Wnt/β-catenin通路在胰腺癌的表达及干扰CXCR4基因对其抑制作用的体外实验研究
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摘要
目的:
胰腺癌恶性度高,发病率逐年上升。据报道,80%的胰腺癌患者由于转移的发生失去手术机会。Wnt/β-catenin通路在消化道肿瘤进展中具有重要作用。此外,肿瘤转移的趋向性学说以及趋化因子受体CXCR4的作用备受关注。据此,设计该实验,旨在
1.明确Wnt/β-catenin经典通路在胰腺癌细胞中的活性,以及Wnt/β-catenin通路靶基因在胰腺癌细胞中的表达;
2.观察Wnt拮抗剂DKK-1和sFRPs因子在胰腺癌中的表达,探讨Wnt拮抗剂表遗传学改变对胰腺癌Wnt/β-catenin通路的作用机制;
3.观察胰腺癌组织中CXCR4的表达变化以及与临床病理特征之间的关系;
4.观察胰腺癌组织中β-catenin的表达变化,探讨其与CXCR4的相互关系;
5.利用RNAi技术构建CXCR4 shRNA表达载体,观察Wnt/β-catenin通路活性及靶基因的变化,验证CXCR4调控Wnt/β-catenin通路抑制胰腺癌的假设;
6.观察CXCR4基因干扰后胰腺癌细胞增殖、细胞周期以及侵袭能力的变化。
方法:
1.通过PGL3-OT/OF荧光素酶检测方法观察Wnt/β-catenin通路在胰腺癌细胞中的活性,应用Real-time PCR技术检测Wnt通路下游靶基因及Wnt拮抗剂DKK-1和sFRPs mRNA水平的表达,观察Wnt/β-catenin经典通路在胰腺癌细胞中的变化;
2.通过MSP方法和5-氮杂-脱氧胞苷酸处理分析Wnt拮抗剂sFRPs、DKK-1在胰腺癌细胞中的甲基化,并应用基因重组技术双向调节DKK-1在不同胰腺癌细胞中的表达,观察Wnt通路活性的变化,分析Wnt拮抗剂的甲基化对Wnt通路的调节作用;
3.通过免疫组化分析CXCR4、β-catenin在胰腺癌组织中的表达,以及与临床病理特征之间的关系,探讨CXCR4与β-catenin的相关性;
4.通过RNAi技术构建CXCR4 shRNA表达载体,转染胰腺癌CXCR4高表达细胞,通过Realtime-PCR、Western Blot实验观察RNAi对CXCR4基因的抑制效率;
5.通过PGL3-OT/OF荧光素酶检测方法观察CXCR4基因沉默后对胰腺癌Wnt/β-catenin经典通路活性的影响,并通过Realtime-PCR、Western Blot实验观察CXCR4 shRNA对Wnt/β-catenin通路下游靶基因以及侵袭转移相关蛋白的影响;
6.通过MTT实验、流式细胞术、软琼脂克隆形成实验检测CXCR4基因干扰后胰腺癌细胞增殖、细胞周期以及细胞成瘤性的变化,并应用Transwell侵袭小室观察CXCR4干扰对胰腺癌细胞侵袭转移力的影响。
结果:
1.与正常胰腺细胞相比,多数胰腺癌细胞表现为Wnt/β-catenin通路的异常激活,此外,Wnt/β-catenin下游靶基因的mRNA水平也具有不同程度的升高,通过对β-catenin Exon 3的测序分析,未发现胰腺癌具有β-catenin Exon 3的突变;
2. Wnt拮抗剂sFRPs和DKK-1因子在胰腺癌细胞中表达水平较低,MSP分析发现sFRPs和DKK-1启动子区在胰腺癌细胞中发生甲基化,5-氮杂-脱氧胞苷酸处理后能显著提高DKK-1和sFRP-1的表达(P<0.01)。当RNAi技术干扰DKK-1表达后,Wnt通路活性上升;当PCMV-XL5-DKK1转染细胞上调DKK-1表达时,Wnt通路活性下降(P<0.05);
3. CXCR4与β-catenin在胰腺癌组织中具有异常表达,两者之间有显著相关性(rs=0.443, P=0.002)。此外,CXCR4表达高的胰腺癌患者临床预后差;
4.特异性CXCR4基因沉默能够在基因和蛋白水平稳定、高效、特异地抑制CXCR4的表达,抑制效率达70%以上,同时,磷酸化CXCR4蛋白表达水平也有明显的下降(P<0.05);
5.特异性CXCR4干扰后能够显著抑制PGL3-OT转染后的荧光素活性(P<0.05)以及Wnt/β-catenin靶基因的表达;
6.特异性CXCR4 shRNA能够抑制细胞生长,细胞达到对数生长期的时间延长(P<0.05),同时CXCR4基因沉默能够阻滞Miapaca-2细胞于G0/G1期,减少S期细胞的数目,抑制细胞的增殖能力。同时,软琼脂克隆形成实验发现CXCR4干扰后,细胞克隆数目显著减少(P<0.01)。Transwell实验证明CXCR4基因干扰后细胞侵袭能力减低(P<0.05),即使SDF-1的刺激也不能够使细胞侵袭能力增加。结论:
1.胰腺癌细胞中存在Wnt/β-catenin通路的异常激活。Wnt通路的激活与β-catenin Exon 3的突变无关;
2.胰腺癌中Wnt拮抗剂发生表遗传学变化,启动子区的甲基化引起DKK-1表达沉默可能是胰腺癌Wnt通路异常激活的原因,DKK-1的表达与胰腺癌Wnt经典通路的活性呈负相关;
3. CXCR4在胰腺癌中异常表达,与β-catenin表达显著相关,CXCR4表达高的胰腺癌患者临床预后差;
4. CXCR4基因沉默能抑制Wnt/β-catenin经典通路在胰腺癌中的活性,阻遏Wnt/β-catenin通路下游靶基因表达,抑制胰腺癌细胞增殖、侵袭和成瘤能力。
Objectives:
Extra-pancreatic metastasis is a difficult problem for surgical intervention on pancreatic cancer. CXC chemokine receptor 4 (CXCR4) was considered as an important role in this process. Wnt pathway activity is abnormal in gastrointestinal cancer but poorly understood on pancreatic cancers. Previous study on neural development confirmed that SDF-1/CXCR4 could effectively modulate the Wnt/β-catenin signaling. We hypothesized that CXCR4 functional study may contribute to the pancreatic cancer progression through inhibiting canonical Wnt pathway. Here we study:
