Annexin A7和CAP1对肿瘤细胞粘附分子表达调控及生物学行为的影响

发布时间：2018-05-03 17:48

本文选题：Annexin + A7　；参考：《大连医科大学》2017年硕士论文

【摘要】：背景:原发性肝细胞癌(Hepatocellular carcinoma,HCC)是目前致死率最高的上皮来源恶性肿瘤之一,近年来在我国的发病率依然居高不下。淋巴道转移是恶性肿瘤最早的出现的转移方式,是造成肝癌高侵袭、高复发以及预后差的主要原因之一。国内外许多研究结果表明淋巴道转移机制受许多基因调控,它们或被激活或被抑制,或相互协调或互相拮抗,其中包括细胞增殖、粘附、迁移、细胞外基质降解等多个阶段与环节。膜联蛋白A7(Annexin A7)是本课题组前期发现的潜在决定小鼠肝癌淋巴道转移的关键基因之一,对各种肿瘤细胞生长、分化、增殖、分泌、侵袭、迁移乃至凋亡能力均可产生影响。同时Annexin A7基因是ca2+磷脂结合蛋白,具有ca2+依赖膜融合活性,可促进膜聚集、融合、粘着、运输等,还可激活GTP酶,介导ca2+/GTP信号转导通路。腺苷酸环化酶联合蛋白(adenylate cyclase-associated protein 1,CAP1)最早发现于酵母,在其中可调节Ras/c AMP信号通路,并且与哺乳动物CAP1具有相同作用调节肌动蛋白的周转与重组,对细胞迁移产生影响。近年来关于CAP1与肿瘤转移关系的研究逐渐深入,但似乎在不同肿瘤细胞中对于细胞生物学功能的影响不尽相同。黏着斑激酶(focal adhesion kinase,FAK)是一种非受体型蛋白酪氨酸激酶,在许多上皮来源的肿瘤细胞中过表达,如乳腺癌、结肠癌、口腔癌、甲状腺癌、卵巢癌和胃癌等,可能在肿瘤生长、增殖、侵袭、转移及血管形成中产生影响。Src为SRC酪氨酸激酶家族成员,参与细胞生长、分化、粘着、增殖、凋亡及细胞运动等过程。桩蛋白(Paxillin)是一种主要定位于黏着斑的关键衔接蛋白信号分子,是FAK下游重要的焦点粘附调节蛋白之一,其异常表达与肿瘤的发生、增殖、粘附、浸润、骨架重组及转移能力的改变密切关联。E-钙黏蛋白(E-cadherin,CDH1)是与肿瘤转移侵袭密切相关的一种钙粘蛋白,可参与细胞极性、细胞形态与组织结构完整性的维持,与肿瘤细胞的良恶性及转移程度呈负相关。目的:通过细胞脂质体转染、qRT-PCR、Western Blot等技术观察对比下调小鼠肝癌Hca-P细胞Annexin A7与CAP1基因后观察其对细胞黏附相关分子FAK、Src、Paxillin、E-cadherin基因及蛋白表达水平的调控。通过CCK-8增殖检测、流式细胞仪凋亡检测、小鼠淋巴结粘附以及Transwell小室等实验方法观察CAP1下调对Hca-P细胞生物学行为的影响。对比Annexin A7与CAP1下调后对彼此基因和蛋白表达水平的影响以及二者在Hca-P细胞的表达定位。方法:1.构建PGPUP-GFP-neo-sh RNA-Annexin A7和PGPUP-GFP-neo-sh NC表达载体,稳定转染至小鼠Hca-P细胞,获得Annexin A7下调表达及无关序列Hca-P细胞株,应用qRT-PCR、Western Blot技术验证Annexin A7基因下调对CAP1、FAK、SRC、E-cadherin基因及蛋白表达水平的影响。2.构建PGPUP-GFP-neo-sh RNA-CAP1和PGPUP-GFP-neo-sh NC表达载体,稳定转染至Hca-P细胞,获得CAP1下调表达及无关序列Hca-P细胞株,应用qRT-PCR、Western Blot技术验证CAP1基因下调对FAK、Src、Paxillin、E-cadherin基因及蛋白表达水平的影响。3.获得CAP1下调表达及无关序列Hca-P细胞株后,应用CCK-8增殖检测、流式细胞仪凋亡检测、小鼠淋巴结粘附以及Transwell小室等实验方法,对比CAP1下调组与对照组增殖、凋亡、粘附、侵袭、迁移能力,分析CAP1对Hca-P细胞生物学行为的影响。4.应用qRT-PCR、Western Blot技术验证Annexin A7基因下调对CAP1基因及蛋白表达水平的影响。采用细胞免疫荧光技术检测Annexin A7与CAP1表达的共定位。结果:1、在mRNA表达水平上,Annexin A7在ShRNA-A7细胞组较A7无关序列细胞组及Hca-P细胞组分别降低82.86%和82.78%(P0.05),而在A7无关序列组及Hca-P组间差异不明显(P0.05)。FAK、Src基因在ShRNA-A7细胞组表达分别为A7无关序列组及Hca-P组的1.88倍、1.67倍和1.78倍、1.54倍(P0.05),二者在A7无关序列组及Hca-P组间差异不明显(P0.05)。E-cadherin基因在ShRNA-A7细胞组较A7无关序列细胞组及Hca-P细胞组分别降低49.88%和43.01%(P0.05),在A7无关序列组及Hca-P组间差异不明显(P0.05)。在蛋白质表达水平上,Annexin A7在ShRNA-A7细胞组中蛋白表达水平较A7无关序列组及Hca-P细胞组降低62.75%和60.34%(P0.05),而在Hca-P与A7无关序列组间不存在明显差异(P0.05)。FAK及Src在ShRNA-A7细胞组较A7无关序列组及Hca-P组蛋白表达量上升1.80倍、1.52倍及1.59、1.58倍(P0.05),E-cadherin蛋白在ShRNA-A7细胞组较A7无关序列组与Hca-P组下降54.27%和59.11%(P0.05),但三种蛋白在Hca-P与A7无关序列组间不存在明显差异(P0.05)。2、在mRNA表达水平上,CAP1在ShRNA-CAP1细胞组较CAP1无关序列细胞组及Hca-P细胞组分别降低60.73%和65.25%(P0.05),而在CAP1无关序列组及Hca-P组间差异不明显(P0.05)。FAK、Src、Paxillin基因在ShRNA-A7细胞组表达分别为A7无关序列组及Hca-P组的2.47倍、2.18倍和1.64倍、1.76倍及1.67倍、1.58倍,三者在CAP1无关序列组及Hca-P组间差异不明显(P0.05)。E-cadherin基因在ShRNA-CAP1细胞组较CAP1无关序列细胞组及Hca-P细胞组分别降低40.11%和43.01%(P0.05),在CAP1无关序列组及Hca-P组间差异不明显(P0.05)。在蛋白质表达水平上,CAP1在ShRNA-CAP1细胞组中蛋白表达水平较CAP1无关序列组与Hca-P组降低50.83%和50.29%(P0.05),而在Hca-P与CAP1无关序列组间差异不明显(P0.05)。FAK、SRC及Paxillin在ShRNA-CAP1细胞组较CAP1无关序列组和Hca-P组蛋白表达量分别升高1.64倍和1.60倍、1.67倍和1.61倍、1.43倍和1.39倍(P0.05),E-cadherin蛋白表达水平在ShRNA-CAP1组较CAP1无关序列组及Hca-P组均有下降,下降幅度为39.6%及48.22%(P0.05),但FAK、SRC、Paxillin及E-cadherin四者在Hca-P细胞组与CAP1无关序列细胞组之间表达量不存在明显差异(P0.05)。