WTX基因在结肠癌中的表达及对结肠癌Lovo细胞株生长的影响
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摘要
研究目的
1.研究抑癌基因WTX在新鲜结肠癌、癌旁组织及正常组织中的表达水平及定位。分析比较WTX基因表达水平与结肠癌临床病理特征之间的关系,以研究WTX基因表达异常与结肠癌之间的关联性。
2.研究WTX在新鲜结肠癌、癌旁组织、正常组织及结肠癌Lovo细胞株中的甲基化水平。
3.构建WTX慢病毒载体,体外转染结肠癌Lovo细胞株。检测WTX在转录及蛋白的表达水平及对细胞增殖、凋亡的影响。
方法
1.对接受根治性手术,并经病理检测证实的50例结肠癌患者的新鲜癌组织、癌旁组织和正常组织标本进行取材,分为癌组织组(C组)、癌旁组织组(CB组)和正常组织组(N组)。提取组织RNA, qPCR检测不同组织中WTX基因转录水平的表达。提取组织总蛋白,Western blot检测不同组织中WTX基因蛋白水平的表达。免疫组织化学法检测WTX在不同组中的表达定位情况并进行免疫组化评分(IHCS)。将C组的WTX表达水平与其临床病理特征进行分析比较。
2.提取样本组织及结肠癌Lovo细胞株DNA,亚硫酸氢钠对DNA进行修饰,Wizard纯化树脂对DNA进行纯化。设计4条MSP特异性引物,甲基化特异性PCR (MSP)检测WTX在样本组织及结肠癌细胞中的甲基化水平。
3.根据Invitrogen公司的Gateway技术构建携带WTX基因的慢病毒载体PLV.Ex3d.null-EF1A>WTX>IRES/eGFP,挑取阳性克隆质粒,送交阳性克隆测序。将慢病毒载体与慢病毒包装质粒同时转染293FT细胞,离心获得病毒上清,计算慢病毒滴度。将结肠癌Lovo细胞株分为空白细胞组(Cell组)、空载体组和WTX转染组(WTX组)行病毒转导,48-72小时后荧光显微镜观察转导效率。qPCR及Western blot分别检测各组细胞WTX转录及蛋白表达；流式细胞仪Annexin V/PI双染法检测细胞凋亡水平；CCK8法检测细胞抑制率并绘制细胞生长曲线；流式细胞仪测定细胞周期。
结果
1.所有标本均有WTX表达。qPCR及Western blot法对标本进行WTX的mRNA及蛋白表达水平检测显示,C组WTX基因的平均表达水平低于N组和CB组,CB组WTX表达低于N组(p<0.05)。免疫组化结果显示WTX在N组、CB组及C组均主要为胞浆表达,在胞膜有少量表达。C组IHCS评分低于N组及CB组(p<0.05)。N组与CB组IHCS评分无明显差异。C组IHCS评分与患者性别(r=—0.168，p=0.245)、年龄(r=—0.170,p=0.238)、肿瘤部位(r=0.022,p=0.879)无关；而与肿瘤分化程度(r=0.711,p<0.01)呈正相关(即肿瘤分化程度越高,IHCS评分越高)；与肿瘤大小(r=—0.510,p<0.01)呈负相关(即肿瘤越大,IHCS评分越低)；与肿瘤血管浸润(r=—0.804,p<0.01)呈负相关(即有血管浸润的病例,IHCS评分越低)；与肿瘤神经浸润(r=—0.785,p<0.01)呈负相关(即有神经浸润的病例,IHCS评分越低)；与肿瘤的TNM分期(r=—0.948,p<0.01)呈负相关(即TNM分级越高,IHCS评分越低)。
2.组织MSP检测显示WTX基因在C组中的甲基化阳性率高于CB组和N组(p<0.05)；在Lovo细胞中MSP检测未发现WTX基因甲基化。
3.成功通过Gateway技术构建携带WTX基因的慢病毒载体PLV.Ex3d.null-EF1A>WTX>IRES/eGFP,鉴定正确；慢病毒包装成功,滴度符合实验要求；病毒转导Lovo细胞48-72小时后荧光显微镜下观察慢病毒载体组(WTX组)及空载体组荧光均较弱,通过挑克隆培养法建成稳定表达WTX的Lovo细胞系Lovo/WTX-eGFP。采用WTX质粒及空载对照质粒进行瞬时转染,48-72小时后WTX组观察荧光转染效率约35%,空载体组约70%。qPCR及Western blot检测均提示转染后WTX表达水平增高；流式细胞仪检测WTX过表达对Lovo细胞凋亡水平无明显影响；CCK8法检测：96孔板转染镜下可见转染后随时间增长,WTX组细胞数较Cell组及空载体组减少,荧光逐渐增强,但WTX组荧光仍少于空载体组。WTX过表达明显抑制Lovo细胞增殖水平。细胞周期检测发现转染WTX后Lovo细胞被阻滞在G0/G1期。
结论
1.抑癌基因WTX结肠癌中表达降低与结肠癌的恶性程度及周围组织浸润密切相关,WTX表达缺失可能是结肠癌发生的重要原因之一。
2.WTX在C组中甲基化阳性率高于CB组和N组,而在结肠癌Lovo细胞株中WTX甲基化呈阴性,尚需对其它结肠癌细胞株进行MSP检测以明确甲基化是否是导致WTX在结肠癌组织中表达降低的原因。
3.WTX慢病毒载体构建成功,但细胞转导效率低,与WTX基因CDS区较长,影响绿色荧光蛋白eGFP表达有关。成功建立表达WTX的Lovo细胞系Lovo/WTX-eGFP。WTX质粒瞬时转染后WTX转录及蛋白水平表达明显升高。WTX转染后Lovo细胞增殖受到明显抑制,对细胞凋亡无明显影响。
Objective
1. To study expression level and location of tumor suppressor gene WTX in fresh colon cancer tissues, peficancerous tissues and normal colon tissues. The relationship between gene expression level and clinical and pathological features of colon cancer were analysed and compared in order to study the relevance between abnormal gene expression of WTX and colon cancer.
