ANXA2及相关miRNAs在结直肠癌发生发展中的作用及与肝癌靶向治疗关系的研究

【作者】 肖丽；

【导师】 侯颖春；

【作者基本信息】 陕西师范大学 ， 细胞生物学， 2019， 博士

【摘要】 背景与目的:结直肠癌(Colorectal cancer,CRC)和肝癌(Hepatocellular cancer,HCC)是世界上常见的恶性肿瘤,在中国发病率和死亡率均很高。膜联蛋白A2(Annexin A2,ANXA2)在多种肿瘤组织高表达,促进肿瘤的恶性发展特质如癌细胞增殖及侵袭转移强化等。研究发现ANXA2也在CRC和HCC中高表达,ANXA2在CRC的发展过程中可能是一个关键性基因表达异化事件之一,而且可能存在一条ANXA2信号通路。ANXA2在29.5%的被调查CRC病例中高表达,并且与肿瘤生长和转移侵袭等密切相关。ANXA2活化是其发挥信号转导作用的基础,文献表明ANXA2活化的重要功能域位于其N末端,但是,其N端关键性氨基酸位点在肿瘤发生、发展过程中的作用机制报道极少。进一步深入研究ANXA2蛋白N端关键位点对理解其信号通路调节及其与肿瘤发生发展的关系具有重要意义。在前期构建的ANXA2人结直肠癌敲除细胞系(ANXA2-/-caco2)的基础之上,我们以单点突变(Site directed mutation)和多点突变(Multiple sites directed mutation)技术构建了其N端有关关键位点单点、多点突变子表达载体,将这些突变子在ANXA2-/-caco2细胞表达,探究ANXA2 N端关键位点突变对细胞恶性表型和行为的调节作用,从而评价ANXA2的分子构效与肿瘤细胞恶性度的关系。同时,预测了与ANXA2可能有靶向关系的miRNAs,选出可以最高分值结合到ANXA2 3,UTR 的 3 个 miRNAs(miR-1-3p、miR-206 和 miR-613),以双荧光素酶报告基因技术证明miR-206与ANXA2靶向关系最好,随后进一步研究了miR-206对caco2细胞恶性表型和行为的调控。HCC发病隐匿,手术治愈率低,化疗效果和预后不理想。目前广泛采用的脂质体阿霉素(Lipo-DOX)为临床常用的广谱抗癌一线化疗药物,但Lipo-DOX缺乏靶向性,药物的毒副作用仍然很大,并伴随快速形成的多药抗药性(Mutiple drug resistance,MDR)。所以,目前肿瘤临床化疗面临两大瓶颈问题:即药物的靶向性不足和MDR形成。这两大瓶颈问题导致了癌症化疗效果不佳、副作用巨大、复发转移率和病人死亡率居高不下。因此,研发具有足够肿瘤靶向性,同时可以抑制甚至逆转MDR的抗癌新药迫在眉睫。我们前期筛选获得的特异、敏感靶向HCC的12肽HCSP4,具有良好的HCC细胞/组织结合特异性和敏感性,同时结合文献及数据库分析预测,发现miR-101在HCC表达极低,并可能靶向ANXA2及MDR相关基因,具有抑制对化疗药物MDR的潜力。在上述ANXA2在CRC发生发展中信号通路调节的研究基础上,以HCSP4和miR-101为药物靶向和MDR抑制元件,制备了 HCSP4-Lipo-DOX-miR101肝癌靶向药物递送体系,对该体系的体外(本室其他研究生课题)、体内HCC靶向、抗MDR作用进行研究,同时对ANXA2基因表达在该载药体系的体内治疗过程中可能的作用进行了分析。研究方法:1.借助 cBio Cancer Genomics Portal 和 GEPIA 等数据库,分析 ANXA2 在 CRC和HCC临床样本中的变异情况以及在其临床发展中的作用。2.以结肠癌和肝癌临床组织和组织芯片为材料,利用免疫荧光法,研究ANXA2的表达水平变化。3.设计构建ANXA2 N端各关键位点单点、多点突变子表达载体,转染ANXA2-/-caco2 细胞。4.以 ANXA2+/+caco2、ANXA2-/-caco2和转染 NM 质粒的 ANXA2-/-caco2为对照组,以分别转染 S1、S11、S25、Y23、S1-Y23、S11-Y23 和 S25-Y23 的ANXA2-/-caco2为实验组,采用MTT、损伤修复、Transwell、流式细胞术、免疫荧光染色等方法,检测ANXA2 N端关键位点对caco2细胞增殖、周期、运动、凋亡、细胞整体骨架以及与运动相关微结构的影响。5.通过权威网站预测与ANXA2有靶向关系的miRNAs并以双荧光素酶报告基因实验验证靶向关系。6.将miR-206表达载体转入野生型caco2细胞,检测ANXA2被抑制率、肿瘤细胞有关行为、形态的指标及EMT相关基因表达水平。7.通过ANXA2及MDR相关基因靶向分析,设计构建miR-101表达载体。8.采用薄膜超声分散法制备脂质体,硫酸铵pH梯度法载药,利用阳离子脂质体带正电荷的特性,与带负电的DNA,即miR-101表达载体,加载miR-101质粒,制备 HCSP4-Lipo-DOX-miR101。9.应用荧光分光光度计、激光粒度仪、扫描电镜和透射电镜等检测HCSP4-Lipo-DOX-miR1 01的特性及药物包封率。10.优化HCSP4-Lipo-DOX-miR101的转染效率并评估其靶向性。11.建立HCC移植瘤裸鼠模型,并通过HCSP4-Lipo-DOX和HCSP4-Lipo-DOX-miR101给药(尾静脉注射)处理,从肿瘤生长曲线、肿瘤体积、肿瘤重量、肿瘤的肺转移及ANXA2在裸鼠移植瘤中的表达等方面综合评估HCSP4-Lipo-DOX-miR101的体内治疗效果。研究结果:1.ANXA2的表达与结肠癌恶性程度高度相关,尤其在低分化腺癌T3N0Mx期高表达,与大数据分析结果高度吻合。2.ANXA2的表达与肝癌恶性程度高度相关,尤其在中-低分化肝细胞癌高表达,与大数据分析结果高度吻合。3.ANXA2 N端各关键位点突变能够不同程度抑制ANXA2的激活水平。4.ANXA2 N端各关键位点突变能够不同程度抑制caco2细胞的恶性行为。5.ANXA2的N端各关键位点突变能够重塑caco2细胞的运动相关微结构。6.miR-206、miR-1-3p 和 miR-613 均能够靶向 ANXA2,且 miR-206 对其靶向关系最好。7.miR-206通过靶向ANXA2,负调控ANXA2表达。8.体外过表达miR-206抑制caco2细胞的增殖和迁移能力,促进细胞凋亡,抑制肿瘤EMT发生。9.成功构建HCSP4-Lipo-DOX-miR101肝癌靶向药物递送体系,特性良好。10.HCSP4-Lipo-DOX-miR101具有较高的转染效率,对HepG2细胞具有良好的靶向性,并抑制ANXA2表达。11.HCSP4-Lipo-DOX-miR101抑制裸鼠体内移植瘤生长,尤其是在耐药细胞株HepG2/ADR中,并能够抑制裸鼠移植瘤中ANXA2的表达。研究结论:1.ANXA2的表达与结肠癌和肝癌的恶性程度高度相关。2.ANXA2的表达及其N端4个关键位点,尤其是Y23对于维持caco2细胞的恶性表型至关重要。3.miR-206靶向ANXA2并负调控其表达,体外过表达miR-206抑制caco2细胞的恶性表型。4.HCSP4-Lipo-DOX-miR101具有明显的HCC体内靶向治疗效果和MDR抑制特性,其作用机制与miR-101对ANXA2的表达抑制密切相关。更多还原

