钙调神经磷酸酶参与肺癌骨转移的机制研究
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摘要
肺癌是发病率和死亡率都非常高的恶性肿瘤,导致肺癌患者死亡的主要原因是肺癌细胞的远处转移,骨骼是肺癌患者最常见的转移部位,约30%的肺癌患者会发生骨转移。肺癌骨转移可导致疼痛、骨折、脊髓压迫、截瘫、高钙血症等,严重影响着患者的生活质量。目前临床对于肺癌骨转移的治疗效果仍然不令人满意,需要研发新的治疗方法,肺癌骨转移机制研究是探索预防和治疗骨转移有效方法的关键。
钙调神经磷酸酶(Calcineurin, CaN)是能够参与多种细胞功能调节的多功能信号酶。最近研究发现CaN参与了胰腺癌和结肠癌的形成,并且研究发现CaN具有致骨转移的潜能:对肿瘤细胞、破骨细胞和成骨细胞均有影响,而肿瘤骨转移的发生是这三种细胞相互作用的结果。因此,CaN可能在恶性肿瘤骨转移发生中发挥了重要作用。我们前期的研究结果发现钙调神经磷酸酶在骨转移小细胞肺癌细胞系SBC-5和非骨转移小细胞肺癌细胞系SBC-3中表达有明显差异。而此两种肺癌细胞有相同的遗传背景却有不同的骨转移潜能。由此推测该基因可能参与了肺癌骨转移的发生。本研究的目的是讨钙调神经磷酸酶在肺癌骨转移发生中可能的作用。本研究为所获国家自然科学基金课题的一部分。
研究内容和结果如下:
第一部分:CnAα在肺癌组织中的表达
1.采用RT-PCR方法对发生骨转移肺癌组织和非骨转移肺癌组织钙调神经磷酸酶Aα同工酶(CnAα)mRNA水平的研究发现:与非骨转移肺癌组织相比,钙调神经磷酸酶Aα同工酶(CnAα)在发生骨转移的肺癌组织表达增高。
2.通过免疫组织化学检测肺癌组织中CnAα的表达,CnAα在发生骨转移的小细胞肺癌组织中表达增强,且主要表达在肿瘤细胞的胞核中,而在非骨转移的小细胞肺癌组织主要表达在胞浆。
第二部分: CnAα的表达及其对肺癌细胞增殖和侵袭的影响
1.通过RT-PCR和Western blot鉴定SBC-5表达CnAα而SBC-3细胞中未发现CnAα的表达。
2.为了进一步探讨钙调神经磷酸酶在肺癌骨转移中的作用和机制,我们通过稳定转染构建了稳定高表达外源性钙调神经磷酸酶的细胞系SBC-3/CnAα和空载体对照细胞系SBC-3/neo。
3. MTT和软琼脂克隆形成实验提示CnAα提高了肺癌细胞的增殖能力。
4.流式细胞仪检测细胞周期显示与对照细胞相比SBC-3/CnAα细胞在G1期显著减低,在S期比例显著升高。结果提示钙调神经磷酸酶分子能够提高细胞的增殖能力;流式细胞仪检测细胞凋亡结果显示与对照细胞SBC-3/neo相比,高表达钙调神经磷酸酶的细胞SBC-3/CnAα凋亡率显著降低。
5.细胞迁移实验显示:与对照细胞相比,高表达钙调神经磷酸酶细胞的迁移能力显著增强;细胞的侵袭实验结果显示,高表达钙调神经磷酸酶的细胞侵袭能力显著增强。
第三部分:利用NOD/SCID鼠探讨CnAα对肺癌骨转移的影响
1.通过尾静脉注射肿瘤细胞SBC-3/CnAα和SBC-3/neo入NOD/SCID体内,每只NOD/SCID鼠注射2.0×106个肿瘤细胞,注射肿瘤细胞四周后,老鼠被通过注射戊巴比妥进行腹腔麻醉,然后拍摄X光片观察发现了骨转移发生。
综上所述,肺癌组织标本CnAα在肺癌骨转移组显著高于肺癌非骨转移组,并且在骨转移肺癌组织肿瘤细胞主要分布在细胞核,而在非骨转移肺癌组织主要分布在细胞浆;体外是实验CnAα显著促进了肺癌细胞增殖,抑制了细胞的凋亡,并且CnAα显著增强肺癌细胞的迁移和侵袭能力;动物实验发现显著高表达CnAα稳定转染细胞(SBC-3/CnAα)发生了可见骨转移。研究提示,CnAα有促进肺癌骨转移的作用。
Lung cancer is the leading morbidity and mortality of malignance. Distant metastasis of lung cancer cells is the main cause of patient death. Bone is common site in lung cancer patients, and more than 30% patients will happen bone metastasis. Bone metastasis usually leads to pain, fracture, spinal compression, paraplegia and hypercalcinemia. These complications make life quality of patients very poor. Now, clinical treatment effect of bone metastasis still didn’t please patients.Therefore, effective therapeutic methods for bone metastasis of lung cancer are urgently needed. Understanding the mechanism of bone metastases is therefore critical for the design of effective strategies to prevent and treat bone metastasis.