3、CCK-8增殖实验分别检测Hca-P细胞组、ShRNA-CAP1细胞组与CAP1无关序列细胞组在0h、24h、48h、72h、96h等时间段的吸光值,其中ShRNA-CAP1细胞组各时间点吸光值均数±标准差分别为0.106±0.015、0.152±0.010、0.522±0.049、0.651±0.040、0.651±0.040、0.708±0.037;CAP1无关序列细胞组各时间点吸光值均数±标准差分别为0.095±0.017、0.144±0.024、0.374±0.027、0.485±0.018、0.535±0.039;Hca-P细胞组各时间点吸光值均数±标准差分别为0.101±0.014、0.153±0.006、0.410±0.021、0.553±0.022、0.601±0.030,结果提示Hca-P、ShRNA-CAP1、CAP1无关序列三种细胞数量在24小时内不存在显著差异(P0.05),自48小时起开始产生明显差异,每个时间点差异均显著(P0.05),且ShRNA-CAP1细胞增殖能力始终高于CAP1无关序列细胞组及Hca-P细胞组,CAP1无关序列组与Hca-P组的增殖能力在各时间段无显著差异(P0.05)。4、流式细胞仪分别检测Hca-P细胞组、ShRNA-CAP1细胞组与CAP1无关序列细胞组早期凋亡能力,结果显示三组细胞早期凋亡细胞比例分别为6.33%±0.17%,3.41%±0.22%及6.43%±0.25%,提示在CAP1基因低表达后,ShRNA-CAP1细胞组早期凋亡细胞数低于CAP1无关序列及Hca-P细胞组。5、淋巴结粘附实验检测Hca-P组、ShRNA-CAP1组与CAP1无关序列组细胞淋巴结粘附能力,三种细胞组粘附于淋巴结的细胞数目分别为255.67±28.43、734.33±67.50、228.33±20.03,提示在CAP1基因低表达后,ShRNA-CAP1细胞组对于小鼠淋巴结的黏附能力强于Hca-P细胞组与CAP1无关序列细胞组。6、Transwell小室细胞侵袭实验中Hca-P细胞组、ShRNA-CAP1细胞组与N2-Control细胞组穿透小室膜的细胞数目均数±标准差分别为61.00±8.51、173.00±18.03、58.00±6.40,提示ShRNA-CAP1细胞组穿过小室的细胞数目明显多于CAP1无关序列细胞组及Hca-P细胞组,CAP1无关序列细胞组与Hca-P细胞组对比则差异不显著(P0.05)。7、对Annexin A7与CAP1相互关系的分析提示,在Annexin A7基因表达下调后,CAP1基因与蛋白表达水平均降低,其中,ShRNA-A7细胞组与A7无关序列细胞组对比,CAP1基因下调至原水平的17.21%(P0.05),CAP1蛋白表达水平下调至原水平的46.86%(P0.05),表明Annexin A7基因低表达后,CAP1基因与蛋白水平均随之下降。另外通过免疫荧光方法证明Annexin A7与CAP1均主要在在Hca-P细胞浆内表达,少量表达于细胞核,由图像结果推测两种蛋白可能在细胞浆及邻近细胞内膜处共定位。结论:Annexin A7与CAP1基因分别可调控FAK、SRC、Paxillin、E-cadherin的表达,在mRNA和蛋白质水平,FAK、SRC、Paxillin均随Annexin A7与CAP1基因的下调而高表达,而E-cadherin则在上述两基因表达下调后低表达。CAP1低表达后,Hca-P细胞增殖、淋巴结黏附、侵袭能力均有所上升,早期凋亡能力下降;Annexin A7基因下调表达后,CAP1在mRNA和蛋白质水平随之下调,且两基因在Hca-P细胞内表达位置基本一致,可能存在共定位。Annexin A7基因可能与CAP1基因存在一定分子机制的关联,可对细胞粘附转移分子产生一致性影响,并对肝癌Hca-P细胞的生物学行为的产生密切相关。
[Abstract]:Background: Hepatocellular carcinoma (HCC) is one of the most fatal malignant tumors at present. In recent years, the incidence of cancer is still high in our country. Lymphatic metastasis is the earliest metastasis mode of malignant tumor, which is one of the main causes of high invasion, high recurrence and poor prognosis of liver cancer. Many studies at home and abroad have shown that the mechanism of lymphatic metastasis is regulated by many genes, they are activated or suppressed, or are coordinated or antagonistic to each other, including cell proliferation, adhesion, migration, and extracellular matrix degradation. Annexin A7 (A7) is a potential determinant of the early discovery of the group. One of the key genes of lymphatic metastasis of liver cancer can affect the growth, differentiation, proliferation, secretion, invasion, migration and even apoptosis of various tumor cells. At the same time, the Annexin A7 gene is a ca2+ phospholipid binding protein, with ca2+ dependent membrane fusion activity, which can promote membrane aggregation, fusion, adhesion, transport and so on, and can also activate GTP enzyme and mediate ca2+/GTP The signal transduction pathway, adenylate cyclase-associated protein 1 (CAP1), was first found in yeast, which regulates the Ras/c AMP signaling pathway, and has the same effect with mammalian CAP1 to regulate the turnover and recombination of actin, and the effect on cell migration. In recent years, CAP1 and tumor metastasis The study of the relationship is deepening, but it seems that the effect of focal adhesion kinase (FAK) is a kind of non receptor protein tyrosine kinase, which is expressed in many epithelial tumor cells, such as breast cancer, colon cancer, oral cancer, thyroid cancer, ovarian cancer. And gastric cancer, which may affect the.Src