【Abstract】 Background and Purpose:Colorectal cancer(CRC)and Hepatocellular cancer(HCC)are common malignant tumors in the world,and their morbidity and mortality are high in China.Annexin A2(ANXA2)is upregulated in various tumor tissues and promotes the malignant development of tumors such as proliferation,invasion and metastasis of cancer cells.The study found that ANXA2 is also upregulated in CRC and HCC.ANXA2 may be one of the key gene expression alienation events during the CRC progression.There may be an ANXA2 signaling pathway.ANXA2 is upregulated in 29.5%of the investigated CRC cases and is closely related to tumor growth and metastasis.Activation of ANXA2 is the basis of its signal transduction.The reference indicates that the important functional domain of ANXA2 activation is located at its N-terminus.However,the mechanism of key N-terminal amino acid sites in oncogenesis and progression is rarely reported.Further research on the key N-terminal sites of ANXA2 is of great significance for understanding the regulation of its signaling pathway and its relationship with oncogenesis and progression.Based on the previously constructed ANXA2 human colorectal cancer knockout cell line(ANXA2-/-caco2),we constructed the single-point and multi-point mutant expression vectors of its key N-terminal sites using the Site directed mutation and Multiple sites directed mutation.The expression vectors were expressed in ANXA2-/-caco2 cells to explore the regulatory role of key ANXA2 N-terminal sites mutation in the malignant phenotypes and behaviors of cells and to evaluate the relationship between the molecular structure of ANXA2 and the malignancy of tumor cells.The miRNAs that may have a targeted relationship with ANXA2 were predicted and 3 miRNAs(miR-1-3p,miR-206 and miR-613)that bind to the ANXA2 3’UTR with the highest score were selected.We demonstrated that miR-206 had the best targeting relationship with ANXA2 by the dual luciferase reporter assay and further studied the regulation of miR-206 on the malignant phenotypes and behaviors of caco2 cells.The incidence of HCC is concealed,the surgical cure rate is low and the chemotherapy effect and prognosis are not ideal.The currently widely used liposome doxorubicin(Lipo-DOX)is a commonly used broad-spectrum anticancer first-line chemotherapeutic drugs,but Lipo-DOX lacks targeting specificity and the toxic side effects are still large,accompanied by rapid formation of Mutiple drug resistance(MDR).Therefore,the current clinical chemotherapy of tumors faces two major bottlenecks:the lack of targeting specificity and the formation of MDR of drugs.The two