1. Wnt pathway activity in pancreatic cancer cells and its target gene expression;
2. The methylation of Wnt antagonists and its possible function on pancreatic cancers;
3. CXCR4 andβ-catenin expression on pancreatic cancer tissues and their relation with clinical characteristics;
4. The influence of CXCR4 knockdown on the expression of Wnt/β-catenin pathway;
5. The influence of CXCR4 knockdown on pancreatic cancer cell growth, cell cycle, cell tumorigenesis, cell invasive ability.
Methods:
1. To observe the canonical Wnt pathway activity in pancreatic cancer cells, PGL3-OT/OF luciferase activity was applied. The expression of Wnt/β-catenin signaling target genes and Wnt antagonists were detected with Realtime-PCR technique.
2. To observe the methylation of Wnt antagonists, MSP analysis was applied. To modulate the DKK-1 expression on pancreatic cancers, gene recombination technique was applied.
3. To analyze the expression of CXCR4 andβ-catenin expression on pancreatic cancer tissues, immunohistochemical technique was applied. Kaplan-Meier estimated curves, Spearman and Chi-square analysis were used to study the relation between CXCR4 expression and clinical characteristics.
4. RNAi technique was applied to construct the CXCR4 shRNA expression vector. Lipofecatime 2000 was used to transfect the CXCR4 high expression pancreatic cancer cell Miapaca-2. Realtime-PCR and Western Blot were used to observe the inhibitory effect of RNAi on CXCR4 expression.
5. Realtime-PCR and Western Blot were applied to observe the influence of specific CXCR4 shRNA on Wnt signaling target gene expression. PGL3-OT/OF luciferase activity was used to observe the influence of CXCR4 shRNA on Wnt activity.
6. MTT assay was applied to assess the influence of specific CXCR4 shRNA on cell growth; Flow cytometry was applied to analyze the cell cycle change; Soft agar assay was to observe the CXCR4 shRNA influence of cell tumorigenesis; Transwell method was to observe the cancer cell invasive ability.
Results:
1. Abnormal canonical Wnt pathway activity was detected in pancreatic cancer cell lines. Compared with normal pancreas cell lines, Wnt activity and its target gene expression were upregulated in most of pancreatic cancer cells. Moreover, the sequence ofβ-catenin Exon 3 showed that there is no mutation in pancreatic cancer cell lines.
