趋化因子受体CCR9在非小细胞肺癌中的表达及作用研究
摘要
肺癌是世界上最常见的肿瘤,位于肿瘤相关死亡的首位,每年有约160万新增病例,并且其相关死亡率在继续增加。其中非小细胞肺癌(NSCLC)占整个肺癌的85%左右,出于缺少有效的生物靶点,至少有40%的患者确诊时已经处于无法手术的肿瘤晚期。因此,独立可靠的、有预测性的分子靶标对肺癌的干预尤为重要。
趋化因子是一类分子量为8-14kDa的小蛋白,与其同源的受体结合后具有白细胞的激活与趋化作用。趋化因子受体是有7个跨膜结构域的G蛋白偶联受体,其中趋化因子受体9(CC chemokine receptor-9, CCR9)与其特异性配体CCL25/TECK(thymus expressed CC chemokine)结合后与T淋巴细胞的活化和募集相关。趋化因子及其受体在实体瘤的凋亡中也起到重要作用。其中CCR9高表达于部分实体瘤细胞上,可能通过调控PI3K/Akt信号通路促进肿瘤细胞的增殖、抑制肿瘤细胞的凋亡。
目前,CCR9在人NSCLC中的作用尚不明确。本实验旨在评估CCR9在NSCLC中的表达及其在NSCLC发生、发展过程中的生物学作用及可能的作用机制,为临床治疗人NSCLC提供有价值的实验依据。
方法
1.对广西壮族自治区人民医医院的119名NSCLC患者施行电话随访,了解其生存期及术后治疗；使用免疫组织化学技术对119名NSCLC患者癌组织及相应癌旁正常肺组织的石蜡标本中CCR9的表达水平进行检测；结合患者临床资料,分析CCR9表达与NSCLC中临床病理特征、生存率及预后的关系。
2.设计合成3个与CCR9基因(Gen Bank accession number, Gene ID:10803)构建pcDNATM6.2-GW/EmGFPmiR表达载体,筛选出沉默效率最高的表达载体进行慢病毒包装并转染人NSCLC细胞系SK-MES-1和A549,构建能稳定沉默CCR9表达的NSCLC细胞系。
3.运用MTT法实验观察CCR9-CCL25相互作用对NSCLC细胞系SK-MES-1和A549增殖能力影响；通过FACE (Fast-activated cell-based enzyme-linked immunosorbent assay)实验检测对PI3K/Akt信号通路活化的影响；通过PI染色流式细胞仪分析及免疫印迹法(Western blot),检测细胞周期及重要的周期蛋的改变；通过AnnexinV/PI流式双染及免疫印迹法(Western blot),检测细胞凋亡和凋亡蛋白的改变。
4.以BALB/c裸鼠为研究对象,采用皮下注射稳转NSCLC细胞和对照组细胞悬液,建立肺癌细胞的裸鼠动物模型,通过观察肿瘤形成时间,测量肿瘤体积的方法观察肿瘤的形成和肿瘤的生长状况；采用Western blot检测肿瘤组织CCR9和PI3K/Akt信号通路重要蛋白PI3K110、pAkt和Akt蛋白的表达。
结果
1.免疫组化结果显示,CCR9主要定位于细胞膜与细胞质。NSCLC组织中CCR9阳性表达率为52.1%,明显高于正常对照组的10.1%(x2=49.028,P=0.000)。Pearson卡方检验表明,CCR9表达与组织病理分型、淋巴结转移和TNM分期等临床病理变量密切相关。CCR9阳性组患者的生存时间明显短于CCR9阴性组。多因素Cox比例风险模型分析显示,CCR9是NSCLC患者总体生存时间的独立预后因素。
2.针对CCR9基因构建3个miRNA干扰载体和1个阴性对照载体,转化至感受态细胞DH-5a后,经测序表明插入的片段序列与设计合成的CCR9的RNA编码序列完全一致,证明miRNA干扰载体全部构建成功。根据RT-PCR和Western blot检测结果筛选出瞬时共转染SK-MES-1和A549细胞48小时后抑制作用最为显著的载体pcDNA6.2-GW/EmGFP-miR-MR1与pDNOR221和plenti6/v5-DEST连接,构建plenti6/v5慢病毒表达载体。经RT-PCR和Western blot验证与阴性病毒感染对照细胞SK-MES-1/Lv-NC和A549/Lv-NC以及未转染SK-MES-1和A549细胞相比,沉默细胞中CCR9的表达明显减少。经6ug/ml Blasticidin(杀稻瘟菌素)抗生素筛选后,用3ug/ml Blasticidin对慢病毒转染的细胞进行维持,最终构建能够稳定沉默CCR9表达的NSCLC细胞SK-MES-1/Lv-miCCR9和A549/Lv-miCCR9。
3.体外实验表明,CCR9-CCL25轴相互作用以PI3K/Akt依赖的方式,上调重要的细胞周期蛋白和抗凋亡蛋白的表达同时抑制凋亡蛋白的表达,促进NSCLC细胞的增殖,抑制NSCLC细胞的凋亡,同时上述效应可以被wortmannin(一种P13K酶的特异性抑制剂)或用CCR9的沉默阻断CC9-CCL25轴的相互作用所逆转。此表明CCR9-CCL25轴主要是通过PI3K/Akt信号通路改变NSCLC的生物学特性的。
