胃癌新辅助化疗的疗效评价和MicroRNA及其靶基因在胃癌中的作用机制研究

发布时间：2018-04-30 08:16

本文选题：胃癌 + 新辅助化疗　；参考：《北京协和医学院》2016年博士论文

【摘要】：背景：胃癌是常见的消化道恶性肿瘤,以手术和化疗为主的综合治疗是胃癌的标准治疗方式。其中,新辅助化疗是进展期胃癌综合治疗的重要组成部分。但是目前在进展期胃癌的诊断和治疗过程中,仍缺乏对肿瘤情况及新辅助化疗的疗效进行实时准确评价的有效评估手段。多数患者在治疗过程中出现化疗耐药,甚至在术前新辅助化疗期间即出现原发耐药,从而导致肿瘤进展,这是进展期胃癌预后不良的主要原因。因此,探索敏感性和特异性更高的胃癌诊疗相关标志物,对于准确判断肿瘤进展、实时评价疗效有重要意义,也是进一步探索胃癌发生发展机制,寻找有效治疗靶点的重要手段。MicroRNA (miRN A)是一类内源性非编码小分子单链RNA,通过与靶基因mRNA的3’UTR碱基互补结合,促进靶基因mRNA的降解或抑制其翻译,导致靶基因表达下调,从而影响细胞的增殖、分化和凋亡等过程。miRNA参与肿瘤细胞的活动及相邻微环境的变化,在多种肿瘤中异常表达,与肿瘤的发生发展密切相关。外周循环血中也存在miRNA,其含量可在一定程度上反映肿瘤大小、浸润深度等情况；同时,肿瘤组织中往往存在特异性miRNA表达量的上调或下调。因此,miRNA可以作为肿瘤诊断和治疗的靶标。MiR-301a在结直肠癌、胰腺癌等肿瘤中呈现异常高表达,与多种恶性生物学行为及预后密切相关。研究表明,miR-301a在胃癌组织中的表达较配对的癌旁组织明显升高,与胃癌的分化程度、增殖、侵袭、转移密切相关,在胃癌的发生、发展过程中具有重要作用。但是miR-301a在胃癌发生发展中的作用及机制尚不明确,其与胃癌新辅助化疗的相关性也不明确。深入研究其作用机制,对于探索胃癌新辅助化疗的疗效评价相关标志物具有重要意义。在胃癌的发生发展过程中有许多因素参与,其中microRNA也发挥了重要作用。miR-32在许多恶性肿瘤中表现为“癌基因”的作用,但在胃癌中的作用尚不清楚。我们发现miR-32在胃癌患者的外周血和胃癌组织中呈异常高表达,可能促进了胃癌的发生发展,miR-32通过调控其靶基因Kruppel样因子-4(KLF-4)促进胃癌细胞的增殖、侵袭和迁移。探索miR-32在胃癌发生发展中的作用机制,为胃癌的临床诊治提供新的理论依据和潜在靶点。胃印戒细胞癌是胃癌的一种特殊病理类型,具有独特的临床病理特征。例如,早期诊断率低、对化疗不敏感、易于向胃壁发生浸润性生长等。目前对于胃印戒细胞癌尚无针对性的个体化治疗方案,患者预后极差。利用microRNA芯片发现miR-935在胃印戒细胞癌中较非印戒细胞癌表达明显降低,miR-935可能通过调控靶基因Notch1在胃印戒细胞癌中发挥作用。研究miR-935在胃印戒细胞癌中的作用机制,利于我们更好地了解这种特殊类型的胃癌,为提高此类患者的诊疗效果提供新的思路。目的：(1)研究新辅助化疗及营养支持治疗对进展期胃癌患者肿瘤缓解及预后的意义;(2)研究miR-301a对进展期胃癌新辅助化疗疗效评价的意义,明确miR-301a及其靶基因在胃癌新辅助化疗中的作用机制；(3)研究miR-32及其靶基因在胃癌发生发展中的作用机制；(4)研究miR-935及其靶基因在胃印戒细胞癌中的作用机制。方法：(1)收集在我院接受新辅助化疗及手术治疗的147例进展期胃癌患者的临床病理资料,分析患者在接受新辅助化疗及营养支持治疗后的肿瘤缓解情况及预后。(2)通过实时定量PCR法检测miR-301a在新辅助化疗患者胃癌组织及外周血中的表达水平,并对新辅助化疗前后CA199、CEA、CA724的表达变化、肿瘤的缓解情况进行对比；在胃癌细胞AGS和MGC8-03中发现miR-301a抑制顺铂、5-氟尿嘧啶诱导的细胞凋亡；预测并确认谷氨酰胺合成酶(GLUL)为miR-301a的靶基因为；采用qPCR检测胃癌组织和配对癌旁中GLUL的表达,并分析其与miR-301a的相关性。(3)采用实时定量PCR检测miR-32在胃癌组织及其配对的癌旁组织中的表达情况；在胃癌细胞系HGC-27和AGS中瞬时转染miR-32 mimic, MGC8-03MKN-45中瞬时转染miR-32 inhibitor,通过xCELLigence RTCA MP系统检测miR-32对胃癌细胞增殖能力的影响,通过transwe11实验检测miR-32对胃癌细胞侵袭和迁移能力的影响;通过Targetscan7.0软件预测、双荧光酶报告基因确认miR-32与KLF4的3’UTR结合;观察KLF4基因对胃癌细胞增殖、侵袭和迁移的作用；采用qPCR检测胃癌组织和配对癌旁中KLF4的表达情况,并分析其与miR-32的相关性。(4)在非印戒细胞癌(MGC8-03, SGC-7901, AGS)和印戒细胞癌(MKN-45和KATO-Ⅲ)中进行Affymetrix miRNA芯片分析,筛选出功能尚未被报道的miR-935；在健康人胃组织、永生化的正常胃粘膜上皮细胞株GES-1及9株胃癌细胞系中检测miR-935的表达情况；在MKN-45和KATO-Ⅲ中瞬时转染miR-935 mimic, MGC8-03和HGC-27中瞬时转染miR-935 inhibitor:通过xCELLigence RTCA MP系统检测miR-935对胃癌细胞增殖能力的影响；通过transwell实验检测miR-935对胃癌细胞侵袭和迁移能力的影响；通过Targetscan7.0软件预测、双荧光酶报告基因验证Notch 1是miR-935的靶基因；采用qPCR检测胃癌组织和配对癌旁中Notch 1的表达情况,并分析其与miR-935的相关性。结果：(1)147例进展期胃癌患者中,男性占66%(97/147),女性占34%(50/147)。治疗前病变部位位于胃上部者占23%(34/147),胃体中部者占31%(46/147),胃窦部者占46%(67/147)。R0切除者135例,占91.8%。患者平均接受3.7周期的新辅助化疗。术中清扫淋巴结15-77枚,平均28枚,淋巴结阳性者占68.7%(101/147)。147例患者新辅助化疗后评价为CR(完全缓解)者2例,PR(部分缓解)者74例,SD(稳定)者55例,PD(进展)者16例,化疗有效率(CR+PR)为51.7%(76/147),疾病控制率为89.1%(131/147)。45.5%(67/147)的患者N分期发生降期：55.6%(5/9)的T2期患者T分期发生降期；42.6%(29/68)的T3期患者T分期发生降期；58.6%(41/70)的T4期患者T分期发生降期。新辅助化疗后的病理TNM分期与预后显著相关(P＜0.01)。(2)miR-301a在胃癌组织及外周血中的表达较正常人明显升高。相比于CA199、CEA、 CA724, miR-301a在新辅助化疗前后的表达水平变化更加符合肿瘤缓解情况。在胃癌细胞中过表达miR-301a可降低细胞的化疗敏感性。