兰州大学机构库 >第二临床医学院
FEZF1-AS1 调控PI3K/AKT/mTOR信号通路介导自噬在结肠癌发展中的作用机制研究
Alternative TitleSTUDY ON THE MECHANISM OF FEZF1-AS1 REGULATING PI3K/AKT/mTOR SIGNALING PATHWAY TO MEDIATE AUTOPHAGY IN THE DEVELOPMENT OF COLON CANCER
杨小萍
Subtype博士
Thesis Advisor张德奎
2023-09-03
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name医学博士
Degree Discipline内科学
KeywordFEZF1-AS1 FEZF1-AS1 结肠癌 colon cancer 自噬 autophagy PI3K/AKT/mTOR PI3K/AKT/mTOR
Abstract

背景

结肠癌是世界范围内发病率和死亡率均位于前列的恶性肿瘤。结肠癌的治疗 主要有内镜治疗、手术治疗、辅助化疗、靶向治疗以及免疫治疗。虽然现阶段结 肠癌的诊断和治疗已经取得了长足的进步,但是由于大多数患者就诊时已经处于 进展期、对化疗耐药性的产生以及发生转移,使得结肠癌患者的预后很差。目前 针对结肠癌的靶向治疗和生物标志物驱动疗法的研究并不乐观。因此,寻找结肠 癌诊断和发展的关键生物学标志物,阐明结肠癌发展的分子机制并开发更有效的 治疗方法迫在眉睫。 FEZ 家族锌指 1 反义 RNA 1(FEZ family zinc finger 1-antisense RNA 1, FEZF1-AS1)是一个反义长链非编码RNA(long non-coding RNA,lncRNA),长度为2653bp。FEZF1-AS1 在胃癌、食管癌和卵巢癌等多个肿瘤中显著高表达, 与患者的临床病理特征(如肿瘤大小、TNM 分期)及预后相关,且参与肿瘤的 发生发展过程。 自噬通过回收细胞内不需要的或受损的细胞器和蛋白质,用于再生各种前体, 维持细胞的生物合成和生存。在正常细胞中,自噬是防止肿瘤发生的“刹车”。 但是一旦肿瘤形成后,肿瘤细胞通常利用自噬作为一种促生存机制,促进自身的 生长增殖和获得性耐药。有研究报道,FEZF1-AS1 通过激活自噬促进胃癌的进 展,诱导胃癌细胞发生多重耐药。研究表明,激活自噬能够抑制结肠癌细胞凋亡, 提高结肠癌细胞对化疗药物的耐药性。基于以上理论,我们提出科学假设: FEZF1-AS1 可能通过自噬参与结肠癌的进展,降低结肠癌细胞对化疗药物的敏感性。

