Beclin 1沉默对维生素K_3诱导的人肝癌细胞损伤的影响
摘要
肝癌是我国常见的恶性肿瘤之一,近年来其发病率有上升的趋势。因此,深入研究肝癌的分子机制,从分子水平和基因水平上发展出更有效的治疗手段越来越受到重视。本研究探讨不同浓度VK3对人肝癌细胞SMMC-7721损伤影响,以及Bcl-2和Bax表达的变化。利用RNAi技术,建立自噬基因Beclin 1沉默的人肝癌细胞SMMC-7721模型系统。进一步研究自噬抑制对VK3诱导的人肝癌细胞SMMC-7721损伤的影响以及在此过程中自噬和凋亡的关系。
研究结果表明:VK3能够诱导人肝癌SMMC-7721细胞损伤,并呈剂量相关性。机制可能与下调Bcl-2、上调Bax mRNA和蛋白表达有关;利用RNAi技术成功构建pSH1Si-Beclin 1表达载体,转染人肝癌SMMC-7721细胞后,可显著抑制Beclin1 mRNA和蛋白表达;RNAi沉默Beclin1表达以及应用自噬特异性抑制剂3-MA,显著降低人肝癌SMMC-7721细胞自噬能力,促进VK3诱导的SMMC-7721细胞凋亡,提示自噬在VK3诱导人肝癌细胞SMMC-7721损伤中很可能是一种细胞的自我保护机制。
这一实验结果对探讨Beclin 1在肝癌发病过程中所发挥的作用进行了新的探索,也为寻找肝癌基因治疗新的作用靶点进行了有益的尝试,细胞通过自噬提供能量促进细胞生存,能在一定程度上拮抗氧化应激造成的细胞损伤,从而推测肝癌治疗中利用自噬抑制结合抗癌药物的方法很可能是一种新的治疗手段。
Background:
Hepatocellular carcinoma (HCC) is one of the most common tumors in our country and some Asian and African countries, and the incidence is still rising. Because of its rapid development, homo-malignancy, the average survival time is only 3 to 6 months, although some patients with HCC and even a small number of advanced or metastatic patients benefit from surgical resection, liver transplantation, as well as a comprehensive treatment, achieved long-term survival, but the overall long-term effect is still not satisfactory, the mortality rate remained high. To elucidate the molecular mechanism of HCC will contribute to the development of more effective treatment methods. Now many studies have shown that programmed cell death (PCD) in tumor occurrence and development of the process is playing an important role. Therefore,it is not surprising that manipulation of PCD has emerged as a new therapeutic strategy to help eliminate cancer cells.
Form of cell death can be divided into apoptotic programmed cell death-apoptosis (typeⅠ), autophagy-autophagic programmed cell death (typeⅡ) and cell necrosis, all participated cell death regulation with their own various genetic signal pathways. The function of apoptosis is well demonstrated by lots of research that play a significant role in carcinogenesis, cell differentitation, and chemotherapy resistance. Autophagy is a type of programmed cell death which is caspase-independent and different from apoptosis. Autophagy is an evolutionarily conserved mechanism of protein and organelle degradation which has been observed in organisms as diverse as yeast and mammals, as a normal physiological process to maintain homeostasis and enhance survival. Some studies have shown that autophagy suppression may result in malignant transformation. However, the roles and relationships of autophagy and apoptosis in HCC are not yet very clear, the relations between autophagy and cell survival or autophagy and cell death are still debated.
Synthetic vitamin K3 (2-methyl-1,4-naphthoquinone, VK3) are commonly used in clinical as a procoagulant drug. Early fifties in the last century, people began to study the antineoplastic effects of vitamin K. A number of studies have confirmed it in vivo or in vitro had a variety of tumor cell growth inhibition and demonstrated a broad-spectrum antineoplastic activity. Now it is considered to be an oxidative stress stimuli and shown antineoplastic activity.
RNA interference is double-stranded RNA-induced post-transcriptional gene silencing phenomenon, the mechanism can reduce the specificity of target gene expression. This technology has been continuously improved and widely used in gene function and gene therapy research recently.
Beclin1 is the only one founded as a mammals"autophagy gene" so far, it is a homolog of Atg6/Vps30 yeast gene in mammalian. At formation process of autophagic vacuoles(AV), Beclin1 plays an important role,which is also a candidate as a tumor suppressing-gene. So Beclin 1 might be a more directly objective gene for gene therapy on HCC. The study on Beclin 1 has great significance in finding a new treatment strategy and the pathogenesis of HCC, it’s important for the investigation on the role of HCC and search for a new therapic strategy in tumorigenesis and development of HCC.
Objective:
(1)To investigate the influence of injury in human hepatoma SMMC-7721 cells treated with different concentrations of VK3,and the changes of Bcl-2 and Bax expressions in this process.
(2)To establish human hepatoma SMMC-7721 cells model of autophagic gene-Beclin 1 silencing by RNA interference technique.
