HDGF基因在子宫内膜癌中的功能和分子机制研究

发布时间：2018-06-25 13:10

本文选题：HDGF + 子宫内膜癌　；参考：《南方医科大学》2014年硕士论文

【摘要】：研究背景与目的子宫内膜癌是妇科常见的恶性肿瘤之一,且随着经济社会发展,发病率在全世界范围内呈上升趋势。与其他腺癌的发展一样,子宫内膜癌的发展经过一系列复杂变化,包括从不典型增生到高分化癌。在临床上,年龄、肥胖、不孕、长期高雌激素水平等都可能成为子宫内膜癌发生的危险因素,而长期暴露于高雌激素水平被认为是最危险的因素。子宫内膜癌的分类方法很多种,目前根据临床症状及病理类型把子宫内膜癌分为两种类型,即Ⅰ型和Ⅱ型。第一种类型,Ⅰ型子宫内膜癌即雌激素依赖型子宫内膜癌,常发生于围绝经期妇女,尤其是子宫内膜长期处于高雌激素水平刺激而无孕激素抵抗的妇女,在白人妇女尤为常见。这种类型的子宫内膜癌病理类型类似正常子宫内膜。Ⅱ型子宫内膜癌,即非雌激素依赖型,发病与雌激素无明显关系。此类型常发生于老年、绝经后的妇女,常见于非裔美国人。这类子宫内膜癌的病理形态属少见类型,如子宫内膜浆液性乳头癌、透明细胞癌等,且肿瘤分化差,恶性程度高,常侵润到子宫肌层深部,预后不良。子宫内膜癌的发生、发展是多种因素参与、多步骤的复杂过程,多数学者认为是下列因素相互作用的结果：激素的调节,基因突变,粘附分子,细胞凋亡等。若子宫内膜癌患者在早期诊断并经正规治疗,5年生存率为80%。但不是所有患者能在早期阶段发现,因此,转移是子宫内膜癌患者死亡的主要原因。然而,子宫内膜癌发生发展的分子机制尚不明确。肝癌衍生生长因子(Hepatoma-derived growth factor,HDGF)是一种肝素结合的生长因子,最初从人肝癌HuH-7细胞系无血清培养的上清液中分离得到。HDGF在胚胎组织表达最高,且与多器官如心血管系统、肝脏及肾脏等器官发育密切相关。HDGF在血管增生、内皮细胞增殖、迁移等细胞活动中发挥着重要作用。最开始认为HDGF定位于包浆,但后来证实是一个含有典型的双向核定位序列的核定位蛋白,它通过核定位序列转移到细胞核内而发挥维持细胞活性的功能。最近有研究发现,有在几种小鼠和人的癌中HDGF的表达高于癌旁组织。还有研究发现,HDGF表达与肿瘤的恶性生长有关,如增殖、侵袭及转移。因此,HDGF可能是一个与肿瘤预后相关的因子。磷脂酰肌醇3一激酶(PI3Ks),因具有通过磷酸化肌醇环的3'-OH基团的肌醇磷脂,以产生第二信使磷脂酰肌醇-3,4,5-三磷酸酯的能力(PI-3,4,5-P(3))而构成脂质激酶家族。包浆膜上的PI3K通过激活RPTK产生PI(3,4,5)P(3)和PI(3,4)P(2),这些脂质与AKT结合后转移到包膜内侧,磷酸化后并被PDKl和PDK2激活。活化的Akt调节涉及细胞存活,细胞周期和细胞生长的调节等众多功能。近年来国内外研究发现,很多肿瘤的发生发展与PI3K/AKT信号通路的异常激活相关。PI3K和Akt活性的异常增高与肿瘤细胞的血管形成、侵袭转移、抗凋亡、异常增殖等生物学行为密切相关。目前,尚无有关HDGF与子宫内膜癌的文献报道。本课题的研究目的为明确HDGF基因在子宫内膜癌中的功能,探讨其与临床参数的关系及参与的信号转导通路,为子宫内膜癌发生发展的分子机制研究提供新思路。研究内容与方法 1.HDGF基因在子宫内膜癌组织中表达及与临床参数之间的关系 (1)本研究所采用的122例子宫内膜癌、20例正常子宫内膜及22例不典型增生内膜组织切片均来自于广州医科大学(原广州医学院)第三附属医院2002-2008年间住院患者。子宫内膜癌患者在手术前均未接受化疗或放疗。患者平均年龄51.3岁(30-82岁)。临床随访时间是5至96月。所有标本均经病理检查确诊,术后根据FIGO2009对患者进行手术-病理分期。 (2)采用SP免疫组织化学方法检测122例子宫内膜癌、22例子宫内膜不典型增生、20例正常子宫内膜组织的HDGF表达情况,并分析HDGF的表达与临床参数之间的关系。 (3)由两位病理专家对所有免疫组化染色的切片进行双盲分析。HDGF蛋白表达定位于胞核,按阳性细胞个数占比例计算,其中根据阳性细胞个数比例≥80%为高表达,80%为低表达。 2.HDGF基因在子宫内膜癌中功能初步研究 (1)构建靶向HDGF的shRNA慢病毒干扰载体,包装成成熟颗粒,感染高成瘤细胞,建立稳定干扰HDGF的子宫内膜癌细胞株,以空载作为对照。 (2)以空载作为对照,利用MTT法检测干扰HDGF的子宫内膜癌细胞的增殖能力。 (3)以空载作为对照,利用平板克隆法检测干扰HDGF的子宫内膜癌细胞的生长能力。 (4)以空载作为对照,Transwell小室迁移实验检测干扰HDGF的子宫内膜癌细胞的迁移能力。 (5)以空载作为对照,Boyden小室侵袭实验检测干扰HDGF的子宫内膜癌细胞的侵袭能力。 (6)检测稳定干扰HDGF的子宫内膜癌细胞裸鼠皮下成瘤能力的改变。 3.HDGF基因介导的分子基础初步研究 (1)利用western blot检测干扰HDGF后PI3K/Akt信号通路相关基因PI3K、pPBK、pAkt、Akt的变化。 (2)Western blot检测细胞周期相关基因CDK4、CCND1、P15、P21及EMT标志物N-cadherin、Vimentin的表达。 4.统计分析所有数据均用SPSS17.0统计软件处理,当P0.05时认为差异具有统计学意义。结果 1.HDGF基因在子宫内膜癌组织中表达及与临床参数之间的关系 (1)应用免疫组织化学染色法检测122例子宫内膜癌石蜡切片,22例不典型增生内膜及20例正常子宫内膜石蜡切片的HDGF蛋白在细胞中的表达位置及过表达率。HDGF在EC组织及非肿瘤组织中均表达于胞核,在子宫内膜癌,不典型增生内膜及正常子宫内膜的高表达率分别为25.5%(31/122),13.6%(3/122),5%(1/20),差别有显著性(χ2=115.03,P0.001),子宫内膜癌组织中HDGF的表达显著高于非肿瘤组织。 (2)分析HDGF表达水平与子宫内膜癌患者临床病理参数的关系。HDGF高表达在年龄、月经状态、组织分型、淋巴转移以及肌层浸润的深度中无统计学意义,在FIGO分期差异有显著意义(P=0.032)。 (3)我们用Kaplan-Meier法及log-rank检验进行生存率的估计与生存曲线的分析,在122例子宫内膜癌患者组织中,发现HDGF的阳性表达与患者生存率呈负相关(P=0.001)。多因素分析显示,HDGF不是EC患者预后的独立影响因素(P=0.111)。 2.HDGF基因在子宫内膜癌细胞功能的初步研究 (1)抑制HDGF表达对子宫内膜癌细胞生物学特性的影响。构建靶向HDGF的ShRNA1慢病毒干扰载体,稳定干扰子宫内膜癌细胞HEC-1B和Ishikawa,由于感染效率达到90%以上,western blot检测干扰后HDGF蛋白的表达明显下降,可直接用于后续实验。阳性干扰多克隆命名为HEC-1B shHDGF、Ishikawa shHDGF,阴性对照多克隆为HEC-1B PLV-Ctr、Ishikawa PLV-Ctr. (2)MTT法检测细胞增殖情况,与空载对照细胞HEC-1B PLV-Ctr、Ishikawa PLV-Ctr相比,干扰细胞HEC-1B shHDGF、Ishikawa shHDGF生长速度明显减慢,差异有统计学意义(HEC-1B细胞株F=259.5, P0.001;Ishikawa细胞株F=38.324,P0.001) (3)平板克隆法检测细胞生长情况,与空载对照细胞HEC-1B PLV-Ctr、Ishikawa PLV-Ctr相比,干扰细胞HEC-1B shHDGF、Ishikawa shHDGF生长速度明显减慢,差异有统计学意义(HEC-1B细胞株t=3.74, P=0.02;Ishikawa细胞株t=4.9,P=0.008)。 (4)Transwell小室迁移实验,细胞培养12h后HEC-1B shHDGF、Ishikawa shHDGF穿过聚碳酸酯膜的数量少于HEC-1B PLV-Ctr、Ishikawa PLV-Ctr (HEC-IB细胞株t=7.17,P=0.002;Ishikawa细胞株t=7.89,P0.001)。提示干扰HDGF后,细胞迁移能力减弱。 (5)Boyden小室侵袭实验,与空载细胞相比,HEC-1B shHDGF、Ishikawa shHDGF穿过聚碳酸酯膜的数量明显减少,差异具有统计学意义(HEC-1B细胞株t=9.04,P0.001;Ishikawa细胞株t=8.48,P0.001),提示干扰HDGF后,细胞侵袭能力减弱。 (6)裸鼠皮下成瘤,与空载对照组细胞相比,HEC-1B shHDGF、Ishikawa shHDGF两处理组细胞的裸鼠成瘤速度显著减慢,差异有统计学意义(HEC-1BF=255.9,P0.001;Ishikawa F=216.5,P0.001)。 3. HDGF在子宫内膜癌中介导的分子机制研究 (1)采用western blot检测了干扰HDGF后PI3K/Akt信号通路相关基因P13K、pPI3K、pAkt、Akt的表达。结果显示,与空载对照组细胞相比,HEC-1B shHDGF、 Ishikawa shHDGF组的PI3K、Akt无明显变化,pPI3K、pAkt表达下调。 (2)干扰HDGF后细胞周期相关基因及EMT标志物的表达变化。结果显示,与空载对照组细胞相比,HEC-1B shHDGF、Ishikawa shHDGF组的P15、P21在干扰细胞中表达上调,CCND1、CDK4表达下降。EMT相关N-cadherin、 Vimentin表达下调。结论 1.HDGF蛋白在子宫内膜癌的核高表达率明显高于子宫内膜不典型增生及正常子宫内膜组织,且HDGF的核高表达率与子宫内膜癌病人的FIGO分期正相关,与病人的生存时间负相关。因此,HDGF很可能是一个子宫内膜癌预后不良的影响因素。 2.干扰HDGF后,子宫内膜癌细胞的增殖、迁移、侵袭能力显著减弱,进一步提示HDGF在子宫内膜癌中发挥了癌基因的功能。 3. HDGF基因通过介导PI3K/AKT信号通路影响子宫内膜癌的发生发展。
[Abstract]:Research background and purpose
Endometrial carcinoma is one of the most common malignant tumors in gynecology, and with the development of economy and society, the incidence of endometrium is rising worldwide. As with the development of other adenocarcinoma, the development of endometrial cancer has undergone a series of complex changes, including untypical hyperplasia to highly differentiated carcinoma. In clinical, age, obesity, infertility, long term high female irritable disease Hormone levels may be a risk factor for endometrial carcinogenesis, and long-term exposure to high estrogen levels is considered as the most dangerous factor. Endometrial carcinoma is classified into two types according to clinical