BCL6B对人结直肠癌细胞SW480和LoVo增殖和迁移的影响及其机制探讨
发布时间：2018-07-31 09:52
【摘要】：研究背景与目的结直肠癌(Colorectal cancer,CRC)为常见的消化道肿瘤,在全球每年约有100万新增病例及50万死亡病例。在我国,随人口老龄化及致癌因素的增加,CRC发病率呈逐年上升趋势。现如今,CRC难于早期诊断,其治疗以手术切除为主,转移和复发仍是导致CRC患者低生存率的主要原因。因此,阐明CRC发病的分子机制尤为重要,可为其早期诊断、治疗及预后等提供新的思路。B细胞淋巴瘤6B(B cell lymphoma 6 member B,BCL6B)基因位于人17号染色体短臂1区3带1亚带,属于B细胞淋巴瘤6(B cell lymphoma 6,BCL6)超家族成员,其广泛表达于人体正常组织,然而在多种肿瘤中则发生不同位点的缺失或突变;因启动子区DNA高甲基化导致的BCL6B表达缺失或下调普遍存在于胃癌、肝癌及CRC中,且其启动子甲基化程度与肿瘤的恶性程度及患者不良预后呈正相关;在CRC中,BCL6B的低表达与肿瘤TNM分期、淋巴转移及放化疗敏感性相关,增加CRC细胞系中BCL6B的表达可激活p53信号通路发挥抗CRC的作用。这些都提示BCL6B可能是一个抑癌基因。目前,关于BCL6B在肿瘤中的作用及机制的报道尚少。本研究拟检测和比较人正常肠上皮细胞系FHC及具有高转移潜能的CRC细胞系SW480和Lo Vo中BCL6B的表达水平,研究BCL6B对CRC细胞增殖、迁移及侵袭能力的影响,并进一步探讨其相关分子机制,为阐明BCL6B在CRC发生发展中的作用和CRC发生发展的机制积累实验依据,也为CRC的诊治提供新的思路。方法1.人正常肠上皮细胞及人CRC细胞系中BCL6B内源性表达的检测:通过RT-PCR、q PCR和Western blot检测人正常肠上皮细胞系FHC及CRC细胞系SW480和Lo Vo中BCL6B的表达,比较癌细胞与正常细胞之间的表达差异。2.重组质粒pc DNA3.1-BCL6B和pc DNA3.1的扩增和验证:将携带人BCL6B基因编码序列的重组质粒pc DNA3.1-BCL6B及其对照质粒pc DNA3.1分别化转至感受态大肠杆菌DH5α中,在LB培养基中培养扩增后提取质粒,分光光度仪测定质粒的浓度,分装后于-20°C保存备用。将扩增所得pc DNA3.1-BCL6B转染SW480和Lo Vo细胞,经RT-PCR、q PCR和Western blot验证该质粒的转染效果。3.BCL6B对SW480和Lo Vo细胞增殖能力的影响:用pc DNA3.1-BCL6B转染SW480和Lo Vo细胞后,经MTT法和克隆形成试验检测其对细胞增殖能力的影响。4.BCL6B对SW480和Lo Vo细胞周期及凋亡的影响:用pc DNA3.1-BCL6B转染SW480和Lo Vo细胞后,经流式细胞术(flow cytometry,FCM)检测和比较细胞周期及凋亡的变化。5.BCL6B对SW480和Lo Vo细胞迁移及侵袭能力的影响:用pc DNA3.1-BCL6B转染SW480和Lo Vo细胞后,经划痕愈合试验及Transwell试验分别检测其对细胞迁移及侵袭的影响。6.BCL6B对SW480和Lo Vo细胞中PI3K/AKT信号通路活性的影响:用pc DNA3.1-BCL6B处理SW480和Lo Vo细胞后,经Western blot检测细胞中p-AKT的蛋白水平;经RT-PCR和Western blot检测细胞中PI3K/AKT通路下游与细胞增殖及迁移相关靶基因Cyclin D1、E-cadherin和MMP-9的表达。7.PI3K/AKT信号通路在BCL6B诱导的CRC细胞增殖和迁移中的作用:联合应用pc DNA3.1-BCL6B和PI3K/AKT抑制剂LY294002处理Lo Vo细胞,经MTT法和Transwell试验分别检测细胞增殖和迁移能力的变化;同时,经Western blot检测Lo Vo细胞中Cyclin D1、E-cadherin和MMP-9蛋白水平的变化。结果1.q RT-PCR结果显示FHC、SW480和Lo Vo细胞中BCL6B的m RNA相对表达量依次为0.93±0.60、0.04±0.05和0.06±0.04。Western blot结果显示这三株细胞中BCL6B蛋白的校正灰度值分别为0.74±0.20、0.06±0.04和0.10±0.03。即与人正常肠上皮细胞系FHC相比,BCL6B在人CRC细胞系SW480和Lo Vo中呈明显低表达。2.pc DNA3.1-BCL6B转染SW480和Lo Vo细胞48小时后,SW480细胞中BCL6B的m RNA和蛋白的相对灰度值分别是对照组的16.7倍(P0.01)和10.5倍(P0.01),Lo Vo细胞中BCL6B的m RNA和蛋白的相对灰度值分别是对照组的12.7倍(P0.01)和7.3倍(P0.05),提示:该重组质粒转染能够上调BCL6B在CRC细胞SW480和Lo Vo中的表达,可用于后续研究。3.BCL6B抑制SW480和Lo Vo细胞的增殖能力。1)MTT检测结果:BCL6B组SW480和Lo Vo细胞的OD值在1d及2d时与各自对照组的OD值之间无明显差异,在3d时则分别是对照组的65.6%(P0.05)和70.7%(P0.05),在4d时分别是对照组的61.2%(P0.05)和52.7%(P0.01)。2)克隆形成试验结果:BCL6B组SW480和Lo Vo细胞形成的克隆数分别是对照组的20.3%(P0.01)和42.2%(P0.05)。4.BCL6B可使SW480和Lo Vo细胞周期阻滞在G1期,且促进细胞凋亡。流式细胞术检测结果:BCL6B组SW480和Lo Vo细胞G1期细胞百分比分别为对照组的1.63倍(P0.01)和1.67倍(P0.01),S期细胞百分比分别为对照组的61.1%(P0.01)和67.9%(P0.05)。同时,与对照组相比,BCL6B组SW480和Lo Vo细胞的凋亡细胞数分别增加了的0.75倍(P0.05)和2.12倍(P0.01)。5.BCL6B抑制SW480和Lo Vo细胞的迁移和侵袭能力。1)划痕愈合试验结果显示,BCL6B组SW480和Lo Vo细胞48h划痕愈合率分别为对照组的61.1%(P0.05)和55.6%(P0.05),72h划痕愈合率分别为对照组的68.8%(P0.05)和63.9%(P0.05)。2)Transwell结果显示,质粒转染后48h,对照组SW480和Lo Vo细胞的穿膜细胞数分别为177±40和143±31,BCL6B组两种细胞的穿膜细胞数分别为69±37个和48±24,实验组穿膜细胞数明显低于对照组,差异具统计学显著性(P0.05)。6.在pc DNA3.1-BCL6B转染的SW480和Lo Vo细胞中:1)p-AKT较对照组分别下调67.7%(P0.05)和45.2%(P0.05)。2)SW480细胞中PI3K/AKT通路下游与增殖和迁移相关的靶基因Cyclin D1、E-cadherin和MMP-9的m RNA水平分别为对照组的63.3%(P0.05)、2.54倍(P0.05)和29.4%(P0.05),Lo Vo细胞中这三者的水平分别为对照组的32.2%(P0.01)、3.32倍(P0.05)和51.8%(P0.05);3)与之一致的是,SW480细胞中Cyclin D1、E-cadherin和MMP-9的蛋白水平分别为对照组的54.4%(P0.05)、2.05倍(P0.05)和50.6%(P0.05),Lo Vo细胞中三者的表达分别为对照组的18.9%(P0.01)、2.04倍(P0.05)和46.4%(P0.01)。