目的

探讨FEZF1-AS1 在结肠癌中的作用及可能的分子机制,为结肠癌的诊断和治疗提供新的方法和理论依据。

方法

1. 从 TCGA 肿瘤数据库中下载结肠癌患者的 RNA 测序数据和对应的临床病理 信息,通过R语言中的“limma”包获取差异性表达的lncRNA。通过综合分析, 我们选取FEZF1-AS1作为本课题研究的目的分子。 2. 分析NCBI、NONCODE、UCSC数据库中FEZF1-AS1在多种正常组织中的表 达;我们可视化了TCGA,GEPIA和lnCAR数据库中结肠癌及结肠正常组织中 FEZF1-AS1 的表达;通过收集临床上病理诊断为结肠腺癌的36例结肠癌组织和 配对的结肠正常组织,我们运用qRT-PCR进一步检测所收集组织中FEZF1-AS1 的表达;通过cbioportal数据库,分析结肠癌中FEZF1-AS1的突变情况。 3. 根据R语言中的“clusterProfiler”包,基因探针富集分析(Gene Set Enrichment Analysis,GSEA)以及 lnCAR 数据库,对FEZF1-AS1及与其共表达的基因集进 行功能富集分析,初步获得FEZF1-AS1可能参与结肠癌发展的相关信号通路。 4. 采用 R 语言中“pROC”包对结肠癌中的FEZF1-AS1进行接受者操作特性曲 线(receiver operating characteristic curve,ROC)分析;采用 Mann-Whitney U 检验和Kruskal-Wallis 检验分析 TCGA 数据库中 FEZF1-AS1 与结肠癌患者临床病 理参数之间的关系;在TCGA数据库中,采用COX回归分析结肠癌相关危险因 素;通过GEPIA数据库,分析FEZF1-AS1与结肠癌患者生存之间的关系。 5. 培养结肠癌细胞(RKO、caco2、SW620和SW480)和结肠上皮细胞(NCM460), qRT-PCR 检测细胞中FEZF1-AS1的表达,根据结果选择caco2和RKO,分别用 来构建FEZF1-AS1过表达和敲减的慢病毒转染细胞株;运用qRT-PCR检测慢病 毒转染细胞中FEZF1-AS1 的表达,验证过表达和敲减效率;运用 CCK8、平板 克隆实验、划痕实验、transwell实验和流式细胞术分析FEZF1-AS1对细胞增殖、 迁移、侵袭、周期和凋亡等生物学的影响。 6. 对敲减 FEZF1-AS1 的 RKO 细胞和对照细胞进行全转录组测序,每组三次重 复。根据测序结果分析FEZF1-AS1敲减前后的差异基因及通路富集,确认FEZF1 AS1 可能参与结肠癌发展的相关信号通路。 7. 通过蛋白免疫印迹和免疫荧光方法验证FEZF1-AS1可能参与结肠癌发展的相 关信号通路中关键蛋白的表达情况;采用一定浓度的自噬抑制剂(氯喹)和mTOR 抑制剂(雷帕霉素)分别作用上述细胞,再一次验证信号通路中关键蛋白的表达 情况。 8. 将FEZF1-AS1过表达和敲减的慢病毒转染细胞及相应的对照细胞分别注射到裸鼠皮下,构建荷瘤小鼠移植瘤模型;测量瘤体体积,验证FEZF1-AS1影响结 肠癌细胞的成瘤能力;免疫组化检测移植瘤中FEZF1-AS1可能参与结肠癌发展的相关信号通路关键蛋白的表达情况。 9. 采用一定浓度的氯喹和雷帕霉素分别作用或不作用FEZF1-AS1过表达和敲减的慢病毒转染细胞及相应对照细胞,再将一定浓度的化疗药物(奥沙利铂)作用 上述细胞,采用CCK8和流式细胞术检测药物敏感性。

