MiR-200a对人胰腺癌干细胞上皮—间质转化（EMT）及体外生物学行为影响的实验研究
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摘要
目的
研究miR-200a对从人胰腺癌细胞系PANC-1分选出的胰腺癌干细胞（Pancreaticcancer stem cells，PCSCs）EMT及体外生物学行为的影响。探索靶向调控胰腺癌干细胞中miR-200a的表达在预防及治疗胰腺癌方面的可行性。
方法
(1)细胞培养和流式细胞仪分选
将人胰腺癌细胞系PANC-1细胞接种于由含10％胎牛血清的DMEM高糖培养基、青霉素(10U/ml)和链霉素(100μg/ml)组成的培养基中，在5％CO2，饱和湿度，恒温37°C培养箱中培养。分散后的细胞进行计数，移至5ml的试管中，用PBS洗涤两次，以1×106/100μl重新悬浮于PBS中。分别加入抗人CD24-PE、抗人CD44-APC和抗人ESA-FITC抗体（稀释度均为1:40），避光冰上孵育20min。根据厂商说明书将各自同型对照抗体配成相同浓度使用。PBS洗涤2次，样本用500μl PBS重悬，进行流式细胞仪分选。细胞常规分选两次，并重新分析的纯度，保证分选后的细胞纯度>97％。数据用BD FACS Diva的软件进行分析。
(2)转染miR-200模拟物
将CD24+CD44+ESA+胰腺癌干细胞接种在6孔板中并孵育过夜。将含miR-200模拟物或者阴性对照序列的EntransterTM-R转染液（配制成终浓度25nM）转染细胞。转染6小时后更换培养液以避免细胞死亡。
(3)实时定量RT-PCR
根据说明书用Trizol法提取总RNA，使用Power SYBR_green PCR mastermix试剂盒进行实时定量RT-PCR分析E-cadherin、N-cadherin、vimentin、ZEB1、Oct4和Nanog的mRNA表达。以GAPDH为内参照。
(4) microRNA表达分析
使用miScript Reverse Transcription Kit逆转录细胞总RNA。使用miScript PCR Kit进行RT-qPCR分析转染组与非转染组、阴性对照组胰腺癌干细胞miR-200a表达水平。以U6为内参照。
(5) Western blot检测
用RIPA裂解缓冲液裂解细胞并测定蛋白质浓度。SDS-PAGE分离总蛋白并转移到聚偏氟乙烯膜。配制含5%脱脂奶粉、0.1%Tween20的TBST缓冲溶液封闭膜，加一抗（兔抗人Oct4, l:2000；兔抗人Nanog,1:2000；兔抗人N-cadherin,1:1000；鼠抗人Vimentin,1:500；兔抗人E-cadherin,1:1000；兔抗人ZEB1,1:1000）室温孵育2小时。再加入HRP-偶联的二抗（HRP-羊抗兔IgG抗体，1:5000；HRP-羊抗小鼠IgG抗体，1:5000）室温孵育1小时，增强化学发光检测系统检测结果。提取未转染和已转染miR-200a模拟物的细胞总蛋白进行Western blot，检测E-cadherin、N-cadherin、ZEB1、vimentin、Oct4和Nanog蛋白表达水平。以β-actin为内参照。
(6) Transwell小室迁移实验
1×105PCSCs被平铺于非涂覆膜的上室，使其可以朝向下室中含血清的培养基迁移。孵育48小时后多聚甲醛固定细胞，以0.1％结晶紫（2mg/ml）染色。在光学显微镜下计数通过膜孔迁移的细胞数量。每孔观察3个随机视野。
(7) Transwell小室侵袭实验
1×105PCSCs置于Matrigel涂覆膜的上室，检测前每孔涂覆Matrigel60mg。细胞平铺在不含血清或生长因子的培养基中，而添加血清的培养基被用作化学诱导剂置于下室中。将细胞孵育48小时后，用棉签除去未通过膜孔侵入下室的细胞。收集膜下表面的细胞，用多聚甲醛固定和0.1%结晶紫染色。在光学显微镜下计数侵入到膜下表面的细胞数量。每孔观察3个随机视野。
(8) CCK法检测细胞增殖能力和对吉西他滨敏感性
调整各组细胞密度，按照每孔1000-3000个细胞/100μl培养基接种于96孔板中，各孔在不同时间点（24h、48h、72h、96h）或者在加入不同浓度（50ng/ml、100ng/ml、200ng/ml、400ng/ml、800ng/ml、2000ng/ml）吉西他滨后72h加入10μl CCK-8，37°C、5%CO2孵育3h。用酶标检测仪（测定波450nm）测定各孔吸光密度值。
结果
(1)在PNC-1细胞中有（1.442±0.532）%细胞呈CD44+CD24+，而仅有（0.492±0.334）%细胞呈CD44+CD24+ESA+。Oct4和Nanog基因在CD44+CD24+ESA+细胞中表达显著上升。
(2)定时RT-PCR显示miR-200a、 E-cadherin在PCSCs中表达显著下调，而N-cadherin、vimentin和ZEB1表达则显著上调。
(3)在PCSCs中过表达miR-200a导致在mRNA水平上上皮表型标志物E-cadherin表达上调，而间质表型标志物N-cadherin、Vimentin和ZEB1和胚胎干性基因Oct4和Nanog表达下调。Western blot检测结果显示PCSCs过表达miR-200a后E-cadherin蛋白表达上调，ZEB1、N-cadherin、Oct4和Nanog蛋白表达下调，而Vimentin蛋白表达未见下调。
(4) Transwell实验显示转染组相比非转染组或阴性对照组迁移和侵袭至膜下表面的细胞数目显著减少，分别下降了0.33倍和0.22倍。
