通过改变Smad4的表达研究BMP7-Smad4-Id2信号通路在大肠癌发生发展中的作用及其机制
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摘要
第一部分Smad4蛋白在大肠癌组织中的表达及临床意义
目的：探讨Smad4蛋白在大肠癌组织中的表达及其与患者临床、病理之间的关系。方法：利用免疫组化S-P法检测60例大肠癌组织、27例癌旁正常组织中Smad4的表达；回顾性收集相应患者的临床病理资料。结果：Smad4在大肠癌原发灶中的免疫组化切片评分,与癌旁正常组织相比,采用Mann-Whitney检验,U=294.50,有显著性差异(P<0.001)；Smad4的表达情况与肿瘤的浸润层次,淋巴结转移,Dukes分期有关(P<0.05)。结论：Smad4的低表达可能与大肠癌发生发展有关,Smad4可以作为大肠癌的预后指标和基因靶向治疗的有效靶点。
第二部分Smad4表达下降对大肠癌细胞HCT116增殖性、迁移性、侵袭性以及BMP7-Smad4-Id2通路的影响(体外实验)
目的：本部分实验研究HCT116细胞中的Smad4被小干扰RNA沉默后,是否影响了该细胞的增殖性、迁移性、侵袭性及细胞周期,是否影响了BMP7-Smad4-Id2信号通路的作用。方法：构建Smad4-shRNA质粒,并通过转染和筛选形成该质粒稳定转染的HCT116细胞。通过PCR, Western实验来验证Smad4-shRNA质粒的作用,并测量Id2的表达情况。通过MTT来评价细胞的增殖性的改变,通过Transwell实验来评价细胞迁移性和侵袭性的改变,通过流式细胞技术来评价细胞周期的改变。结果：成功构建Smad4-shRNA质粒,并形成该质粒稳定转染的HCT116细胞株。通过PCR和Western实验分别验证了Smad4在基因和蛋白水平的降低。同时,通过Western实验,得到干扰质粒对Smad4蛋白的抑制率为47.17%。干扰后的HCT116细胞,增殖性、迁移性和侵袭性均增强(P<0.05),细胞周期发生改变。干扰后的HCT116细胞中Id2的表达增加,但在该类细胞中BMP7增多不能引起Id2的改变。结论：在HCT116细胞中,Smad4的降低可以使细胞的增殖性、迁移性、侵袭性增加,Smad4是一种抑癌基因。BMP7通过Smad4改变Id2的表达来影响细胞的活性。BMP7-Smad4-Id2在大肠癌的发生发展中发挥作用。
第三部分Smad4表达下降对大肠癌细胞HCT116裸鼠植瘤生长以及BMP7-Smad4-Id2通路的影响(体内实验)
目的：研究Smad4表达降低的HCT116细胞在裸鼠皮下植瘤后肿瘤的生长情况以及BMP7细胞因子对各种细胞所植肿瘤的影响。方法：用HCT116细胞,HCT116-HK细胞,HCT116-Smad4-shRNA细胞构建裸鼠皮下成瘤模型,5天后肿瘤形成,将裸鼠分为(1)HCT116组：(2)HCT116-HK组：(3)HCT116+BMP7组：(4)HCT116-Smad4-shRNA组：和(5)HCT116-Smad4-shRNA+BMP7组。五个实验组,每个实验组每隔2天瘤周注射0.2ml100ng／ml的BMP7或生理盐水,重复4次。观察肿瘤的大小和重量。通过PCR和免疫组化,测量每组肿瘤中Smad4、Id2在基因和蛋白水平上的表达量。通过TUNEL实验,评估每组肿瘤细胞凋亡的情况。结果：5天后,裸鼠植瘤均成功,肿瘤5-7mm大小,HCT116-Smad4-shRNA组肿瘤生长明显快于HCT116组和HK组。PCR提示HCT116-Smad4-shRNA组肿瘤中Smad4mRNA表达降低,Id2mRNA表达增加。对于OT116-Smad4-shRNA组细胞所成肿瘤,有无BMP7作用,不影响Id2的表达和肿瘤的生长。TUNEL实验提示HCT116-Smad4-shRNA组肿瘤中细胞凋亡降低。结论：Smad4-shRNA可以在体内实验中抑制大肠肿瘤的生长。BMP7-Smad4-Id2细胞信号通路在大肠癌的发生发展中起作用。
第四部分Smad4再表达对大肠癌细胞SW480增殖性、迁移性、侵袭性以及BMP7-Smad4-Id2通路的影响(体外实验)
目的：研究Smad4再表达对大肠癌细胞SW480增殖性和侵袭性的影响；探索BMP7是通过BMP7-Smad4通路,还是直接对Id2起调节作用,从而使Id2发生改变而影响细胞的活性。方法：成功构建Smad4-cDNA质粒并形成该质粒稳定转染的SW480细胞。通过Western实验来验证Smad4的再表达,并测量Id2的蛋白表达情况。通过MTT来评价细胞的增殖性的改变,通过Transwell实验来评价细胞迁移性和侵袭性的改变,通过流式细胞技术来评价细胞周期的改变。结果：成功构建Smad4-cDNA质粒,并形成该质粒稳定转染的SW480细胞株。通过Western实验验证了Smad4在蛋白水平的再表达。再表达后的SW480细胞,增殖性、迁移性和侵袭性均减弱(P<0.05),细胞周期发生改变,静止期细胞增多。再表达后的SW480细胞中Id2的表达降低,而且在该类细胞中BMP7增多可以使该类细胞的活性增强且引起Id2的增加。结论：在SW480细胞中,Smad4的再表达可以使细胞的增殖性、迁移性、侵袭性降低,进一步证实Smad4是一种抑癌基因。BMP7通过Smad4改变Id2的表达来影响细胞的活性。BMP7-Smad4-Id2在大肠癌的发生发展中发挥作用。
PART1. Smad4protein expression in colorectal cancer and its clinical significance