(3)To explore the effect of autophagic inhibition in the injury of human hepatoma cells induced by Vitamin K3, and the relationship of autophagy and apoptosis in this process.
Methods :
(1)SMMC-7721 cells were treated with different concentrations of VK3. The cell viability was measured by MTT assay; LDH release rate was detected by ultraviolet spectrophotometry; the expressions of Bcl-2 and Bax mRNA were determined using RT-PCR, and the expressions of Bcl-2 and Bax protein were assayed by Western blot.
(2)The recombinant plasmid pSilencer TM 3.1-H1 neo- Beclin 1 siRNA was transfected into SMMC- 7721 hepatoma cells by eukaryotic cell transfection technique. The cells were collected 48h later to extract cell RNA and total protein, to detect Beclin 1 gene expression by RT-PCR and Western blot.
(3)With measure of MDC staining, the accumulation of autophagic vaculos was analyzed quantitatively.
(4)Hoechst 33342 staining was used for the determination of the cell chromatin condensation. FCM was used to detected apotosis by PI staining.
Results:
(1)VK3 induced cells viability decreased and the LDH release rate increased, 40μM is the optimum dose.
(2)VK3 induced the expression of Bcl-2 mRNA and protein decresed, the expression of Bax mRNA and protein increased.
(3)The synthetic siRNA significantly inhibited Beclin 1 decreased the levels of Beclin 1 mRNA and protein expression.
(4)VK3 induced autophagic gene Beclin 1 activation in SMMC-7721 cells.
(5)pSH1Si-Beclin 1 transfection inhibited the activation of Beclin 1 and decreased the average fluorescence intensity of MDC staining.
(6)pSH1Si-Beclin 1 transfection increased the percentage of apoptosis cells in Hoechst 33342 and PI staining.
(7)VK3 combinated with autophagy inhibitor 3-MA accelerated the apoptosis induced by VK3.
Conclusion:
(1)VK3 could induce SMMC-7721 cells injury in a dose-dependent way.
(2)The mechanism of VK3 induced cell injury may be related to down-regulated the expression of Bcl-2 and up-regulation of Bax.
(3)VK3 induced SMMC-7721 cells injury involved in apoptosis and autophagy simultaneously.
(4)The transfection of SMMC-7721 hepatoma cells by pSH1Si-Beclin 1 could effectively inhibit the expression of Beclin 1 mRNA and protein.
(5)RNA interference of Beclin 1 or autophagy inhibitor 3-MA could degrade the autophagy activity, aggravated the apoptosis induced by VK3, suggested that autophagy in the injury induced by VK3 may be a cell survival mechanism.
(6)The results of RNA interference of Beclin 1 accelarated apoptosis induced by VK3 proceded new research of the effect of Beclin 1 in the development of hepatoma, and also a beneficial attempt to search new taget of cancer genetic therapy.
研究结果表明:VK3能够诱导人肝癌SMMC-7721细胞损伤,并呈剂量相关性。机制可能与下调Bcl-2、上调Bax mRNA和蛋白表达有关;利用RNAi技术成功构建pSH1Si-Beclin 1表达载体,转染人肝癌SMMC-7721细胞后,可显著抑制Beclin1 mRNA和蛋白表达;RNAi沉默Beclin1表达以及应用自噬特异性抑制剂3-MA,显著降低人肝癌SMMC-7721细胞自噬能力,促进VK3诱导的SMMC-7721细胞凋亡,提示自噬在VK3诱导人肝癌细胞SMMC-7721损伤中很可能是一种细胞的自我保护机制。
这一实验结果对探讨Beclin 1在肝癌发病过程中所发挥的作用进行了新的探索,也为寻找肝癌基因治疗新的作用靶点进行了有益的尝试,细胞通过自噬提供能量促进细胞生存,能在一定程度上拮抗氧化应激造成的细胞损伤,从而推测肝癌治疗中利用自噬抑制结合抗癌药物的方法很可能是一种新的治疗手段。
Background:
Hepatocellular carcinoma (HCC) is one of the most common tumors in our country and some Asian and African countries, and the incidence is still rising. Because of its rapid development, homo-malignancy, the average survival time is only 3 to 6 months, although some patients with HCC and even a small number of advanced or metastatic patients benefit from surgical resection, liver transplantation, as well as a comprehensive treatment, achieved long-term survival, but the overall long-term effect is still not satisfactory, the mortality rate remained high. To elucidate the molecular mechanism of HCC will contribute to the development of more effective treatment methods. Now many studies have shown that programmed cell death (PCD) in tumor occurrence and development of the process is playing an important role. Therefore,it is not surprising that manipulation of PCD has emerged as a new therapeutic strategy to help eliminate cancer cells.