以上结果提示BCL6B可通过抑制SW480和Lo Vo细胞中PI3K/AKT通路活性,进而上调E-cadherin的表达和下调Cyclin D1和MMP-9的表达,从而实现其抑制CRC细胞增殖和迁移的作用。7.在Lo Vo细胞中,PI3K抑制剂LY294002显著增强了BCL6B对细胞增殖和迁移的抑制(MTT和Transwell,P0.05),同时也增强BCL6B引起的Cyclin D1和MMP-9的下调和E-cadherin的上调(P0.05)。提示PI3K/AKT信号通路的抑制参与介导BCL6B对CRC细胞增殖和迁移的抑制。结论1.BCL6B在人CRC细胞系SW480和Lo Vo中的表达明显低于人正常肠上皮细胞系FHC。2.BCL6B抑制SW480和Lo Vo细胞的增殖、迁移和侵袭,同时促进其凋亡。3.BCL6B抑制SW480和Lo Vo细胞中PI3K/AKT通路活性,并上调E-cadherin的表达和下调Cyclin D1和MMP-9的表达。4.PI3K/AKT通路的抑制参与介导BCL6B对Lo Vo细胞增殖和迁移的抑制及对Cyclin D1、E-cadherin和MMP-9表达的调节。
[Abstract]:Background and objective Colorectal cancer (CRC) is a common digestive tract tumor. There are about 1 million new cases and 500 thousand death cases in the world each year. In China, the incidence of CRC is increasing year by year with the increase of population aging and carcinogenic factors. Now, CRC is difficult to diagnose early, and the treatment is mainly surgical excision. Migration and recurrence are still the main causes of low survival in CRC patients. Therefore, it is particularly important to elucidate the molecular mechanism of the pathogenesis of CRC, which provides a new idea for the early diagnosis, treatment and prognosis of the.B cell lymphoma 6B (B cell lymphoma 6 member B, BCL6B), which is located in the 3 band 1 subband of the short arm of human chromosome 1, and belongs to 6 of B cell lymphoma (B). Cell lymphoma 6, BCL6) a member of the superfamily, which is widely expressed in normal tissues of the human body. However, the deletion or mutation of different loci occurs in a variety of tumors; the deletion or downregulation of BCL6B expression resulting from the hypermethylation of the promoter region is commonly found in gastric cancer, liver cancer and CRC, and the degree of promoter methylation and the malignancy of the tumor and the degree of malignancy of the tumor. The poor prognosis of patients is positively correlated; in CRC, the low expression of BCL6B is associated with TNM staging, lymphatic metastasis and chemosensitivity. The increase of the expression of BCL6B in the CRC cell line activates the anti CRC effect of the p53 signaling pathway. These suggest that BCL6B may be a tumor suppressor gene. Currently, the role and mechanism of BCL6B in the tumor The purpose of this study is to detect and compare the expression level of BCL6B in human normal intestinal epithelial cell line FHC and the high metastatic potential CRC cell line SW480 and Lo Vo, to study the effect of BCL6B on the proliferation, migration and invasion of CRC cells, and to further explore its molecular mechanism, in order to clarify the role and CRC of BCL6B in the development of CRC. The mechanism accumulates the experimental basis for the development and provides new ideas for the diagnosis and treatment of CRC. Methods the endogenous expression of BCL6B in 1. normal intestinal epithelial cells and human CRC cell lines was detected by RT-PCR, Q PCR and Western blot to detect the expression of FHC and CRC cell lines in normal intestinal epithelial cell lines and CRC cell lines. The amplification and verification of the.2. recombinant plasmid PC DNA3.1-BCL6B and PC DNA3.1: the recombinant plasmid PC DNA3.1-BCL6B and its control plasmid PC DNA3.1 were transferred to the DH5 alpha of the receptive Escherichia coli respectively, and