2. To study methylation level of WTX gene in fresh colon cancer tissues, peficancerous tissues, normal colon tissues and colon cancer cell strain Lovo.
3. To establish lentiviral vector of WTX and transfect WTX into colon cancer cell line Lovo in vitro. Detecting expression of WTX in transcription and protein level and its influence to cell proliferation and apoptosis.
Methods
1. Fresh colon cancer tissues, peficancerous tissues and normal colon tissues of50colon cancer patients who had been accepted radical surgery and pathological confirmed were collected. These50specimens were divided into3groups including group of cancer tissues (group C), group of peficancerous tissues (group CB) and normal colon tissues (group N). RNA of these tissues was extracted and qPCR was used to detect transcription level of WTX in different tissues. Total proteins of these tissues were extracted and Western blot was used to detect protein level of WTX in different tissues. Immunohistochemistry was used to detect location of WTX in different tissues and immunohistochemistry score (IHCS) was analyzed. The relationship between WTX expression level and clinical and pathological features of group C were analysed and compared.
2. DNA of all specimens an colon cancer cell strain Lovo were extracted and modified by sodium hydrogensulfite. We utilized Wizard purified resin to purifiy DNA.4methylation specific PCR (MSP) specific primers were designed for detecting methylation level of WTX in all specimens and Lovo cells by MSP.
3. The lentiviral vector of PLV.Ex3d.null-EF1A>WTX>IRES/eGFP was constructed by Gateway Technology (Invitrogen Company). The positive cloning plasmid was picked for positive cloning sequencing. The lentiviral vector and lentiviral packaging plasmid were co-trasfected into293FT cells. Lentivirus titer was caculated after virus supernatant was obtained by centrifugation. Lovo cells were devided into3groups including blank cells (group Cell), blank vector (group Blank Vector) and WTX vector (group WTX) and were trsducted by lentivirus. Fluorescence microscope was used to observe transducting efficiency at48-72h after transducting.We utilized qPCR and Western blot to detect expression of WTX in transcription and protein level of each cell groups. Flow cytometry was used to detect cell apoptosis level and cell cycle. Regent CCK8was used to detect cell inhibition ratio and draw cell growth curve.