Calcineurin(CaN) is multifunctional signal enzyme which involves in regulating function of various cells. A novel role for calcineurin (CaN) has been reported recently regarding the oncogenic potential in pancreatic and colorectal cancer. Recently, an emerging role for CaN is provided with bone metastasis potential: it affects tumor cells, osteoclast and osteoblast. It is the interaction of these three kinds of cells leading to bone metastasis. So, CaN may play important roles in bone metastasis of different human malignancies. Our study previously demonstrated that the distribution of CaN was significantly different between SBC-5 cells which can lead to bone metastasis and SBC-3 cells which can not lead to bone metastasis. The SBC-5 cells and SBC-3 cells have similar genetic background but different potential of bone metastasis. Thus, we speculated that CaN might be responsible for bone metastasis in small cell lung cancer.The aim of this study was to investigate the putative causal role CaN could play in the development of lung cancer with bone metastases. This study is a part of subject financed by National Natural Science Foundation of China.
The objectives and results of the study are as follows:
Part one: CnAαexpression in lung cancer tissues.
1. CnAαwas overexpressed in small cell lung cancer tissue with bone metastasis as compared to small cell lung cancer with non-bone metastasis control samples by RT-PCR.
2. We performed immunostaining on small cell lung cancer tissue sections. Strong nuclear staining of tumor cells was observed in small cell lung cancer tissues with bone metastasis. Conversely, cytoplasm staining of tumor cells was observed in small cell lung cancer tissues with non-bone metastasis.
Part two: CnAαexpression in lung cancer cells and its effects on biological behavior of lung cancer cell in vitro.
1. CnAαwas expressed in small cell lung cancer with bone metastasis cell line (SBC-5), but not in small cell lung cancer with non-bone metastasis cell line (SBC-3) by RT-PCR and Western blot.
2. In order to further illustrate the function and mechanism of CnAαon bone metastasis, we established two clones, SBC-3/CnAαwhich expressed exogenous CnAαand SBC-3/neo as empty vector (control) by stable transfection.
3. MTT assay and soft agar colony formation assay indicate that CnAαenhanced proliferation ability of lung cancer cells.
4. Flow cytometry indicated SBC-3/CnAαcells had a relative lower proportion in the G1 phase and a higher proportion in S phase, as compared to SBC-3/neo cells. Results indicated that CnAαincrease cell proliferation ability. Cell apoptosis was detected by flow cytometry. The number of apoptotic cells of SBC-3/CnAαwas significantly lower compared to SBC-3/neo cells
5. Cell migration assay indicated that CnAα-transfected cells increased migration compared with control-transfected cells. Cell invasion assay indicated CnAαtransfectants increased invasion compared with control transfectants.
Part three: CnAαeffect on lung cancer with bone metastasis was examined by NOD/SCID mice.
1. NOD/SCID mice were inoculated with SBC-3/CnAαand SBC-3/neo, 2.0×106 mouse into the tail vein. Four weeks after tumor cell inoculation, the mice were anesthetized by i.p. injection of pentobarbital and X-ray photographs of the mice were taken to determine bone metastasis. Bone metastasis was discovered.
In conclusion, CnAαwas overexpressed in lung cancer tissues with bone metastasis as compared to tumors with non-bone metastases. Strong nuclear staining of tumor cells was observed in small cell lung cancer tissues with bone metastasis. Conversely, cytoplasmic staining of tumor cells was observed in small cell lung cancer tissues with non-bone metastasis. In vitro it was demonstrated that the CnAαgene obviously promoted cell proliferation and inhibited cell apoptosis. Transfection with the CnAαgene promoted cell migration and invasion. Animal experiment found bone metastasis by intravenous injection of SBC-3/CnAαcells in a mouse model. The study indicated that CnAαpromoted generation of bone metastasis.