the plasmids were extracted and expanded in the medium of the LB culture, and the plasmids were determined by spectrophotometer. The effect of PC DNA3.1-BCL6B transfection on SW480 and Lo Vo cells was tested by RT-PCR, Q PCR and Western blot. The effect of the transfection of the plasmid on the proliferation ability of the plasmid was verified by RT-PCR, Q PCR and Western blot. The effect of.4.BCL6B on the cycle and apoptosis of SW480 and Lo Vo cells: the effects of PC DNA3.1-BCL6B transfection on SW480 and Lo Vo cells, through flow cytometry (flow cytometry, FCM) detection and comparison of cell cycle and apoptosis After 480 and Lo Vo cells, the effects of.6.BCL6B on the activity of PI3K/AKT signaling pathway in SW480 and Lo Vo cells were detected by scratch healing and Transwell test. The role of Cyclin D1, E-cadherin and MMP-9 signaling pathway in the proliferation and migration of CRC cells induced by BCL6B, the downstream of PI3K/AKT pathway and cell proliferation and migration related target genes,.7.PI3K/AKT signaling pathway in BCL6B induced CRC cell proliferation and migration. At the same time, the changes in the level of Cyclin D1, E-cadherin and MMP-9 protein in Lo Vo cells were detected by Western blot. The results of 1.q RT-PCR showed FHC, and the relative expression of SW480 and 0.06 + three cells showed the three cells. The corrected gray value of BCL6B protein was 0.74 + 0.20,0.06 + 0.04 and 0.10 + 0.03., respectively, compared with human normal intestinal epithelial cell line FHC, BCL6B was obviously low expression in SW480 and Lo Vo of human CRC cell line,.2.pc DNA3.1-BCL6B transfection SW480 and FHC cells were respectively the relative gray value of the protein. 16.7 times (P0.01) and 10.5 times (P0.01), the relative gray value of M RNA and protein in Lo Vo cells was 12.7 times (P0.01) and 7.3 times (P0.05) in the control group, suggesting that the recombinant plasmid transfection could up regulate the expression of BCL6B in CRC cell SW480 and the proliferation ability. .1) MTT detection results: the OD values of SW480 and Lo Vo cells in group BCL6B were not significantly different from those of the control groups at 1D and 2D, while at 3D, they were 65.6% (P0.05) and 70.7% (P0.05) in the control group, respectively, at 61.2% of the control group. The 20.3% (P0.01) and 42.2% (P0.05).4.BCL6B of the control group could block the SW480 and Lo Vo cell cycle in G1 phase and promote cell apoptosis. The percentage of SW480 and Lo Vo cells in the BCL6B group was 1.63 times as high as that of the control group and 1.67 times, respectively, and the percentage of the cells in the BCL6B group was 6 of the control group, respectively. 1.1% (P0.01) and 67.9% (P0.05). Compared with the control group, the number of apoptotic cells in the SW480 and Lo Vo cells of the BCL6B group increased by 0.75 times (P0.05) and 2.12 times (P0.01).5.BCL6B inhibition SW480 and Lo Vo cells. The healing rate of the 61.1% (P0.05) and 55.6% (P0.05) of the group was 68.8% (P0.05) and 63.9% (P0.05).2) in the control group, respectively, Transwell results showed that the number of membrane cells of the plasmid transfected 48h, SW480 and Lo Vo cells in the control group were 177 + 40 and 143 + 31 respectively. The number of membrane