p53诱导的蛋白磷酸酶1（Wip1）表达特性与非小细胞肺癌的相关性研究

发布时间：2018-06-17 20:58

本文选题：p53诱导的蛋白磷酸酶1 + 非小细胞肺癌　；参考：《河北医科大学》2016年博士论文

【摘要】：肺癌已经成为癌症相关病死率最高的恶性肿瘤,2012年赫捷院士报道了中国排名前十位的肿瘤发病率和死亡率,肺癌无论从发病率还是死亡率来讲,都是名列第一位,其中80%以上病例为非小细胞肺癌(non-small cell lung cancer,NSCLC),确诊为NSCLC时,尽管近来年采用了多种综合预防和治疗手段,仍然难以显著降低其发病率和改善其生存率,肺癌的5年生存率约为15%,因此,系统的研究NSCLC的发生、发展机制,筛选有效的治疗靶点和治疗药物,一直是肿瘤研究中面临的巨大挑战。作为抑癌基因p53的负性调控因子,p53诱导的蛋白磷酸酶1(Wild-type p53-induced phosphatase 1,Wip1)位于人染色体17q23/q24区域,属于丝氨酸/苏氨酸蛋白磷酸酶PP2C(PP2C type protein phosphatase)家族成员,由PPM1D编码,人的Wip1蛋白分子量约为61k D,由605个氨基酸组成,能够通过依赖于p53蛋白途径和非依赖于p53蛋白途径,促进肿瘤进展。近年来,肺癌、乳腺癌、胰腺癌、膀胱、肝、脑膜、和神经母细胞瘤细胞系中发现了Wip1的扩增。此外,许多人类原发性肿瘤(如神经母细胞瘤、卵巢透明细胞腺癌、乳腺癌、胰腺腺癌)包含扩增的Wip1基因和高表达的Wip1蛋白质,并且显示出与癌症患者预后不佳有关。Naoyuki Satoh等发现原发性肺腺癌患者,Wip1在肿瘤上皮细胞表达的增加,与肿瘤进展和临床预后具有重要意义。因此本研究拟观察Wip1在NSCLC中表达的临床病理意义和其判断预后的价值,并分析Wip1具体作用机制,为将其作为一个NSCLC候选治疗靶点提供进一步的理论和实验依据。第一部分p53诱导的蛋白磷酸酶1(Wip1)在非小细胞肺癌中表达的临床病理意义及其预后价值分析目的:研究Wip1在非小细胞肺癌中表达的临床病理意义,并分析其预后价值。方法:收集河北省胸科医院2001年-2010年期间117例NSCLC患者临床样本,均具有完整的随访资料,10例来自于支气管扩张等手术中切除的正常肺组织及癌旁肺组织作为对照组。采用免疫组织化学方法,检测Wip1在正常肺组织和非小细胞肺癌组织中的表达。采用SPSS 13.0统计软件进行数据分析,应用Pearsonχ2检验和t检验分析其表达的临床病理意义。使用Kaplan-Meier法绘制生存曲线并计算生存率,单因素分析应用Log rank检验进行相关性判断,多因素分析使用Cox比例风险模型,采用逐步回归分析进行危险因素判断。以α=0.05为检验水平,以P0.05为差异具有统计学意义。结果:1 Wip1阳性表达定位于胞浆和细胞核,呈浅黄色、棕黄色或黄褐色。在非小细胞肺癌组织中阳性表达率为69.2%(81/117),与正常肺组织中Wip1蛋白的阳性表达率0.00%(0/10)比较具有统计学差异(χ2=19.114,P=0.000)。2 Wip1阳性表达组年龄与对照组比较无统计学意义(P=0.268)。非小细胞肺癌组织Wip1蛋白阳性表达组中,男性占70.4%(57/81),女性占29.6%(24/81),Wip1蛋白阴性表达组中,男性占83.3%(30/36),女性占16.7%(6/36),采用χ2检验差异无统计学意义(χ2=2.197,P=0.138)。Wip1在肺腺癌组织中的阳性表达率为88.7%(47/53),在鳞状细胞癌中的表达率为56.8%(25/44),在其他类型非小细胞肺癌中的表达率为45%(9/20),采用χ2检验进行统计,发现肺腺癌中Wip1表达高于鳞状细胞癌和其他类型非小细胞肺癌(χ2=18.106,P0.001)在非小细胞肺癌组织Wip1蛋白阳性表达组中,病理分化程度高分化组占29.6%(24/81),中低分化组占70.4%(57/81),Wip1蛋白阴性表达组中,高分化组占19.4%(7/36),中低分化组占80.6%(29/36),采用χ2检验进行统计,非小细胞肺癌组织中Wip1蛋白表达与分化程度之间无相关性,差异无统计学意义(χ2=1.328,P=0.249)。Wip1蛋白阳性表达组中,肿瘤大小≤3 cm病例占14.8%(12/81),肿瘤大小3 cm病例占85.2%(69/81),Wip1蛋白阴性表达组中,肿瘤大小≤3 cm占38.9%(14/36),肿瘤体积3 cm病例占61.1%(22/36),采用χ2检验进行统计,发现非小细胞肺癌组织中Wip1蛋白表达与肿瘤大小相关,差异有统计学意义(χ2=8.357,P=0.004)。Wip1蛋白阳性表达组中,淋巴结无转移病例占55.6%(45/81),淋巴结转移病例占44.4%(36/81),Wip1蛋白阴性表达组中,淋巴结无转移病例占69.4%(25/36),淋巴结转移病例占30.6%(11/36),采用χ2检验进行统计,发现非小细胞肺癌组织中Wip1蛋白表达与淋巴结转移不相关,差异无统计学意义(χ2=2.000,P=0.157)。非小细胞肺癌Wip1蛋白阳性表达组织中,临床分期I-II期占55.6%(45/81),III-IV期占44.4%(36/81),Wip1蛋白阴性表达组中,临床分期I-II期占75.0%(27/36),III-IV期占25.0%(9/36),采用χ2检验进行统计,发现非小细胞肺癌组织中Wip1蛋白表达与临床分期相关,差异有统计学意义(χ2=3.98,P=0.046)。3单因素Log-rank检验分析显示Wip1表达与患者预后差相关,Wip1阳性表达组总体生存率低于Wip1阴性表达组。Cox回归模型分析显示高Wip1表达相对危险度为5.096(95%可信区间=1.501-17.303),临床分期相对危险度为0.159(95%可信区间=0.037-0.680),均具有统计学差异。小结:1 Wip1在NSCLC组织中高表达。2 Wip1表达水平与非小细胞肺癌病理分型、肿瘤大小、临床分期关系密切,而与患者的年龄、性别、组织分化程度、淋巴结转移无关。3 Wip1阳性表达组总体生存期低于Wip1阴性表达组,其高表达是预后不良的高风险因子。第二部分Wip1-si RNA对非小细胞肺癌细胞增殖的影响目的:研究基因沉默Wip1对NSCLC A549细胞、NCI-H1299细胞增殖的抑制作用及其调节机制。方法:研究采用RNA干扰技术基因沉默Wip1。采用real-time PCR技术和Western blot技术从三个设计的si RNA中筛选出最有效的si RNA。通过MTT技术观察细胞增殖情况,流式细胞术观察细胞周期,并应用Western blot检测细胞内增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、P21及P27的表达。结果:1三种Wip1-si RNA(40 n M)转染A549细胞、NCI-H1299细胞株48h,对Wip1的表达均具有一定的沉默作用,其中Wip1-si RNA-3的沉默效率最高,达到90%以上;而空白对照组及阴性转染组Wip1 m RNA及蛋白表达水平无明显变化。2与阴性转染组细胞相比,基因沉默Wip1对非小细胞肺癌细胞A549、NCI-H1299的细胞增殖具有显著的抑制作用,明显高于Wip1-si RNA转染组,差异具有统计学意义(P0.05);而空白对照组及阴性转染组细胞之间增殖率无明显差异。3敲低Wip1对非小细胞肺癌细胞A549、NCI-H1299的细胞周期具有显著作用,表现为S期的细胞明显减少,而处于G0/G1期的细胞显著增加。4 Wip1-si RNA转染A549、NCI-H1299细胞48 h后,与Non-Target-si RNA和对照组细胞相比,PCNA的蛋白表达明显下调,而P21、P27的蛋白表达显著升高(P0.05)。