(5) CCK-8法检测结果显示转染组胰腺癌干细胞增殖能力下降，对吉西他滨的敏感性增强。
结论
在本研究中，我们发现miRNA在调控肿瘤干细胞EMT特征方面发挥了重要的作用，通过过表达miR-200a能够逆转胰腺癌干细胞的EMT表型和抑制胰腺癌干细胞的体外恶性生物学行为，为未来设计以miRNA为基础的胰腺癌靶向治疗方法提供了重要的理论和实验依据。
Objective To evaluate the role of miR-200a in the epithelial-mesenchymal transitioncharacteristics and biological characteristics in vitro of pancreatic cancer stem cells(PCSCs) isolated from human pancreatic cancer cell line PANC-1and to confer thepossibility of regulation of miR-200a in PCSCs as a new approach for prevention and/ortreatment of pancreatic cancer.
Methods
(1) Cell Culture and Flow cytometry
Human pancreatic adenocarcinoma cell line, PANC-1was cultured in DMEMsupplemented with10%fetal bovine serum (FBS),100U/ml penicillin G, and100Ug/mlstreptomycin. Dissociated cells were counted and transferred to a5ml tube, washed twicewith PBS, counted and resuspended in PBS at1×106cell/100μl. Then, the antibodies APCanti-human CD44, PE anti-human CD24and FITC anti-human ESA (each at a dilution of1:40) were added and incubated for20min on ice in dark. The respective isotype controlantibodies were used at the same concentrations according to the manufacturer'sinstructions. After washing twice with PBS, samples were resuspended in500μl PBS andanalyzed on a flow cytometer. Side-scatter and forward-scatter profiles were used toeliminate cell doublets. Cells were routinely sorted twice, and the cells were reanalyzed forpurity, which typically was>97%.Data were analyzed with BD FACS Diva software.
(2) MiR-200a mimic Transfection
The CD24+CD44+ESA+populations of PANC-1cells were plated in6well plates andincubated overnight. Cells were transfected with either control or miR-200a mimic at afinal concentration of25nM using EntransterTM-R transfection reagent. After6h oftransfection the medium was changed to avoid cell death during transfection.
(3) Real-Time Reverse Transcriptase-PCR
Total RNA was extracted using Trizol according to the manufacturer’s instructions.E-cadherin, N-cadherin, vimentin and ZEB1, Oct4and Nanog mRNA were analysed byreal-time PCR with Power SYBR_green PCR master mix and datas were normalized toGAPDH expression.
(4) miR expression analysis
Total RNA was reverse transcribed using the miScript Reverse Transcription KitMiR-200a, miR-200b, miR-200c were analysed by RT-qPCR using the miScript PCR Kit.Experiments were normalized to U6.