结果

1. 在 TCGA 数据库中,我们获得了371 例结肠癌患者的RNA测序数据和对应的临床病理信息;获得507个在结肠癌和结肠正常组织中差异性表达的lncRNA。 我们最终选取FEZF1-AS1作为目的分子。 2. NCBI、NONCODE 以及UCSC数据库显示,FEZF1-AS1在结肠正常组织中处 于低表达模式;TCGA,GEPIA,lnCAR数据库和qRT-PCR结果显示,相比于结 肠正常组织,FEZF1-AS1在结肠癌中高表达;cbioportal数据库显示,FEZF1-AS1 在结肠癌中呈现出扩增突变。 3. 功能富集表明,FEZF1-AS1及其共表达的基因集富集到细胞外基质、ABC转 运体、VEGF信号通路、PI3K/AKT信号通路、MAPK信号通路、溶酶体和肿瘤相关信号通路等。 4. 在结肠癌中,FEZF1-AS1的ROC曲线下面积为0.949;FEZF1-AS1在结肠癌患者的T分期、年龄、肿瘤发生部位和种族等不同临床参数分组之间的表达存在差异;COX回归生存分析表明,结肠癌患者的年龄、病理T分期、N分期、临 床分期、淋巴结转移与患者的生存密切相关,可作为结肠癌的独立预后因素; GEPIA 数据库生存分析表明,FEZF1-AS1与结肠癌患者的生存无明显相关性。 5. qRT-PCR 检测结肠癌细胞中FEZF1-AS1的表达,结果显示,相对于NCM460 细胞,FEZF1-AS1在caco2细胞中表达量相对较低,在RKO细胞中表达量相对较高,因此我们选择caco2 成功构建FEZF1-AS1 过表达的慢病毒转染细胞株, 选择RKO成功构建FEZF1-AS1敲减的慢病毒转染细胞株。相比对照细胞,过表 达FEZF1-AS1 的caco2细胞的增殖、迁移、侵袭、周期(S期细胞比例)显著增加,凋亡明显降低;相比对照细胞,敲减FEZF1-AS1的 RKO 细胞的增殖、迁 移、侵袭、周期(S期比例)显著减低,凋亡明显增加。 6. 对敲减 FEZF1-AS1 的 RKO 及对照细胞进行全转录组测序分析,将获取得到 的差异表达基因进行功能富集分析,结果富集到PI3K/AKT信号通路、溶酶体、 自噬和细胞外基质等肿瘤相关通路。我们认为 FEZF1-AS1 可能通过调控PI3K/AKT 信号通路介导自噬促进结肠癌的发展。 7. 蛋白免疫印迹结果显示,相比于对照细胞,PI3K、AKT、p-AKT、mTOR、p mTOR 和p62 等蛋白在过表达FEZF1-AS1 的 caco2 细胞中表达均降低,LC3 II 表达增加;相反的,相比于对照细胞,PI3K、AKT、p-AKT、mTOR、p-mTOR和 p62 等蛋白在敲减FEZF1-AS1的RKO细胞中表达均升高,LC3 II表达降低。 300μmol/L 氯喹作用细胞 12h 后,相比于对照细胞,PI3K、AKT、p-AKT、 mTOR、p-mTOR 和 p62 等蛋白在过表达 FEZF1-AS1 的 caco2 细胞中表达均降 低,LC3 II 表达增加;与未经氯喹干预的过表达FEZF1-AS1的caco2细胞和对 照细胞相比,经过氯喹干预的过表达FEZF1-AS1的caco2细胞和对照细胞中LC3 II 表达显著升高。 0.62nmol/L 的雷帕霉素作用细胞 5h 后,相比于对照细胞,PI3K、AKT、p AKT、mTOR、p-mTOR和p62等蛋白在敲减FEZF1-AS1的RKO细胞中表达均升高,LC3 II 表达降低;与未利用雷帕霉素干预的敲减FEZF1-AS1的RKO和对 照细胞相比,经过雷帕霉素干预的敲减 FEZF1-AS1 的 RKO 和对照细胞中 p mTOR 显著降低,LC3 II 表达显著升高。免疫荧光检测LC3的表达结果与蛋白 免疫印迹检测结果相一致。因此,我们得出 FEZF1-AS1 可能通过抑制 PI3K/AKT/mTOR 信号通路激活自噬促进结肠癌的发展。 8.荷瘤小鼠实验结果表明,过表达FEZF1-AS1的caco2细胞在裸鼠皮下成瘤的体 积显著大于对照组裸鼠;敲减FEZF1-AS1 的 RKO 细胞在裸鼠皮下成瘤的体积 显著小于对照组裸鼠;免疫组化所得结果与上述进行的蛋白免疫印迹实验所得结 果一致。 9. 药物敏感性实验结果表明,相比于对照细胞,过表达FEZF1-AS1的caco2细 胞的死亡率明显更低;与没有经过氯喹干预的过表达FEZF1-AS1的caco2细胞 相比,经过氯喹干预的过表达FEZF1-AS1 的caco2 细胞的死亡率明显更高。相比于对照细胞,敲减FEZF1-AS1的RKO细胞的死亡率明显增加;与未利用雷帕 霉素干预的敲减FEZF1-AS1的RKO细胞相比,经过雷帕霉素干预的敲减FEZF1 AS1 的RKO细胞的死亡率明显更低。

结论

FEZF1-AS1 在结肠癌中显著高表达可能是由于FEZF1-AS1在基因水平发生 扩增突变引起的;FEZF1-AS1可能具有成为结肠癌诊断的生物学标志物的潜力; FEZF1-AS1 可能通过抑制 PI3K/AKT/mTOR 信号通路激活自噬促进结肠癌的发 展,降低结肠癌细胞对奥沙利铂的药物敏感性。总之,FEZF1-AS1可能有望成为结肠癌疾病诊断、进展及疗效评估的一个生物学标志物。