4.与对照组相比,稳转CCR9RNAi的NSCLC细胞接种裸鼠皮下后,肿瘤生长明显减慢,肿瘤体积明显缩小。同时,CCR9蛋白下调,肿瘤内PI3K/Akt信号通路蛋白PI3K110、pAkt和Akt蛋白表达均明显降低。
结论
1.我们的免疫组化实验证实,CCR9在NSCLC组织中高表达,并且癌组织中表达显著高于相应癌旁正常肺组织；CCR9表达与患者组织学类型、是否合并淋巴结转移、临床分期以及预后密切相关。CCR9可以作为NSCLC患者预后的独立预测因素。
2.我们的体内外实验证实,CCR9-CCL25轴的相互作用,通过活化PI3K/Akt信号通路,促进NSCLC细胞的增殖,抑(?)NSCLC细胞的凋亡。这可能是NSCLC进展的重要原因。
Background
Lung cancer is the leading cause of cancer-related mortality worldwide, Approximately1.6million new cases of lung cancer are diagnosed each year throughout the world, the mortality related to lung cancer continues to rise. Non-small cell lung cancer (NSCLC) is the most frequent (approximately85%) type of lung cancer. Due to the lack of effective biomarkers, at least40%of patients with lung cancer are diagnosed in an advanced stage. Therefore, reliable and independent prognostic or predictive markers are needed for further intervention.
Chemokines are small (8-14kDa) proteins that play key roles in development and leukocyte trafficking through interactions with cognate cell surface seven-transmembrane G protein-coupled receptors(GPCRs). CC chemokine receptor-9(CCR9) is a GPCRs and plays an important role in T-cell development and tissue-specific homing when binding to its specific ligand CCL25, which is also known as thymus expressed chemokine (TECK). Chemokines are also important for solid tumor apoptosis. CCR9is expressed in some carcinoma cells, and may promote proliferation and suppress apoptosis of cancer cells by activating the PI3K/Akt pathway.
We investigated the expression status and functional significance of CCR9in NSCLC tissue and cell lines, and then explored the probable mechanism in vitro and in vivo, so as to provide experimental evidence on searching novel therapeutic targets of NSCLC.
Methods
1. Telephone following up regarding on life span and postoperative treatment was carried out with119patients who visited at The People's Hospital of Guangxi Zhuang Autonomous Region; Immunohistochemistry was used on119NSCLC pathological sections to detect the expression of CCR9; the correlation of CCR9expression with clinic pathologic variables and prognosis was analyzed.
