Notch信号通路在胰腺癌中的表达及其作用研究
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摘要
目的研究Notch信号通路成员在胰腺癌组织中的异常表达及其在胰腺癌发生发展中的重要作用。方法应用Affymetrix基因表达谱芯片筛选10例胰腺癌组织及其癌旁组织中Notch信号通路存在差异表达的成员,并应用实时定量PCR和Western blot印迹法加以验证;利用携锯齿2(Jagged 2,JAG2)基因siRNA片段的慢病毒表达载体转染胰腺癌原代细胞构建JAG2基因阻遏表达胰腺癌细胞株,并采用MTT法、流式细胞术、侵袭小室实验法观察与分析该细胞株增殖、细胞周期及侵袭转移能力的变化。结果 Affymetrix基因表达谱芯片共检测到差异表达基因512个,其中表达上调的基因419个,表达下调的基因93个; Notch信号通路中存在差异表达的为JAG2(上调表达8. 20倍)、NOTCH1(上调表达3. 74倍)、HES1(上调表达3. 27倍)、NOTCH2(上调表达3. 16倍); PCR和Western blot法验证结果与基因芯片结果相符;与对照组相比,JAG2基因阻遏表达胰腺癌细胞株的生长明显受到抑制;经流式细胞仪分析两组细胞周期示JAG2基因阻遏表达胰腺癌细胞株的凋亡与S期阻滞明显增强;侵袭小室侵实验结果示JAG2基因阻遏表达胰腺癌细胞株袭转移能力明显减弱(P <0. 05)。结论 Notch信号通路部分成员在胰腺癌组织中存在显著差异表达,且阻遏该成员表达可影响胰腺癌细胞生长、细胞周期及侵袭转移。
Objective The objective of this study was to investigate the abnormal expression of Notch signaling pathway members in pancreatic cancer and its important effect on the development of pancreatic carcinoma. Methods Affymetrix gene expression microarray was used to screen the differentially expressed members of Notch signal pathway in 10 cases of pancreatic carcinoma and its adjacent tissues,and verified by real-time quantitative PCR and Western blotting. The lentivirus expression vector carrying the siRNA fragment of Jagged 2( JAG2) gene was transfected into the pancreatic cancer primary cells to construct the JAG2 gene repression-expressing pancreatic cancer cell line. MTT,flow cytometry and Transwell assays were used to analyze cell proliferation,changes of cell cycle and invasive transfer capabilities. Results A total of 512 differentially expressed genes were detected by Affymetrix gene expression microarray,including 419 up-regulated genes and 93 down-regulated genes. JAG2( up-regulated expression 8. 20 times),NOTCH1( up-regulated expression 3. 74 times),HES1( up-regulated expression 3. 27 times),and NOTCH2( up-regulated expression 3. 16 times) were differentially expressed in Notch signaling pathway. The results of PCR and Western blotting were consistent with those of gene chip. The growth curves of JAG2 gene repressed pancreatic cancer cells and pancreatic cancer primary cells were drawn by the standard OD490 value of d1-d5 by MTT assay. JAG2 gene repressor expression vector could significantly inhibit the proliferation of pancreatic cancer cells. The cell cycle analysis showed that the apoptosis and the arrested cell cycle at the S phase were significantly increased in pancreatic cancer cells with JAG2 gene repressor expression. The invasive ability was significantly reduced in JAG2 gene repressor expression pancreatic cancer cells( P < 0. 05). Conclusion Some members of the Notch signaling pathway are significantly differentially expressed in pancreatic cancer tissues,and repression of this member can affect the growth,cell cycle,invasion and metastasis of pancreatic cancer cells.
Objective The objective of this study was to investigate the abnormal expression of Notch signaling pathway members in pancreatic cancer and its important effect on the development of pancreatic carcinoma. Methods Affymetrix gene expression microarray was used to screen the differentially expressed members of Notch signal pathway in 10 cases of pancreatic carcinoma and its adjacent tissues,and verified by real-time quantitative PCR and Western blotting. The lentivirus expression vector carrying the siRNA fragment of Jagged 2( JAG2) gene was transfected into the pancreatic cancer primary cells to construct the JAG2 gene repression-expressing pancreatic cancer cell line. MTT,flow cytometry and Transwell assays were used to analyze cell proliferation,changes of cell cycle and invasive transfer capabilities. Results A total of 512 differentially expressed genes were detected by Affymetrix gene expression microarray,including 419 up-regulated genes and 93 down-regulated genes. JAG2( up-regulated expression 8. 20 times),NOTCH1( up-regulated expression 3. 74 times),HES1( up-regulated expression 3. 27 times),and NOTCH2( up-regulated expression 3. 16 times) were differentially expressed in Notch signaling pathway. The results of PCR and Western blotting were consistent with those of gene chip. The growth curves of JAG2 gene repressed pancreatic cancer cells and pancreatic cancer primary cells were drawn by the standard OD490 value of d1-d5 by MTT assay. JAG2 gene repressor expression vector could significantly inhibit the proliferation of pancreatic cancer cells. The cell cycle analysis showed that the apoptosis and the arrested cell cycle at the S phase were significantly increased in pancreatic cancer cells with JAG2 gene repressor expression. The invasive ability was significantly reduced in JAG2 gene repressor expression pancreatic cancer cells( P < 0. 05). Conclusion Some members of the Notch signaling pathway are significantly differentially expressed in pancreatic cancer tissues,and repression of this member can affect the growth,cell cycle,invasion and metastasis of pancreatic cancer cells.
引文
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2王瑞芝,王小林.胰腺癌分子靶向治疗的研究进展[J].复旦学报(医学版),2018,45(1):77-86.
