慢病毒介导的mTOR靶向抑制对肺腺癌A549细胞生物学功能的影响
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摘要
1目的
肺癌是最常见的肺原发性恶性肿瘤,近年来肺癌的发病率和病死率均迅速上升,其中男性患者中肺癌的死亡率已居首位。其中非小细胞肺癌的发病率在所有肺癌中比例最高。肺癌的发生及发展与肿瘤相关基因的异常表达存在着密切的关系。哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)作为一种高度保守的蛋白激酶,可以通过整合生长因子和营养信号来调节肿瘤细胞生长,是肿瘤生物调节中的关键性基因。目前mTOR已成为肿瘤治疗的生物靶点。
慢病毒介导的RNA干扰是目前公认的最有效的基因沉默手段之一。本文拟通过利用慢病毒介导的RNA干扰技术,以非小细胞肺癌细胞A549为研究对象,以mTOR为目的靶基因。利用MTT、流式细胞术、克隆形成、Transwell及裸鼠体内试验等技术,分别在体外及体内环境中探讨降低mTOR的表达对A549细胞生物学行为的影响。此外通过检测P70S6K, MT1-MMP, HIF-1a, VEGFA, cyclinD1等基因的表达变化,进一步分析mTOR调控肿瘤生物学功能的分子机制。本研究旨在通过以上实验探讨mTOR在肺癌细胞中的作用及其机理,同时为mTOR作为治疗性靶基因在临床方面的应用、为肺癌的基因治疗、基因药物的研制提供必要的实验依据。
2方法
2.1根据mTOR编码区基因序列,设计合成4条shRNA,并克隆到慢病毒表达载体。转染A549细胞后,应用RT-PCR及western blot检测其干扰效果,并确定干扰效率最好的一条shRNA。最后分别将其包装成带有GFP标签及Puro抗性基因的慢病毒颗粒。
2.2体外实验中,将带有GFP绿色荧光标签的慢病毒感染A549细胞。应用MTT、细胞生长曲线、克隆形成、流式细胞术检测A549细胞的增殖和凋亡能力；应用transwell检测细胞侵袭能力；利用western blot检测mTOR的表达降低对P70S6K,MT1-MMP, HIF-1a, VEGFA, cyclinD1蛋白表达的影响。
2.3体内实验中,利用带有Puro抗性基因的慢病毒感染A549细胞并获得持续表达mTOR shRNA及对照病毒的稳定细胞株。将获得的稳定细胞株扩增并以107/只的细胞浓度接种裸鼠。接种后对裸鼠的体重及肿瘤形成进行监测,分析mTOR的低表达对A549细胞成瘤性的影响。
2.4统计学处理：应用SPSS16.0统计分析软件进行统计学处理,计量资料采用均数士标准差(X±s)表示,多样本均数比较采用方差分析,两两比较采用LSD比较,率的比较采用X2检验等进行统计学处理,显著性检验水准取a=0.05。
3结果3.1成功将四条mTOR shRNA克隆至pSIHl-H1-copGFP慢病毒克隆载体,分别为Sh-mTOR-6506, Sh-mTOR-3266, Sh-mTOR-4995, Sh-mTOR-6216。分别转染A549细胞后,通过RT-PCR及western blot验证mTOR的表达水平后发现,Sh-mTOR-6216对A549细胞中mTOR的抑制率可达70%以上,干扰效率明显高于其他干扰组及对照载体组。然后成功包装含有该shRNA序列的带有GFP荧光标记的慢病毒lenti-GFP-mTOR及Puro抗性基因的慢病毒lenti-Puro-mTOR,分别用于以下的体外实验及体内试验。
3.2体外实验中,利用lenti-GFP-mTOR慢病毒感染A549细胞。感染后48h,MTT检测结果显示：空细胞组,对照慢病毒组及lenti-GFP-mTOR组在570nm的吸光度值分别为：0.687±0.103,0.667±0.037,0.356±0.059。lenti-GFP-mTOR组的细胞活力较其它两组有明显下降,差异具有统计学意义(P<0.05)；对照病毒组与空细胞组之间无明显差异,不具有统计学意义。
3.3病毒感染后,A549细胞克隆形成结果显示：空细胞组克隆形成的数量为63±10.536,对照病毒组克隆形成的数量为59.333±9.713,lenti-GFP-mTOR组克隆形成的数量为16±4.583。以上数据说明mTOR的表达降低能够有效地抑制A549细胞克隆形成能力与其余两组相比明显变弱,形成的克隆数量明显变少,差异具有统计学意义(P<0.05)。对照病毒组与空细胞组之间虽略有差异,但不具有统计学意义。
3.4Transwell实验显示,病毒感染后24h,空细胞组穿膜细胞数为124.333±6.110,对照病毒组穿膜细胞数为109.333±11.504,lenti-GFP-mTOR组穿膜细胞数为18±5.568。lenti-GFP-mTOR穿膜细胞数较空细胞组及对照病毒组相比明显减少,差异具有统计学意义(P<0.05)。对照病毒组穿膜细胞数较空细胞组有所下降,但不具有统计学意义。