小结:1设计的Wip1-si RNA能够特异、有效的抑制Wip1 m RNA和蛋白的表达水平。2 Wip1-si RNA能够显著抑制NSCLC细胞株A549和NCI-H1299细胞的增殖;3 Wip1-si RNA能够显著抑制NSCLC细胞株A549和NCI-H1299细胞G1→S的转换;4 Wip1-si RNA能够显著抑制NSCLC细胞株A549和NCI-H1299细胞PCNA蛋白表达水平,促进抑癌基因p21和p27的蛋白表达水平。第三部分Wip1-si RNA对与非小细胞肺癌细胞迁移、侵袭行为的影响目的:探讨Wip-si RNA敲低Wip基因表达对非小细胞肺癌细胞迁移和侵袭能力的影响。方法:本部分研究采用RNA干扰技术敲低肺腺癌细胞A549、非小细胞肺癌NCI-H1299细胞Wip1的表达,通过细胞划痕和Transwell实验观察肿瘤细胞的迁移和侵袭能力,并应用Western blot方法检测基质金属蛋白酶(matrix metalloprotease,MMP)-2、MMP-9和组织金属蛋白酶抑制剂(tissue inhibitor of metalloproteinase,TIMP)-2蛋白表达情况。结果:1 Wip1-si RNA(40 n M)转染A549、NCI-H1299细胞48 h后,与non-Target-si RNA及对照组相比,其迁移到划痕区域的细胞数目明显减少(P0.05)。non-target si RNA组与空白对照组相比,其侵袭率无统计学差异(P0.05)。2与non-Target-si RNA组相比,Wip1-si RNA(40 n M)转染A549、NCI-H1299细胞48 h后,与non-target si RNA及空白对照组相比,Wip1敲低的A549和NCI-H1299细胞,其侵袭到膜下的细胞数目明显减少(P0.05)。non-Target-si RNA组与对照组相比,其侵袭抑制率无统计学差异。3将40 n M的Wip1-si RNA转染A549、NCI-H1299细胞48 h后,Non-target-si RNA组相比,MMP2、MMP9蛋白表达明显下调,而TIMP2蛋白表达显著增加,non-Target-si RNA组与对照组相比无明显差异。小结:1 Wip1敲低显著抑制A549和NCI-H1299细胞的迁移能力。2 Wip1敲低显著抑制A549和NCI-H1299细胞的侵袭能力。3 A549和NCI-H1299细胞中敲低Wip1,可以下调MMP2和MMP9的蛋白表达,促进TIMP2的蛋白表达。结论:1 Wip1在人非小细胞肺癌组织中高表达,与肿瘤病理分型、p T分期、临床分期及预后相关,可能参与了对NSCLC发生、发展的调控,其可做为判断NSCLC患者预后的一个较好的指标。2基因沉默Wip1能够通过下调PCNA,上调p21和p27蛋白表达,抑制了G1/S期转换,从而抑制了NSCLC细胞增殖。3基因沉默Wip1能够通过下调MMP2、MMP9,上调TIMP2的表达,从而抑制了NSCLC细胞的迁移和侵袭。
[Abstract]:Lung cancer has become the most fatal malignant tumor associated with cancer. In 2012, the academician reported the incidence and mortality of the top ten in China. Lung cancer ranked first in terms of morbidity and mortality, and more than 80% of them were non small cell lung cancer (non-small cell lung cancer, NSCLC), and NSCL was diagnosed as NSCL. C, although a variety of comprehensive prevention and treatment methods have been adopted in recent years, it is still difficult to significantly reduce its incidence and improve its survival rate. The 5 year survival rate of lung cancer is about 15%. Therefore, systematic research on the development of NSCLC, the mechanism of development, the screening of effective therapeutic targets and the treatment of drugs have been a great challenge in cancer research. The negative regulatory factor of the tumor suppressor gene p53, p53 induced protein phosphatase 1 (Wild-type p53-induced phosphatase 1, Wip1), is located in the 17q23/q24 region of the human chromosome, belonging to the serine / threonine protein phosphatase PP2C (PP2C type protein phosphatase) family. In recent years, the amplification of Wip1 has been found in lung, breast, pancreatic, bladder, liver, meningeal, and neuroblastoma cells in lung cancer, breast, liver, and neuroblastoma cell lines. In addition, many human primary swelling tumors (such as neuroblastoma, ovarian clear cell adenocarcinoma, breast cancer, pancreas) Adenocarcinoma) contains the amplified Wip1 gene and the high expression of Wip1 protein, and shows that the expression of primary lung adenocarcinoma in patients with.Naoyuki Satoh is not associated with the prognosis of cancer patients. The increase in the expression of Wip1 in the epithelial cells of the tumor is important to the progression of the tumor and the prognosis of the cancer. Therefore, this study is to observe the expression of Wip1 in NSCLC. The value of clinicopathological significance and its