Galectin-3特异性siRNA对肺癌肿瘤干细胞生物学功能影响的研究
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摘要
目的：肿瘤干细胞对化疗药物的敏感性差,往往成为肿瘤复发的根源,研发特异性针对肿瘤干细胞的新型药物是目前临床治疗肿瘤的最具潜力的研究方向。肺癌干细胞分离后,通过表面标记纯化肺癌干细胞,研究它们的基因表达模式,找到肺癌发生过程中必需的功能蛋白,把该蛋白作为新的药物靶标来治疗肺癌,是目前肺癌防治的一个新的研究方向。本研究检测了肺腺癌CD133+细胞中Galectin-3的表达情况,并利用体外化学合成的特异性针对galectin-3的siRNA研究了CD133阳性肺腺癌肿瘤干细胞中表达的Galectin-3的生物学功能。
方法：第一部分实验是从10例肺腺癌患者切除的肿瘤标本中通过磁珠分选技术分选出CD133+肺腺癌细胞,通过流式细胞术检测分选出的细胞中CD133+细胞百分率。第二部分实验是采用FQRT-PCR和Westernblot的方法分别在转录和翻译两个水平检测了CD133+肺腺癌细胞中Galectin-3的表达,同时流式细胞术检测了CD133+细胞中galectin-3的分布情况。第三部分研究内容旨在深入研究Galectin-3在CD133+细胞中高表达的生物学意义。采用针对galectin-3的特异性siRNA来研究降低CD133+细胞中galectin-3的表达对CD133+肺腺癌细胞的增殖,转移能力及其诱导CD8+T细胞凋亡能力的影响。首先通过FQRT-PCR和Westernblot检测siRNA对galectin-3的干扰效率,随后单克隆形成实验以及MTT法检测了特异性针对galectin-3的siRNA对CD133+肺腺癌细胞增殖的影响,Boyden小室模型检测了siRNA对CD133+肺腺癌细胞转移能力的影响,最后AnnexinV和PI双染检测转染siRNA的CD133+肺腺癌细胞上清诱导CD8+T细胞凋亡能力。
结果：第一部分的实验结果表明,磁珠分选出的细胞中CD133+细胞数达到了90%,体外培养2-3天后经过磁珠分选出来的CD133+细胞能形成典型的肿瘤干细胞球。第二部分FQRT-PCR和Westernblot结果发现Galectin-3在CD133+细胞中的表达量分别为CD133-细胞中的1.24倍和1.5倍,差异有统计学意义(p<0.05)。流式细胞仪检测结果表明CD133+肺腺癌肿瘤干细胞胞内以及细胞膜上均有galectin-3的分布,且平均荧光强度统计结果显示CD133+细胞内(17.8±2.5)以及细胞膜(25.6±5.1)上的galectin-3的表达量均较CD133-细胞内(7.3±3.1)以及细胞膜(10.3±3.4)上galectin-3的表达量高,并且差异具有统计学意义(p<0.05)。第三部分的实验结果显示,针对galectin-3的siRNA平均干扰效率为80.3%,明显降低CD133+细胞中galectin-3的表达,并抑制CD133+细胞增殖,转染96h后细胞活率为未转染组的(75±3.5)%,转染siRNA后的CD133+细胞单克隆形成率(60.12±4.32)%明显较未转染组(87.34±4.89)%下降,差异具有统计学意义(p<0.05)。Boyden小室模型检测结果表明：转染siRNA后,CD133+细胞的转移侵袭能力(68±7)明显较未转染组(100±8)下降,差异具有统计学意义(p<0.05),凋亡检测结果表明CD133+细胞的上清诱导CD8+T细胞凋亡率为(18.6±3.5)%,并且这种诱导凋亡的能力能被乳糖以及抗Galectin-3的抗体中和,转染siRNA的CD133+肺腺癌细胞上清诱导CD8+T细胞凋亡率为8.2%,与未转染组凋亡率18.6%相比诱导凋亡的能力明显下降,差异有统计学意义(p<0.05)。
结论：以上结果表明磁珠分选技术能够有效地从肿瘤标本中分选出CD133+的肺癌肿瘤干细胞,分选效率高,所分选的细胞体外培养能形成典型的肿瘤干细胞球。Galectin-3在CD133+肺腺癌细胞中高表达,在其细胞内以及细胞膜上均有表达且均高于CD133-细胞细胞内以及细胞膜的表达量。CD133+细胞表达出的Galectin-3具有很强的生物学活性,在体外具有明显的诱导CD8+T细胞凋亡的功能,针对galectin-3的siRNA能高效抑制galectin-3在CD133+细胞中的的表达,并能降低CD133+细胞的增殖能力、单克隆形成能力、体外转移能力及诱导CD8+T细胞凋亡的能力,具有潜在的临床应用价值。本研究进一步阐明了Galectin-3在肿瘤的发生发展中的作用,为抗肿瘤药物的研发提供了一条新的思路。
Purpose Cancer stem cells show poor sensitivity to chemotherapeutics, which often become the root of tumor recurrence. So far, numerous studies have focused on development of novel drugs specific to tumor stem cells, which would be one of the most effective treatment of cancers. Of note, to separate and purify lung cancer stem cells by surface marker, study their gene expression patterns and then find out the essential functional protein for oncogenesis as new drug targets for lung cancer treatment, is a dynamic research direction and may provide important information for developing novel therapy. In this study, we detect the expression of Galectin-3 in CD133+ pulmonary adenocarcinoma cells and investigate its biological functions by synthesized galectin-3-specific siRNA.