2. Three precursor microRNAs (Pre-miRNA) sequences targeting to CCR9and a negative control were designed and inserted into pcDNA6.2-GW/EmGFP-miR expression Vector to construct recombinant plasmid. To verify the recombinants, the recombinants were extracted for sequence detection. Using the instruction for lipofectamine2000, we transient transfected the recombinant plasmids in cultured SK-MES-1and A549cells. After48hours, RT-PCR and Western blot testing were used to identify the target site with the highest interfering efficiency. We packaged the recombinant lentiviral vector for CCR9RNAi with the highest interfering efficiency using the BLOCK-iTTPol Ⅱ miR RNAi Expression Vector Kit. The lentiviral vectors were transfceted into SK-MES-1and A549cells. To produce stable transfection cell lines, the cells were cultured in a selection medium.
3. MTT assay was used to assess the proliferation ability of NSCLC cell lines SK-MES-1and A549with CCR9-CCL25interaction; FACE (Fast-activated cell-based enzyme-linked immunosorbent) assay was used to assess the influence of activation of PI3K/Akt signal pathway; Cell cycle distributions and related proteins were analyzed by flowcytometry and Western blot; Cell apoptosis and related proteins were analyzed by Annexin V-A and Western blot.
4. BALB/c nude mice were injected subcutaneously with stably transfected NSCLC cells groups and control cell groups respectively to construct nude mouse of lung cancer animal models. Times of tumor formation of each group were obtained. The formation and growth of the tumors were observed by measuring. CCR9, PI3K110, pAkt and Akt proteins expression were detected by Western blot test.
Results
1. The results of immunohistochemistry showed that CCR9was mainly localized in cell membrane and cytoplasm. The positive rate of CCR9in lung cancer was significantly higher than that of CCR9in normal lung tissues (52.1%vs.10.1%,/2=49.028, P=0.000). CCR9expression closely correlated with histopathologic classification, lymph node metastasis and TNM stage in NSCLC by Pearson Chi-square test. The survival time in the CCR9expression group was shorter than those in the none expression group. CCR9was adopted as an independent prognostic factor for survival of NSCLC patients though multivariate Cox proportional hazard model analysis.
2. Three recombinant plasmid expression vectors encoding Pre-miRNA against CCR9and a negative control were constructed correctly and veritified by sequencing. GFP was observed under the fluorescence microscope after the plasmids were transfected into the SK-MES-1and A549cells24hours later. Then pcDNA-CCR9-miR-MR-1was identified to have the highest interfering efficiency by RT-PCR and Western blot. We packaged the plenti6/v5recombinant lentiviral expression vector by pDNOR221and plenti6/v5-DEST for CCR9RNA interference with pcDNA-CCR9-miR-MR-1. To construct the stable transfectants, the cells were cultured in a selection medium containing6ug/ml of blasticidin for14days and the stable cell lines after selection were obtained with3ug/ml of blasticidin. Of note, about85-90%knockdown of CCR9expression in the SK-MES-1and A549cells were obtained by miRNA tcehnique.
3. Compromised CCR9-CCL25interaction induced cell cycle arrest, reduced proliferation and promoted apoptosis in NSCLC cells in vitro. Importantly, CCR9-CCL25interaction resulted in a significant increase in phosphoinositide3-kinase (PI3K)/Akt activity. In addition, we showed that CCR9-CCL25interaction mediated the activation of the PI3K/Akt pathway in NSCLC cells, resulting in the up-regulation of important cell cycle proteins and antiapoptotic proteins, as well as the down-regulation of apoptotic proteins in a PI3K/Akt-dependent manner. These CCR9-CCL25mediated effects were abrogated in the presence of a PI3K inhibitor (wortmannin) or by inhibiting the CCR9-CCL25interaction through CCR9silencing, which also suggested that the biological function of CCR9-CCL25was mainly regulated by PI3K.
4. After lung cancer cells transfected with CCR9RNAi were inoculated subcutaneously in nude mice, tumor growth were inhibited and tumor volume reduced significantly, compared with the control group. Expression of CCR9protein reduced. The expression of activated PI3K/Akt signaling pathway proteins were significantly down regulated.
Conclusion
1. Our IHC data indicated that CCR9is over-expressed in NSCLC. The expression of CCR9is correlated to histological types, with or without lymphatic metastasis and TNM stages. The survival time in the CCR9expression group was shorter than those in the none expression group. CCR9was adopted as an independent prognostic factor for survival of NSCLC patients.