谷氨酰胺合成酶(GLUL)是miR-301a的靶基因。在胃癌组织中miR-301a与GLUL的表达呈负相关。(3)miR-32在胃癌组织、胃癌患者血浆以及胃癌细胞中呈高表达。过表达miR-32可促进胃癌细胞的增殖、侵袭和迁移。KLF4是miR-32的靶基因。在胃癌组织中,miR-32与KLF4的mRNA水平呈负相关。(4)与非印戒细胞癌及正常胃癌组织相比,miR-935在胃印戒细胞癌细胞系、胃印戒细胞癌组织中呈低表达。miR-935可抑制胃印戒细胞癌细胞的增殖、侵袭和迁移。Notch 1是miR-935的靶基因,miR-935通过Notch 1在胃癌中发挥类似抑癌基因的作用。在胃癌组织中,miR-935与Notch 1的mRNA水平呈负相关。结论：(1)新辅助化疗对进展期胃癌患者具有良好的化疗有效率和疾病控制率,肿瘤分期与新辅助化疗患者的预后具有相关性,新辅助化疗后TNM分期较早的患者预后较好。营养支持治疗可改善患者的营养状况,维持体重,降低化疗不良反应发生率。(2) miR-301a比传统肿瘤标志物更有效地评价胃癌新辅助化疗的疗效,可作为胃癌诊断和化疗疗效评价的标志物；miR-30la可通过调控靶基因GLUL降低胃癌细胞对化疗药物的敏感性。(3)miR-32在胃癌患者的血浆、组织及胃癌细胞系中高表达；miR-32可通过靶基因KLF4促进胃癌细胞的增殖、侵袭和迁移,发挥癌基因的作用。(4)miR-935在胃印戒细胞癌细胞系、胃印戒细胞癌组织中呈低表达。miR-935可通过靶基因Notch 1抑制胃印戒细胞癌细胞的增殖、侵袭和迁移,在胃癌中发挥类似抑癌基因的作用。
[Abstract]:Background: gastric cancer is a common malignant tumor of the digestive tract. Comprehensive treatment based on surgery and chemotherapy is the standard treatment for gastric cancer. Among them, neoadjuvant chemotherapy is an important part of the comprehensive treatment of advanced gastric cancer. However, in the diagnosis and treatment of advanced gastric cancer, there is still a lack of treatment for tumor and neoadjuvant chemotherapy. Most patients have chemoresistance in the course of treatment, even in the course of neoadjuvant chemotherapy, which may lead to the development of the tumor, which is the main cause of poor prognosis in advanced gastric cancer. Therefore, to explore the related markers of sensitivity and specificity for the diagnosis and treatment of gastric cancer. It is of great significance to accurately judge the progression of tumor and evaluate the curative effect in real time. It is an important means to further explore the development mechanism of gastric cancer and to find effective therapeutic targets,.MicroRNA (miRN A) is a class of endogenous non coding small molecule single strand RNA, which can promote the degradation of target gene mRNA by combining with the 3 'UTR base of the target gene mRNA. Or inhibition of its translation, which leads to down regulation of target gene expression, which affects cell proliferation, differentiation and apoptosis, and.MiRNA participates in the activity of tumor cells and changes in adjacent microenvironment. Abnormal expression in various tumors is closely related to the occurrence and development of tumors. There are also miRNA in peripheral circulation blood, which can be reflected to a certain extent. Tumor size, depth of infiltration and so on. At the same time, specific miRNA expression is often up or down in tumor tissue. Therefore, miRNA can be used as a target for diagnosis and treatment of cancer,.MiR-301a is abnormal high expression in colorectal cancer, pancreatic cancer and other tumors, and is closely related to many malignant biological behavior and prognosis. The expression of miR-301a in gastric cancer tissue is significantly higher than that of para cancerous tissue, which is closely related to the differentiation, proliferation, invasion