2. Wnt antagonists including sFRPs and DKK-1 mRNA level were obviously downregulated in pancreatic cancers. MSP analysis confirmed that methylation of Wnt antagonist was detected in pancreatic cancer cells. The percentage of methylated DKK-1 in pancreatic cancer cells was 6/17. Moreover, the percentage of sFRP-1, 2, 4, 5 is 11/17, 17/17, 8/17, 5/17, respectively. With 5-aza-2'-deoxycytidine treatment, the expression of Wnt antagonists especially DKK-1 and sFRP-1 could be repaired (P<0.01).
3. With the upregulation of DKK-1 expression, the Wnt pathway activity was inhibited. On the contrary, with the knockdown of DKK-1 expression, the Wnt pathway activity was increased (P<0.05).
4. Abnormal expression of CXCR4 andβ-catenin were detected on pancreatic cancer tissues. There is obvious relation between CXCR4 andβ-catenin expression (rs=0.443, P=0.002). Moreover, Kaplan-Meier estimated curve showed that CXCR4 expression could influence the clinical survival of pancreatic cancer patients.
5. Through RNAi technique, the CXCR4 shRNA expression plasmid was successfully constructed. After the transfection on CXCR4 high expression pancreatic cancer cell line Miapaca-2, one plasmid which has the inhibitoriest effect was selected in the following experiment in vitro.
6. CXCR4 gene interference could effectively inhibit Wnt pathway activity and Wnt/β-catenin target gene expression on pancreatic cancer cell lines.
7. In CXCR4 shRNA transfected cells, cell growth was slower than that of control cells (P<0.05). A prolonged and prominent delay in progression from the G0 to G1 phase, a decrease at the S phase and G2-M phase were detected. Moreover, CXCR4 shRNA could decrease the cell tumorigenesis and cell invasive ability.
Conclusions:
1. The canonical Wnt/β-catenin pathway was deregulated in pancreatic cancer cells, which has no relation withβ-catenin Exon 3 mutation.
2. Epigenetic Modification of the Wnt antagonists was considered to contribute to active Wnt pathway in pancreatic cancers. The modulation of DKK-1 could influence the Wnt pathway activity in pancreatic cancer cells.
3. CXCR4 was abnormally expressed in pancreatic cancers. There is some relation between CXCR4 andβ-catenin expression. High CXCR4 expression was a predictor of poor clinical survival.
4. CXCR4 knockdown could inhibit Wnt/β-catenin pathway and its target gene expression, which could result in the decrease of pancreatic cancer cell growth, cell tumorigenesis and invasive ability.
胰腺癌恶性度高,发病率逐年上升。据报道,80%的胰腺癌患者由于转移的发生失去手术机会。Wnt/β-catenin通路在消化道肿瘤进展中具有重要作用。此外,肿瘤转移的趋向性学说以及趋化因子受体CXCR4的作用备受关注。据此,设计该实验,旨在
1.明确Wnt/β-catenin经典通路在胰腺癌细胞中的活性,以及Wnt/β-catenin通路靶基因在胰腺癌细胞中的表达;
2.观察Wnt拮抗剂DKK-1和sFRPs因子在胰腺癌中的表达,探讨Wnt拮抗剂表遗传学改变对胰腺癌Wnt/β-catenin通路的作用机制;
3.观察胰腺癌组织中CXCR4的表达变化以及与临床病理特征之间的关系;
4.观察胰腺癌组织中β-catenin的表达变化,探讨其与CXCR4的相互关系;
5.利用RNAi技术构建CXCR4 shRNA表达载体,观察Wnt/β-catenin通路活性及靶基因的变化,验证CXCR4调控Wnt/β-catenin通路抑制胰腺癌的假设;