Other Abstract

Background

Colon cancer is a malignant tumor with the highest incidence and mortality in the world. The treatment options for colon cancer mainly include endoscopic therapy, conventional surgery, adjuvant chemotherapy, targeted therapy and immunotherapy. Although the diagnosis and treatment of colon cancer have greatly improved, most colon cancer patients are in the advanced stage once diagnosed, the development of chemotherapy resistance, as well as occurrence of metastasis, making the prognosis of colon cancer patients poor. Current researches on targeted therapies or biomarker driven therapies for this malignancy are not promising. Therefore, it is urgent to discover the critical biomarkers for the early diagnosis and development of colon cancer, elucidate the molecular mechanism of colon cancer and develop more effective therapeutic methods. FEZ family zinc finger 1-antisense RNA 1 (FEZF1-AS1) is an antisense long non coding RNA (lncRNA) with a length of 2653bp. FEZF1-AS1 is highly expressed in gastric cancer, esophageal cancer, and ovarian cancer, which is correlated with prognosis and clinicopathological features of tumor patients (such as tumor size, TNM stage), and is involved in the occurrence and development of tumors. Autophagy recycles unwanted or damaged cellular organelles and proteins, regenerates various precursors to maintain cell biosynthesis and viability. In normal cell, autophagy is the “brake” that prevents the oncogenesis. However, once the tumor is formed, tumor cell usually uses autophagy as a survival mechanism to promote its growth and proliferation, and acquires drug resistance. It has been reported that FEZF1 AS1 promotes the progression of gastric cancer by activating autophagy and induces multiple drug resistance. Studies have shown that activation of autophagy can inhibit the apoptosis of colon cancer cells and increase the resistance of colon cancer cells to chemotherapy drugs. Based on the above researches, we hypothesize that FEZF1-AS1 may be involved in the progression of colon cancer through autophagy, reducing the sensitivity of colon cancer cell to chemotherapy drugs.

Objectives

To explore the function of FEZF1-AS1 in colon cancer and its possible molecular mechanism, and to provide new method and theoretical basis for the early diagnosis and treatment of colon cancer.

Methods

1. We downloaded the RNA sequencing data and corresponding clinicopathological information of colon cancer patients from TCGA database, and obtained differentially expressed lncRNA through the “limma” package in R language. Through comprehensive analysis, we selected FEZF1-AS1 as the target molecule of this study. 2. In NCBI, NONCODE and UCSC databases, we analyzed the expression of FEZF1 AS1 in various normal tissues. In TCGA, GEPIA and lnCAR databases, we visualized the expression of FEZF1-AS1 in colon cancer and normal colon tissues. The expression of FEZF1-AS1 in 36 colon cancer and paired normal colon tissues was further detected by qRT-PCR. Mutations in FEZF1-AS1 in colon cancer were analyzed using the cbioportal database. 3. We used the “clusterProfiler” package in R language, Gene Set Enrichment Analysis (GSEA) and lnCAR database to conduct functional enrichment analysis of FEZF1-AS1 and its co-expressed genes. Therefore, we preliminarily acquired the related signaling pathways that FEZF1-AS1 may participate in the development of colon cancer. 4. The “pROC” package in R language was used to analyze the receiver operating characteristic curve (ROC) of FEZF1-AS1 in colon cancer. Mann-Whitney U test and Kruskal-Wallis test were used to analyze the relationship between FEZF1-AS1 and clinicopathological parameters of colon cancer patients. In the TCGA database, COX regression was used to analyze the risk factors associated with colon cancer. The association between FEZF1-AS1 and survival of colon cancer patients was analyzed using the GEPIA database. 5. We cultured colon cancer cells (RKO, caco2, SW620, SW480) and colon epithelial cell (NCM460), detected the expression of FEZF1-AS1 in the cells by qRT-PCR. Based on the result, caco2 and RKO were selected to construct FEZF1-AS1 overexpressed and knocked down cell lines lentivirus transfected, respectively. We used qRT-PCR to detect the expression of FEZF1-AS1 in cell lines lentivirus transfected to verify the efficiencies of overexpression and knockdown. The effects of FEZF1-AS1 on cell proliferation, migration, invasion, cycle and apoptosis were analyzed by CCK8, plate cloning assay, scratch assay, transwell assay and flow cytometry. 6. RKO cell knocked down FEZF1-AS1 and control cell were performed RNA sequencing, each group was repeated three times. According to the sequencing results, differential genes and pathway enrichment before and after FEZF1-AS1 knocked down were analyzed. The related signaling pathway of FEZF1-AS1 might involve in colon cancer development were confirmed. 7. The expression of critical proteins in the signaling pathway of FEZF1-AS1 might involve in the development of colon cancer was verified by western blot and immunofluorescence methods. Then, the above cells were treated with a certain concentration of autophagy inhibitor (chloroquine) and mTOR inhibitor (rapamycin) to verify the expression of critical proteins in the signaling pathway again. 8. Coco2 cell overexpressed FEZF1-AS1, RKO cell knocked down FEZF1-AS1and corresponding control cells were injected into the subcutaneous skin of nude mice to construct tumor transplantation models of tumor-bearing mice. The tumor volumes were measured to verify the effect of FEZF1-AS1 on tumorigenic ability of colon cancer cells. Immunohistochemistry was used to detect the expression of critical proteins of the related signaling pathway of FEZF1-AS1 might involve in colon cancer development in transplanted tumors. 9. Coco2 cell overexpressed FEZF1-AS1, RKO cell knocked down FEZF1-AS1 and corresponding control cells were treated with or without chloroquine and rapamycin at certain concentrations, respectively. Then, a certain concentration of the chemotherapy drug (oxaliplatin) was applied to the above cells. CCK8 and flow cytometry were used to detect drug sensitivity.