(5) Western Blot Analysis
Cells were lysed in RIPA lysis buffer and the protein concentration was determined.Total proteins were fractionated using SDS-PAGE and transferred onto a polyvinylidenefluoride membrane. The membranes were blocked in5%skim milk in TBST buffercontaining0.1%Tween20and then incubated with indicated primary antibodies (Rabbitanti Oct4, l:2000; Rabbit anti Nanog,1:2000; Rabbit anti-N-cadherin,1:1000; Mouseanti-Vimentin,1:500; Rabbit anti-E-cadherin,1:1000; Rabbit Anti-ZEB1,1:1000) for2hat room temperature. HRP-conjugated secondary antibodies (HRP Goat anti-Rabbit IgGAntibody,1:5000; HRP Goat anti-Mouse IgG Antibody,1:5000) were incubated at roomtemperature for1h and detected using the enhanced chemiluminesence detectionsystem.Total protein was extracted from untreated and the cells treated by transfectingmiR-200a and subjected to western blot analysis as described to evaluate the expression ofE-cadherin, N-cadherin, ZEB1and vimentin. The data was adjusted against loading controlusing β-actin.
(6) Transwell migration assay
1×105PCSCs were plated in the top chamber onto the noncoated membrane andallowed to migrate toward serum-containing medium in the lower chamber. Cells werefixed after48hours of incubation with methanol and stained with0.1%crystal violet (2mg/ml). The number of cells invading through the membrane was counted under a lightmicroscope (three random fields per well)
(7) Transwell invasion assay
1×105cells were plated in the top chamber onto the Matrigel coated Membrane. Eachwell was coated freshly with Matrigel (60mg) before the invasion assay. Cells were platedin medium without serum or growth factors, and medium supplemented with serum wasused as a chemo-attractant in the lower chamber. The cells were incubated for48hours.Cells which did not invade through the pores were removed by a cotton swab. Cells on thelower surface of the membrane were fixed with methanol and stained with0.1%crystalviolet. The number of cells invading through the membrane was counted under a lightmicroscope (three random fields per well)
(8) Cell Counting Kit-8（CCK-8）assay for cell proliferation and chemotherapysensitivity to gemcitabine
The cells of each group were planted at a density of1000-3000cells per well in96-well plates.10μl CCK-8solution was added into each well at a given time point (24h、48h、72h、96h) or at72h after different concentrations (50ng/ml、100ng/ml、200ng/ml、400ng/ml、800ng/ml、2000ng/ml) of gemcitabine added in. Then all groups were incubatedat37°C,5%CO2for3h. Absorbance was measured at450nm on a microplate reader.
Results
(1) In PANC-1,(1.442±0.532)%of cells were CD44+CD24+, and (0.492±0.334)%of cells were CD44+CD24+ESA+. The results showed that Oct4and Nanog expressionssignificantly increased in CD44+CD24+ESA+cells.
(2) Real time RT-PCR result showed that miR-200a was significantly downregulated in PCSCs. The expressions of N-cadherin, vimentin and ZEB1were up-regulated, whilethe expression of E-cadherin exhibited down-regulation at mRNA levels in PCSCs.
(3) It suggested that the overexpression of miR-200a in the PCSCs resulted in theup-regulation of epithelial marker E-cadherin while down-regulation of mesenchymalmarkers ZEB1, N-cadherin, Vimentin and human embryonic stem cell genes Oct4, Nanogat mRNA leve. By westernblot analysis, the results showed that the overexpression ofmiR-200a in the PCSCs had led to the up-regulation of E-cadherin and down-regulation ofZEB1, N-cadherin and Oct4, Nanog, but not Vimentin at protein level.
(4) The Transwell inserts results showed that the number of PCSCs transfected withmiR-200a mimic which invaded and migrated to the lower side of the membrane wassignificantly decreased than PCSCs without transfection or transfected with NC mimic byabout0.33fold in invasion and0.22fold in migration, respectively.
(5) The CCK-8assays demonstrated that the PCSCs treated by transfection ofmiR-200a mimic displayed decreasing cell proliferation and increasing sensitivity togemcitabine.
Conclusions
In this study, we found that miRNA played an important role in regulating thecharacteristics of cancer stem cells with EMT signatures. To up-regulate miR-200a maylead to the reversal of EMT phenotype and inhibit malignant biological characteristics ofPCSCs in vitro. It will provide theoretical and experimental evidence for designingmiRNA-based target therapies for pancreatic cancer in the future.