Objective To investigate the expression of the Smad4protein in colorectal cancer tissues and the relationship between the expression of Smad4and the clinical pathological data of the patient. Methods Detected60cases of colorectal carcinoma tissue and27cases of the corresponding adjacent normal tissue expression of Smad4by the Immunohistochemistry S-P method, collected the corresponding clinicopathological data of patients. Results Compared with normal tissues, the immunohistochemical biopsy score of Smad4in colorectal cancer primary tumors, was a significant difference (Mann-Whitney test, u=294.50, P<0.001). Smad4expression was relationship with the tumor invasion, lymph node metastasis, and Dukes staging (P <0.05). Conclusion Low expression of Smad4may be related to the progression of the colorectal cancer. The Smad4can be used as predictors of the prognosis of colorectal cancer, and the gene can be an effective target in gene therapy.
PART2. Down-regulation of Smad4affected proliferation and invasion of colorectal carcinoma HCT116cells and BMP7-Smad4-Id2pathway
Objective The aim of this study was to determine whether the suppression of Smad4by short hairpin RNA (shRNA) regulates the proliferation and invasion of colorectal carcinoma HCT116cells and BMP7-Smad4-Id2pathway. Methods The Smad4-shRNA expression vectors were constructed and stably transfected to HCT116cells. The expression of mRNA and protein of Smad4and Id2was detected using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Cellular proliferation inhibitory activity was determined by methyl thiazolyl tetrazolium (MTT) assay. Transwell assay was used to detect the effect of the inhibition of Smad4-shRNA on migration and invasion.Tht flow cytometry was used to assess the change of the cell cycle. Results The Smad4-shRNA vector, which inhibited Smad4expression, was constructed and successfully transfected to HCT116cells. The levels of mRNA and protein expression of Smad4were markedly decreased following transfection of shRNA compared with the control groups (P<0.05). The abilities of proliferation, migration and invasion were increased following transfection of shRNA (P<0.05). The expression of Id2was increased following transfection of shRNA (P<0.05). For the Smad4-down-regulated HCT116cells, treated with or without BMP7(25ng/ml), no difference was found. Conclusion shRNA-mediated silencing of Smad4was able to enhance the abilities of proliferation, migration and invasion in the HCT116cell line. Therefore, Smad4may act as a tumor-suppressor gene in colorectal carcinoma. BMP7affects Id2by Smad4to change the activity of the HCT116cell.
