LIF在胃癌中的表达及对胃癌细胞生物学行为的影响

发布时间：2018-06-06 01:02

本文选题：白血病抑制因子 + 胃癌　；参考：《中国人民解放军医学院》2016年博士论文

【摘要】：背景近年来随着胃癌相关诊疗技术的不断进步,胃癌患者的总体生存率得到了显著提高,但其整体治疗效果仍不十分理想。肿瘤的增殖、侵袭和转移是其重要的恶性生物学行为,从根本上寻找肿瘤发生、发展及恶性生物学行为的调节机制,可以有效提高胃癌的治疗效果。目的研究LIF在胃癌中的表达情况与病理学参数及预后的相关性,评价其在胃癌诊断方面的价值,探索LIF对胃癌细胞增殖、侵袭、转移等生物学行为的影响,并进一步探讨具体作用机制。方法1.利用ELISA技术检测208例胃癌患者、67例慢性萎缩性胃炎患者和70例正常人LIF因子外周血表达水平。免疫组织化学染色检测128例胃癌石蜡切块LIF和LIFR在组织中的表达。通过Western blot和qRT-PCR方法检测40例胃癌新鲜组织样本及配对的癌旁正常组织中LIF和LIFR蛋白和mRNA的表达情况,并与临床病理学参数及预后进行相关性分析。2.筛选LIF低表达、LIFR高表达的胃癌细胞株(Western blot和qRT-PCR方法)。利用RNA干扰技术,借助质粒为载体,敲低胃癌细胞株中LIFR的表达。通过CCK-8、EdU和平板克隆实验检测加入外源性LIF因子及沉默LIFR基因后,对胃癌细胞增殖能力的影响。采用流式细胞技术检测LIF及沉默LIFR对胃癌细胞的细胞周期分布和凋亡的影响。采用Transwell实验观察LIF及沉默LIFR对胃癌细胞侵袭和迁移能力的影响。通过裸鼠体内成瘤模型探讨LIF及沉默LIFR后对肿瘤生长的影响。3.Western blot法和细胞免疫荧光染色技术检测LIF对Hippo-YAP信号通路中核心元件的影响及相互关系。利用siRNA沉默信号通路下游YAP蛋白表达后,通过CCK-8实验检测LIF是否主要通过Hippo-YAP信号对胃癌细胞增殖产生影响。结果1.胃癌患者、慢性萎缩性胃炎患者、正常人外周血LIF水平分别为8.650±6.282 pg/ml、4.325±1.824 pg/ml、3.344±1.918 pg/ml。胃癌患者LIF水平明显高于慢性萎缩性胃炎患者(P=0.003)和正常人(P0.001)。LIF诊断胃癌的界值为5.585 pg/ml,诊断敏感度：88.14%,特异度：81.82%,准确度：85.64%。免疫组化结果显示：LIF和LIFR在胃癌中的阳性表达率明显高于正常粘膜(P0.001),与肿瘤分化程度(P=0.021)、脉管浸润(P=0.001)、T分期(P=0.001)、淋巴结转移(P=0.001)及pTNM分期(P=0.012)存在相关性。相同肿瘤组织中LIF与LIFR的蛋白表达量及mRNA含量呈正性相关(P0.001,P0.001)。利用Cox回归多因素生存模型分析患者的预后相关因素发现LIF及LIFR与预后紧密相关,是独立的预后风险因素(P=0.009, P=0.034)。 2.MGC803细胞在几种常见胃癌细胞系中LIF的表达量最低,LIFR的表达量最高。加入外源性LIF可以显著增强细胞的增殖、侵袭、迁移、抗凋亡能力和裸鼠体内成瘤速度(P0.001)。沉默LIFR后,与阴性对照组相比,细胞的上述恶性生物学行为能力被明显减弱,且在LIFR被沉默的细胞中再次加入LIF无法发挥上述LIF的肿瘤促进作用。3.LIF可以显著降低Hippo-YAP信号通路中核心元件的磷酸化水平(P0.001),并促进YAP蛋白的核内转移。另外,LIF无法促进YAP-siRNA细胞的增殖。结论1.LIF参与了胃癌内在的发生过程,能够用于胃癌的诊断,并且与胃癌的增殖、侵袭、转移及患者预后紧密相关。2.LIF可以促进胃癌细胞的增殖、侵袭和转移等恶性生物学行为,而沉默LIFR可以抑制肿瘤的发生、发展。在LIFR表达被沉默后,再次加入LIF无法发挥其作用,提示LIF是与其受体LIFR结合,增强肿瘤的恶性生物学行为能力。3.LIF可以有效降低细胞内MST1/2、LATS1和YAP磷酸化蛋白的含量,从而抑制了Hippo-YAP通路中的核心元件,最终促进YAP的核内转移。4.LIF在胃癌的发生、进展、侵袭和转移中起到了重要作用,能够成为未来胃癌靶向治疗的重要研究方向。运用LIF抗体或沉默LIFR的表达可以成为一种新型的胃癌个体化治疗手段。
[Abstract]:Background in recent years, with the continuous progress of gastric cancer related diagnosis and treatment technology, the overall survival rate of gastric cancer patients has been significantly improved, but its overall treatment effect is still not very ideal. Tumor proliferation, invasion and metastasis are its important malignant biological behavior, fundamentally looking for the occurrence of swelling, development and malignant biological behavior of the regulator The purpose of this study is to effectively improve the therapeutic effect of gastric cancer. Objective to study the correlation between the expression of LIF in gastric cancer and the pathological parameters and prognosis, evaluate its value in the diagnosis of gastric cancer, explore the effect of LIF on the biological behavior of gastric cancer cell proliferation, invasion and metastasis, and further explore the specific mechanism of action. Method 1. using ELISA technique The expression of LIF factor LIF factor expression in 208 cases of gastric cancer, 67 cases of chronic atrophic gastritis and 70 normal people was detected. Immunohistochemical staining was used to detect the expression of LIF and LIFR in 128 cases of paraffin section of gastric cancer. The Western blot and qRT-PCR methods were used to detect the fresh tissue samples in 40 cases of gastric cancer and the LIF in the normal tissues adjacent to the cancer. The expression of LIFR protein and mRNA, and the correlation with the clinicopathological parameters and prognosis analysis.2. screening LIF low expression, LIFR high expression of gastric cancer cell line (Western blot and qRT-PCR method). Using RNA interference technique and plasmid as carrier, the expression of LIFR in low gastric cancer cell lines was knocked down by CCK-8, EdU and flat clones. The effects of exogenous LIF factor and silencing of LIFR gene on the proliferation of gastric cancer cells were measured. Flow cytometry