钙调神经磷酸酶(Calcineurin, CaN)是能够参与多种细胞功能调节的多功能信号酶。最近研究发现CaN参与了胰腺癌和结肠癌的形成,并且研究发现CaN具有致骨转移的潜能:对肿瘤细胞、破骨细胞和成骨细胞均有影响,而肿瘤骨转移的发生是这三种细胞相互作用的结果。因此,CaN可能在恶性肿瘤骨转移发生中发挥了重要作用。我们前期的研究结果发现钙调神经磷酸酶在骨转移小细胞肺癌细胞系SBC-5和非骨转移小细胞肺癌细胞系SBC-3中表达有明显差异。而此两种肺癌细胞有相同的遗传背景却有不同的骨转移潜能。由此推测该基因可能参与了肺癌骨转移的发生。本研究的目的是讨钙调神经磷酸酶在肺癌骨转移发生中可能的作用。本研究为所获国家自然科学基金课题的一部分。
研究内容和结果如下:
第一部分:CnAα在肺癌组织中的表达
1.采用RT-PCR方法对发生骨转移肺癌组织和非骨转移肺癌组织钙调神经磷酸酶Aα同工酶(CnAα)mRNA水平的研究发现:与非骨转移肺癌组织相比,钙调神经磷酸酶Aα同工酶(CnAα)在发生骨转移的肺癌组织表达增高。
2.通过免疫组织化学检测肺癌组织中CnAα的表达,CnAα在发生骨转移的小细胞肺癌组织中表达增强,且主要表达在肿瘤细胞的胞核中,而在非骨转移的小细胞肺癌组织主要表达在胞浆。
第二部分: CnAα的表达及其对肺癌细胞增殖和侵袭的影响
1.通过RT-PCR和Western blot鉴定SBC-5表达CnAα而SBC-3细胞中未发现CnAα的表达。
2.为了进一步探讨钙调神经磷酸酶在肺癌骨转移中的作用和机制,我们通过稳定转染构建了稳定高表达外源性钙调神经磷酸酶的细胞系SBC-3/CnAα和空载体对照细胞系SBC-3/neo。
3. MTT和软琼脂克隆形成实验提示CnAα提高了肺癌细胞的增殖能力。
4.流式细胞仪检测细胞周期显示与对照细胞相比SBC-3/CnAα细胞在G1期显著减低,在S期比例显著升高。结果提示钙调神经磷酸酶分子能够提高细胞的增殖能力;流式细胞仪检测细胞凋亡结果显示与对照细胞SBC-3/neo相比,高表达钙调神经磷酸酶的细胞SBC-3/CnAα凋亡率显著降低。
5.细胞迁移实验显示:与对照细胞相比,高表达钙调神经磷酸酶细胞的迁移能力显著增强;细胞的侵袭实验结果显示,高表达钙调神经磷酸酶的细胞侵袭能力显著增强。
第三部分:利用NOD/SCID鼠探讨CnAα对肺癌骨转移的影响
1.通过尾静脉注射肿瘤细胞SBC-3/CnAα和SBC-3/neo入NOD/SCID体内,每只NOD/SCID鼠注射2.0×106个肿瘤细胞,注射肿瘤细胞四周后,老鼠被通过注射戊巴比妥进行腹腔麻醉,然后拍摄X光片观察发现了骨转移发生。
综上所述,肺癌组织标本CnAα在肺癌骨转移组显著高于肺癌非骨转移组,并且在骨转移肺癌组织肿瘤细胞主要分布在细胞核,而在非骨转移肺癌组织主要分布在细胞浆;体外是实验CnAα显著促进了肺癌细胞增殖,抑制了细胞的凋亡,并且CnAα显著增强肺癌细胞的迁移和侵袭能力;动物实验发现显著高表达CnAα稳定转染细胞(SBC-3/CnAα)发生了可见骨转移。研究提示,CnAα有促进肺癌骨转移的作用。
Lung cancer is the leading morbidity and mortality of malignance. Distant metastasis of lung cancer cells is the main cause of patient death. Bone is common site in lung cancer patients, and more than 30% patients will happen bone metastasis. Bone metastasis usually leads to pain, fracture, spinal compression, paraplegia and hypercalcinemia. These complications make life quality of patients very poor. Now, clinical treatment effect of bone metastasis still didn’t please patients.Therefore, effective therapeutic methods for bone metastasis of lung cancer are urgently needed. Understanding the mechanism of bone metastases is therefore critical for the design of effective strategies to prevent and treat bone metastasis.