3.5流式细胞术检测细胞凋亡结果显示：空细胞组早期凋亡细胞占有比例为0.25%,对照病毒组早期凋亡细胞占有比例为3.2%,lenti-GFP-mTOR组早期凋亡细胞占有比例为62.3%。该实验数据提示,利用慢病毒降低A549细胞中mTOR的表达后,lenti-GFP-mTOR能够有效地促进细胞凋亡,细胞凋亡数量较空细胞组及对照病毒组相比明显上升,差异具有统计学意义(P<0.05)。流式细胞术检测细胞周期结果显示：感染lenti-GFP-mTOR后,相比较空细胞组及对照病毒组,细胞出现G1期阻滞。
3.6病毒感染后48h,应用western blot检测A549细胞中mTOR, P70S6K, MT1-MMP, HIF-1a, VEGFA, cyclinD1蛋白的表达变化。结果显示：mTOR的表达降低能够抑制P70S6K, MT1-MMP, HIF-la, VEGFA and cyclinD1等基因的蛋白表达。
3.7体内试验中,将lenti-Puro-mTOR组稳定细胞株、对照病毒组稳定细胞株及空细胞接种于裸鼠体内。裸鼠处死后观察肿瘤大小。结果显示：空细胞组肿瘤大小为72.767±14.677mm3,对照病毒组肿瘤大小为62.625±6.358mm3,lenti-Puro-mTOR组肿瘤大小为28.308±3.521mm3。lenti-Puro-mTOR组形成的肿瘤体积明显小于空细胞组及对照病毒组,差异具有统计学意义(P<0.05)。对照病毒组肿瘤体积小于空细胞组,但不具有统计学意义。
4结论
4.1成功构建分别带有绿色荧光标记及嘌呤霉素抗性的特异性靶向mTOR shRNA的慢病毒,将其感染A549细胞后,可有效降低mTOR的蛋白表达。
4.2体外实验证实利用慢病毒靶向携带mTOR RNA干扰技术降低A549细胞中mTOR的表达,可以抑制细胞的增殖、转移,以及促进细胞的凋亡。通过western blot实验发现,mTOR的低表达导致其下游一些分子的表达降低,包括P70S6K、HIF-1、MT1-MNP、VEGFA、cyclinD1。
4.3成功建立A549细胞裸鼠模型并应用于裸鼠体内实验。实验结果显示降低mTOR的表达可以抑制裸鼠体内肿瘤的生长。
Objective
Lung cancer is an aggressive malignancy with the higher incidence in men than that in women. Laryngal squamous cell carcinoma(LSCC) is the main kind of lung cancer. Gene deregulation in patiens associates with lung caner risk. mTOR as one kind of protein kinase has been confirmed to control cancer progression by regulating cancer relative genes. Early studies also shown that mTOR is crucial to the development of cancer and also provides a new, potential therapeutic target for treating cancer.
RNA interference(RNAi) is a popular technique that can down-regulate gene expression specifically. In this study, RNAi was performed to regulate mTOR expression in A549cells. After mTOR down-regulation, MTT assay, colony formation assay, Transwell and Murine xenograft model were performed to test the influence of mTOR on A549cell progression. In addition, to further understand the
mechanism of mTOR in lung cancer regulation, the expression of P70S6K, MT1-MMP, HIF-1a, VEGFA and cyclinD1were analysised by western blot.