judgement of prognosis, and analysis of the specific mechanism of Wip1 to provide further theoretical and experimental basis for the targeting of NSCLC as a candidate for treatment. The clinical pathological significance and prognostic value of p53 induced protein phosphatase 1 (Wip1) in non small cell lung cancer and its prognostic value: study Wip 1 the clinicopathological significance of expression in non-small cell lung cancer and its prognostic value. Methods: the clinical samples of 117 patients with NSCLC in Hebei thoracic hospital during -2010 2001 were collected, all of which had complete follow-up data, 10 cases of normal lung tissue and paracancerous lung tissue removed from bronchiectasis were used as control group. Immunohistochemical method was used to detect the expression of Wip1 in normal lung tissues and non-small cell lung cancer tissues. The clinical and pathological significance of the expression was analyzed by SPSS 13 statistical software and Pearson chi 2 test and t test. The survival curve was plotted by Kaplan-Meier method and the survival rate was calculated. The single factor analysis was applied to Log rank examination. The Cox proportional hazard model was used to judge the risk factors using the stepwise regression analysis. The level of alpha =0.05 as the test level and the difference of P0.05 were statistically significant. Results: the positive expression of 1 Wip1 was located in the cytoplasm and nucleus, in the light yellow color, brown or yellow brown. The positive expression rate was 69.2% (81/117), and the positive expression rate of Wip1 protein in normal lung tissues was compared with 0% (0/10) (x 2=19.114, P=0.000). The age of.2 Wip1 positive expression group was not statistically significant (P=0.268). 70.4% (57/81) in the positive group of non small cell lung cancer, the male was 70.4% (57/81), female. In the 29.6% (24/81), 83.3% (30/36) and 16.7% (6/36) in the negative expression group of Wip1 protein, the positive rate of.Wip1 in lung adenocarcinoma was 88.7% (47/53), and the expression rate in squamous cell carcinoma was 56.8% (25/44), and the table in other types of non-small cell lung cancer was found to be 88.7% (47/53). The rate of arrival was 45% (9/20), and the statistics showed that the expression of Wip1 in lung adenocarcinoma was higher than that of squamous cell carcinoma and other types of non small cell lung cancer (x 2=18.106, P0.001) in the positive expression group of Wip1 protein in non small cell lung cancer, the high differentiation group was 29.6% (24/81), the middle and low differentiation group was 70.4% (57/81), and the Wip1 protein was negative. In the expression group, the high differentiation group was 19.4% (7/36), the middle and low differentiation group accounted for 80.6% (29/36), and the Wip1 protein expression in the non small cell lung cancer tissues was not correlated with the degree of differentiation, the difference was not statistically significant (x 2=1.328, P=0.249).Wip1 positive expression group, the tumor size less than 3 cm cases accounted for 14.8% (12/81), the tumor was large. 