symptoms and pathological types, i. e. type I and type II. Type I, type I uteri Membrane cancer is estrogen dependent endometrial cancer, often occurring in perimenopausal women, especially in women who have long estrogen levels in the endometrium without progestin resistance, especially in white women. This type of endometrial carcinoma is similar to normal endometrium. Type II endometrial carcinoma, that is, non estrogen This type is often found in elderly, postmenopausal women and common in African Americans. This type of endometrial carcinoma is a rare type of endometrial carcinoma, such as serous papillary carcinoma of the endometrium, clear cell carcinoma and so on, and the tumor is poorly differentiated and has a high degree of evil, often embellish into the deep myometrium of the uterus and has poor prognosis.
The development of endometrial carcinoma is a complex process of multiple factors and multiple steps. Most scholars believe that the following factors interact: hormone regulation, gene mutation, adhesion molecules, cell apoptosis and so on. If endometrial cancer patients are diagnosed early and are treated with regular therapy, the 5 year survival rate is 80%. but not all patients can be in In the early stage, metastasis is the main cause of death in endometrial cancer. However, the molecular mechanism of endometrial cancer is unclear.
Hepatoma-derived growth factor (HDGF) is a heparin binding growth factor. It was initially isolated from the serum-free supernatant of human hepatocellular carcinoma HuH-7 cell line and obtained the highest expression of.HDGF in the embryonic tissue, and it was closely related to the proliferation of multiple organs such as the cardiovascular system, the liver and kidney and other organs, and.HDGF was increased in blood vessels. Life, endothelial cell proliferation, migration and other cellular activities play an important role. First, HDGF is located in the plasma, but later proved to be a nuclear localizing protein containing a typical bi-directional nuclear location sequence, which is transferred into the nucleus by nuclear location sequence and plays the function of maintaining the cell activity. The expression of HDGF in mice and human cancers is higher than that in paracancerous tissues. Other studies have found that the expression of HDGF is associated with the malignant growth of the tumor, such as proliferation, invasion and metastasis. Therefore, HDGF may be a factor associated with the prognosis of the tumor.