Methods
1According to coding region of mTOR, mTOR specific shRN A expression plasmids were constructed and transfected into A549cells, western blot was performed to detect the expression of mTOR. After the most efficiency shRNA selected, lentivirus with GFP (lenti-GFP-mTOR) or Puro (lenti-Puro-mTOR) resistance marker were packaged.
2After treated with lenti-GFP-mTOR, MTT assay, colony formation assay, flow assay and Transwell were performed to analysis the proliferation and invasion ability of A549cells.
3In vivo, stable cell lines were established by treated A549cells with lenti-Puro-mTOR and control lentivirus.1×107cells were injected into athymic nude mice. The mice were monitored and tumor size was measured, and tumor volumes were calculated as width (mm)×width (mm) X length (mm)×0.5.23days after injection, the mice were killed.
4Statistical analysis:Data are expressed as means±SEM. Statistical analysis was performed using the SPSS software version16.0. All the data were analyzed by ANOVA. P<0.05was considered statistically significant.
Results
1Four mTOR specific shRNA lentivirus expression plasmids were constructed, Sh-mTOR-6506, Sh-mTOR-3266, Sh-mTOR-4995, Sh-mTOR-6216. After trasfection, Sh-mTOR-6216was selected to be the most inhibition rate of mTOR in A549cells by western blot. Then lentivirus with GFP (lenti-GFP-mTOR) or Puro (lenti-Puro-mTOR) resistance marker containing mTOR-6216shRNA were packaged.
2In vitro, MTT assay was performed to detect A549cell vability after being treated with lenti-GFP-mTOR and control lentivirus. The absorbance at570nm was as followed, Mock,0.687+0.103, control group,0.667±0.037, lenti-GFP-mTOR,0.356±0.059. Compared with control, lenti-GFP-mTOR had significant inhibition on A549cell(P<0.05). There is no significant difference between mock group and control group.
3Colony formation assay was performed to detect A549cell proliferation after treated with lenti-GFP-mTOR and control lentivirus. The clone number were culculated, Mock,63±10.536, control group,59.333±9.713, lenti-GFP-mTOR,16±4.583. lenti-GFP-mTOR had greater influence on A549cell colony formation ability compared with control group. Clone numbers between lenti-GFP-mTOR and control group had significant difference(.P<0.05).
4Transwell was performed to detect A549cell invasion24h after being treated with lenti-GFP-mTOR and control lentivirus. The results were shown that after treated with lenti-GFP-mTOR, A549cell invasion ability was decreased compared with control goup, and there is significant difference (P<0.05), but no significant difference between Mock and control group. The passed cells were shown, Mock,124.333±6.110, control group,109.333±11.504, lenti-GFP-mTOR,18±5.568.
5The results of flow cytometry analysis shown that down-regulation of mTOR expression by lenti-GFP-mTOR leaded to G1phase arrest and contributed to A549cell apoptosis compared with control group. The proportion of apoptosis cell was as followed, Mock,0.337±0.103, control group,2.473±0.634, lenti-GFP-mTOR,54.973±6.377. 6The expressions of P70S6K, MT1-MMP, HIF-la, VEGFA and cyclinDl were analyzed by western blot after mTOR down-regulated. Data shown that, mTOR is the positate regulator of these genes, down-regulation of mTOR decreased the expression of P70S6K, MT1-MMP, HIF-la, VEGFA and cyclinD1.7In vivo, tumor volume was recorded after mice were killed. Tumor volume in the group of lenti-Puro-mTOR was28.308+3.521mm3, which was significantly less than that in control group (P<0.05). Tumor volume of Mock group and control group were,72.767±14.677mm3and62.625±6.358mm3respectivly, and there is no significant difference between them.
Conclusions
1mTOR specific silencing lentivirus with GFP of Puro selecting marker was successfully packaged,
2In vitro, down-regulation of mTOR expression by lenti-GFP-mTOR inhibited A549cell proliferation, invasion ability, and contributed to cell apoptosis. Western blot analysis shown that the expression of P70S6K, HIF-la, VEGFA and cyclinDl were decreased after mTOR silenced.
3In vivo, compared with control group, down-regulation of mTOR in A549cells inhibited tumor genesis, the tumor volume in lenti-Puro-mTOR group was significantly less than that in control group.