bottlenecks have led to poor cancer chemotherapy,high side effects,recurrence and metastasis rates and high mortality.It is extremely urgent to develop new anticancer drugs with sufficient tumor targeting specificity and MDR inhibition or even reverse.The 12-peptide HCSP4 which targeted to HCC with specificity and sensitivity has good HCC cell/tissue binding specificity and sensitivity.It is found that miR-101 is extremely low in HCC and may target ANXA2 and MDR-related genes by the references and database analysis.It is speculated that miR-101 has the potential to inhibit MDR against chemotherapeutic drugs.Based on the above-mentioned research on the regulation of ANXA2 signaling pathway in the oncogenesis and progression of CRC,HCSP4-Lipo-DOX-miR101 liver cancer targeted drug delivery system was prepared by using HCSP4 and miR-101 as drug targeting and MDR inhibitory elements.The targeting specificity and anti-MDR effects of the drug delivery system were studied in vitro(the other postgraduate research in our lab)and in vivo.The possible role of ANXA2 gene expression in the in vivo treatment of the drug delivery system was analyzed.Methods:1.Analyze the variation of ANXA2 in CRC and HCC clinical samples and its role in clinical development with databases such as cBio Cancer Genomics Portal and GEPIA.2.Using colon cancer and liver cancer clinical tissues and tissue chips as materials,the expression level of ANXA2 was studied by immunofluorescence.3.Design and construct the single-point,multi-point mutant expression vectors of ANXA2 key N-terminal sites and transfect ANXA2-/-caco2 cells.4.ANXA2+/+caco2,ANXA2-/-caco2 and ANXA2-/-caco2 transfected with NM plasmids as control groups,ANXA2-/-caco2 transfected with S1,S11,S25,Y23,S1-Y23,S11-Y23 and S25-Y23 as the experimental groups,the methods of MTT,wound healing,Transwell,flow cytometry and immunofluorescence staining were used to detect the effects of ANXA2 key N-terminal sites on caco2 cell proliferation,cell cycle,motility,apoptosis,overall cell skeleton and motility assosiated with microstructures.5.Predict miRNAs that have the targeting relationships with ANXA2 by the authoritative websites.The targeting relationships were confirmed by the dual luciferase reporter assay.6.The miR-206 expression vector was transfected into wild-type caco2 cells and the inhibition rate of ANXA2,the related behavior and morphology of tumor cells and the expression level of EMT-related genes were detected.7.The miR-101 expression vector was designed and constructed by the analysis of ANXA2 and MDR-related gene targeting relationships.8.The liposomes were prepared by thin film ultrasonic dispersion method.The drug was loaded by ammonium sulfate pH gradient method.miR-101 were linked to liposomes by the positive polarity of cationic liposomes interacting with negatively charged