cells in the two cells of the BCL6B group were 69 + 37 and 48 + 24, the experimental group was dressed. The number of cells was significantly lower than that of the control group. The difference was statistically significant (P0.05).6. in SW480 and Lo Vo cells transfected by PC DNA3.1-BCL6B: 1) p-AKT decreased by 67.7% (P0.05) and 45.2% (P0.05).2), respectively. For 63.3% (P0.05), 2.54 times (P0.05) and 29.4% (P0.05) and Lo Vo cells, the levels of these three were 32.2% (P0.01), 3.32 times (P0.05) and 51.8% (P0.05) and 3) in the control group, and the Cyclin D1 in SW480 cells was 54.4% (2.05) and 50.6% (2.05 times) and 50.6% (2.05 times) and 50.6% (2.05 times), respectively, respectively. The expression of the three in the Vo cells was 18.9% (P0.01), 2.04 times (P0.05) and 46.4% (P0.01) in the control group. These results suggest that BCL6B can inhibit the PI3K/AKT pathway activity in SW480 and Lo Vo cells, and then up regulate the expression of E-cadherin and down regulation of Cyclin D1 and expression, thus realizing its inhibition of cell proliferation and migration. In o Vo cells, the PI3K inhibitor LY294002 significantly enhanced the inhibition of BCL6B on cell proliferation and migration (MTT and Transwell, P0.05), and also enhanced Cyclin D1 and MMP-9 down and up regulation of BCL6B. The expression in human CRC cell line SW480 and Lo Vo was significantly lower than that in human normal intestinal epithelial cell line FHC.2.BCL6B inhibiting the proliferation, migration and invasion of SW480 and Lo Vo cells, and promoting its apoptosis in SW480 and Lo Vo cells. Inhibition of the pathway mediates the inhibition of BCL6B on proliferation and migration of Lo Vo cells and the regulation of Cyclin D1, E-cadherin and MMP-9 expression.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34
本文编号：2155214
[Abstract]:Background and objective Colorectal cancer (CRC) is a common digestive tract tumor. There are about 1 million new cases and 500 thousand death cases in the world each year. In China, the incidence of CRC is increasing year by year with the increase of population aging and carcinogenic factors. Now, CRC is difficult to diagnose early, and the treatment is mainly surgical excision. Migration and recurrence are still the main causes of low survival in CRC patients. Therefore, it is particularly important to elucidate the molecular mechanism of the pathogenesis of CRC, which provides a new idea for the early diagnosis, treatment and prognosis of the.B cell lymphoma 6B (B cell lymphoma 6 member B, BCL6B), which is located in the 3 band 1 subband of the short arm of human chromosome 1, and belongs to 6 of B cell lymphoma (B). Cell lymphoma 6, BCL6) a member of the superfamily, which is widely expressed in normal tissues of the human body. However, the deletion or mutation of different loci occurs in a variety of tumors; the deletion or downregulation of BCL6B expression resulting from the hypermethylation of the promoter region is commonly found in gastric cancer, liver cancer and CRC, and the degree of promoter methylation and the malignancy of the tumor and the degree of malignancy of the tumor. The poor prognosis of patients is positively correlated; in CRC, the low expression of BCL6B is associated with TNM