肺癌是最常见的肺原发性恶性肿瘤,近年来肺癌的发病率和病死率均迅速上升,其中男性患者中肺癌的死亡率已居首位。其中非小细胞肺癌的发病率在所有肺癌中比例最高。肺癌的发生及发展与肿瘤相关基因的异常表达存在着密切的关系。哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)作为一种高度保守的蛋白激酶,可以通过整合生长因子和营养信号来调节肿瘤细胞生长,是肿瘤生物调节中的关键性基因。目前mTOR已成为肿瘤治疗的生物靶点。
慢病毒介导的RNA干扰是目前公认的最有效的基因沉默手段之一。本文拟通过利用慢病毒介导的RNA干扰技术,以非小细胞肺癌细胞A549为研究对象,以mTOR为目的靶基因。利用MTT、流式细胞术、克隆形成、Transwell及裸鼠体内试验等技术,分别在体外及体内环境中探讨降低mTOR的表达对A549细胞生物学行为的影响。此外通过检测P70S6K, MT1-MMP, HIF-1a, VEGFA, cyclinD1等基因的表达变化,进一步分析mTOR调控肿瘤生物学功能的分子机制。本研究旨在通过以上实验探讨mTOR在肺癌细胞中的作用及其机理,同时为mTOR作为治疗性靶基因在临床方面的应用、为肺癌的基因治疗、基因药物的研制提供必要的实验依据。
2方法
2.1根据mTOR编码区基因序列,设计合成4条shRNA,并克隆到慢病毒表达载体。转染A549细胞后,应用RT-PCR及western blot检测其干扰效果,并确定干扰效率最好的一条shRNA。最后分别将其包装成带有GFP标签及Puro抗性基因的慢病毒颗粒。
2.2体外实验中,将带有GFP绿色荧光标签的慢病毒感染A549细胞。应用MTT、细胞生长曲线、克隆形成、流式细胞术检测A549细胞的增殖和凋亡能力；应用transwell检测细胞侵袭能力；利用western blot检测mTOR的表达降低对P70S6K,MT1-MMP, HIF-1a, VEGFA, cyclinD1蛋白表达的影响。
2.3体内实验中,利用带有Puro抗性基因的慢病毒感染A549细胞并获得持续表达mTOR shRNA及对照病毒的稳定细胞株。将获得的稳定细胞株扩增并以107/只的细胞浓度接种裸鼠。接种后对裸鼠的体重及肿瘤形成进行监测,分析mTOR的低表达对A549细胞成瘤性的影响。
2.4统计学处理：应用SPSS16.0统计分析软件进行统计学处理,计量资料采用均数士标准差(X±s)表示,多样本均数比较采用方差分析,两两比较采用LSD比较,率的比较采用X2检验等进行统计学处理,显著性检验水准取a=0.05。
3结果3.1成功将四条mTOR shRNA克隆至pSIHl-H1-copGFP慢病毒克隆载体,分别为Sh-mTOR-6506, Sh-mTOR-3266, Sh-mTOR-4995, Sh-mTOR-6216。分别转染A549细胞后,通过RT-PCR及western blot验证mTOR的表达水平后发现,Sh-mTOR-6216对A549细胞中mTOR的抑制率可达70%以上,干扰效率明显高于其他干扰组及对照载体组。然后成功包装含有该shRNA序列的带有GFP荧光标记的慢病毒lenti-GFP-mTOR及Puro抗性基因的慢病毒lenti-Puro-mTOR,分别用于以下的体外实验及体内试验。
3.2体外实验中,利用lenti-GFP-mTOR慢病毒感染A549细胞。感染后48h,MTT检测结果显示：空细胞组,对照慢病毒组及lenti-GFP-mTOR组在570nm的吸光度值分别为：0.687±0.103,0.667±0.037,0.356±0.059。lenti-GFP-mTOR组的细胞活力较其它两组有明显下降,差异具有统计学意义(P<0.05)；对照病毒组与空细胞组之间无明显差异,不具有统计学意义。
3.3病毒感染后,A549细胞克隆形成结果显示：空细胞组克隆形成的数量为63±10.536,对照病毒组克隆形成的数量为59.333±9.713,lenti-GFP-mTOR组克隆形成的数量为16±4.583。以上数据说明mTOR的表达降低能够有效地抑制A549细胞克隆形成能力与其余两组相比明显变弱,形成的克隆数量明显变少,差异具有统计学意义(P<0.05)。对照病毒组与空细胞组之间虽略有差异,但不具有统计学意义。
3.4Transwell实验显示,病毒感染后24h,空细胞组穿膜细胞数为124.333±6.110,对照病毒组穿膜细胞数为109.333±11.504,lenti-GFP-mTOR组穿膜细胞数为18±5.568。lenti-GFP-mTOR穿膜细胞数较空细胞组及对照病毒组相比明显减少,差异具有统计学意义(P<0.05)。对照病毒组穿膜细胞数较空细胞组有所下降,但不具有统计学意义。