2.Our in vitro and in vivo data indicated that CCR9-CCL25interaction, via activation of the PI3K/Akt signaling pathway, significantly promoted NSCLC cell proliferation and suppressed NSCLC cells apoptosis, which might be a possible cause of NSCLC tumor progression.
趋化因子是一类分子量为8-14kDa的小蛋白,与其同源的受体结合后具有白细胞的激活与趋化作用。趋化因子受体是有7个跨膜结构域的G蛋白偶联受体,其中趋化因子受体9(CC chemokine receptor-9, CCR9)与其特异性配体CCL25/TECK(thymus expressed CC chemokine)结合后与T淋巴细胞的活化和募集相关。趋化因子及其受体在实体瘤的凋亡中也起到重要作用。其中CCR9高表达于部分实体瘤细胞上,可能通过调控PI3K/Akt信号通路促进肿瘤细胞的增殖、抑制肿瘤细胞的凋亡。
目前,CCR9在人NSCLC中的作用尚不明确。本实验旨在评估CCR9在NSCLC中的表达及其在NSCLC发生、发展过程中的生物学作用及可能的作用机制,为临床治疗人NSCLC提供有价值的实验依据。
方法
1.对广西壮族自治区人民医医院的119名NSCLC患者施行电话随访,了解其生存期及术后治疗；使用免疫组织化学技术对119名NSCLC患者癌组织及相应癌旁正常肺组织的石蜡标本中CCR9的表达水平进行检测；结合患者临床资料,分析CCR9表达与NSCLC中临床病理特征、生存率及预后的关系。
2.设计合成3个与CCR9基因(Gen Bank accession number, Gene ID:10803)构建pcDNATM6.2-GW/EmGFPmiR表达载体,筛选出沉默效率最高的表达载体进行慢病毒包装并转染人NSCLC细胞系SK-MES-1和A549,构建能稳定沉默CCR9表达的NSCLC细胞系。
3.运用MTT法实验观察CCR9-CCL25相互作用对NSCLC细胞系SK-MES-1和A549增殖能力影响；通过FACE (Fast-activated cell-based enzyme-linked immunosorbent assay)实验检测对PI3K/Akt信号通路活化的影响；通过PI染色流式细胞仪分析及免疫印迹法(Western blot),检测细胞周期及重要的周期蛋的改变；通过AnnexinV/PI流式双染及免疫印迹法(Western blot),检测细胞凋亡和凋亡蛋白的改变。
4.以BALB/c裸鼠为研究对象,采用皮下注射稳转NSCLC细胞和对照组细胞悬液,建立肺癌细胞的裸鼠动物模型,通过观察肿瘤形成时间,测量肿瘤体积的方法观察肿瘤的形成和肿瘤的生长状况；采用Western blot检测肿瘤组织CCR9和PI3K/Akt信号通路重要蛋白PI3K110、pAkt和Akt蛋白的表达。
结果
1.免疫组化结果显示,CCR9主要定位于细胞膜与细胞质。NSCLC组织中CCR9阳性表达率为52.1%,明显高于正常对照组的10.1%(x2=49.028,P=0.000)。Pearson卡方检验表明,CCR9表达与组织病理分型、淋巴结转移和TNM分期等临床病理变量密切相关。CCR9阳性组患者的生存时间明显短于CCR9阴性组。多因素Cox比例风险模型分析显示,CCR9是NSCLC患者总体生存时间的独立预后因素。
2.针对CCR9基因构建3个miRNA干扰载体和1个阴性对照载体,转化至感受态细胞DH-5a后,经测序表明插入的片段序列与设计合成的CCR9的RNA编码序列完全一致,证明miRNA干扰载体全部构建成功。根据RT-PCR和Western blot检测结果筛选出瞬时共转染SK-MES-1和A549细胞48小时后抑制作用最为显著的载体pcDNA6.2-GW/EmGFP-miR-MR1与pDNOR221和plenti6/v5-DEST连接,构建plenti6/v5慢病毒表达载体。经RT-PCR和Western blot验证与阴性病毒感染对照细胞SK-MES-1/Lv-NC和A549/Lv-NC以及未转染SK-MES-1和A549细胞相比,沉默细胞中CCR9的表达明显减少。经6ug/ml Blasticidin(杀稻瘟菌素)抗生素筛选后,用3ug/ml Blasticidin对慢病毒转染的细胞进行维持,最终构建能够稳定沉默CCR9表达的NSCLC细胞SK-MES-1/Lv-miCCR9和A549/Lv-miCCR9。
3.体外实验表明,CCR9-CCL25轴相互作用以PI3K/Akt依赖的方式,上调重要的细胞周期蛋白和抗凋亡蛋白的表达同时抑制凋亡蛋白的表达,促进NSCLC细胞的增殖,抑制NSCLC细胞的凋亡,同时上述效应可以被wortmannin(一种P13K酶的特异性抑制剂)或用CCR9的沉默阻断CC9-CCL25轴的相互作用所逆转。此表明CCR9-CCL25轴主要是通过PI3K/Akt信号通路改变NSCLC的生物学特性的。
4.与对照组相比,稳转CCR9RNAi的NSCLC细胞接种裸鼠皮下后,肿瘤生长明显减慢,肿瘤体积明显缩小。同时,CCR9蛋白下调,肿瘤内PI3K/Akt信号通路蛋白PI3K110、pAkt和Akt蛋白表达均明显降低。
结论
1.我们的免疫组化实验证实,CCR9在NSCLC组织中高表达,并且癌组织中表达显著高于相应癌旁正常肺组织；CCR9表达与患者组织学类型、是否合并淋巴结转移、临床分期以及预后密切相关。CCR9可以作为NSCLC患者预后的独立预测因素。
2.我们的体内外实验证实,CCR9-CCL25轴的相互作用,通过活化PI3K/Akt信号通路,促进NSCLC细胞的增殖,抑(?)NSCLC细胞的凋亡。这可能是NSCLC进展的重要原因。
Background