The phosphatidyl inositol 3 kinase (PI3Ks) is composed of the 3'-OH group of inositol phosphorylated by the inositol phosphatidylcholine, which produces the second messenger of the phosphatidylinositol -3,4,5- three phosphate (PI-3,4,5-P (3)) and constitutes the lipid kinase family. The PI3K on the plasma membrane generates PI (3,4,5) P (3) and PI (3,4) P (2) by activating RPTK. It is transferred to the inside of the capsule and activated by PDKl and PDK2 after phosphorylation. The activation of Akt regulation involves many functions such as cell survival, cell cycle and cell growth regulation. In recent years, domestic and foreign studies have found that the occurrence and development of many tumors are associated with abnormal activation of.PI3K and Akt associated with abnormal activation of PI3K/AKT signaling pathways and tumor cells Angiogenesis, invasion and metastasis, anti apoptosis, abnormal proliferation and other biological behaviors are closely related.
At present, there is no literature about HDGF and endometrial carcinoma. The purpose of this study is to clarify the function of HDGF gene in endometrial carcinoma, to explore the relationship with the clinical parameters and the signal transduction pathway involved in the study of the molecular mechanism of the development of endometrial cancer.
Research content and method
Expression of 1.HDGF gene in endometrial carcinoma and its relationship with clinical parameters
(1) 122 cases of endometrial carcinoma, 20 cases of normal endometrium and 22 cases of atypical hyperplasia of endometrium were from 2002-2008 years of hospitalization in Third Affiliated Hospitals of Guangzhou Medical University (former Guangzhou Medical College). Patients with endometrial cancer were not treated with chemotherapy or radiotherapy before operation. The average age of the patients was 51.3 years (30-82). The clinical follow-up time was from 5 to 96 months. All the specimens were confirmed by pathological examination. Postoperative pathological stages were performed according to FIGO2009.
(2) SP immunohistochemical method was used to detect the HDGF expression of endometrial carcinoma in 122 cases, 22 cases of endometrial atypical hyperplasia and 20 cases of normal endometrium, and the relationship between the expression of HDGF and the clinical parameters was analyzed.
(3) a double blind analysis of all immunohistochemical staining by two pathological experts was used to determine the expression of.HDGF protein in the nucleus, which was calculated according to the number of positive cells, among which the number of positive cells was more than 80%, and 80% was low.
Preliminary study on the function of 2.HDGF gene in endometrial carcinoma
(1) construction of shRNA lentivirus interference carrier for target HDGF, packed into mature particles, infected high tumor cells, and established endometrial carcinoma cell lines that were stable to interfere with HDGF, and used no load as control.
(2) using no-load as control, MTT assay was used to detect the proliferation ability of endometrial cancer cells interfering with HDGF.
(3) using no-load as control, the growth ability of endometrial cancer cells interfering with HDGF was detected by plate cloning assay.
(4) with no load as a control, Transwell cell migration assay was used to detect the migration ability of HDGF endometrial cancer cells.
(5) with no load as a control, Boyden cell invasion assay was used to detect the invasion ability of HDGF endometrial cancer cells.
(6) to detect the change of subcutaneous tumor formation ability of endometrial carcinoma cells with stable HDGF interference in nude mice.
A preliminary study on the molecular basis mediated by 3.HDGF gene
(1) using Western blot to detect the changes of PI3K/Akt signaling pathway related genes PI3K, pPBK, pAkt and Akt after interfering with HDGF.
(2) Western blot was used to detect the expression of CDK4, CCND1, P15, P21 and EMT markers N-cadherin and Vimentin in cell cycle related genes.
4. statistical analysis
All data were processed by SPSS17.0 statistical software. When P0.05 was considered, the difference was statistically significant.
Result
Expression of 1.HDGF gene in endometrial carcinoma and its relationship with clinical parameters
(1) the expression and overexpression of HDGF protein in 122 cases of endometrial carcinoma, 22 cases of atypical endometrium and 20 normal endometrium paraffin sections were detected by immunohistochemical staining, and the expression rate of.HDGF was expressed in the nucleus in EC tissue and non tumor tissues, in endometrial carcinoma, atypical hyperplasia endometrium and positive. The high expression rate of normal endometrium was 25.5% (31/122), 13.6% (3/122), 5% (1/20), and the difference was significant (x 2=115.03, P0.001). The expression of HDGF in endometrial carcinoma was significantly higher than that of non tumor tissue.