Methods In the first part, we sepatated CD133+ pulmonary adenocarcinoma cells from excised pulmonary adenocarcinoma specimens of 10 patients by magnetic activated cell sorting (MACS) and determined the percentage of CD133+ cells in different cell populations by flow cytometry(FCM). In the second part, we detected the expression of Galectin-3 in lung cancer cells on both transcriptional and translational level by FQRT-PCR and Western blot respectively, whereas extracellular expression by ELISA.In the third part, the studies were aimed to investigate the biological functions of Galectin-3 in CD133+ cells. CD133+ cells were transfected with galectin-3-specific siRNA to explore the affects of galectin-3 inhibition on cancer cell growth, metastasis and its ability of inducing CD8+T cell apoptosis. The interference efficiency of galectin-3 expression by siRNA was determined by FQRT-PCR and Western blot, and then the proliferation rate of CD133+ lung cancer cells post-transfected was detected by colony formation assay and MTT test. Boyden chamber model was used to observe the metastasis ability of CD133+ lung cancer cells. Finally, Annexin V and PI double staining flow cytometry was performed to determine CD8+T cell apoptosis induced by supernatant of CD133+ lung cancer cells
Results Cells expressing CD133 constituted 90% of the CD133+subpopulation after separation by MACS whereas only 1.2% in the unsorted cells. Expression of galectin-3 was 1.24 folds,1.5 folds and 2 folds higher in CD133+cells than in CD133-cells detected by FQRT-PCR, Western Blot and ELISA respectively(p<0.05). siRNA was able to interfere with galectin-3 and the interference efficiency was 80.3%, showed by FQRT-PCR and Western Blot. The MTT assay showed that proliferation rate of CD133+ cell treated with siRNA was decreased correlated with incubation time after transfection. At the point 96h after transfection, the viability rate of CD133+ cell treated with siRNA was significantly lower than CD133+ cell without treatment(p<0.05), the former 75 percents of the later, whereas there was no much difference between the viability rates of siRNA group and siRNA mutant group at the point 48h after transfection. The colony formation rate of CD133+ cells post-transfected (60.12%±4.32%) was lower than that of non-treated group (87.34%±4.89%). Boyden chamber model showed that siRNA transfected CD133+ lung cancer cells were less invasive than non-treated group,68±7 cells compared with 100±8 cells (p<0.05). Apoptosis detection by flow cytometry showed that the supernatants of CD133+ cells induced more efficiently apoptosis of CD8+T cells (18.6±3.5) compared with siRNA transfected cells(8.2%), which could be also inhibited by lactose, anti-galectin-3 polyclonal antibody.