PART3. Down-regulation of Smad4affected human colorectal carcinoma HCT116growth and BMP7-Smad4-Id2pathway in vivo
Objective The reduction of Smad4by short hairpin RNA (shRNA) resulted growth Promotation of cancer cells. The present study was to evaluate the effect of reduction of Smad4expression by shRNA on growth of human colorectal carcinoma (CRC) in tumor-bearing nude mice in vivo. Methods To establish HCT116cell, HCT116-HK cell,HCT116-Smad4-shRNA cell transplantable model, the nude mice were subcutaneously inoculated with1.0×10(7) cells and kept growing till the tumor xenografts reached5-7mm in diameter. Then the mice were randomly assigned to five groups(three mice in each group:(1) HCT116cell group;(2) HCT116-HK group;(3) HCT116+BMP7group;(4) HCT116-Smad4-shRNA group; and (5) HCT116-Smad4-shRNA+BMP7group.0.2ml/mouse BMP7(100ng/ml) or Saline was injected intratumorally four times once every two day. The weight and volumes of tumor xenografts were recorded. The levels of Smad4and Id2mRNA transcripts and proteins in tumor xenografts were detected by reverse quantitative transcription polymerase chain reaction (QRT-PCR) and immunohistochemical staining respectively. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to detect the death of cells. Results The xenografts in mice could be seen at5th day from the implantation of HCT116cells and all had reached5-7mm in size. After injection intratumorally, the growth speed of tumor xenografts in HCT116-Smad4-shRNA group was significantly fast compared with those in HCT116and HK group(P<0.05). The results of QRT-PCR showed that mRNA levels of Smad4reduced more in HCT116-Smad4-shRNA group than those in HCT116and HK group. The mRNA levels of Id2increased more in HCT116-Smad4-shRNA group than those in HCT116and HK group. For the tumor which was growing from the Smad4-down-regulated HCT116cells, treated with or without BMP7, no difference was found. Immunohistochemical analyses of tumor xenograft sections also revealed the decreased Smad4expression in HCT116-Smad4-shRNA group and the change of Id2. TUNEL assay also showed lower death of tumor xenograft tissue cells in HCT116-Smad4-shRNA group. Conclusion Smad4-shRNA may inhibit the growth of human colorectal tumor xenografts in vivo. BMP7-Smad4-Id2pathway was working on the colorectal tumor.
PART4. Re-expression of Smad4affected proliferation and invasion of colorectal carcinoma sw480cells and BMP7-Smad4-Id2pathway
:Objective The bone morphogenetic proteins (BMPs) Smad4and Id2exert their effect on colorectal carcinoma via several uncharacterized mechanisms. In this study, we determine whether the re-expression of Smad4by cDNA regulates the proliferation and invasion of colorectal carcinoma SW480cells, and investigated whether the transcription factor Id2, which has been implicated in colorectal carcinoma proliferation and metastasis, is involved in BMP7-Smad4signaling, or whether it is regulated by BMP7via another mechanism in this cell type. Methods A Smad4-cDNA vector was constructed and stably transfected into SW480cells. Protein levels of Smad4and Id2were examined by Western blotting. Inhibitory effects on cellular proliferation activity were determined by the methyl thiazolyl tetrazolium (MTT) assay, and invasion and migration potential was detected using the in vitro Matrigel-coated invasion and migration assay. Results Levels of Smad4protein were significantly increased in SW480cells transfected with Smad4-cDNA, compared to those transfected with empty vector. Growth curve analysis revealed that live cell numbers were lower in the Smad4-expressing group than in the control group after36h, and that BMP7treatment caused an increase in live cell numbers in Smad4-expressing cells. Transwell chamber analysis revealed that migration/invasion activity was significantly suppressed when Smad4was expressed. Finally, Smad4-expressing cells treated with BMP7expressed a higher level of Id2protein than the controls. Conclusion The results indicate that Smad4expression may inhibit the growth and invasion of SW480cells, and that BMP7affects Id2levels through Smad4. Therefore, BMP7-Smad4-Id2signaling may play a significant role in the development of colorectal carcinoma.