Form of cell death can be divided into apoptotic programmed cell death-apoptosis (typeⅠ), autophagy-autophagic programmed cell death (typeⅡ) and cell necrosis, all participated cell death regulation with their own various genetic signal pathways. The function of apoptosis is well demonstrated by lots of research that play a significant role in carcinogenesis, cell differentitation, and chemotherapy resistance. Autophagy is a type of programmed cell death which is caspase-independent and different from apoptosis. Autophagy is an evolutionarily conserved mechanism of protein and organelle degradation which has been observed in organisms as diverse as yeast and mammals, as a normal physiological process to maintain homeostasis and enhance survival. Some studies have shown that autophagy suppression may result in malignant transformation. However, the roles and relationships of autophagy and apoptosis in HCC are not yet very clear, the relations between autophagy and cell survival or autophagy and cell death are still debated.
Synthetic vitamin K3 (2-methyl-1,4-naphthoquinone, VK3) are commonly used in clinical as a procoagulant drug. Early fifties in the last century, people began to study the antineoplastic effects of vitamin K. A number of studies have confirmed it in vivo or in vitro had a variety of tumor cell growth inhibition and demonstrated a broad-spectrum antineoplastic activity. Now it is considered to be an oxidative stress stimuli and shown antineoplastic activity.
RNA interference is double-stranded RNA-induced post-transcriptional gene silencing phenomenon, the mechanism can reduce the specificity of target gene expression. This technology has been continuously improved and widely used in gene function and gene therapy research recently.
Beclin1 is the only one founded as a mammals"autophagy gene" so far, it is a homolog of Atg6/Vps30 yeast gene in mammalian. At formation process of autophagic vacuoles(AV), Beclin1 plays an important role,which is also a candidate as a tumor suppressing-gene. So Beclin 1 might be a more directly objective gene for gene therapy on HCC. The study on Beclin 1 has great significance in finding a new treatment strategy and the pathogenesis of HCC, it’s important for the investigation on the role of HCC and search for a new therapic strategy in tumorigenesis and development of HCC.
Objective:
(1)To investigate the influence of injury in human hepatoma SMMC-7721 cells treated with different concentrations of VK3,and the changes of Bcl-2 and Bax expressions in this process.
(2)To establish human hepatoma SMMC-7721 cells model of autophagic gene-Beclin 1 silencing by RNA interference technique.
(3)To explore the effect of autophagic inhibition in the injury of human hepatoma cells induced by Vitamin K3, and the relationship of autophagy and apoptosis in this process.
Methods :
(1)SMMC-7721 cells were treated with different concentrations of VK3. The cell viability was measured by MTT assay; LDH release rate was detected by ultraviolet spectrophotometry; the expressions of Bcl-2 and Bax mRNA were determined using RT-PCR, and the expressions of Bcl-2 and Bax protein were assayed by Western blot.
(2)The recombinant plasmid pSilencer TM 3.1-H1 neo- Beclin 1 siRNA was transfected into SMMC- 7721 hepatoma cells by eukaryotic cell transfection technique. The cells were collected 48h later to extract cell RNA and total protein, to detect Beclin 1 gene expression by RT-PCR and Western blot.
(3)With measure of MDC staining, the accumulation of autophagic vaculos was analyzed quantitatively.
(4)Hoechst 33342 staining was used for the determination of the cell chromatin condensation. FCM was used to detected apotosis by PI staining.
Results:
(1)VK3 induced cells viability decreased and the LDH release rate increased, 40μM is the optimum dose.
(2)VK3 induced the expression of Bcl-2 mRNA and protein decresed, the expression of Bax mRNA and protein increased.
(3)The synthetic siRNA significantly inhibited Beclin 1 decreased the levels of Beclin 1 mRNA and protein expression.
(4)VK3 induced autophagic gene Beclin 1 activation in SMMC-7721 cells.
(5)pSH1Si-Beclin 1 transfection inhibited the activation of Beclin 1 and decreased the average fluorescence intensity of MDC staining.
(6)pSH1Si-Beclin 1 transfection increased the percentage of apoptosis cells in Hoechst 33342 and PI staining.
(7)VK3 combinated with autophagy inhibitor 3-MA accelerated the apoptosis induced by VK3.
Conclusion:
(1)VK3 could induce SMMC-7721 cells injury in a dose-dependent way.
(2)The mechanism of VK3 induced cell injury may be related to down-regulated the expression of Bcl-2 and up-regulation of Bax.
(3)VK3 induced SMMC-7721 cells injury involved in apoptosis and autophagy simultaneously.
(4)The transfection of SMMC-7721 hepatoma cells by pSH1Si-Beclin 1 could effectively inhibit the expression of Beclin 1 mRNA and protein.
(5)RNA interference of Beclin 1 or autophagy inhibitor 3-MA could degrade the autophagy activity, aggravated the apoptosis induced by VK3, suggested that autophagy in the injury induced by VK3 may be a cell survival mechanism.
(6)The results of RNA interference of Beclin 1 accelarated apoptosis induced by VK3 proceded new research of the effect of Beclin 1 in the development of hepatoma, and also a beneficial attempt to search new taget of cancer genetic therapy.
引文
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