研究miR-200a对从人胰腺癌细胞系PANC-1分选出的胰腺癌干细胞（Pancreaticcancer stem cells，PCSCs）EMT及体外生物学行为的影响。探索靶向调控胰腺癌干细胞中miR-200a的表达在预防及治疗胰腺癌方面的可行性。
方法
(1)细胞培养和流式细胞仪分选
将人胰腺癌细胞系PANC-1细胞接种于由含10％胎牛血清的DMEM高糖培养基、青霉素(10U/ml)和链霉素(100μg/ml)组成的培养基中，在5％CO2，饱和湿度，恒温37°C培养箱中培养。分散后的细胞进行计数，移至5ml的试管中，用PBS洗涤两次，以1×106/100μl重新悬浮于PBS中。分别加入抗人CD24-PE、抗人CD44-APC和抗人ESA-FITC抗体（稀释度均为1:40），避光冰上孵育20min。根据厂商说明书将各自同型对照抗体配成相同浓度使用。PBS洗涤2次，样本用500μl PBS重悬，进行流式细胞仪分选。细胞常规分选两次，并重新分析的纯度，保证分选后的细胞纯度>97％。数据用BD FACS Diva的软件进行分析。
(2)转染miR-200模拟物
将CD24+CD44+ESA+胰腺癌干细胞接种在6孔板中并孵育过夜。将含miR-200模拟物或者阴性对照序列的EntransterTM-R转染液（配制成终浓度25nM）转染细胞。转染6小时后更换培养液以避免细胞死亡。
(3)实时定量RT-PCR
根据说明书用Trizol法提取总RNA，使用Power SYBR_green PCR mastermix试剂盒进行实时定量RT-PCR分析E-cadherin、N-cadherin、vimentin、ZEB1、Oct4和Nanog的mRNA表达。以GAPDH为内参照。
(4) microRNA表达分析
使用miScript Reverse Transcription Kit逆转录细胞总RNA。使用miScript PCR Kit进行RT-qPCR分析转染组与非转染组、阴性对照组胰腺癌干细胞miR-200a表达水平。以U6为内参照。
(5) Western blot检测
用RIPA裂解缓冲液裂解细胞并测定蛋白质浓度。SDS-PAGE分离总蛋白并转移到聚偏氟乙烯膜。配制含5%脱脂奶粉、0.1%Tween20的TBST缓冲溶液封闭膜，加一抗（兔抗人Oct4, l:2000；兔抗人Nanog,1:2000；兔抗人N-cadherin,1:1000；鼠抗人Vimentin,1:500；兔抗人E-cadherin,1:1000；兔抗人ZEB1,1:1000）室温孵育2小时。再加入HRP-偶联的二抗（HRP-羊抗兔IgG抗体，1:5000；HRP-羊抗小鼠IgG抗体，1:5000）室温孵育1小时，增强化学发光检测系统检测结果。提取未转染和已转染miR-200a模拟物的细胞总蛋白进行Western blot，检测E-cadherin、N-cadherin、ZEB1、vimentin、Oct4和Nanog蛋白表达水平。以β-actin为内参照。
(6) Transwell小室迁移实验
1×105PCSCs被平铺于非涂覆膜的上室，使其可以朝向下室中含血清的培养基迁移。孵育48小时后多聚甲醛固定细胞，以0.1％结晶紫（2mg/ml）染色。在光学显微镜下计数通过膜孔迁移的细胞数量。每孔观察3个随机视野。
(7) Transwell小室侵袭实验
1×105PCSCs置于Matrigel涂覆膜的上室，检测前每孔涂覆Matrigel60mg。细胞平铺在不含血清或生长因子的培养基中，而添加血清的培养基被用作化学诱导剂置于下室中。将细胞孵育48小时后，用棉签除去未通过膜孔侵入下室的细胞。收集膜下表面的细胞，用多聚甲醛固定和0.1%结晶紫染色。在光学显微镜下计数侵入到膜下表面的细胞数量。每孔观察3个随机视野。
(8) CCK法检测细胞增殖能力和对吉西他滨敏感性
调整各组细胞密度，按照每孔1000-3000个细胞/100μl培养基接种于96孔板中，各孔在不同时间点（24h、48h、72h、96h）或者在加入不同浓度（50ng/ml、100ng/ml、200ng/ml、400ng/ml、800ng/ml、2000ng/ml）吉西他滨后72h加入10μl CCK-8，37°C、5%CO2孵育3h。用酶标检测仪（测定波450nm）测定各孔吸光密度值。
结果
(1)在PNC-1细胞中有（1.442±0.532）%细胞呈CD44+CD24+，而仅有（0.492±0.334）%细胞呈CD44+CD24+ESA+。Oct4和Nanog基因在CD44+CD24+ESA+细胞中表达显著上升。
(2)定时RT-PCR显示miR-200a、 E-cadherin在PCSCs中表达显著下调，而N-cadherin、vimentin和ZEB1表达则显著上调。
(3)在PCSCs中过表达miR-200a导致在mRNA水平上上皮表型标志物E-cadherin表达上调，而间质表型标志物N-cadherin、Vimentin和ZEB1和胚胎干性基因Oct4和Nanog表达下调。Western blot检测结果显示PCSCs过表达miR-200a后E-cadherin蛋白表达上调，ZEB1、N-cadherin、Oct4和Nanog蛋白表达下调，而Vimentin蛋白表达未见下调。
(4) Transwell实验显示转染组相比非转染组或阴性对照组迁移和侵袭至膜下表面的细胞数目显著减少，分别下降了0.33倍和0.22倍。