3 cm cases accounted for 85.2% (69/81), Wip1 protein negative expression group, tumor size less than 3 cm accounted for 38.9% (14/36), tumor volume 3 cm cases accounted for 61.1% (22/36). Chi chi 2 test was used to determine the expression of Wip1 protein in non small cell lung tissues related to tumor size, the difference was statistically significant (x 2=8.357, P=0.004).Wip1 protein expression group 55.6% (45/81), 44.4% (36/81) of lymph node metastases, 69.4% (25/36) in Wip1 negative expression group, 30.6% (11/36) in lymph node metastases (11/36), and there was no correlation between the expression of Wip1 protein and lymph node metastasis in non small cell lung cancer. There was no statistical significance (x 2=2.000, P=0.157). In the Wip1 protein positive tissues of non small cell lung cancer, the clinical stage I-II period accounted for 55.6% (45/81), III-IV period accounted for 44.4% (36/81), Wip1 protein negative expression group, clinical stage I-II period 75% (27/36), III-IV phase 25% (9/36), the use of chi 2 test for statistics, found non-small cell lung cancer tissue The expression of P1 protein was correlated with clinical staging. The difference was statistically significant (x 2=3.98, P=0.046).3 single factor Log-rank test analysis showed that the expression of Wip1 was related to the prognosis of patients, and the overall survival rate of Wip1 positive expression group was lower than that of Wip1 negative expression group.Cox regression model analysis showed that the relative risk of high Wip1 expression was 5.096 (95% confidence interval =1.501-17.) 303) the relative risk of clinical staging was 0.159 (95% confidence interval =0.037-0.680), and there were statistical differences. Conclusion: the high expression of.2 Wip1 in NSCLC tissues in 1 Wip1 was closely related to the pathological classification of non-small cell lung cancer, the size of the tumor, and the clinical stage, but it was not related to the age, sex, the degree of differentiation of the tissue and the lymph node metastasis of the patient's.3 Wi. The overall survival time of P1 positive expression group was lower than that of Wip1 negative expression group, and its high expression was a high risk factor for poor prognosis. Second part of the effect of Wip1-si RNA on the proliferation of non-small cell lung cancer cells: the study of the inhibitory effect of gene silencing Wip1 on NSCLC A549 cells and NCI-H1299 cell proliferation and its regulation mechanism. Methods: the study was conducted using RNA dry. Wip1. real-time PCR technology and Western blot technology were used to screen the most effective Si RNA. from three designed Si RNA to observe cell proliferation by MTT technology. Flow cytometry was used to observe cell cycle, and cell proliferation cell nuclear antigen was detected by Western blot. The expression of P21 and P27. Results: 1 three kinds of Wip1-si RNA (40 n M) transfected A549 cells, NCI-H1299 cell line 48h, the expression of Wip1 has a certain silence effect, and Wip1-si RNA-3 has the highest silence efficiency, reaching more than 90%, but there is no significant change in the blank control group and negative transfection group. Compared with the cells, gene silencing Wip1 had a significant inhibitory effect on the proliferation of A549 and NCI-H1299 cells in non small cell lung cancer cells, obviously higher than that of Wip1-si RNA transfection group, the difference was statistically significant (P0.05), but the proliferation rate between the blank control