3.5流式细胞术检测细胞凋亡结果显示：空细胞组早期凋亡细胞占有比例为0.25%,对照病毒组早期凋亡细胞占有比例为3.2%,lenti-GFP-mTOR组早期凋亡细胞占有比例为62.3%。该实验数据提示,利用慢病毒降低A549细胞中mTOR的表达后,lenti-GFP-mTOR能够有效地促进细胞凋亡,细胞凋亡数量较空细胞组及对照病毒组相比明显上升,差异具有统计学意义(P<0.05)。流式细胞术检测细胞周期结果显示：感染lenti-GFP-mTOR后,相比较空细胞组及对照病毒组,细胞出现G1期阻滞。
3.6病毒感染后48h,应用western blot检测A549细胞中mTOR, P70S6K, MT1-MMP, HIF-1a, VEGFA, cyclinD1蛋白的表达变化。结果显示：mTOR的表达降低能够抑制P70S6K, MT1-MMP, HIF-la, VEGFA and cyclinD1等基因的蛋白表达。
3.7体内试验中,将lenti-Puro-mTOR组稳定细胞株、对照病毒组稳定细胞株及空细胞接种于裸鼠体内。裸鼠处死后观察肿瘤大小。结果显示：空细胞组肿瘤大小为72.767±14.677mm3,对照病毒组肿瘤大小为62.625±6.358mm3,lenti-Puro-mTOR组肿瘤大小为28.308±3.521mm3。lenti-Puro-mTOR组形成的肿瘤体积明显小于空细胞组及对照病毒组,差异具有统计学意义(P<0.05)。对照病毒组肿瘤体积小于空细胞组,但不具有统计学意义。
4结论
4.1成功构建分别带有绿色荧光标记及嘌呤霉素抗性的特异性靶向mTOR shRNA的慢病毒,将其感染A549细胞后,可有效降低mTOR的蛋白表达。
4.2体外实验证实利用慢病毒靶向携带mTOR RNA干扰技术降低A549细胞中mTOR的表达,可以抑制细胞的增殖、转移,以及促进细胞的凋亡。通过western blot实验发现,mTOR的低表达导致其下游一些分子的表达降低,包括P70S6K、HIF-1、MT1-MNP、VEGFA、cyclinD1。
4.3成功建立A549细胞裸鼠模型并应用于裸鼠体内实验。实验结果显示降低mTOR的表达可以抑制裸鼠体内肿瘤的生长。
Objective
Lung cancer is an aggressive malignancy with the higher incidence in men than that in women. Laryngal squamous cell carcinoma(LSCC) is the main kind of lung cancer. Gene deregulation in patiens associates with lung caner risk. mTOR as one kind of protein kinase has been confirmed to control cancer progression by regulating cancer relative genes. Early studies also shown that mTOR is crucial to the development of cancer and also provides a new, potential therapeutic target for treating cancer.
RNA interference(RNAi) is a popular technique that can down-regulate gene expression specifically. In this study, RNAi was performed to regulate mTOR expression in A549cells. After mTOR down-regulation, MTT assay, colony formation assay, Transwell and Murine xenograft model were performed to test the influence of mTOR on A549cell progression. In addition, to further understand the
mechanism of mTOR in lung cancer regulation, the expression of P70S6K, MT1-MMP, HIF-1a, VEGFA and cyclinD1were analysised by western blot.