and metastasis of gastric cancer, and plays an important role in the occurrence and development of gastric cancer. However, the role and mechanism of miR-301a in the development of gastric cancer are not clear, and the correlation with the neoadjuvant chemotherapy of gastric cancer is not It is of great significance to explore the mechanism of the action of the new adjuvant chemotherapy for gastric cancer. There are many factors involved in the development of gastric cancer, and microRNA also plays an important role in the role of "oncogene" in many malignant tumors, but the role of.MiR-32 in gastric cancer. It is not clear that miR-32 is highly expressed in the peripheral blood and gastric cancer tissues of the patients with gastric cancer, which may promote the development of gastric cancer. MiR-32 can promote the proliferation, invasion and migration of gastric cancer cells by regulating the target gene Kruppel like factor -4 (KLF-4), and explore the mechanism of miR-32 in the development of gastric cancer for gastric cancer. The diagnosis and treatment of the bed provides a new theoretical basis and potential targets. Gastric signet ring cell carcinoma is a special pathological type of gastric cancer and has unique clinicopathological features. For example, early diagnosis rate is low, chemotherapy is insensitive to chemotherapy, and the invasive growth of gastric wall is easy to occur. At present, there is no targeted individualized treatment for gastric signet ring cell carcinoma, patients The prognosis is very poor. The expression of miR-935 in gastric signet ring cell carcinoma is obviously lower than that of non signet ring cell carcinoma by microRNA chip. MiR-935 may play a role in the cancer of gastric signet ring cell by regulating target gene Notch1. The mechanism of miR-935 in gastric signet ring cell carcinoma is helpful for us to better understand this special type of gastric cancer, To improve the diagnosis and treatment effect of such patients. Objective: (1) to study the significance of neoadjuvant chemotherapy and nutrition support therapy on the tumor remission and prognosis of advanced gastric cancer patients; (2) to study the significance of miR-301a on the evaluation of the therapeutic effect of neoadjuvant chemotherapy on advanced gastric cancer, and to clarify the role of miR-301a and its target genes in neoadjuvant chemotherapy for gastric cancer. Mechanism; (3) study the mechanism of miR-32 and its target gene in the development of gastric cancer; (4) study the mechanism of miR-935 and its target gene in gastric signet ring cell carcinoma. Methods: (1) collect the clinicopathological data of 147 cases of gastric cancer patients receiving neoadjuvant chemotherapy and surgical treatment in our hospital, and analyze the patients receiving new AIDS Tumor remission and prognosis after chemotherapy and nutritional support. (2) the expression of miR-301a in gastric