Results 

1. In the TCGA database, we obtained RNA sequencing data and corresponding clinicopathological information of 371 patients with colon cancer. 507 lncRNA were differentially expressed in colon cancer and normal colon tissues. We finally selected FEZF1-AS1 as the target molecule. 2. The results of NCBI, NONCODE and UCSC databases showed that FEZF1-AS1 was in a low expression mode in normal colon tissues. The results of TCGA, GEPIA, lnCAR databases and qRT-PCR showed that FEZF1-AS1 was highly expressed in colon cancer compared with normal colon tissues. The cbioportal database showed that FEZF1-AS1 presented an amplified mutation in colon cancer. 3. Functional enrichment showed that FEZF1-AS1 and its co-expressed genes were enriched into extracellular matrix, ABC transporters, VEGF signaling pathway, PI3K/AKT signaling pathway, MAPK signaling pathway, lysosome, and tumor-related signaling pathways. 4. In colon cancer, the area under ROC curve of FEZF1-AS1 was 0.949. The expression of FEZF1-AS1 was different among different clinical parameter groups of colon cancer patients, such as T stage, age, tumor site and race. COX regression analysis showed that the age, pathological T stage, N stage, clinical stage and lymph node metastasis of patients with colon cancer were correlated with overall survival, and could be used as independent prognostic factors for colon cancer. GEPIA database showed that FEZF1 AS1 was not significantly associated with the survival of colon cancer patients. 5. We used qRT-PCR to detect the expression of FEZF1-AS1 in colon cancer cell lines. It showed that compared with NCM460 cell, the expression of FEZF1-AS1 in caco2 cell was relatively lower, and in RKO cell was relatively higher. Therefore, caco2 was selected to successfully construct FEZF1-AS1 overexpressed cell line lentivirus transfected, and RKO was selected to successfully construct FEZF1-AS1 knocked down cell line lentivirus transfected. Compared with the control cell, the proliferation, migration, invasion and cycle (S phase cell ratio) of caco2 cell overexpressed FEZF1 AS1 were significantly increased, and the apoptosis was significantly decreased. Compared with control cell, the proliferation, migration, invasion and cycle (S phase cell ratio) of RKO cell knocked down FEZF1-AS1 were significantly reduced, and apoptosis was significantly increased. 6. RKO cell knocked down FEZF1-AS1 and control cell were analyzed by RNA sequencing, and the obtained differentially expressed genes were enriched into PI3K/AKT signaling pathway, lysosome, autophagy, extracellular matrix and other tumor-related pathways. We suggested that FEZF1-AS1 may promote the development of colon cancer by regulating the PI3K/AKT signaling pathway to mediate autophagy. 7. The results of western blot showed that compared with control cell, the proteins expression of PI3K, AKT, p-AKT, mTOR, p-mTOR, p62 was decreased in caco2 cell overexpressed FEZF1-AS1, while LC3 II expression was increased. On the contrary, compared with control cell, the proteins expression of PI3K, AKT, p-AKT, mTOR, p mTOR, and p62 was increased in RKO cell knocked down FEZF1-AS1, while LC3 II expression was decreased. After 12h treatment with 300μmol/L chloroquine, compared with control cell, the proteins expression of PI3K, AKT, p-AKT, mTOR, p-mTOR, and p62 was decreased in caco2 cell overexpressed FEZF1-AS1, while LC3 II expression was increased. The protein expression of LC3 II in chloroquine-treated caco2 cell overexpressed FEZF1 AS1 and control cell was significantly higher than that in non-chloroquine-treated caco2 cell overexpressed FEZF1-AS1 and control cell. After 5h treatment with 0.62nmol/L rapamycin, compared with control cell, the proteins expression of PI3K, AKT, p-AKT, mTOR, p-mTOR, and p62 was increased in RKO cell knocked down FEZF1-AS1, while LC3 II expression was decreased. The protein expression of p-mTOR was significantly decreased and LC3 II expression was significantly increased in rapamycin-treated RKO cell knocked down FEZF1-AS1 and control cell, compared with non-rapamycin-treated RKO cell knocked down FEZF1 AS1 and control cell. The results of immunofluorescence detection of LC3 were consistent with those of western blot. We believed that FEZF1-AS1 may inhibit PI3K/AKT/mTOR signaling pathway to activate autophagy and promote the development of colon cancer. 8. The results of tumor-bearing mice experiment showed that the tumor formation volumes of caco2 cell overexpressed FEZF1-AS1 in nude mice were significantly larger than those in control mice. The tumor formation volumes of RKO cell knocked down FEZF1-AS1 in nude mice were significantly smaller than those in control mice. The results of immunohistochemistry were consistent with those of western blot. 9. The results of drug sensitivity test indicated that caco2 cell overexpressed FEZF1 AS1 had significantly lower mortality than that of control cell. Compared with non- chloroquine-treated caco2 cell overexpressed FEZF1-AS1, the mortality of caco2 cell overexpressed FEZF1-AS1 treated with chloroquine was significantly higher. RKO cell knocked down FEZF1-AS1 had significantly higher mortality than that of control cell. Compared with non-rapamycin-treated RKO cell knocked down FEZF1-AS1, the mortality of RKO cell knockdown FEZF1-AS1 treated with rapamycin was significantly lower.