Methods
1According to coding region of mTOR, mTOR specific shRN A expression plasmids were constructed and transfected into A549cells, western blot was performed to detect the expression of mTOR. After the most efficiency shRNA selected, lentivirus with GFP (lenti-GFP-mTOR) or Puro (lenti-Puro-mTOR) resistance marker were packaged.
2After treated with lenti-GFP-mTOR, MTT assay, colony formation assay, flow assay and Transwell were performed to analysis the proliferation and invasion ability of A549cells.
3In vivo, stable cell lines were established by treated A549cells with lenti-Puro-mTOR and control lentivirus.1×107cells were injected into athymic nude mice. The mice were monitored and tumor size was measured, and tumor volumes were calculated as width (mm)×width (mm) X length (mm)×0.5.23days after injection, the mice were killed.
4Statistical analysis:Data are expressed as means±SEM. Statistical analysis was performed using the SPSS software version16.0. All the data were analyzed by ANOVA. P<0.05was considered statistically significant.
Results
1Four mTOR specific shRNA lentivirus expression plasmids were constructed, Sh-mTOR-6506, Sh-mTOR-3266, Sh-mTOR-4995, Sh-mTOR-6216. After trasfection, Sh-mTOR-6216was selected to be the most inhibition rate of mTOR in A549cells by western blot. Then lentivirus with GFP (lenti-GFP-mTOR) or Puro (lenti-Puro-mTOR) resistance marker containing mTOR-6216shRNA were packaged.
2In vitro, MTT assay was performed to detect A549cell vability after being treated with lenti-GFP-mTOR and control lentivirus. The absorbance at570nm was as followed, Mock,0.687+0.103, control group,0.667±0.037, lenti-GFP-mTOR,0.356±0.059. Compared with control, lenti-GFP-mTOR had significant inhibition on A549cell(P<0.05). There is no significant difference between mock group and control group.
3Colony formation assay was performed to detect A549cell proliferation after treated with lenti-GFP-mTOR and control lentivirus. The clone number were culculated, Mock,63±10.536, control group,59.333±9.713, lenti-GFP-mTOR,16±4.583. lenti-GFP-mTOR had greater influence on A549cell colony formation ability compared with control group. Clone numbers between lenti-GFP-mTOR and control group had significant difference(.P<0.05).
4Transwell was performed to detect A549cell invasion24h after being treated with lenti-GFP-mTOR and control lentivirus. The results were shown that after treated with lenti-GFP-mTOR, A549cell invasion ability was decreased compared with control goup, and there is significant difference (P<0.05), but no significant difference between Mock and control group. The passed cells were shown, Mock,124.333±6.110, control group,109.333±11.504, lenti-GFP-mTOR,18±5.568.
5The results of flow cytometry analysis shown that down-regulation of mTOR expression by lenti-GFP-mTOR leaded to G1phase arrest and contributed to A549cell apoptosis compared with control group. The proportion of apoptosis cell was as followed, Mock,0.337±0.103, control group,2.473±0.634, lenti-GFP-mTOR,54.973±6.377. 6The expressions of P70S6K, MT1-MMP, HIF-la, VEGFA and cyclinDl were analyzed by western blot after mTOR down-regulated. Data shown that, mTOR is the positate regulator of these genes, down-regulation of mTOR decreased the expression of P70S6K, MT1-MMP, HIF-la, VEGFA and cyclinD1.7In vivo, tumor volume was recorded after mice were killed. Tumor volume in the group of lenti-Puro-mTOR was28.308+3.521mm3, which was significantly less than that in control group (P<0.05). Tumor volume of Mock group and control group were,72.767±14.677mm3and62.625±6.358mm3respectivly, and there is no significant difference between them.
Conclusions
1mTOR specific silencing lentivirus with GFP of Puro selecting marker was successfully packaged,
2In vitro, down-regulation of mTOR expression by lenti-GFP-mTOR inhibited A549cell proliferation, invasion ability, and contributed to cell apoptosis. Western blot analysis shown that the expression of P70S6K, HIF-la, VEGFA and cyclinDl were decreased after mTOR silenced.
3In vivo, compared with control group, down-regulation of mTOR in A549cells inhibited tumor genesis, the tumor volume in lenti-Puro-mTOR group was significantly less than that in control group.
引文
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