cancer tissue and peripheral blood in neoadjuvant chemotherapy patients was detected by real-time quantitative PCR method, and the expression changes of CA199, CEA, CA724 before and after neoadjuvant chemotherapy were compared, and the tumor remission situation was compared, and in AGS and MGC8-03 of gastric cancer cells. MiR-301a inhibits the apoptosis induced by cisplatin and 5- fluorouracil; predicts and confirms that glutamine synthetase (GLUL) is a target for miR-301a; qPCR is used to detect the expression of GLUL in gastric cancer tissue and para cancerous, and to analyze its correlation with miR-301a. (3) detection of miR-32 in gastric cancer tissue and its paired cancer by real time quantitative PCR Transient transfection of miR-32 mimic in gastric cancer cell lines HGC-27 and AGS, transient transfection of miR-32 inhibitor in MGC8-03MKN-45, and the effect of miR-32 on the proliferation of gastric cancer cells through xCELLigence RTCA MP system, and the effect of transwe11 experiments on the invasion and migration of gastric cancer cells; Targetscan7.0 software predicted that the double fluorescent enzyme reporter gene confirmed the combination of miR-32 with 3 'UTR of KLF4, observed the effect of KLF4 gene on the proliferation, invasion and migration of gastric cancer cells, and used qPCR to detect the expression of KLF4 in gastric cancer tissues and paired cancerous tissues, and to analyze its correlation with miR-32. (4) in non signet ring cell carcinoma (MGC8-03, SGC-7901, AGS). The analysis of Affymetrix miRNA chip in MKN-45 and KATO- III of signet ring cell carcinoma (MKN-45 and KATO- III) screened the miR-935 that had not been reported. The expression of miR-935 was detected in healthy human gastric tissue, GES-1 of immortalized normal gastric mucosa epithelial cell strain and 9 lines of gastric cancer cell lines; miR-935 mimic was transfected in MKN-45 and KATO- III, MGC8-0. The effect of miR-935 on the proliferation of gastric cancer cells was detected by transient transfection of miR-935 inhibitor: in 3 and HGC-27 through xCELLigence RTCA MP system, and the effect of miR-935 on the invasion and migration of gastric cancer cells was detected by Transwell test. The target gene of Notch 1 was verified by the Targetscan7.0 software, and the double fluorescent enzyme reported that Notch 1 was the target gene of miR-935. QPCR was used to detect the expression of Notch 1 in gastric cancer tissue and paracanal para cancer, and the correlation with miR-935 was analyzed. Results: (1) among 147 patients with gastric cancer, 66% (97/147) and 34% (50/147) were male, 23% (34/147), 31% (46/147) in the middle of the stomach, and 46% in the gastric antrum (67/1). 