蛋白酶体亚基PSMA7在结直肠癌中的表达及其功能研究
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摘要
蛋白酶体亚基PSMA7参与组成20S蛋白酶体核心复合体,其编码基因定位于20q13.33染色体。有报道在不同肿瘤中发现PSMA7的表达上调,一些研究表明PSMA7参与了多种重要的肿瘤相关蛋白的表达及活性调节,但PSMA7本身在肿瘤的发生发展中的作用尚未明确。
目的:1.探讨PSMA7在结直肠癌组织中的表达情况及其与各临床病理因素的相关性。2.构建PSMA7 shRNA表达质粒,评价其RNA干扰的有效性。3.探讨PSMA7在结直肠癌发生发展中的作用及可能的分子机制。
材料与方法:1.采用半定量RT-PCR方法检测32对结直肠癌组织及其对应正常组织中的PSMA7 mRNA表达水平;采用免疫组化方法检测62例结直肠癌原发灶、34例淋巴结转移灶和13例行手术切除的肝转移灶组织中PSMA7的蛋白表达情况,与对应的正常组织进行比较,并分析结直肠癌原发灶组织PSMA7蛋白的表达水平与结直肠癌不同临床病理因素的相关性。2.构建PSMA7 shRNA表达质粒,通过脂质体Lipofectamine 2000介导转染结直肠癌RKO细胞,并通过蛋白免疫印迹验证RNA干扰的有效性。3.对PSMA7 shRNA转染RKO细胞和对照细胞进行细胞表型的对比研究,包括细胞形态、细胞增殖、细胞凋亡、细胞周期、非锚定生长能力、迁移侵袭能力、细胞肌动蛋白骨架和裸鼠皮下移植瘤生长情况的检测;通过RT-PCR检测基质金属蛋白酶(MMP)和组织金属蛋白酶抑制剂(TIMP)的表达,通过RT-PCR和蛋白免疫印迹检测CD44mRNA和CD44蛋白的表达。
结果:1.PSMA7在结直肠癌原发灶、淋巴结转移灶和肝转移灶组织中的表达高于对应的正常组织,结直肠癌原发灶组织的PSMA7表达与肝转移密切相关,结直肠癌原发灶PSMA7高表达患者的生存率明显低于低表达患者,生存率多因素分析显示结直肠癌原发灶组织PSMA7的表达为独立预后因素。2.成功构建PSMA7 shRNA表达质粒,转染RKO细胞,并筛选出PSMA7 shRNA表达克隆,蛋白免疫印迹检测表明PSMA7 shRNA转染明显下调PSMA7的蛋白表达。
3.PSMA7 shRNA转染RKO细胞和对照细胞的对比研究结果表明:PSMA7 shRNA转染对RKO细胞的增殖、凋亡和细胞周期无明显影响,但可以影响细胞形态,抑制RKO细胞的非锚定生长能力和迁移侵袭能力,影响肌动蛋白细胞骨架的组装,抑制RKO细胞在裸鼠体内的成瘤能力。RT-PCR检测表明PSMA7 shRNA转染不影响MMP和TIMP的表达,RT-PCR和蛋白免疫印迹检测表明PSMA7 shRNA转染抑制CD44的表达。
结论:PSMA7在结直肠癌组织中高表达,其表达与肝转移密切相关,是结直肠癌患者独立预后因素。通过构建PSMA7 shRNA表达质粒和脂质体介导转染RKO细胞成功实施RNAi,进行PSMA7的功能研究结果表明:PSMA7shRNA转染可以影响RKO细胞的细胞形态,抑制其非锚定生长能力和迁移侵袭能力,影响肌动蛋白细胞骨架的组装,抑制RKO细胞在裸鼠体内的成瘤能力,提示我们PSMA7可能在结直肠癌的进展中发挥重要作用。PSMA7作用分子机制初步研究结果表明:PSMA7 shRNA转染抑制RKO细胞CD44的表达,调控CD44的表达可能是PSMA7的部分作用机制。
The proteasome subunit PSMA7 located on the 20q13.33 is one of the 7 proteasomeα-subunits. PSMA7 was reported to be overexpressed in several malignant tumors, and play important roles in regulation of some important cancer-associated
proteins. However, the role of PSMA7 in the development of cancer is still unknown. Objective:
1. To investigate the expression of PSMA7 in colorectal cancer and clarify the
correlations between of this expression and the clincopathologic factors.
2. To construct the shorthairpin RNA (shRNA) expressing plasmids which target to PSMA7 and evaluate its effectiveness of RNA interference.
3. To explore the roles and possible mechanisms of PSMA7 in the progression of colorectal cancer.
Material and methods:
1. RT-PCR to evaluate the level of PSMA7 mRNA was carried out in 32 cases colorectal cancerous tissues and matched normal colorectal mucosa. Immunohistiochemistry to evaluate the level of PSMA7 protein in the colorectal cancer was performed in tissues from 62 primary sites, 34 metastatic lymph nodes and 13 metastatic liver lesions as well as matched normal colorectal mucosa. The correlations between the level of PSMA7 protein in the primary sites of colorectal cancer and the clincopathologic factors were analyzed.