staging, lymphatic metastasis and chemosensitivity. The increase of the expression of BCL6B in the CRC cell line activates the anti CRC effect of the p53 signaling pathway. These suggest that BCL6B may be a tumor suppressor gene. Currently, the role and mechanism of BCL6B in the tumor The purpose of this study is to detect and compare the expression level of BCL6B in human normal intestinal epithelial cell line FHC and the high metastatic potential CRC cell line SW480 and Lo Vo, to study the effect of BCL6B on the proliferation, migration and invasion of CRC cells, and to further explore its molecular mechanism, in order to clarify the role and CRC of BCL6B in the development of CRC. The mechanism accumulates the experimental basis for the development and provides new ideas for the diagnosis and treatment of CRC. Methods the endogenous expression of BCL6B in 1. normal intestinal epithelial cells and human CRC cell lines was detected by RT-PCR, Q PCR and Western blot to detect the expression of FHC and CRC cell lines in normal intestinal epithelial cell lines and CRC cell lines. The amplification and verification of the.2. recombinant plasmid PC DNA3.1-BCL6B and PC DNA3.1: the recombinant plasmid PC DNA3.1-BCL6B and its control plasmid PC DNA3.1 were transferred to the DH5 alpha of the receptive Escherichia coli respectively, and the plasmids were extracted and expanded in the medium of the LB culture, and the plasmids were determined by spectrophotometer. The effect of PC DNA3.1-BCL6B transfection on SW480 and Lo Vo cells was tested by RT-PCR, Q PCR and Western blot. The effect of the transfection of the plasmid on the proliferation ability of the plasmid was verified by RT-PCR, Q PCR and Western blot. The effect of.4.BCL6B on the cycle and apoptosis of SW480 and Lo Vo cells: the effects of PC DNA3.1-BCL6B transfection on SW480 and Lo Vo cells, through flow cytometry (flow cytometry, FCM) detection and comparison of cell cycle and apoptosis After 480 and Lo Vo cells, the effects of.6.BCL6B on the activity of PI3K/AKT signaling pathway in SW480 and Lo Vo cells were detected by scratch healing and Transwell test. The role of Cyclin D1, E-cadherin and MMP-9 signaling pathway in the proliferation and migration of CRC cells induced by BCL6B, the downstream of PI3K/AKT pathway and cell proliferation and migration related target genes,.7.PI3K/AKT signaling pathway in BCL6B induced CRC cell proliferation and migration. At the same time, the changes in the level of Cyclin D1, E-cadherin and MMP-9 protein in Lo Vo cells were detected by Western blot. The results of 1.q RT-PCR showed FHC, and the relative expression of SW480 and 0.06 + three cells showed the three cells. The corrected gray value of BCL6B protein was 0.74 + 0.20,0.06 + 0.04 and 0.10 + 0.03., respectively, compared with human normal intestinal epithelial cell line FHC, BCL6B was obviously low expression in SW480 and Lo Vo of human CRC cell line,.2.pc DNA3.1-BCL6B transfection SW480 and FHC cells were respectively the relative gray value of the protein. 