47) 135 cases of.R0 resection, accounting for 3.7 cycles of neoadjuvant chemotherapy in patients with 91.8%., 15-77 of lymph nodes, 28, 68.7% (101/147), 2 cases of CR (complete remission), 74 cases of PR (partial remission), 55 cases of SD (stable), 16 cases of PD (progressing), and effective rate of chemotherapy (CR) +PR) for 51.7% (76/147), the disease control rate was 89.1% (131/147).45.5% (67/147). The stage of N stage occurred: 55.6% (5/9) of T2 stage patients with T staging; 42.6% (29/68) T3 phase of the stage of T; 58.6% (2) (0.01) (2) the expression of miR-301a in gastric cancer tissue and peripheral blood was significantly higher than that of normal people. Compared to CA199, CEA, CA724, miR-301a was more in line with the tumor remission situation before and after the neoadjuvant chemotherapy. The overexpression of miR-301a in gastric cancer cells could reduce the chemosensitivity of the cell cell. The glutamine synthetase (GLUL) was miR-3 The target gene of 01A is negatively correlated with the expression of miR-301a and GLUL in gastric cancer. (3) miR-32 is highly expressed in gastric cancer tissue, gastric cancer plasma and gastric cancer cells. Overexpression of miR-32 can promote the proliferation of gastric cancer cells, invasion and migration of.KLF4 are the target genes of miR-32. In gastric cancer, miR-32 and KLF4 mRNA levels are negatively correlated. (4) Compared with non signet ring cell carcinoma and normal gastric cancer tissue, miR-935 in gastric signet ring cell carcinoma cell line and low expression of.MiR-935 in gastric signet ring cell carcinoma can inhibit the proliferation of gastric signet ring cell carcinoma cells, invasion and migration of.Notch 1 is the target gene of miR-935, and miR-935 plays a role similar to tumor suppressor gene in gastric cancer through Notch 1. In the tissue, miR-935 was negatively correlated with the mRNA level of Notch 1. Conclusion: (1) neoadjuvant chemotherapy has good Chemotherapy Effectiveness and disease control rate for advanced gastric cancer patients. Tumor staging is related to the prognosis of neoadjuvant chemotherapy patients. After neoadjuvant chemotherapy, the prognosis of patients with early TNM staging is better. Nutrition support therapy can improve the patient's prognosis. (2) miR-301a is more effective than traditional tumor markers to evaluate the curative effect of neoadjuvant chemotherapy for gastric cancer, which can be used as a marker for the evaluation of gastric cancer diagnosis and chemotherapy, and miR-30la can reduce the sensitivity of gastric cancer cells to chemotherapeutic drugs by regulating target gene GLUL. (3) miR-32 MiR-32 can promote the proliferation, invasion and migration of gastric cancer cells and play the role of oncogene through target gene KLF4. (4) miR-935 in gastric signet ring cell carcinoma cell line, low expression of.MiR-935 in gastric signet ring cell carcinoma can inhibit gastric signet ring cell carcinoma by target gene Notch 1 Cell proliferation, invasion and migration play a similar role as tumor suppressor genes in gastric cancer.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

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