SRC tyrosine kinase family in tumor growth, proliferation, invasion, metastasis and angiogenesis, and participate in the process of cell growth, differentiation, adhesion, proliferation, apoptosis and cell movement. Paxillin is a key cohesive protein signal molecule located in the sticky spot, which is the important downstream of FAK. One of the focal adhesion regulatory proteins, whose abnormal expression is closely related to the changes in tumor occurrence, proliferation, adhesion, infiltration, cytoskeleton recombination and metastasis,.E- calcium mucin (E-cadherin, CDH1) is a kind of calcic protein closely related to tumor metastasis and invasion, which can be involved in cell polarity, cell morphology and structural integrity maintenance, and the maintenance of cell morphology and tissue structure. Objective: To observe and compare the regulation of cell adhesion related molecule FAK, Src, Paxillin, E-cadherin gene and protein expression through the transfection of cell liposomes, qRT-PCR, Western Blot and other techniques, and to observe and control the Annexin A7 and CAP1 genes of mouse liver cancer Hca-P cells. The effect of CAP1 down regulation on biological behavior of Hca-P cells was observed by colonization detection, flow cytometry apoptosis, mouse lymph node adhesion and Transwell compartment. The effect of Annexin A7 and CAP1 on the expression level of each other gene and protein and the expression of two in Hca-P fine cell were compared. Method: 1. construction of PGPUP-GFP-neo-s H RNA-Annexin A7 and PGPUP-GFP-neo-sh NC expression vector, stable transfection into mouse Hca-P cells to obtain Annexin A7 down-regulation and independent sequence Hca-P cell lines. QRT-PCR, Western Blot technique was used to verify the effect of the down regulation of the gene and the expression level of egg white. And PGPUP-GFP-neo-sh NC expression vector, stable transfection into Hca-P cells to obtain CAP1 down expression and unrelated sequence Hca-P cell lines. QRT-PCR, Western Blot technique was used to verify the effect of CAP1 down regulation on FAK, Src, Paxillin, gene and protein expression. CK-8 proliferation detection, flow cytometry apoptosis detection, mouse lymph node adhesion and Transwell chamber and other experimental methods, compare the proliferation, apoptosis, adhesion, invasion and migration ability of CAP1 down-regulation group and control group, analyze the effect of CAP1 on the biological behavior of Hca-P cells,.4. application qRT-PCR, Western Blot technique to verify Annexin A7 gene downregulation to CAP1 genes. The co localization of the expression of Annexin A7 and CAP1 was detected by cell immunofluorescence. Results: 1, at the level of mRNA expression, Annexin A7 decreased by 82.86% and 82.78% (P0.05) in the ShRNA-A7 cell group compared with the A7 independent sequence cell group and the Hca-P cell group respectively, but there was no significant difference between the A7 independent sequence group and the Hca-P group (P0.05). 