6.观察CXCR4基因干扰后胰腺癌细胞增殖、细胞周期以及侵袭能力的变化。
方法:
1.通过PGL3-OT/OF荧光素酶检测方法观察Wnt/β-catenin通路在胰腺癌细胞中的活性,应用Real-time PCR技术检测Wnt通路下游靶基因及Wnt拮抗剂DKK-1和sFRPs mRNA水平的表达,观察Wnt/β-catenin经典通路在胰腺癌细胞中的变化;
2.通过MSP方法和5-氮杂-脱氧胞苷酸处理分析Wnt拮抗剂sFRPs、DKK-1在胰腺癌细胞中的甲基化,并应用基因重组技术双向调节DKK-1在不同胰腺癌细胞中的表达,观察Wnt通路活性的变化,分析Wnt拮抗剂的甲基化对Wnt通路的调节作用;
3.通过免疫组化分析CXCR4、β-catenin在胰腺癌组织中的表达,以及与临床病理特征之间的关系,探讨CXCR4与β-catenin的相关性;
4.通过RNAi技术构建CXCR4 shRNA表达载体,转染胰腺癌CXCR4高表达细胞,通过Realtime-PCR、Western Blot实验观察RNAi对CXCR4基因的抑制效率;
5.通过PGL3-OT/OF荧光素酶检测方法观察CXCR4基因沉默后对胰腺癌Wnt/β-catenin经典通路活性的影响,并通过Realtime-PCR、Western Blot实验观察CXCR4 shRNA对Wnt/β-catenin通路下游靶基因以及侵袭转移相关蛋白的影响;
6.通过MTT实验、流式细胞术、软琼脂克隆形成实验检测CXCR4基因干扰后胰腺癌细胞增殖、细胞周期以及细胞成瘤性的变化,并应用Transwell侵袭小室观察CXCR4干扰对胰腺癌细胞侵袭转移力的影响。
结果:
1.与正常胰腺细胞相比,多数胰腺癌细胞表现为Wnt/β-catenin通路的异常激活,此外,Wnt/β-catenin下游靶基因的mRNA水平也具有不同程度的升高,通过对β-catenin Exon 3的测序分析,未发现胰腺癌具有β-catenin Exon 3的突变;
2. Wnt拮抗剂sFRPs和DKK-1因子在胰腺癌细胞中表达水平较低,MSP分析发现sFRPs和DKK-1启动子区在胰腺癌细胞中发生甲基化,5-氮杂-脱氧胞苷酸处理后能显著提高DKK-1和sFRP-1的表达(P<0.01)。当RNAi技术干扰DKK-1表达后,Wnt通路活性上升;当PCMV-XL5-DKK1转染细胞上调DKK-1表达时,Wnt通路活性下降(P<0.05);
3. CXCR4与β-catenin在胰腺癌组织中具有异常表达,两者之间有显著相关性(rs=0.443, P=0.002)。此外,CXCR4表达高的胰腺癌患者临床预后差;
4.特异性CXCR4基因沉默能够在基因和蛋白水平稳定、高效、特异地抑制CXCR4的表达,抑制效率达70%以上,同时,磷酸化CXCR4蛋白表达水平也有明显的下降(P<0.05);
5.特异性CXCR4干扰后能够显著抑制PGL3-OT转染后的荧光素活性(P<0.05)以及Wnt/β-catenin靶基因的表达;
6.特异性CXCR4 shRNA能够抑制细胞生长,细胞达到对数生长期的时间延长(P<0.05),同时CXCR4基因沉默能够阻滞Miapaca-2细胞于G0/G1期,减少S期细胞的数目,抑制细胞的增殖能力。同时,软琼脂克隆形成实验发现CXCR4干扰后,细胞克隆数目显著减少(P<0.01)。Transwell实验证明CXCR4基因干扰后细胞侵袭能力减低(P<0.05),即使SDF-1的刺激也不能够使细胞侵袭能力增加。结论:
1.胰腺癌细胞中存在Wnt/β-catenin通路的异常激活。Wnt通路的激活与β-catenin Exon 3的突变无关;
2.胰腺癌中Wnt拮抗剂发生表遗传学变化,启动子区的甲基化引起DKK-1表达沉默可能是胰腺癌Wnt通路异常激活的原因,DKK-1的表达与胰腺癌Wnt经典通路的活性呈负相关;
3. CXCR4在胰腺癌中异常表达,与β-catenin表达显著相关,CXCR4表达高的胰腺癌患者临床预后差;
4. CXCR4基因沉默能抑制Wnt/β-catenin经典通路在胰腺癌中的活性,阻遏Wnt/β-catenin通路下游靶基因表达,抑制胰腺癌细胞增殖、侵袭和成瘤能力。
Objectives:
Extra-pancreatic metastasis is a difficult problem for surgical intervention on pancreatic cancer. CXC chemokine receptor 4 (CXCR4) was considered as an important role in this process. Wnt pathway activity is abnormal in gastrointestinal cancer but poorly understood on pancreatic cancers. Previous study on neural development confirmed that SDF-1/CXCR4 could effectively modulate the Wnt/β-catenin signaling. We hypothesized that CXCR4 functional study may contribute to the pancreatic cancer progression through inhibiting canonical Wnt pathway. Here we study:
1. Wnt pathway activity in pancreatic cancer cells and its target gene expression;
2. The methylation of Wnt antagonists and its possible function on pancreatic cancers;
3. CXCR4 andβ-catenin expression on pancreatic cancer tissues and their relation with clinical characteristics;
4. The influence of CXCR4 knockdown on the expression of Wnt/β-catenin pathway;
5. The influence of CXCR4 knockdown on pancreatic cancer cell growth, cell cycle, cell tumorigenesis, cell invasive ability.