2. The shRNA expressing plasmids which target to PSMA7 was constructed and transfected to human colorectal cancer line RKO by cationic liposome, Lipofectamine 2000. The effectiveness of RNA interference was evaluated by Western Blot.
3. Comparative studies between PSMA7 shRNA transfected RKO cell and control cells were carried out, including cell morphology, proliferation, apoptosis, cell cycle, anchorage-independent growth, invasion and migration, actin cytoskeleton and the growth of human colorectal RKO xenograft in nude mice. Expression of MMPs and TIMPs were evaluated by RT-PCR, expression of CD44 was evaluated by RT-PCR and
Western Blot.
Results:
1. PSMA7 was found to be overexpressed in colorectal cancer when compared to
matched normal mucosa. Moreover, expression of PSMA7 in primary tumor was significantly correlated with liver metastasis. High expression of PSMA7 was associated with poor survival, and multivariate analysis revealed expression of PSMA7 to be an independent prognosis factor.
2. The shRNA expressing plasmids which target to PSMA7 was constructed and transfected to human colorectal cancer line RKO by cationic liposome, Lipofectamine 2000, successfully. The stable PSMA7 shRNA transfected clone was selected and efficient inhibition of PSMA7 expression was confirmed by Western Blot.
3. Comparative studies between PSMA7 shRNA transfected RKO cell and control cells revealed that knockdown of PSMA7 affected the cell morphology, inhibited the anchorage-independent growth, suppressed cell invasion and migration, affected the organization of actin cytoskeleton as well as suppressed the tumorigenicity in vivo without significant influence on the proliferation, apoptosis and cell cycle transition. PSMA7 shRNA transfection resulted in significant inhibition of CD44 expression without obvious effect on the expression of MMPs and TIMPs.
Conclusions:
PSMA7 was found to be overexpressed in colorectal cancer when compared to matched normal mucosa. Moreover, expression of PSMA7 in primary tumor was significantly correlated with liver metastasis and was an independent prognosis factor. RNAi was successfully carried out by construction of PSMA7 shRNA expressing plasmid and transfection to RKO cell by liposome, and the functional study of PSMA7 revealed that: knockdown of PSMA7 affected the cell morphology, inhibited the anchorage-independent growth, suppressed cell invasion and migration, affected the organization of actin cytoskeleton as well as suppressed the tumorigenicity in vivo. These results indicated that PSMA7 may play an important role in the progression of colorectal cancer. The initiative study on molecular mechanism of PSMA7 revealed that PSMA7 shRNA transfection inhibited the expression of CD44, regulation of CD44 expression may contribute to the effects of PSMA7.
目的:1.探讨PSMA7在结直肠癌组织中的表达情况及其与各临床病理因素的相关性。2.构建PSMA7 shRNA表达质粒,评价其RNA干扰的有效性。3.探讨PSMA7在结直肠癌发生发展中的作用及可能的分子机制。
材料与方法:1.采用半定量RT-PCR方法检测32对结直肠癌组织及其对应正常组织中的PSMA7 mRNA表达水平;采用免疫组化方法检测62例结直肠癌原发灶、34例淋巴结转移灶和13例行手术切除的肝转移灶组织中PSMA7的蛋白表达情况,与对应的正常组织进行比较,并分析结直肠癌原发灶组织PSMA7蛋白的表达水平与结直肠癌不同临床病理因素的相关性。2.构建PSMA7 shRNA表达质粒,通过脂质体Lipofectamine 2000介导转染结直肠癌RKO细胞,并通过蛋白免疫印迹验证RNA干扰的有效性。3.对PSMA7 shRNA转染RKO细胞和对照细胞进行细胞表型的对比研究,包括细胞形态、细胞增殖、细胞凋亡、细胞周期、非锚定生长能力、迁移侵袭能力、细胞肌动蛋白骨架和裸鼠皮下移植瘤生长情况的检测;通过RT-PCR检测基质金属蛋白酶(MMP)和组织金属蛋白酶抑制剂(TIMP)的表达,通过RT-PCR和蛋白免疫印迹检测CD44mRNA和CD44蛋白的表达。
结果:1.PSMA7在结直肠癌原发灶、淋巴结转移灶和肝转移灶组织中的表达高于对应的正常组织,结直肠癌原发灶组织的PSMA7表达与肝转移密切相关,结直肠癌原发灶PSMA7高表达患者的生存率明显低于低表达患者,生存率多因素分析显示结直肠癌原发灶组织PSMA7的表达为独立预后因素。2.成功构建PSMA7 shRNA表达质粒,转染RKO细胞,并筛选出PSMA7 shRNA表达克隆,蛋白免疫印迹检测表明PSMA7 shRNA转染明显下调PSMA7的蛋白表达。
3.PSMA7 shRNA转染RKO细胞和对照细胞的对比研究结果表明:PSMA7 shRNA转染对RKO细胞的增殖、凋亡和细胞周期无明显影响,但可以影响细胞形态,抑制RKO细胞的非锚定生长能力和迁移侵袭能力,影响肌动蛋白细胞骨架的组装,抑制RKO细胞在裸鼠体内的成瘤能力。RT-PCR检测表明PSMA7 shRNA转染不影响MMP和TIMP的表达,RT-PCR和蛋白免疫印迹检测表明PSMA7 shRNA转染抑制CD44的表达。
结论:PSMA7在结直肠癌组织中高表达,其表达与肝转移密切相关,是结直肠癌患者独立预后因素。通过构建PSMA7 shRNA表达质粒和脂质体介导转染RKO细胞成功实施RNAi,进行PSMA7的功能研究结果表明:PSMA7shRNA转染可以影响RKO细胞的细胞形态,抑制其非锚定生长能力和迁移侵袭能力,影响肌动蛋白细胞骨架的组装,抑制RKO细胞在裸鼠体内的成瘤能力,提示我们PSMA7可能在结直肠癌的进展中发挥重要作用。PSMA7作用分子机制初步研究结果表明:PSMA7 shRNA转染抑制RKO细胞CD44的表达,调控CD44的表达可能是PSMA7的部分作用机制。