group and the negative transfected group had no significant difference between.3 knockout and low Wip1 against non small cell lung cancer cells A549, NC. The cell cycle of I-H1299 had a significant effect, showing that the cells in the S phase decreased significantly, while the cells in the G0/G1 phase significantly increased the.4 Wip1-si RNA transfection to A549. After NCI-H1299 cells 48 h, the protein expression of PCNA was significantly down compared with Non-Target-si RNA and the control group. Wip1-si RNA can inhibit the proliferation of A549 and NCI-H1299 cells of NSCLC cell lines, which can inhibit the proliferation of NSCLC cell lines A549 and NCI-H1299 cells effectively, and the expression level of Wip1 m RNA and protein can significantly inhibit the proliferation of A549 and NCI-H1299 cells in NSCLC cell lines. The expression level of PCNA protein, promoting the protein expression level of tumor suppressor gene p21 and p27. Third the effect of Wip1-si RNA on the migration and invasion of non small cell lung cancer cells: To explore the effect of Wip-si RNA knocking low Wip gene expression on the migration and invasiveness of non-small cell lung cancer cells. Methods: this part of this study uses RNA interference. The technique knocks low lung adenocarcinoma cell A549 and the expression of Wip1 in non small cell lung cancer NCI-H1299 cells. The migration and invasion ability of tumor cells are observed by cell scratch and Transwell test, and Western blot method is used to detect matrix metalloproteinases (matrix metalloprotease, MMP) -2, MMP-9 and tissue inhibitor of metalloproteinase (tissue) Of metalloproteinase, TIMP) -2 protein expression. Results: 1 Wip1-si RNA (40 n M) transfected A549, NCI-H1299 cells 48 h, compared with non-Target-si and control group, the number of cells migrated to the scratch area significantly decreased. Compared with the arget-si RNA group, Wip1-si RNA (40 n M) transfected A549, NCI-H1299 cells after 48 h, compared with non-target Si RNA and blank control group, the number of cells invaded under the membrane decreased significantly. Ip1-si RNA transfected A549, NCI-H1299 cells 48 h, Non-target-si RNA group compared, MMP2, MMP9 protein expression obviously down, and TIMP2 protein expression significantly increased, non-Target-si RNA group compared with the control group, no significant difference. The invasive ability of 99 cells in.3 A549 and NCI-H1299 cells knocks low Wip1, which can reduce the protein expression of MMP2 and MMP9 and promote the protein expression of TIMP2. Conclusion: the high expression of 1 Wip1 in human non-small cell lung cancer tissues is related to the pathological classification of tumor, P T staging, clinical staging and prognosis, and may be involved in the regulation of NSCLC occurrence and development, which can be done In order to judge the prognosis of NSCLC patients, a better indicator of.2 gene silencing Wip1 can inhibit the G1/S phase conversion by down regulation of PCNA, up regulation of p21 and p27 protein expression, thus inhibiting the.3 gene silencing of NSCLC cells can reduce the expression of MMP2, MMP9, and up-regulation, thus inhibiting the migration and invasion of NSCLC cells.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

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