Lung cancer is the leading cause of cancer-related mortality worldwide, Approximately1.6million new cases of lung cancer are diagnosed each year throughout the world, the mortality related to lung cancer continues to rise. Non-small cell lung cancer (NSCLC) is the most frequent (approximately85%) type of lung cancer. Due to the lack of effective biomarkers, at least40%of patients with lung cancer are diagnosed in an advanced stage. Therefore, reliable and independent prognostic or predictive markers are needed for further intervention.
Chemokines are small (8-14kDa) proteins that play key roles in development and leukocyte trafficking through interactions with cognate cell surface seven-transmembrane G protein-coupled receptors(GPCRs). CC chemokine receptor-9(CCR9) is a GPCRs and plays an important role in T-cell development and tissue-specific homing when binding to its specific ligand CCL25, which is also known as thymus expressed chemokine (TECK). Chemokines are also important for solid tumor apoptosis. CCR9is expressed in some carcinoma cells, and may promote proliferation and suppress apoptosis of cancer cells by activating the PI3K/Akt pathway.
We investigated the expression status and functional significance of CCR9in NSCLC tissue and cell lines, and then explored the probable mechanism in vitro and in vivo, so as to provide experimental evidence on searching novel therapeutic targets of NSCLC.
Methods
1. Telephone following up regarding on life span and postoperative treatment was carried out with119patients who visited at The People's Hospital of Guangxi Zhuang Autonomous Region; Immunohistochemistry was used on119NSCLC pathological sections to detect the expression of CCR9; the correlation of CCR9expression with clinic pathologic variables and prognosis was analyzed.
2. Three precursor microRNAs (Pre-miRNA) sequences targeting to CCR9and a negative control were designed and inserted into pcDNA6.2-GW/EmGFP-miR expression Vector to construct recombinant plasmid. To verify the recombinants, the recombinants were extracted for sequence detection. Using the instruction for lipofectamine2000, we transient transfected the recombinant plasmids in cultured SK-MES-1and A549cells. After48hours, RT-PCR and Western blot testing were used to identify the target site with the highest interfering efficiency. We packaged the recombinant lentiviral vector for CCR9RNAi with the highest interfering efficiency using the BLOCK-iTTPol Ⅱ miR RNAi Expression Vector Kit. The lentiviral vectors were transfceted into SK-MES-1and A549cells. To produce stable transfection cell lines, the cells were cultured in a selection medium.