(5) CCK-8法检测结果显示转染组胰腺癌干细胞增殖能力下降，对吉西他滨的敏感性增强。
结论
在本研究中，我们发现miRNA在调控肿瘤干细胞EMT特征方面发挥了重要的作用，通过过表达miR-200a能够逆转胰腺癌干细胞的EMT表型和抑制胰腺癌干细胞的体外恶性生物学行为，为未来设计以miRNA为基础的胰腺癌靶向治疗方法提供了重要的理论和实验依据。
Objective To evaluate the role of miR-200a in the epithelial-mesenchymal transitioncharacteristics and biological characteristics in vitro of pancreatic cancer stem cells(PCSCs) isolated from human pancreatic cancer cell line PANC-1and to confer thepossibility of regulation of miR-200a in PCSCs as a new approach for prevention and/ortreatment of pancreatic cancer.
Methods
(1) Cell Culture and Flow cytometry
Human pancreatic adenocarcinoma cell line, PANC-1was cultured in DMEMsupplemented with10%fetal bovine serum (FBS),100U/ml penicillin G, and100Ug/mlstreptomycin. Dissociated cells were counted and transferred to a5ml tube, washed twicewith PBS, counted and resuspended in PBS at1×106cell/100μl. Then, the antibodies APCanti-human CD44, PE anti-human CD24and FITC anti-human ESA (each at a dilution of1:40) were added and incubated for20min on ice in dark. The respective isotype controlantibodies were used at the same concentrations according to the manufacturer'sinstructions. After washing twice with PBS, samples were resuspended in500μl PBS andanalyzed on a flow cytometer. Side-scatter and forward-scatter profiles were used toeliminate cell doublets. Cells were routinely sorted twice, and the cells were reanalyzed forpurity, which typically was>97%.Data were analyzed with BD FACS Diva software.
(2) MiR-200a mimic Transfection
The CD24+CD44+ESA+populations of PANC-1cells were plated in6well plates andincubated overnight. Cells were transfected with either control or miR-200a mimic at afinal concentration of25nM using EntransterTM-R transfection reagent. After6h oftransfection the medium was changed to avoid cell death during transfection.
(3) Real-Time Reverse Transcriptase-PCR
Total RNA was extracted using Trizol according to the manufacturer’s instructions.E-cadherin, N-cadherin, vimentin and ZEB1, Oct4and Nanog mRNA were analysed byreal-time PCR with Power SYBR_green PCR master mix and datas were normalized toGAPDH expression.
(4) miR expression analysis
Total RNA was reverse transcribed using the miScript Reverse Transcription KitMiR-200a, miR-200b, miR-200c were analysed by RT-qPCR using the miScript PCR Kit.Experiments were normalized to U6.