Conclusions

FEZF1-AS1 was highly expressed in colon cancer, which may be caused by the amplification mutation of FEZF1-AS1 at the gene level. FEZF1-AS1 had the potential to be a biomarker in the diagnosis of colon cancer. FEZF1-AS1 may activate autophagy through inhibiting PI3K/AKT/mTOR signaling pathway to promote the development of colon cancer, and reduce the drug sensitivity of colon cancer cells to oxaliplatin. In conclusion, FEZF1-AS1 was expected to be a biomarker for the diagnosis, progression and efficacy evaluation of colon cancer.

Subject Area结肠癌进展的分子机制研究
MOST Discipline Catalogue医学 - 临床医学 - 内科学
URL查看原文
Language中文
Other Code262010_120200901050
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/539762
Collection第二临床医学院
Affiliation
兰州大学第二临床医学院
Recommended Citation
GB/T 7714
杨小萍. FEZF1-AS1 调控PI3K/AKT/mTOR信号通路介导自噬在结肠癌发展中的作用机制研究[D]. 兰州. 兰州大学,2023.
Files in This Item:
There are no files associated with this item.
Related Services
Recommend this item
Bookmark
Usage statistics
Export to Endnote
Altmetrics Score
Google Scholar
Similar articles in Google Scholar
[杨小萍]'s Articles
Baidu academic
Similar articles in Baidu academic
[杨小萍]'s Articles
Bing Scholar
Similar articles in Bing Scholar
[杨小萍]'s Articles
Terms of Use
No data!
Social Bookmark/Share
No comment.
Items in the repository are protected by copyright, with all rights reserved, unless otherwise indicated.