目的：探讨Smad4蛋白在大肠癌组织中的表达及其与患者临床、病理之间的关系。方法：利用免疫组化S-P法检测60例大肠癌组织、27例癌旁正常组织中Smad4的表达；回顾性收集相应患者的临床病理资料。结果：Smad4在大肠癌原发灶中的免疫组化切片评分,与癌旁正常组织相比,采用Mann-Whitney检验,U=294.50,有显著性差异(P<0.001)；Smad4的表达情况与肿瘤的浸润层次,淋巴结转移,Dukes分期有关(P<0.05)。结论：Smad4的低表达可能与大肠癌发生发展有关,Smad4可以作为大肠癌的预后指标和基因靶向治疗的有效靶点。
第二部分Smad4表达下降对大肠癌细胞HCT116增殖性、迁移性、侵袭性以及BMP7-Smad4-Id2通路的影响(体外实验)
目的：本部分实验研究HCT116细胞中的Smad4被小干扰RNA沉默后,是否影响了该细胞的增殖性、迁移性、侵袭性及细胞周期,是否影响了BMP7-Smad4-Id2信号通路的作用。方法：构建Smad4-shRNA质粒,并通过转染和筛选形成该质粒稳定转染的HCT116细胞。通过PCR, Western实验来验证Smad4-shRNA质粒的作用,并测量Id2的表达情况。通过MTT来评价细胞的增殖性的改变,通过Transwell实验来评价细胞迁移性和侵袭性的改变,通过流式细胞技术来评价细胞周期的改变。结果：成功构建Smad4-shRNA质粒,并形成该质粒稳定转染的HCT116细胞株。通过PCR和Western实验分别验证了Smad4在基因和蛋白水平的降低。同时,通过Western实验,得到干扰质粒对Smad4蛋白的抑制率为47.17%。干扰后的HCT116细胞,增殖性、迁移性和侵袭性均增强(P<0.05),细胞周期发生改变。干扰后的HCT116细胞中Id2的表达增加,但在该类细胞中BMP7增多不能引起Id2的改变。结论：在HCT116细胞中,Smad4的降低可以使细胞的增殖性、迁移性、侵袭性增加,Smad4是一种抑癌基因。BMP7通过Smad4改变Id2的表达来影响细胞的活性。BMP7-Smad4-Id2在大肠癌的发生发展中发挥作用。
第三部分Smad4表达下降对大肠癌细胞HCT116裸鼠植瘤生长以及BMP7-Smad4-Id2通路的影响(体内实验)
目的：研究Smad4表达降低的HCT116细胞在裸鼠皮下植瘤后肿瘤的生长情况以及BMP7细胞因子对各种细胞所植肿瘤的影响。方法：用HCT116细胞,HCT116-HK细胞,HCT116-Smad4-shRNA细胞构建裸鼠皮下成瘤模型,5天后肿瘤形成,将裸鼠分为(1)HCT116组：(2)HCT116-HK组：(3)HCT116+BMP7组：(4)HCT116-Smad4-shRNA组：和(5)HCT116-Smad4-shRNA+BMP7组。五个实验组,每个实验组每隔2天瘤周注射0.2ml100ng／ml的BMP7或生理盐水,重复4次。观察肿瘤的大小和重量。通过PCR和免疫组化,测量每组肿瘤中Smad4、Id2在基因和蛋白水平上的表达量。通过TUNEL实验,评估每组肿瘤细胞凋亡的情况。结果：5天后,裸鼠植瘤均成功,肿瘤5-7mm大小,HCT116-Smad4-shRNA组肿瘤生长明显快于HCT116组和HK组。PCR提示HCT116-Smad4-shRNA组肿瘤中Smad4mRNA表达降低,Id2mRNA表达增加。对于OT116-Smad4-shRNA组细胞所成肿瘤,有无BMP7作用,不影响Id2的表达和肿瘤的生长。TUNEL实验提示HCT116-Smad4-shRNA组肿瘤中细胞凋亡降低。结论：Smad4-shRNA可以在体内实验中抑制大肠肿瘤的生长。BMP7-Smad4-Id2细胞信号通路在大肠癌的发生发展中起作用。
第四部分Smad4再表达对大肠癌细胞SW480增殖性、迁移性、侵袭性以及BMP7-Smad4-Id2通路的影响(体外实验)
目的：研究Smad4再表达对大肠癌细胞SW480增殖性和侵袭性的影响；探索BMP7是通过BMP7-Smad4通路,还是直接对Id2起调节作用,从而使Id2发生改变而影响细胞的活性。方法：成功构建Smad4-cDNA质粒并形成该质粒稳定转染的SW480细胞。通过Western实验来验证Smad4的再表达,并测量Id2的蛋白表达情况。通过MTT来评价细胞的增殖性的改变,通过Transwell实验来评价细胞迁移性和侵袭性的改变,通过流式细胞技术来评价细胞周期的改变。结果：成功构建Smad4-cDNA质粒,并形成该质粒稳定转染的SW480细胞株。通过Western实验验证了Smad4在蛋白水平的再表达。再表达后的SW480细胞,增殖性、迁移性和侵袭性均减弱(P<0.05),细胞周期发生改变,静止期细胞增多。再表达后的SW480细胞中Id2的表达降低,而且在该类细胞中BMP7增多可以使该类细胞的活性增强且引起Id2的增加。结论：在SW480细胞中,Smad4的再表达可以使细胞的增殖性、迁移性、侵袭性降低,进一步证实Smad4是一种抑癌基因。BMP7通过Smad4改变Id2的表达来影响细胞的活性。BMP7-Smad4-Id2在大肠癌的发生发展中发挥作用。
PART1. Smad4protein expression in colorectal cancer and its clinical significance