Results
1. All specimens have WTX expression. Expression of WTX in transcription and protein level detected by qPCR and Western blot showed that mean expression level of WTX in group C was lower than group N and CB, mean expression level of WTX in group CB was lower than group N. The difference was statistically significant (p<0.05). Results of immunohistochemistry showed that expression of WTX was located at cytoplasm and little at cytomembrane. IHCS of group C was lower than group N and CB (p<0.05). IHCS of group N and CB had no obvious difference. IHCS of group C has no correlation with sex (r=-0.168,p=0.245), age (r=-0.170,p=0.238) and tumor position (r=0.022, p=0.879). IHCS of group C has positive correlation with tumor differentiation degree (the higher level of tumor differentiation, the higher of IHCS, r=0.711, p<0.01), and has negative correlation with tumor size (the bigger of tumor size, the lower of IHCS, r=-0.510,p<0.01), and has negative correlation with tumor vessel infiltration (when there is tumor vessel infiltration, the lower of IHCS, r=-0.804,p<0.01), and has negative correlation with tumor nerve infiltration (when there is tumor nerve infiltration, the lower of IHCS, r=-0.785, p<0.01), and has negative correlation with tumor TNM stages (the higher level of TNM stages, the lower of IHCS, r=-0.948,p<0.01).
2. MSP detecting of tissues showed that positive rate of WTX methylation in group C was higher than group CB and N (p<0.05), but there was no WTX methylation found in colon cancer cell strain Lovo.
3. The lentiviral vector of PLV.Ex3d.null-EF1A>WTX>IRES/eGFP was succesfully constructed by Gateway Technology and identified perfectly correct. Lentivirus was successfully packaged and titer was accorded with the requirement of experiment. Fluorescence of group WTX and Blank Vector were weak at48-72h after transducting WTX into Lovo cells. The Lovo/WTX-eGFP colon cancer cell line stably transducting WTX was established by picking clone several times and cell culture. Then we proceeded transient transfection of WTX plasmid and contract blank plasmid. Fluorescence can be observed at48-72h after transient transfecting and transfection efficiency of group WTX and Blank Vector was about35%and70%respectively. Results of qPCR and Western blot showed that expression level rised after transfecting. Results of flow cytometry showed that excessive expression of WTX had no obvious influence to cell apoptosis level of Lovo cells. Results of CCK8test showed that with time increasing, cell population of group WTX reduced but fluorescence increased compared with group cell and group Blank Vector in96-well plates. Fluorescence cell population of group WTX was lesser than group Blank Vector. Excessive expression of WTX could obviously suppress cell proliferation. Cell cycle detection indicates that Lovo cells were blocked in G0/G1phase.
Conclusions
1. Lower expression level of tumor suppressor gene WTX in colon cancer is closely ralated to grade malignancy and infiltration of surrounding tissues. Loss of WTX expression maybe a important reason of occurring of colon cancer.
2. Although positive rate of WTX methylation in group C was higher than group CB and N, but WTX methylation was negative in colon cancer cell strain Lovo. More MSP should be done to detect whether methylation is exist in other colon cancer cell strains in order to clear whether methylation is a reason of lower expression of WTX in colon cancer tissues.
3. The lentiviral vector of PLV.Ex3d.null-EF1A>WTX>IRES/eGFP was succesfully constructed but cell trasducting efficiency was low. Long CDS region of WTX may influence eGFP expression, and this may be the reason that why transducting efficiency was low. The Lovo/WTX-eGFP colon cancer cell line stably transducting WTX was established successfully. Transient transfection of WTX plasmid lead higher WTX expression in transcription and protein level. Transfection of WTX could obviously suppress cell proliferation but had no influence to cell apoptosis.