DNA.HCSP4-Lipo-DOX-miR101 was obtained.9.The characteristics and drug encapsulation efficiency of HCSP4-Lipo-DOX-miR101 were detected by fluorescence spectrophotometer,laser particle size analyzer,scanning electron microscope and transmission electron microscope.10.Optimize the transfection efficiency of the HCSP4-Lipo-DOX-miR101 and evaluate the targeting specificity.11.Establish the nude mouse model of HCC xenografts and treat them with HCSP4-Lipo-DOX and HCSP4-Lipo-DOX-miR101(tail vein injection).The therapeutic effect of HCSP4-Lipo-DOX-miR101 in vivo was comprehensively evaluated from the aspects of tumor growth curve,tumor volume,tumor weight and lung metastasis.Results:1.The expression of ANXA2 was highly correlated with the degree of colon cancer malignancy,especially upregulated in the poorly differentiated adenocarcinoma at the T3N0Mx stage,which was highly consistent with the results of the database analysis.2.The expression of ANXA2 was highly correlated with the degree of liver cancer malignancy,especially upregulated in the moderate-poorly differentiated hepatocellular carcinoma,which was highly consistent with the results of the database analysis.3.The activation levels of ANXA2 can be differentially inhibited by the mutation of key ANXA2 N-terminal sites.4.The malignant behaviors of caco2 cells can be differentially inhibited by the mutation of key ANXA2 N-terminal sites.5.Motility-associated microstructures of caco2 cells can be remodeled by the mutation of key ANXA2 N-terminal sites.6.miR-206,miR-1-3p and miR-613 can target ANXA2.miR-206 had the best targeting relationship.7.miR-206 negatively regulated ANXA2 expression by targeting ANXA2.8.The forced expression of miR-206 inhibited the proliferation and migration of caco2 cells,promoted apoptosis and inhibited tumor EMT in vitro.9.HCSP4-Lipo-DOX-miR101 liver cancer targeted drug delivery system was successfully constructed with good characteristics.10.The transfection efficiency of HCSP4-Lipo-DOX-miR101 was high.The system can target to HepG2 cells and inhibit the expression of ANXA2.11.HCSP4-Lipo-DOX-miR101 inhibited the growth of xenografts in nude mice,especially in the resistant cell line HepG2/ADR,and inhibited the expression of ANXA2 in xenografts.Conclusions:1.The expression of ANXA2 is highly correlated with the degree of colon cancer and liver cancer malignancy.2.The expression of ANXA2 and its 4 key N-terminal sites,especially Y23,are essential for maintaining the malignant phenotypes of caco2 cells.3.miR-206 targets ANXA2 and negatively regulates its expression.The forced expression of miR-206 inhibits the malignant phenotypes of caco2 cells in vitro.4.HCSP4-Lipo-DOX-miR101 has obvious HCC targeted therapeutic effects in vivo and MDR inhibitory property.Its mechanism is closely related to the inhibition of ANXA2 expression by miR-101.更多还原