Methods:
1. To observe the canonical Wnt pathway activity in pancreatic cancer cells, PGL3-OT/OF luciferase activity was applied. The expression of Wnt/β-catenin signaling target genes and Wnt antagonists were detected with Realtime-PCR technique.
2. To observe the methylation of Wnt antagonists, MSP analysis was applied. To modulate the DKK-1 expression on pancreatic cancers, gene recombination technique was applied.
3. To analyze the expression of CXCR4 andβ-catenin expression on pancreatic cancer tissues, immunohistochemical technique was applied. Kaplan-Meier estimated curves, Spearman and Chi-square analysis were used to study the relation between CXCR4 expression and clinical characteristics.
4. RNAi technique was applied to construct the CXCR4 shRNA expression vector. Lipofecatime 2000 was used to transfect the CXCR4 high expression pancreatic cancer cell Miapaca-2. Realtime-PCR and Western Blot were used to observe the inhibitory effect of RNAi on CXCR4 expression.
5. Realtime-PCR and Western Blot were applied to observe the influence of specific CXCR4 shRNA on Wnt signaling target gene expression. PGL3-OT/OF luciferase activity was used to observe the influence of CXCR4 shRNA on Wnt activity.
6. MTT assay was applied to assess the influence of specific CXCR4 shRNA on cell growth; Flow cytometry was applied to analyze the cell cycle change; Soft agar assay was to observe the CXCR4 shRNA influence of cell tumorigenesis; Transwell method was to observe the cancer cell invasive ability.
Results:
1. Abnormal canonical Wnt pathway activity was detected in pancreatic cancer cell lines. Compared with normal pancreas cell lines, Wnt activity and its target gene expression were upregulated in most of pancreatic cancer cells. Moreover, the sequence ofβ-catenin Exon 3 showed that there is no mutation in pancreatic cancer cell lines.
2. Wnt antagonists including sFRPs and DKK-1 mRNA level were obviously downregulated in pancreatic cancers. MSP analysis confirmed that methylation of Wnt antagonist was detected in pancreatic cancer cells. The percentage of methylated DKK-1 in pancreatic cancer cells was 6/17. Moreover, the percentage of sFRP-1, 2, 4, 5 is 11/17, 17/17, 8/17, 5/17, respectively. With 5-aza-2'-deoxycytidine treatment, the expression of Wnt antagonists especially DKK-1 and sFRP-1 could be repaired (P<0.01).
3. With the upregulation of DKK-1 expression, the Wnt pathway activity was inhibited. On the contrary, with the knockdown of DKK-1 expression, the Wnt pathway activity was increased (P<0.05).
4. Abnormal expression of CXCR4 andβ-catenin were detected on pancreatic cancer tissues. There is obvious relation between CXCR4 andβ-catenin expression (rs=0.443, P=0.002). Moreover, Kaplan-Meier estimated curve showed that CXCR4 expression could influence the clinical survival of pancreatic cancer patients.
5. Through RNAi technique, the CXCR4 shRNA expression plasmid was successfully constructed. After the transfection on CXCR4 high expression pancreatic cancer cell line Miapaca-2, one plasmid which has the inhibitoriest effect was selected in the following experiment in vitro.
6. CXCR4 gene interference could effectively inhibit Wnt pathway activity and Wnt/β-catenin target gene expression on pancreatic cancer cell lines.
7. In CXCR4 shRNA transfected cells, cell growth was slower than that of control cells (P<0.05). A prolonged and prominent delay in progression from the G0 to G1 phase, a decrease at the S phase and G2-M phase were detected. Moreover, CXCR4 shRNA could decrease the cell tumorigenesis and cell invasive ability.
Conclusions:
1. The canonical Wnt/β-catenin pathway was deregulated in pancreatic cancer cells, which has no relation withβ-catenin Exon 3 mutation.
2. Epigenetic Modification of the Wnt antagonists was considered to contribute to active Wnt pathway in pancreatic cancers. The modulation of DKK-1 could influence the Wnt pathway activity in pancreatic cancer cells.
3. CXCR4 was abnormally expressed in pancreatic cancers. There is some relation between CXCR4 andβ-catenin expression. High CXCR4 expression was a predictor of poor clinical survival.
4. CXCR4 knockdown could inhibit Wnt/β-catenin pathway and its target gene expression, which could result in the decrease of pancreatic cancer cell growth, cell tumorigenesis and invasive ability.
引文
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