1.研究抑癌基因WTX在新鲜结肠癌、癌旁组织及正常组织中的表达水平及定位。分析比较WTX基因表达水平与结肠癌临床病理特征之间的关系,以研究WTX基因表达异常与结肠癌之间的关联性。
2.研究WTX在新鲜结肠癌、癌旁组织、正常组织及结肠癌Lovo细胞株中的甲基化水平。
3.构建WTX慢病毒载体,体外转染结肠癌Lovo细胞株。检测WTX在转录及蛋白的表达水平及对细胞增殖、凋亡的影响。
方法
1.对接受根治性手术,并经病理检测证实的50例结肠癌患者的新鲜癌组织、癌旁组织和正常组织标本进行取材,分为癌组织组(C组)、癌旁组织组(CB组)和正常组织组(N组)。提取组织RNA, qPCR检测不同组织中WTX基因转录水平的表达。提取组织总蛋白,Western blot检测不同组织中WTX基因蛋白水平的表达。免疫组织化学法检测WTX在不同组中的表达定位情况并进行免疫组化评分(IHCS)。将C组的WTX表达水平与其临床病理特征进行分析比较。
2.提取样本组织及结肠癌Lovo细胞株DNA,亚硫酸氢钠对DNA进行修饰,Wizard纯化树脂对DNA进行纯化。设计4条MSP特异性引物,甲基化特异性PCR (MSP)检测WTX在样本组织及结肠癌细胞中的甲基化水平。
3.根据Invitrogen公司的Gateway技术构建携带WTX基因的慢病毒载体PLV.Ex3d.null-EF1A>WTX>IRES/eGFP,挑取阳性克隆质粒,送交阳性克隆测序。将慢病毒载体与慢病毒包装质粒同时转染293FT细胞,离心获得病毒上清,计算慢病毒滴度。将结肠癌Lovo细胞株分为空白细胞组(Cell组)、空载体组和WTX转染组(WTX组)行病毒转导,48-72小时后荧光显微镜观察转导效率。qPCR及Western blot分别检测各组细胞WTX转录及蛋白表达；流式细胞仪Annexin V/PI双染法检测细胞凋亡水平；CCK8法检测细胞抑制率并绘制细胞生长曲线；流式细胞仪测定细胞周期。
结果
1.所有标本均有WTX表达。qPCR及Western blot法对标本进行WTX的mRNA及蛋白表达水平检测显示,C组WTX基因的平均表达水平低于N组和CB组,CB组WTX表达低于N组(p<0.05)。免疫组化结果显示WTX在N组、CB组及C组均主要为胞浆表达,在胞膜有少量表达。C组IHCS评分低于N组及CB组(p<0.05)。N组与CB组IHCS评分无明显差异。C组IHCS评分与患者性别(r=—0.168，p=0.245)、年龄(r=—0.170,p=0.238)、肿瘤部位(r=0.022,p=0.879)无关；而与肿瘤分化程度(r=0.711,p<0.01)呈正相关(即肿瘤分化程度越高,IHCS评分越高)；与肿瘤大小(r=—0.510,p<0.01)呈负相关(即肿瘤越大,IHCS评分越低)；与肿瘤血管浸润(r=—0.804,p<0.01)呈负相关(即有血管浸润的病例,IHCS评分越低)；与肿瘤神经浸润(r=—0.785,p<0.01)呈负相关(即有神经浸润的病例,IHCS评分越低)；与肿瘤的TNM分期(r=—0.948,p<0.01)呈负相关(即TNM分级越高,IHCS评分越低)。
2.组织MSP检测显示WTX基因在C组中的甲基化阳性率高于CB组和N组(p<0.05)；在Lovo细胞中MSP检测未发现WTX基因甲基化。
3.成功通过Gateway技术构建携带WTX基因的慢病毒载体PLV.Ex3d.null-EF1A>WTX>IRES/eGFP,鉴定正确；慢病毒包装成功,滴度符合实验要求；病毒转导Lovo细胞48-72小时后荧光显微镜下观察慢病毒载体组(WTX组)及空载体组荧光均较弱,通过挑克隆培养法建成稳定表达WTX的Lovo细胞系Lovo/WTX-eGFP。采用WTX质粒及空载对照质粒进行瞬时转染,48-72小时后WTX组观察荧光转染效率约35%,空载体组约70%。qPCR及Western blot检测均提示转染后WTX表达水平增高；流式细胞仪检测WTX过表达对Lovo细胞凋亡水平无明显影响；CCK8法检测：96孔板转染镜下可见转染后随时间增长,WTX组细胞数较Cell组及空载体组减少,荧光逐渐增强,但WTX组荧光仍少于空载体组。WTX过表达明显抑制Lovo细胞增殖水平。细胞周期检测发现转染WTX后Lovo细胞被阻滞在G0/G1期。
结论
1.抑癌基因WTX结肠癌中表达降低与结肠癌的恶性程度及周围组织浸润密切相关,WTX表达缺失可能是结肠癌发生的重要原因之一。
2.WTX在C组中甲基化阳性率高于CB组和N组,而在结肠癌Lovo细胞株中WTX甲基化呈阴性,尚需对其它结肠癌细胞株进行MSP检测以明确甲基化是否是导致WTX在结肠癌组织中表达降低的原因。
3.WTX慢病毒载体构建成功,但细胞转导效率低,与WTX基因CDS区较长,影响绿色荧光蛋白eGFP表达有关。成功建立表达WTX的Lovo细胞系Lovo/WTX-eGFP。WTX质粒瞬时转染后WTX转录及蛋白水平表达明显升高。WTX转染后Lovo细胞增殖受到明显抑制,对细胞凋亡无明显影响。
Objective
1. To study expression level and location of tumor suppressor gene WTX in fresh colon cancer tissues, peficancerous tissues and normal colon tissues. The relationship between gene expression level and clinical and pathological features of colon cancer were analysed and compared in order to study the relevance between abnormal gene expression of WTX and colon cancer.