05).FAK, the expression of Src gene in the ShRNA-A7 cell group was 1.88 times, 1.67 times and 1.78 times, 1.54 times (P0.05), respectively, in the A7 independent sequence group and the Hca-P group, and the two in the A7 independent sequence group and the Hca-P group were not significantly different (P0.05).E-cadherin gene decreased 49.88% and 43.01% respectively in the ShRNA-A7 cell group than that in the A7 independent sequence group and the cell group. The difference between the A7 independent sequence group and the Hca-P group was not obvious (P0.05). In the protein expression level, the protein expression level of Annexin A7 in the ShRNA-A7 cell group decreased by 62.75% and 60.34% (P0.05) in the A7 independent sequence group and Hca-P cell group, but there was no significant difference between the Hca-P and A7 independent sequence groups. The expression of A7 independent sequence group and Hca-P histone increased 1.80 times, 1.52 times and 1.59,1.58 times (P0.05). E-cadherin protein decreased 54.27% and 59.11% (P0.05) in ShRNA-A7 cell group than that in Hca-P group, but there was no obvious difference between Hca-P and A7 independent sequences. The 1 cell group was 60.73% and 65.25% (P0.05) less than that of the CAP1 unrelated sequence cell group and the Hca-P cell group, but the difference between the CAP1 independent sequence group and the Hca-P group was not obvious (P0.05).FAK, Src and Paxillin gene expression in the ShRNA-A7 cell group were 2.47 times, 2.18 and 1.64 times, 1.76 times and 1.67 times, 1.58 times, three in ShRNA-A7 cell group, respectively. The difference between the CAP1 independent sequence group and the Hca-P group was not obvious (P0.05).E-cadherin gene decreased by 40.11% and 43.01% (P0.05) in the ShRNA-CAP1 cell group than in the CAP1 unrelated sequence cell group and the Hca-P cell group, and the difference between the CAP1 independent sequence group and the Hca-P group was not obvious (P0.05). The expression level of CAP1 independent sequence group and Hca-P group decreased 50.83% and 50.29% (P0.05), while the difference between Hca-P and CAP1 independent sequences was not obvious (P0.05).FAK, SRC and Paxillin increased 1.64 times and 1.60 times, 1.67 and 1.61 times, 1.43 times and 1.39 times, respectively, in ShRNA-CAP1 cell group than CAP1 independent sequence group and Hca-P histone protein expression. The expression level of herin protein decreased in group ShRNA-CAP1 and Hca-P group, the decrease was 39.6% and 48.22% (P0.05), but there was no significant difference between the FAK, SRC, Paxillin and E-cadherin four in the Hca-P cell group and the CAP1 sequence cell group (P0.05). The absorbance of A-CAP1 cell group and CAP1 independent sequence cell group at 0h, 24h, 48h, 72h, 96h and so on, in which the mean absorption standard deviation of each time point of ShRNA-CAP1 cell group is 0.106 + 0.015,0.152 + 0.010,0.522 + 0.049,0.651 + 0.040,0.651 + 0.037, respectively. The difference is 0.095 + 0.017,0.144 + 0.024,0.374 + 0.027,0.485 + 0.018,0.535 + 0.039, and the average number of absorbance values of each time point of Hca-P cell group is 0.101 + 0.014,0.153 + 0.021,0.553 + 0.022,0.601 + 0.030 respectively. The results suggest Hca-P, ShRNA-CAP1, and there is no significant difference in the number of three cells in the CAP1 independent sequence in 24 hours. The difference (P0.05) began to produce obvious difference since 48 hours, and the difference of each time point was significant (P0.05), and the proliferation ability of ShRNA-CAP1 cells was