Calcineurin(CaN) is multifunctional signal enzyme which involves in regulating function of various cells. A novel role for calcineurin (CaN) has been reported recently regarding the oncogenic potential in pancreatic and colorectal cancer. Recently, an emerging role for CaN is provided with bone metastasis potential: it affects tumor cells, osteoclast and osteoblast. It is the interaction of these three kinds of cells leading to bone metastasis. So, CaN may play important roles in bone metastasis of different human malignancies. Our study previously demonstrated that the distribution of CaN was significantly different between SBC-5 cells which can lead to bone metastasis and SBC-3 cells which can not lead to bone metastasis. The SBC-5 cells and SBC-3 cells have similar genetic background but different potential of bone metastasis. Thus, we speculated that CaN might be responsible for bone metastasis in small cell lung cancer.The aim of this study was to investigate the putative causal role CaN could play in the development of lung cancer with bone metastases. This study is a part of subject financed by National Natural Science Foundation of China.
The objectives and results of the study are as follows:
Part one: CnAαexpression in lung cancer tissues.
1. CnAαwas overexpressed in small cell lung cancer tissue with bone metastasis as compared to small cell lung cancer with non-bone metastasis control samples by RT-PCR.
2. We performed immunostaining on small cell lung cancer tissue sections. Strong nuclear staining of tumor cells was observed in small cell lung cancer tissues with bone metastasis. Conversely, cytoplasm staining of tumor cells was observed in small cell lung cancer tissues with non-bone metastasis.
Part two: CnAαexpression in lung cancer cells and its effects on biological behavior of lung cancer cell in vitro.
1. CnAαwas expressed in small cell lung cancer with bone metastasis cell line (SBC-5), but not in small cell lung cancer with non-bone metastasis cell line (SBC-3) by RT-PCR and Western blot.
2. In order to further illustrate the function and mechanism of CnAαon bone metastasis, we established two clones, SBC-3/CnAαwhich expressed exogenous CnAαand SBC-3/neo as empty vector (control) by stable transfection.
3. MTT assay and soft agar colony formation assay indicate that CnAαenhanced proliferation ability of lung cancer cells.
4. Flow cytometry indicated SBC-3/CnAαcells had a relative lower proportion in the G1 phase and a higher proportion in S phase, as compared to SBC-3/neo cells. Results indicated that CnAαincrease cell proliferation ability. Cell apoptosis was detected by flow cytometry. The number of apoptotic cells of SBC-3/CnAαwas significantly lower compared to SBC-3/neo cells
5. Cell migration assay indicated that CnAα-transfected cells increased migration compared with control-transfected cells. Cell invasion assay indicated CnAαtransfectants increased invasion compared with control transfectants.
Part three: CnAαeffect on lung cancer with bone metastasis was examined by NOD/SCID mice.
1. NOD/SCID mice were inoculated with SBC-3/CnAαand SBC-3/neo, 2.0×106 mouse into the tail vein. Four weeks after tumor cell inoculation, the mice were anesthetized by i.p. injection of pentobarbital and X-ray photographs of the mice were taken to determine bone metastasis. Bone metastasis was discovered.
In conclusion, CnAαwas overexpressed in lung cancer tissues with bone metastasis as compared to tumors with non-bone metastases. Strong nuclear staining of tumor cells was observed in small cell lung cancer tissues with bone metastasis. Conversely, cytoplasmic staining of tumor cells was observed in small cell lung cancer tissues with non-bone metastasis. In vitro it was demonstrated that the CnAαgene obviously promoted cell proliferation and inhibited cell apoptosis. Transfection with the CnAαgene promoted cell migration and invasion. Animal experiment found bone metastasis by intravenous injection of SBC-3/CnAαcells in a mouse model. The study indicated that CnAαpromoted generation of bone metastasis.
引文
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