3. MTT assay was used to assess the proliferation ability of NSCLC cell lines SK-MES-1and A549with CCR9-CCL25interaction; FACE (Fast-activated cell-based enzyme-linked immunosorbent) assay was used to assess the influence of activation of PI3K/Akt signal pathway; Cell cycle distributions and related proteins were analyzed by flowcytometry and Western blot; Cell apoptosis and related proteins were analyzed by Annexin V-A and Western blot.
4. BALB/c nude mice were injected subcutaneously with stably transfected NSCLC cells groups and control cell groups respectively to construct nude mouse of lung cancer animal models. Times of tumor formation of each group were obtained. The formation and growth of the tumors were observed by measuring. CCR9, PI3K110, pAkt and Akt proteins expression were detected by Western blot test.
Results
1. The results of immunohistochemistry showed that CCR9was mainly localized in cell membrane and cytoplasm. The positive rate of CCR9in lung cancer was significantly higher than that of CCR9in normal lung tissues (52.1%vs.10.1%,/2=49.028, P=0.000). CCR9expression closely correlated with histopathologic classification, lymph node metastasis and TNM stage in NSCLC by Pearson Chi-square test. The survival time in the CCR9expression group was shorter than those in the none expression group. CCR9was adopted as an independent prognostic factor for survival of NSCLC patients though multivariate Cox proportional hazard model analysis.
2. Three recombinant plasmid expression vectors encoding Pre-miRNA against CCR9and a negative control were constructed correctly and veritified by sequencing. GFP was observed under the fluorescence microscope after the plasmids were transfected into the SK-MES-1and A549cells24hours later. Then pcDNA-CCR9-miR-MR-1was identified to have the highest interfering efficiency by RT-PCR and Western blot. We packaged the plenti6/v5recombinant lentiviral expression vector by pDNOR221and plenti6/v5-DEST for CCR9RNA interference with pcDNA-CCR9-miR-MR-1. To construct the stable transfectants, the cells were cultured in a selection medium containing6ug/ml of blasticidin for14days and the stable cell lines after selection were obtained with3ug/ml of blasticidin. Of note, about85-90%knockdown of CCR9expression in the SK-MES-1and A549cells were obtained by miRNA tcehnique.
3. Compromised CCR9-CCL25interaction induced cell cycle arrest, reduced proliferation and promoted apoptosis in NSCLC cells in vitro. Importantly, CCR9-CCL25interaction resulted in a significant increase in phosphoinositide3-kinase (PI3K)/Akt activity. In addition, we showed that CCR9-CCL25interaction mediated the activation of the PI3K/Akt pathway in NSCLC cells, resulting in the up-regulation of important cell cycle proteins and antiapoptotic proteins, as well as the down-regulation of apoptotic proteins in a PI3K/Akt-dependent manner. These CCR9-CCL25mediated effects were abrogated in the presence of a PI3K inhibitor (wortmannin) or by inhibiting the CCR9-CCL25interaction through CCR9silencing, which also suggested that the biological function of CCR9-CCL25was mainly regulated by PI3K.
4. After lung cancer cells transfected with CCR9RNAi were inoculated subcutaneously in nude mice, tumor growth were inhibited and tumor volume reduced significantly, compared with the control group. Expression of CCR9protein reduced. The expression of activated PI3K/Akt signaling pathway proteins were significantly down regulated.
Conclusion
1. Our IHC data indicated that CCR9is over-expressed in NSCLC. The expression of CCR9is correlated to histological types, with or without lymphatic metastasis and TNM stages. The survival time in the CCR9expression group was shorter than those in the none expression group. CCR9was adopted as an independent prognostic factor for survival of NSCLC patients.
2.Our in vitro and in vivo data indicated that CCR9-CCL25interaction, via activation of the PI3K/Akt signaling pathway, significantly promoted NSCLC cell proliferation and suppressed NSCLC cells apoptosis, which might be a possible cause of NSCLC tumor progression.
引文
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