always higher than that of CAP1 independent sequence cell group and Hca-P cell group. The proliferation ability of CAP1 independent sequence group and Hca-P group had no significant difference (P0.05).4 at each time period, and the flow cytometry was used to detect Hca-P cells respectively. The early apoptotic ability of ShRNA-CAP1 cell group and CAP1 independent sequence cell group showed that the proportion of early apoptotic cells in three groups was 6.33% + 0.17%, 3.41% + 0.22% and 6.43% + 0.25%, suggesting that the number of early apoptotic cells in the ShRNA-CAP1 cell group was lower than that of CAP1 independent sequence and Hca-P cell group.5 after the CAP1 gene was low. The cell lymph node adhesion ability of group Hca-P, ShRNA-CAP1 group and CAP1 independent sequence group was tested. The number of cells adhered to the lymph nodes in the three groups was 255.67 + 28.43734.33 + 67.50228.33 + 20.03 respectively, suggesting that the adhesion ability of ShRNA-CAP1 cell group to mouse lymph node was stronger than that of Hca-P cell group and CAP1 after the CAP1 gene was low. The number of Hca-P cells in the unrelated sequence cell group.6, the Transwell cell cell invasion experiment, the number of cells in the ShRNA-CAP1 cell group and the N2-Control cell group that penetrated the small ventricular membrane were 61 + 8.51173.00 + 6.40, respectively, indicating that the number of cells passing through the compartment in the ShRNA-CAP1 cell group was obviously more than that of the CAP1 independent sequence cell group. And Hca-P cell group, the difference of CAP1 independent sequence cell group and Hca-P cell group was not significantly different (P0.05).7. The analysis of the relationship between Annexin A7 and CAP1 suggested that the CAP1 gene and protein expression level decreased after the Annexin A7 gene expression was down, and the ShRNA-A7 cell group was down to the original. The level of 17.21% (P0.05), the expression level of CAP1 protein was down to 46.86% (P0.05) of the original level, indicating that the CAP1 gene and protein level decreased after the low expression of Annexin A7 gene. In addition, the expression of Annexin A7 and CAP1 were mainly expressed in the Hca-P cytoplasm by immunofluorescence method, which was expressed in a small amount in the nucleus and speculated two from the image results. Conclusion: Annexin A7 and CAP1 gene can regulate the expression of FAK, SRC, Paxillin, E-cadherin, respectively, in mRNA and protein levels, FAK, SRC, and Paxillin are highly expressed with the down regulation of Annexin and the down regulation of the two gene expression. After low expression, Hca-P cell proliferation, lymph node adhesion, invasion ability increased and early apoptosis decreased. After Annexin A7 gene downregulation, CAP1 was down regulated at mRNA and protein levels, and the expression of two gene in Hca-P cells was basically consistent. It may exist in CO location.Annexin A7 gene and may exist with CAP1 gene. The association of molecular mechanisms can have a consistent effect on cell adhesion and transfer molecules, and is closely related to the biological behavior of Hca-P cells.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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