(2) analysis of the relationship between the expression level of HDGF and the clinicopathological parameters of endometriosis patients, the high expression of.HDGF was not statistically significant in age, menstrual state, tissue typing, lymphatic metastasis and the depth of myometrium infiltration, and there was significant difference in FIGO staging (P=0.032).
(3) we used the Kaplan-Meier method and the log-rank test to estimate the survival rate and the survival curve. In 122 endometrium cancer patients, we found that the positive expression of HDGF was negatively correlated with the survival rate of the patients (P=0.001). Multifactor analysis showed that HDGF was not an independent influence factor (P=0.111) of EC patients.
Preliminary study on the function of 2.HDGF gene in endometrial carcinoma cells
(1) to inhibit the effect of HDGF expression on the biological characteristics of endometrial carcinoma cells. To construct the ShRNA1 lentivirus interference carrier for targeting HDGF, to stabilize the endometrial cancer cells HEC-1B and Ishikawa, because the infection efficiency reached more than 90%, and the HDGF protein was obviously decreased after the Western blot detection. The positive interference can be directly used in the follow-up experiment. The polyclonal name was HEC-1B shHDGF, Ishikawa shHDGF, negative control polyclonal HEC-1B PLV-Ctr, Ishikawa PLV-Ctr..
(2) MTT assay was used to detect cell proliferation. Compared with HEC-1B PLV-Ctr, Ishikawa PLV-Ctr, HEC-1B shHDGF and Ishikawa shHDGF growth rate were significantly slowed down, and the difference was statistically significant (HEC-1B cell strain F=259.5, P0.001; Ishikawa cell strain).
(3) cell growth was detected by flat clones. Compared with HEC-1B PLV-Ctr, Ishikawa PLV-Ctr, HEC-1B shHDGF and Ishikawa shHDGF, the growth rate of Ishikawa shHDGF was significantly slowed down, and the difference was statistically significant (t=3.74, P=0.02, Ishikawa fine cell lines).
(4) Transwell cell migration experiment, after cell culture 12h HEC-1B shHDGF, the number of Ishikawa shHDGF through polycarbonate membrane is less than HEC-1B PLV-Ctr, Ishikawa PLV-Ctr (HEC-IB cell strain t=7.17, purified cell strain).
(5) Boyden chamber invasion experiment, compared with empty cells, the number of HEC-1B shHDGF and Ishikawa shHDGF through polycarbonate membrane decreased significantly, the difference was statistically significant (HEC-1B cell line t=9.04, P0.001; Ishikawa cell line t=8.48, P0.001), suggesting that after interference HDGF, the cell invasiveness weakened.
(6) nude mice subcutaneous tumor, compared with the empty control group cells, HEC-1B shHDGF, Ishikawa shHDGF two treated group cells of the tumor growth rate of nude mice significantly slowed, the difference was statistically significant (HEC-1BF=255.9, P0.001; Ishikawa F=216.5, P0.001).
Molecular mechanism of 3. HDGF mediating in endometrial carcinoma
(1) the expression of PI3K/Akt signaling pathway related genes P13K, pPI3K, pAkt, Akt after HDGF interference was detected by Western blot. The results showed that the HEC-1B shHDGF and Ishikawa shHDGF group were not significantly changed compared with those in the empty control group.
(2) changes in the expression of cell cycle related genes and EMT markers after interference with HDGF. The results showed that the expression of P15 in HEC-1B shHDGF and Ishikawa shHDGF group was up regulation, CCND1, CDK4 expression decreased in.EMT related N-cadherin and down regulation compared with those in the empty control group.
conclusion
The high expression rate of 1.HDGF protein in endometrial carcinoma is significantly higher than that of endometrium atypical hyperplasia and normal endometrium, and the high expression of HDGF is positively related to FIGO staging in patients with endometrial cancer and is negatively related to the survival time of patients. Therefore, HDGF is likely to be an influential factor in the poor prognosis of endometrial carcinoma.
2. after interfering with HDGF, the proliferation, migration and invasion ability of endometrial cancer cells were significantly weakened, further suggesting that HDGF played an oncogene function in endometrial carcinoma.
3. HDGF gene mediates the occurrence and development of endometrial carcinoma through mediating PI3K/AKT signaling pathway.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

【参考文献】