16.7 times (P0.01) and 10.5 times (P0.01), the relative gray value of M RNA and protein in Lo Vo cells was 12.7 times (P0.01) and 7.3 times (P0.05) in the control group, suggesting that the recombinant plasmid transfection could up regulate the expression of BCL6B in CRC cell SW480 and the proliferation ability. .1) MTT detection results: the OD values of SW480 and Lo Vo cells in group BCL6B were not significantly different from those of the control groups at 1D and 2D, while at 3D, they were 65.6% (P0.05) and 70.7% (P0.05) in the control group, respectively, at 61.2% of the control group. The 20.3% (P0.01) and 42.2% (P0.05).4.BCL6B of the control group could block the SW480 and Lo Vo cell cycle in G1 phase and promote cell apoptosis. The percentage of SW480 and Lo Vo cells in the BCL6B group was 1.63 times as high as that of the control group and 1.67 times, respectively, and the percentage of the cells in the BCL6B group was 6 of the control group, respectively. 1.1% (P0.01) and 67.9% (P0.05). Compared with the control group, the number of apoptotic cells in the SW480 and Lo Vo cells of the BCL6B group increased by 0.75 times (P0.05) and 2.12 times (P0.01).5.BCL6B inhibition SW480 and Lo Vo cells. The healing rate of the 61.1% (P0.05) and 55.6% (P0.05) of the group was 68.8% (P0.05) and 63.9% (P0.05).2) in the control group, respectively, Transwell results showed that the number of membrane cells of the plasmid transfected 48h, SW480 and Lo Vo cells in the control group were 177 + 40 and 143 + 31 respectively. The number of membrane cells in the two cells of the BCL6B group were 69 + 37 and 48 + 24, the experimental group was dressed. The number of cells was significantly lower than that of the control group. The difference was statistically significant (P0.05).6. in SW480 and Lo Vo cells transfected by PC DNA3.1-BCL6B: 1) p-AKT decreased by 67.7% (P0.05) and 45.2% (P0.05).2), respectively. For 63.3% (P0.05), 2.54 times (P0.05) and 29.4% (P0.05) and Lo Vo cells, the levels of these three were 32.2% (P0.01), 3.32 times (P0.05) and 51.8% (P0.05) and 3) in the control group, and the Cyclin D1 in SW480 cells was 54.4% (2.05) and 50.6% (2.05 times) and 50.6% (2.05 times) and 50.6% (2.05 times), respectively, respectively. The expression of the three in the Vo cells was 18.9% (P0.01), 2.04 times (P0.05) and 46.4% (P0.01) in the control group. These results suggest that BCL6B can inhibit the PI3K/AKT pathway activity in SW480 and Lo Vo cells, and then up regulate the expression of E-cadherin and down regulation of Cyclin D1 and expression, thus realizing its inhibition of cell proliferation and migration. In o Vo cells, the PI3K inhibitor LY294002 significantly enhanced the inhibition of BCL6B on cell proliferation and migration (MTT and Transwell, P0.05), and also enhanced Cyclin D1 and MMP-9 down and up regulation of BCL6B. The expression in human CRC cell line SW480 and Lo Vo was significantly lower than that in human normal intestinal epithelial cell line FHC.2.BCL6B inhibiting the proliferation, migration and invasion of SW480 and Lo Vo cells, and promoting its apoptosis in SW480 and Lo Vo cells. Inhibition of the pathway mediates the inhibition of BCL6B on proliferation and migration of Lo Vo cells and the regulation of Cyclin D1, E-cadherin and MMP-9 expression.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34
【参考文献】
相关期刊论文 前1条
1 李道娟;李倩;贺宇彤;;结直肠癌流行病学趋势[J];肿瘤防治研究;2015年03期
，本文编号：2155214
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