Objective To investigate the expression of the Smad4protein in colorectal cancer tissues and the relationship between the expression of Smad4and the clinical pathological data of the patient. Methods Detected60cases of colorectal carcinoma tissue and27cases of the corresponding adjacent normal tissue expression of Smad4by the Immunohistochemistry S-P method, collected the corresponding clinicopathological data of patients. Results Compared with normal tissues, the immunohistochemical biopsy score of Smad4in colorectal cancer primary tumors, was a significant difference (Mann-Whitney test, u=294.50, P<0.001). Smad4expression was relationship with the tumor invasion, lymph node metastasis, and Dukes staging (P <0.05). Conclusion Low expression of Smad4may be related to the progression of the colorectal cancer. The Smad4can be used as predictors of the prognosis of colorectal cancer, and the gene can be an effective target in gene therapy.
PART2. Down-regulation of Smad4affected proliferation and invasion of colorectal carcinoma HCT116cells and BMP7-Smad4-Id2pathway
Objective The aim of this study was to determine whether the suppression of Smad4by short hairpin RNA (shRNA) regulates the proliferation and invasion of colorectal carcinoma HCT116cells and BMP7-Smad4-Id2pathway. Methods The Smad4-shRNA expression vectors were constructed and stably transfected to HCT116cells. The expression of mRNA and protein of Smad4and Id2was detected using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Cellular proliferation inhibitory activity was determined by methyl thiazolyl tetrazolium (MTT) assay. Transwell assay was used to detect the effect of the inhibition of Smad4-shRNA on migration and invasion.Tht flow cytometry was used to assess the change of the cell cycle. Results The Smad4-shRNA vector, which inhibited Smad4expression, was constructed and successfully transfected to HCT116cells. The levels of mRNA and protein expression of Smad4were markedly decreased following transfection of shRNA compared with the control groups (P<0.05). The abilities of proliferation, migration and invasion were increased following transfection of shRNA (P<0.05). The expression of Id2was increased following transfection of shRNA (P<0.05). For the Smad4-down-regulated HCT116cells, treated with or without BMP7(25ng/ml), no difference was found. Conclusion shRNA-mediated silencing of Smad4was able to enhance the abilities of proliferation, migration and invasion in the HCT116cell line. Therefore, Smad4may act as a tumor-suppressor gene in colorectal carcinoma. BMP7affects Id2by Smad4to change the activity of the HCT116cell.