Conclusion Our data indicate that galectin-3 is expressed at a relatively higher level in CD133+ lung adenocarcinoma cells and could induce CD8+T cell apoptosis in vitro, both of which can be down-regulated by galectin-3 siRNA. Galectin-3-specific siRNA also reduce proliferation and metastasis abilities of CD133+ lung cancer cells, as well as their capacity to induce CD8+T cell apoptosis. These findings indicate Galectin-3 may play an important role during oncogenesis, implying a potential therapeutic target for pulmonary adenocarcinoma.
方法：第一部分实验是从10例肺腺癌患者切除的肿瘤标本中通过磁珠分选技术分选出CD133+肺腺癌细胞,通过流式细胞术检测分选出的细胞中CD133+细胞百分率。第二部分实验是采用FQRT-PCR和Westernblot的方法分别在转录和翻译两个水平检测了CD133+肺腺癌细胞中Galectin-3的表达,同时流式细胞术检测了CD133+细胞中galectin-3的分布情况。第三部分研究内容旨在深入研究Galectin-3在CD133+细胞中高表达的生物学意义。采用针对galectin-3的特异性siRNA来研究降低CD133+细胞中galectin-3的表达对CD133+肺腺癌细胞的增殖,转移能力及其诱导CD8+T细胞凋亡能力的影响。首先通过FQRT-PCR和Westernblot检测siRNA对galectin-3的干扰效率,随后单克隆形成实验以及MTT法检测了特异性针对galectin-3的siRNA对CD133+肺腺癌细胞增殖的影响,Boyden小室模型检测了siRNA对CD133+肺腺癌细胞转移能力的影响,最后AnnexinV和PI双染检测转染siRNA的CD133+肺腺癌细胞上清诱导CD8+T细胞凋亡能力。
结果：第一部分的实验结果表明,磁珠分选出的细胞中CD133+细胞数达到了90%,体外培养2-3天后经过磁珠分选出来的CD133+细胞能形成典型的肿瘤干细胞球。第二部分FQRT-PCR和Westernblot结果发现Galectin-3在CD133+细胞中的表达量分别为CD133-细胞中的1.24倍和1.5倍,差异有统计学意义(p<0.05)。流式细胞仪检测结果表明CD133+肺腺癌肿瘤干细胞胞内以及细胞膜上均有galectin-3的分布,且平均荧光强度统计结果显示CD133+细胞内(17.8±2.5)以及细胞膜(25.6±5.1)上的galectin-3的表达量均较CD133-细胞内(7.3±3.1)以及细胞膜(10.3±3.4)上galectin-3的表达量高,并且差异具有统计学意义(p<0.05)。第三部分的实验结果显示,针对galectin-3的siRNA平均干扰效率为80.3%,明显降低CD133+细胞中galectin-3的表达,并抑制CD133+细胞增殖,转染96h后细胞活率为未转染组的(75±3.5)%,转染siRNA后的CD133+细胞单克隆形成率(60.12±4.32)%明显较未转染组(87.34±4.89)%下降,差异具有统计学意义(p<0.05)。Boyden小室模型检测结果表明：转染siRNA后,CD133+细胞的转移侵袭能力(68±7)明显较未转染组(100±8)下降,差异具有统计学意义(p<0.05),凋亡检测结果表明CD133+细胞的上清诱导CD8+T细胞凋亡率为(18.6±3.5)%,并且这种诱导凋亡的能力能被乳糖以及抗Galectin-3的抗体中和,转染siRNA的CD133+肺腺癌细胞上清诱导CD8+T细胞凋亡率为8.2%,与未转染组凋亡率18.6%相比诱导凋亡的能力明显下降,差异有统计学意义(p<0.05)。
结论：以上结果表明磁珠分选技术能够有效地从肿瘤标本中分选出CD133+的肺癌肿瘤干细胞,分选效率高,所分选的细胞体外培养能形成典型的肿瘤干细胞球。Galectin-3在CD133+肺腺癌细胞中高表达,在其细胞内以及细胞膜上均有表达且均高于CD133-细胞细胞内以及细胞膜的表达量。CD133+细胞表达出的Galectin-3具有很强的生物学活性,在体外具有明显的诱导CD8+T细胞凋亡的功能,针对galectin-3的siRNA能高效抑制galectin-3在CD133+细胞中的的表达,并能降低CD133+细胞的增殖能力、单克隆形成能力、体外转移能力及诱导CD8+T细胞凋亡的能力,具有潜在的临床应用价值。本研究进一步阐明了Galectin-3在肿瘤的发生发展中的作用,为抗肿瘤药物的研发提供了一条新的思路。
Purpose Cancer stem cells show poor sensitivity to chemotherapeutics, which often become the root of tumor recurrence. So far, numerous studies have focused on development of novel drugs specific to tumor stem cells, which would be one of the most effective treatment of cancers. Of note, to separate and purify lung cancer stem cells by surface marker, study their gene expression patterns and then find out the essential functional protein for oncogenesis as new drug targets for lung cancer treatment, is a dynamic research direction and may provide important information for developing novel therapy. In this study, we detect the expression of Galectin-3 in CD133+ pulmonary adenocarcinoma cells and investigate its biological functions by synthesized galectin-3-specific siRNA.