2. To study methylation level of WTX gene in fresh colon cancer tissues, peficancerous tissues, normal colon tissues and colon cancer cell strain Lovo.
3. To establish lentiviral vector of WTX and transfect WTX into colon cancer cell line Lovo in vitro. Detecting expression of WTX in transcription and protein level and its influence to cell proliferation and apoptosis.
Methods
1. Fresh colon cancer tissues, peficancerous tissues and normal colon tissues of50colon cancer patients who had been accepted radical surgery and pathological confirmed were collected. These50specimens were divided into3groups including group of cancer tissues (group C), group of peficancerous tissues (group CB) and normal colon tissues (group N). RNA of these tissues was extracted and qPCR was used to detect transcription level of WTX in different tissues. Total proteins of these tissues were extracted and Western blot was used to detect protein level of WTX in different tissues. Immunohistochemistry was used to detect location of WTX in different tissues and immunohistochemistry score (IHCS) was analyzed. The relationship between WTX expression level and clinical and pathological features of group C were analysed and compared.
2. DNA of all specimens an colon cancer cell strain Lovo were extracted and modified by sodium hydrogensulfite. We utilized Wizard purified resin to purifiy DNA.4methylation specific PCR (MSP) specific primers were designed for detecting methylation level of WTX in all specimens and Lovo cells by MSP.
3. The lentiviral vector of PLV.Ex3d.null-EF1A>WTX>IRES/eGFP was constructed by Gateway Technology (Invitrogen Company). The positive cloning plasmid was picked for positive cloning sequencing. The lentiviral vector and lentiviral packaging plasmid were co-trasfected into293FT cells. Lentivirus titer was caculated after virus supernatant was obtained by centrifugation. Lovo cells were devided into3groups including blank cells (group Cell), blank vector (group Blank Vector) and WTX vector (group WTX) and were trsducted by lentivirus. Fluorescence microscope was used to observe transducting efficiency at48-72h after transducting.We utilized qPCR and Western blot to detect expression of WTX in transcription and protein level of each cell groups. Flow cytometry was used to detect cell apoptosis level and cell cycle. Regent CCK8was used to detect cell inhibition ratio and draw cell growth curve.