PART3. Down-regulation of Smad4affected human colorectal carcinoma HCT116growth and BMP7-Smad4-Id2pathway in vivo
Objective The reduction of Smad4by short hairpin RNA (shRNA) resulted growth Promotation of cancer cells. The present study was to evaluate the effect of reduction of Smad4expression by shRNA on growth of human colorectal carcinoma (CRC) in tumor-bearing nude mice in vivo. Methods To establish HCT116cell, HCT116-HK cell,HCT116-Smad4-shRNA cell transplantable model, the nude mice were subcutaneously inoculated with1.0×10(7) cells and kept growing till the tumor xenografts reached5-7mm in diameter. Then the mice were randomly assigned to five groups(three mice in each group:(1) HCT116cell group;(2) HCT116-HK group;(3) HCT116+BMP7group;(4) HCT116-Smad4-shRNA group; and (5) HCT116-Smad4-shRNA+BMP7group.0.2ml/mouse BMP7(100ng/ml) or Saline was injected intratumorally four times once every two day. The weight and volumes of tumor xenografts were recorded. The levels of Smad4and Id2mRNA transcripts and proteins in tumor xenografts were detected by reverse quantitative transcription polymerase chain reaction (QRT-PCR) and immunohistochemical staining respectively. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to detect the death of cells. Results The xenografts in mice could be seen at5th day from the implantation of HCT116cells and all had reached5-7mm in size. After injection intratumorally, the growth speed of tumor xenografts in HCT116-Smad4-shRNA group was significantly fast compared with those in HCT116and HK group(P<0.05). The results of QRT-PCR showed that mRNA levels of Smad4reduced more in HCT116-Smad4-shRNA group than those in HCT116and HK group. The mRNA levels of Id2increased more in HCT116-Smad4-shRNA group than those in HCT116and HK group. For the tumor which was growing from the Smad4-down-regulated HCT116cells, treated with or without BMP7, no difference was found. Immunohistochemical analyses of tumor xenograft sections also revealed the decreased Smad4expression in HCT116-Smad4-shRNA group and the change of Id2. TUNEL assay also showed lower death of tumor xenograft tissue cells in HCT116-Smad4-shRNA group. Conclusion Smad4-shRNA may inhibit the growth of human colorectal tumor xenografts in vivo. BMP7-Smad4-Id2pathway was working on the colorectal tumor.
PART4. Re-expression of Smad4affected proliferation and invasion of colorectal carcinoma sw480cells and BMP7-Smad4-Id2pathway
:Objective The bone morphogenetic proteins (BMPs) Smad4and Id2exert their effect on colorectal carcinoma via several uncharacterized mechanisms. In this study, we determine whether the re-expression of Smad4by cDNA regulates the proliferation and invasion of colorectal carcinoma SW480cells, and investigated whether the transcription factor Id2, which has been implicated in colorectal carcinoma proliferation and metastasis, is involved in BMP7-Smad4signaling, or whether it is regulated by BMP7via another mechanism in this cell type. Methods A Smad4-cDNA vector was constructed and stably transfected into SW480cells. Protein levels of Smad4and Id2were examined by Western blotting. Inhibitory effects on cellular proliferation activity were determined by the methyl thiazolyl tetrazolium (MTT) assay, and invasion and migration potential was detected using the in vitro Matrigel-coated invasion and migration assay. Results Levels of Smad4protein were significantly increased in SW480cells transfected with Smad4-cDNA, compared to those transfected with empty vector. Growth curve analysis revealed that live cell numbers were lower in the Smad4-expressing group than in the control group after36h, and that BMP7treatment caused an increase in live cell numbers in Smad4-expressing cells. Transwell chamber analysis revealed that migration/invasion activity was significantly suppressed when Smad4was expressed. Finally, Smad4-expressing cells treated with BMP7expressed a higher level of Id2protein than the controls. Conclusion The results indicate that Smad4expression may inhibit the growth and invasion of SW480cells, and that BMP7affects Id2levels through Smad4. Therefore, BMP7-Smad4-Id2signaling may play a significant role in the development of colorectal carcinoma.
引文
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