Methods In the first part, we sepatated CD133+ pulmonary adenocarcinoma cells from excised pulmonary adenocarcinoma specimens of 10 patients by magnetic activated cell sorting (MACS) and determined the percentage of CD133+ cells in different cell populations by flow cytometry(FCM). In the second part, we detected the expression of Galectin-3 in lung cancer cells on both transcriptional and translational level by FQRT-PCR and Western blot respectively, whereas extracellular expression by ELISA.In the third part, the studies were aimed to investigate the biological functions of Galectin-3 in CD133+ cells. CD133+ cells were transfected with galectin-3-specific siRNA to explore the affects of galectin-3 inhibition on cancer cell growth, metastasis and its ability of inducing CD8+T cell apoptosis. The interference efficiency of galectin-3 expression by siRNA was determined by FQRT-PCR and Western blot, and then the proliferation rate of CD133+ lung cancer cells post-transfected was detected by colony formation assay and MTT test. Boyden chamber model was used to observe the metastasis ability of CD133+ lung cancer cells. Finally, Annexin V and PI double staining flow cytometry was performed to determine CD8+T cell apoptosis induced by supernatant of CD133+ lung cancer cells
Results Cells expressing CD133 constituted 90% of the CD133+subpopulation after separation by MACS whereas only 1.2% in the unsorted cells. Expression of galectin-3 was 1.24 folds,1.5 folds and 2 folds higher in CD133+cells than in CD133-cells detected by FQRT-PCR, Western Blot and ELISA respectively(p<0.05). siRNA was able to interfere with galectin-3 and the interference efficiency was 80.3%, showed by FQRT-PCR and Western Blot. The MTT assay showed that proliferation rate of CD133+ cell treated with siRNA was decreased correlated with incubation time after transfection. At the point 96h after transfection, the viability rate of CD133+ cell treated with siRNA was significantly lower than CD133+ cell without treatment(p<0.05), the former 75 percents of the later, whereas there was no much difference between the viability rates of siRNA group and siRNA mutant group at the point 48h after transfection. The colony formation rate of CD133+ cells post-transfected (60.12%±4.32%) was lower than that of non-treated group (87.34%±4.89%). Boyden chamber model showed that siRNA transfected CD133+ lung cancer cells were less invasive than non-treated group,68±7 cells compared with 100±8 cells (p<0.05). Apoptosis detection by flow cytometry showed that the supernatants of CD133+ cells induced more efficiently apoptosis of CD8+T cells (18.6±3.5) compared with siRNA transfected cells(8.2%), which could be also inhibited by lactose, anti-galectin-3 polyclonal antibody.
Conclusion Our data indicate that galectin-3 is expressed at a relatively higher level in CD133+ lung adenocarcinoma cells and could induce CD8+T cell apoptosis in vitro, both of which can be down-regulated by galectin-3 siRNA. Galectin-3-specific siRNA also reduce proliferation and metastasis abilities of CD133+ lung cancer cells, as well as their capacity to induce CD8+T cell apoptosis. These findings indicate Galectin-3 may play an important role during oncogenesis, implying a potential therapeutic target for pulmonary adenocarcinoma.
引文
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