The proteasome subunit PSMA7 located on the 20q13.33 is one of the 7 proteasomeα-subunits. PSMA7 was reported to be overexpressed in several malignant tumors, and play important roles in regulation of some important cancer-associated
proteins. However, the role of PSMA7 in the development of cancer is still unknown. Objective:
1. To investigate the expression of PSMA7 in colorectal cancer and clarify the
correlations between of this expression and the clincopathologic factors.
2. To construct the shorthairpin RNA (shRNA) expressing plasmids which target to PSMA7 and evaluate its effectiveness of RNA interference.
3. To explore the roles and possible mechanisms of PSMA7 in the progression of colorectal cancer.
Material and methods:
1. RT-PCR to evaluate the level of PSMA7 mRNA was carried out in 32 cases colorectal cancerous tissues and matched normal colorectal mucosa. Immunohistiochemistry to evaluate the level of PSMA7 protein in the colorectal cancer was performed in tissues from 62 primary sites, 34 metastatic lymph nodes and 13 metastatic liver lesions as well as matched normal colorectal mucosa. The correlations between the level of PSMA7 protein in the primary sites of colorectal cancer and the clincopathologic factors were analyzed.
2. The shRNA expressing plasmids which target to PSMA7 was constructed and transfected to human colorectal cancer line RKO by cationic liposome, Lipofectamine 2000. The effectiveness of RNA interference was evaluated by Western Blot.
3. Comparative studies between PSMA7 shRNA transfected RKO cell and control cells were carried out, including cell morphology, proliferation, apoptosis, cell cycle, anchorage-independent growth, invasion and migration, actin cytoskeleton and the growth of human colorectal RKO xenograft in nude mice. Expression of MMPs and TIMPs were evaluated by RT-PCR, expression of CD44 was evaluated by RT-PCR and
Western Blot.
Results:
1. PSMA7 was found to be overexpressed in colorectal cancer when compared to
matched normal mucosa. Moreover, expression of PSMA7 in primary tumor was significantly correlated with liver metastasis. High expression of PSMA7 was associated with poor survival, and multivariate analysis revealed expression of PSMA7 to be an independent prognosis factor.
2. The shRNA expressing plasmids which target to PSMA7 was constructed and transfected to human colorectal cancer line RKO by cationic liposome, Lipofectamine 2000, successfully. The stable PSMA7 shRNA transfected clone was selected and efficient inhibition of PSMA7 expression was confirmed by Western Blot.
3. Comparative studies between PSMA7 shRNA transfected RKO cell and control cells revealed that knockdown of PSMA7 affected the cell morphology, inhibited the anchorage-independent growth, suppressed cell invasion and migration, affected the organization of actin cytoskeleton as well as suppressed the tumorigenicity in vivo without significant influence on the proliferation, apoptosis and cell cycle transition. PSMA7 shRNA transfection resulted in significant inhibition of CD44 expression without obvious effect on the expression of MMPs and TIMPs.
Conclusions:
PSMA7 was found to be overexpressed in colorectal cancer when compared to matched normal mucosa. Moreover, expression of PSMA7 in primary tumor was significantly correlated with liver metastasis and was an independent prognosis factor. RNAi was successfully carried out by construction of PSMA7 shRNA expressing plasmid and transfection to RKO cell by liposome, and the functional study of PSMA7 revealed that: knockdown of PSMA7 affected the cell morphology, inhibited the anchorage-independent growth, suppressed cell invasion and migration, affected the organization of actin cytoskeleton as well as suppressed the tumorigenicity in vivo. These results indicated that PSMA7 may play an important role in the progression of colorectal cancer. The initiative study on molecular mechanism of PSMA7 revealed that PSMA7 shRNA transfection inhibited the expression of CD44, regulation of CD44 expression may contribute to the effects of PSMA7.
引文
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