(5) Western Blot Analysis
Cells were lysed in RIPA lysis buffer and the protein concentration was determined.Total proteins were fractionated using SDS-PAGE and transferred onto a polyvinylidenefluoride membrane. The membranes were blocked in5%skim milk in TBST buffercontaining0.1%Tween20and then incubated with indicated primary antibodies (Rabbitanti Oct4, l:2000; Rabbit anti Nanog,1:2000; Rabbit anti-N-cadherin,1:1000; Mouseanti-Vimentin,1:500; Rabbit anti-E-cadherin,1:1000; Rabbit Anti-ZEB1,1:1000) for2hat room temperature. HRP-conjugated secondary antibodies (HRP Goat anti-Rabbit IgGAntibody,1:5000; HRP Goat anti-Mouse IgG Antibody,1:5000) were incubated at roomtemperature for1h and detected using the enhanced chemiluminesence detectionsystem.Total protein was extracted from untreated and the cells treated by transfectingmiR-200a and subjected to western blot analysis as described to evaluate the expression ofE-cadherin, N-cadherin, ZEB1and vimentin. The data was adjusted against loading controlusing β-actin.
(6) Transwell migration assay
1×105PCSCs were plated in the top chamber onto the noncoated membrane andallowed to migrate toward serum-containing medium in the lower chamber. Cells werefixed after48hours of incubation with methanol and stained with0.1%crystal violet (2mg/ml). The number of cells invading through the membrane was counted under a lightmicroscope (three random fields per well)
(7) Transwell invasion assay
1×105cells were plated in the top chamber onto the Matrigel coated Membrane. Eachwell was coated freshly with Matrigel (60mg) before the invasion assay. Cells were platedin medium without serum or growth factors, and medium supplemented with serum wasused as a chemo-attractant in the lower chamber. The cells were incubated for48hours.Cells which did not invade through the pores were removed by a cotton swab. Cells on thelower surface of the membrane were fixed with methanol and stained with0.1%crystalviolet. The number of cells invading through the membrane was counted under a lightmicroscope (three random fields per well)
(8) Cell Counting Kit-8（CCK-8）assay for cell proliferation and chemotherapysensitivity to gemcitabine
The cells of each group were planted at a density of1000-3000cells per well in96-well plates.10μl CCK-8solution was added into each well at a given time point (24h、48h、72h、96h) or at72h after different concentrations (50ng/ml、100ng/ml、200ng/ml、400ng/ml、800ng/ml、2000ng/ml) of gemcitabine added in. Then all groups were incubatedat37°C,5%CO2for3h. Absorbance was measured at450nm on a microplate reader.
Results
(1) In PANC-1,(1.442±0.532)%of cells were CD44+CD24+, and (0.492±0.334)%of cells were CD44+CD24+ESA+. The results showed that Oct4and Nanog expressionssignificantly increased in CD44+CD24+ESA+cells.
(2) Real time RT-PCR result showed that miR-200a was significantly downregulated in PCSCs. The expressions of N-cadherin, vimentin and ZEB1were up-regulated, whilethe expression of E-cadherin exhibited down-regulation at mRNA levels in PCSCs.
(3) It suggested that the overexpression of miR-200a in the PCSCs resulted in theup-regulation of epithelial marker E-cadherin while down-regulation of mesenchymalmarkers ZEB1, N-cadherin, Vimentin and human embryonic stem cell genes Oct4, Nanogat mRNA leve. By westernblot analysis, the results showed that the overexpression ofmiR-200a in the PCSCs had led to the up-regulation of E-cadherin and down-regulation ofZEB1, N-cadherin and Oct4, Nanog, but not Vimentin at protein level.
(4) The Transwell inserts results showed that the number of PCSCs transfected withmiR-200a mimic which invaded and migrated to the lower side of the membrane wassignificantly decreased than PCSCs without transfection or transfected with NC mimic byabout0.33fold in invasion and0.22fold in migration, respectively.
(5) The CCK-8assays demonstrated that the PCSCs treated by transfection ofmiR-200a mimic displayed decreasing cell proliferation and increasing sensitivity togemcitabine.
Conclusions
In this study, we found that miRNA played an important role in regulating thecharacteristics of cancer stem cells with EMT signatures. To up-regulate miR-200a maylead to the reversal of EMT phenotype and inhibit malignant biological characteristics ofPCSCs in vitro. It will provide theoretical and experimental evidence for designingmiRNA-based target therapies for pancreatic cancer in the future.
引文
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