was used to detect the effects of LIF and silent LIFR on the cell cycle distribution and apoptosis of gastric cancer cells. The effects of LIF and silent LIFR on the invasion and migration of gastric cancer cells were observed by Transwell test. The effect of LIF and silencing of LIFR on tumor growth was investigated by the tumor model..3.Western blot method and cell immunofluorescence staining technique were used to detect the influence and relationship of LIF on the core components in the Hippo-YAP signaling pathway. The CCK-8 test was used to detect LIF through the Hippo-YAP signal after the expression of the YAP protein downstream of the siRNA silent signal pathway. Results in 1. gastric cancer patients, chronic atrophic gastritis, normal human peripheral blood LIF levels were 8.650 + 6.282 pg/ml, 4.325 + 1.824 pg/ml, 3.344 + 1.918 pg/ml. gastric cancer patients were significantly higher than those of chronic atrophic gastritis (P=0.003) and normal human (P0.001).LIF for the diagnosis of gastric cancer at the boundary value of 5.585 pg/ Ml, diagnostic sensitivity: 88.14%, specificity: 81.82%, accuracy: the results of 85.64%. immunohistochemical staining showed that the positive expression rate of LIF and LIFR in gastric cancer was significantly higher than that of normal mucosa (P0.001), and the correlation with tumor differentiation (P=0.021), vascular infiltration (P=0.001), T staging (P= 0.001), lymph node metastasis (P=0.001) and pTNM stage (P=0.012). The protein expression of LIF and LIFR in the same tumor tissues was positively correlated with the content of mRNA (P0.001, P0.001). Using Cox regression multivariate survival model to analyze the prognosis related factors of patients, it was found that LIF and LIFR were closely related to prognosis, which was an independent prognostic risk factor (P=0.009, P= 0.034). 2.MGC803 cells in several common gastric cancer cell lines were associated with LI. The expression of F was the lowest and the expression of LIFR was the highest. Adding exogenous LIF could significantly enhance cell proliferation, invasion, migration, anti apoptotic ability and tumorigenic speed (P0.001) in nude mice. After silent LIFR, the above malignant biological behavior of cells was significantly weakened compared with the negative control group, and the cells were added again in the silent cells of LIFR. LIF can not play the tumor promoting effect of the above LIF,.3.LIF can significantly reduce the phosphorylation level of the core components in the Hippo-YAP signaling pathway (P0.001), and promote the intra nuclear transfer of YAP protein. Moreover, LIF can not promote the proliferation of YAP-siRNA cells. Conclusion 1.LIF is involved in the pathogenesis of gastric cancer, and can be used in the diagnosis of gastric cancer, and It is closely related to the proliferation, invasion, metastasis and prognosis of gastric cancer..2.LIF can promote the proliferation, invasion and metastasis of gastric cancer cells, while silence LIFR can inhibit the occurrence and development of tumor. After the expression of LIFR is silenced, LIF can not play its role again, suggesting that LIF is combined with its receptor LIFR to enhance the tumor. The malignant biological behavior.3.LIF can effectively reduce the content of MST1/2, LATS1 and YAP phosphorylated proteins in the cells, thus inhibiting the core elements in the Hippo-YAP pathway, and ultimately promoting the metastasis of YAP in the nuclear transfer of.4.LIF in the occurrence, progression, invasion and metastasis of gastric cancer, which can become a heavy target for the target treatment of gastric cancer in the future. The use of LIF antibody or silencing LIFR expression can become a new personalized treatment for gastric cancer.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

【相似文献】