛋白128 (RNF128)是E3泛素连接酶家族成员之一。其功能的研究主要集中在其对免疫效应T细胞功能的影响，目前已经证实其在平衡外周免疫耐受性和自身免疫疾病方面发挥着关键作用。最新的研究提示其异常表达可能参与肿瘤发生发展。但到目前为止，其对非小细胞肺癌临床进程的影响及其潜在的调节机制仍不清楚。本研究的主要目的是探讨RNF128在NSCLC中表达及与患者预后，并初步探讨其促进NSCLC进展的机制。 资本运营材料和方法：
通过免疫组化、荧光定量PCR和Western blot测定NSCLC组织和相应癌旁组织中RNF128的RNA和蛋白的表达水平。使用208例非小细胞肺癌组织微阵列( TMAs )的方法来评估RNF128表达，结合临床病理与患者随访资料分析RNF128表达与患者临床病理特征及预后的关系；调节RNF128表达后研究其表达与NSCLC细胞侵袭、移动、克隆形成和增殖的关系。Transwell实验与划痕实验被用来评估细胞的侵袭能力。 结果：
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NSCLC组织中RNF128 mRNA的表达高于癌旁组织（2.60±1.25 vs 0.50±0.02），NSCLC组织中RNF128蛋白表达也高于相应癌旁组织（6.25±0.99 vs 3.77±0.55）。免疫组织化学检测组织芯片中RNF128表达后分析显示，RNF128在NSCLC组织中的表达异质性明显，大部分癌组织RNF128表达高于相应癌旁组织；根据RNF128免疫染强度和阳性细胞所占比，NSCLC组织分为RNF128低表达组（52%，108/208）与RNF128高表达组（48%，100/208），结合患者临床病理特征分析显示，RNF128的表达与NSCLC恶性临床病理特征显著相关，如淋巴结转移( P =1.16×10-4)和进展期肿瘤( P=0.012 )，而结合预后分析证实RNF128高表达组的OS明显低于RNF128低表达组( 34.1 % vs 65.9 %，P =1.5×10-5 )。单因素分析显示分化差、淋巴结转移、晚期TNM分期和RNF128过度表达是非小细胞肺癌OS差的显著指标。在多因素分析中，RNF128表达
( HR，1.658；95% CI，1.131 - 2.432；P=0.01 )仍然与5年OS显著相关。细胞中干扰RNF128表达后，细胞侵袭、移动、克隆形成及增殖明显被抑制。
结论：
RNF128高表达促进NSCLC侵袭转移，其表达可能是NSCLC的一个新的预后预测指标及潜在的靶点。
营造地表形态的力量
关键词：RNF128；NSCLC；预后；侵袭转移
ABSTRACT
Objectives：
Ring finger protein 128 (RNF128) is a member of the E3 ubiquitin ligase family. Previous studies have focused on its effects on immunological effect T-cell function, which has been shown to play a key role in balancing peripheral immune tolerance and autoimmune diseases. Recent research suggested that the abnormal expression of RNF128 might be involved in tumorigenesis. So far, its impact on non-small cell lung cancer and its potential mechanisms remains unclear. The main purpose of this study was to investigate the expression of RNF128 in NSCLC and its prognosis, and to explore its mechanism to promote the progression of NSCLC.
Materials and Methods:
The expression levels of RNF128 RNA and protein in NSCLC tissues and corresponding adjacent tissues were determined by immunohistochemistry, real-time PCR and Western blot. 208 cases of non-small cell lung cancer tissue microarrays (TMAs) were used to evaluate the expression of RNF128, and the relationship between RNF128 expression and clinicopathological features and prognosis was analyzed by clinical pathology and patient follow-up data. The expression of RNF128
was regulated to investigate the relationship between its expression and invasion, migration, colony formation and proliferation of NSCLC cells. Transwell experiments and wound healing experiments were used to assess the invasiveness of cells. Results:
The expression of RNF128 mRNA in NSCLC tissues was higher than that in adjacent tissues (2.60 ± 1.25 vs. 0.50 ± 0.02). The expression of RNF128 protein in NSCLC tissues was also higher than that in adjacent tissues (6.25 ± 0.99 vs. 3.77 ±0.55). Immunohistochemistry showed that the expression of RNF128 in tissue microarray showed that the expression heterogeneity of RNF128 in NSCLC tissues was obvious, and the expression of RNF128 in most cancer tissues was higher than农产品网络营销策略
that in adjacent tissues. According to the ratio of RNF128 immunostaining intensity to positive cells, NSCLC tissues were divided into RNF128 low expression group (52%, 108/208) and RNF128 high expression group (48%, 100/208). Combined with the clinicopathological features of patients, the expression of RNF128 was significantly correlated with the malignant clinicopathological features of NSCLC, such as lymph node metastasis (P=1.16 ×10-4) and advanced tumor (P=0.012). Combined with prognostic analysis, the OS of RNF128 high expression group was significantly lower than that of RNF128 low expression group (34.1% vs. 65.9 %, P = 1.5 ×10-5). Univariate analysis showed that poor differentiation, lymph node metastasis, advanced TNM stage, and RNF128 overexpression wer
e significant indicators of poor OS in non-small cell lung cancer. In multivariate analysis, RNF128 expression (HR, 1.658; 95% CI, 1.131-2.432; P = 0.01) remained significantly associated with 5-year OS. After cell interference with RNF128 expression, cell invasion, migration, colony formation and proliferation were significantly inhibited. Conclusions:
High expression of RNF128 promotes invasion and metastasis of NSCLC, and its expression may be a new prognostic indicator and potential therapeutic target for NSCLC.
Key Words: RNF128；NSCLC；Prognosis；Metastasis and invasion
目录
第1章引言 (1)
第2章材料与方法 (3)
2.1 材料 (3)
2.1.1 实验对象 (3)
2.1.2 临床病理资料 (3)
2.1.3实验仪器及试剂 (4)
2.2 实验方法 (6)
2.2.1 随访资料搜集 (6)
2.2.2细胞的复苏及培养 (6)
2.2.3 免疫组化 (7)
2.2.4 Western blot (8)
2.2.5组织和细胞RNA提取，反转录，荧光定量PCR (8)日本参议院选举
星火计划2.2.6 CCK8 法检测细胞增殖 (10)
2.2.7克隆形成实验 (10)
2.2.8细胞划痕实验 (10)
2.2.9Transwell 实验 (11)
2.3 TMA及免疫组织化学染结果判定 (11)
2.4 统计分析 (11)
第3章结果 (12)
3.1 RNF128在NSCLC组织中表达与临床病理因素的关系 (12)
3.2 RNF128在非小细胞肺癌中高度表达 (14)
3.3 RNF128表达与非小细胞肺癌临床病理特征的相关性 (15)
3.4 RNF128参与非小细胞肺癌细胞的增殖 (17)
3.5 RNF128参与了非小细胞肺癌细胞的侵袭 (19)
第4章讨论 (20)
第5章结论 (23)
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