Results
1. All specimens have WTX expression. Expression of WTX in transcription and protein level detected by qPCR and Western blot showed that mean expression level of WTX in group C was lower than group N and CB, mean expression level of WTX in group CB was lower than group N. The difference was statistically significant (p<0.05). Results of immunohistochemistry showed that expression of WTX was located at cytoplasm and little at cytomembrane. IHCS of group C was lower than group N and CB (p<0.05). IHCS of group N and CB had no obvious difference. IHCS of group C has no correlation with sex (r=-0.168,p=0.245), age (r=-0.170,p=0.238) and tumor position (r=0.022, p=0.879). IHCS of group C has positive correlation with tumor differentiation degree (the higher level of tumor differentiation, the higher of IHCS, r=0.711, p<0.01), and has negative correlation with tumor size (the bigger of tumor size, the lower of IHCS, r=-0.510,p<0.01), and has negative correlation with tumor vessel infiltration (when there is tumor vessel infiltration, the lower of IHCS, r=-0.804,p<0.01), and has negative correlation with tumor nerve infiltration (when there is tumor nerve infiltration, the lower of IHCS, r=-0.785, p<0.01), and has negative correlation with tumor TNM stages (the higher level of TNM stages, the lower of IHCS, r=-0.948,p<0.01).
2. MSP detecting of tissues showed that positive rate of WTX methylation in group C was higher than group CB and N (p<0.05), but there was no WTX methylation found in colon cancer cell strain Lovo.
3. The lentiviral vector of PLV.Ex3d.null-EF1A>WTX>IRES/eGFP was succesfully constructed by Gateway Technology and identified perfectly correct. Lentivirus was successfully packaged and titer was accorded with the requirement of experiment. Fluorescence of group WTX and Blank Vector were weak at48-72h after transducting WTX into Lovo cells. The Lovo/WTX-eGFP colon cancer cell line stably transducting WTX was established by picking clone several times and cell culture. Then we proceeded transient transfection of WTX plasmid and contract blank plasmid. Fluorescence can be observed at48-72h after transient transfecting and transfection efficiency of group WTX and Blank Vector was about35%and70%respectively. Results of qPCR and Western blot showed that expression level rised after transfecting. Results of flow cytometry showed that excessive expression of WTX had no obvious influence to cell apoptosis level of Lovo cells. Results of CCK8test showed that with time increasing, cell population of group WTX reduced but fluorescence increased compared with group cell and group Blank Vector in96-well plates. Fluorescence cell population of group WTX was lesser than group Blank Vector. Excessive expression of WTX could obviously suppress cell proliferation. Cell cycle detection indicates that Lovo cells were blocked in G0/G1phase.
Conclusions
1. Lower expression level of tumor suppressor gene WTX in colon cancer is closely ralated to grade malignancy and infiltration of surrounding tissues. Loss of WTX expression maybe a important reason of occurring of colon cancer.
2. Although positive rate of WTX methylation in group C was higher than group CB and N, but WTX methylation was negative in colon cancer cell strain Lovo. More MSP should be done to detect whether methylation is exist in other colon cancer cell strains in order to clear whether methylation is a reason of lower expression of WTX in colon cancer tissues.
3. The lentiviral vector of PLV.Ex3d.null-EF1A>WTX>IRES/eGFP was succesfully constructed but cell trasducting efficiency was low. Long CDS region of WTX may influence eGFP expression, and this may be the reason that why transducting efficiency was low. The Lovo/WTX-eGFP colon cancer cell line stably transducting WTX was established successfully. Transient transfection of WTX plasmid lead higher WTX expression in transcription and protein level. Transfection of WTX could obviously suppress cell proliferation but had no influence to cell apoptosis.
引文
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