3蔡尚党,蔡满地,娄宁,等.miR-15家族促进CD133+胰腺癌干样细胞的迁移和侵袭[J].中国组织工程研究,2018,22(21):3316-3321.
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7徐林,易超,依马木买买提江·阿布拉,等.携Jagged 2基因siRNA慢病毒表达载体的构建及其转染对胰腺癌细胞影响[J].中国普通外科杂志,2017,26(9):1133-1140.
8张波,易超,依马木买买提江·阿布拉,等.Smoothened基因靶向小干扰RNA慢病毒表达载体对人胰腺癌细胞生物学特性的影响[J].中华实验外科杂志,2017,34(12):2158-2161.
9 Haider S,Pollheimer J,Knofler M.Notch signalling in placental development and gestational diseases[J].Placenta,2017,56:65-72.
10易超,依马木买买提江·阿布拉,丁伟,等.Affymetrix基因表达谱芯片技术筛选胰腺癌异常表达基因的研究[J].中国普通外科杂志,2017,26(3):294-303.
11 Yao Y,Ni Y,Zhang J,et al.The role of Notch signaling in gastric carcinoma:molecular pathogenesis and novel therapeutic targets[J].Oncotarget,2017,8(32):53839-53853.
12 Vaish V,Kim J,Shim M.Jagged-2(JAG2)enhances tumorigenicity and chemoresistance of colorectal cancer cells[J].Oncotarget,2017,8(32):53262-53275.
13 Li CY,Miao KL,Chen Y,et al.Jagged2 promotes cancer stem cell properties of triple negative breast cancer cells and paclitaxel resistance via regulating microRNA-200[J].Eur Rev Med Pharmacol Sci,2018,22(18):6008-6014.
14 Hu Y,Su H,Li X,et al.The NOTCH ligand JAGGED2promotes pancreatic cancer metastasis independent of NOTCH signaling activation[J].Mol Cancer Ther,2015,14(1):289-297.
15 Lo JH,Hao L,Muzumdar MD,et al.iRGD-guided tumor-penetrating nanocomplexes for therapeutic siRNA delivery to pancreatic cancer[J].Mol Cancer Ther,2018,17(11):2377-2388.
16 Min HS,Kim HJ,Ahn J,et al.Tuned density of anti-tissue factor antibody fragment onto siRNA-loaded polyion complex micelles for optimizing targetability into pancreatic cancer cells[J].Biomacromolecules,2018,19(6):2320-2329.
2王瑞芝,王小林.胰腺癌分子靶向治疗的研究进展[J].复旦学报(医学版),2018,45(1):77-86.
3蔡尚党,蔡满地,娄宁,等.miR-15家族促进CD133+胰腺癌干样细胞的迁移和侵袭[J].中国组织工程研究,2018,22(21):3316-3321.
4许耀麟,楼文晖.胰腺癌分子病理学进展[J].中华消化外科杂志,2018,17(4):418-422.
5 Unger C,Kofanova O,Sokolowska K,et al.Ultraviolet C radiation influences the robustness of RNA integrity measurement[J].Electrophoresis,2015,36(17):2072-2081.
6 Villegas-Ruiz V,Moreno J,Jacome-Lopez K,et al.Quality control usage in high-density microarrays reveals differential gene expression profiles in ovarian cancer[J].Asian Pac J Cancer Prev,2016,17(5):2519-2525.
7徐林,易超,依马木买买提江·阿布拉,等.携Jagged 2基因siRNA慢病毒表达载体的构建及其转染对胰腺癌细胞影响[J].中国普通外科杂志,2017,26(9):1133-1140.
8张波,易超,依马木买买提江·阿布拉,等.Smoothened基因靶向小干扰RNA慢病毒表达载体对人胰腺癌细胞生物学特性的影响[J].中华实验外科杂志,2017,34(12):2158-2161.
9 Haider S,Pollheimer J,Knofler M.Notch signalling in placental development and gestational diseases[J].Placenta,2017,56:65-72.
10易超,依马木买买提江·阿布拉,丁伟,等.Affymetrix基因表达谱芯片技术筛选胰腺癌异常表达基因的研究[J].中国普通外科杂志,2017,26(3):294-303.
11 Yao Y,Ni Y,Zhang J,et al.The role of Notch signaling in gastric carcinoma:molecular pathogenesis and novel therapeutic targets[J].Oncotarget,2017,8(32):53839-53853.
12 Vaish V,Kim J,Shim M.Jagged-2(JAG2)enhances tumorigenicity and chemoresistance of colorectal cancer cells[J].Oncotarget,2017,8(32):53262-53275.
13 Li CY,Miao KL,Chen Y,et al.Jagged2 promotes cancer stem cell properties of triple negative breast cancer cells and paclitaxel resistance via regulating microRNA-200[J].Eur Rev Med Pharmacol Sci,2018,22(18):6008-6014.
14 Hu Y,Su H,Li X,et al.The NOTCH ligand JAGGED2promotes pancreatic cancer metastasis independent of NOTCH signaling activation[J].Mol Cancer Ther,2015,14(1):289-297.
15 Lo JH,Hao L,Muzumdar MD,et al.iRGD-guided tumor-penetrating nanocomplexes for therapeutic siRNA delivery to pancreatic cancer[J].Mol Cancer Ther,2018,17(11):2377-2388.
16 Min HS,Kim HJ,Ahn J,et al.Tuned density of anti-tissue factor antibody fragment onto siRNA-loaded polyion complex micelles for optimizing targetability into pancreatic cancer cells[J].Biomacromolecules,2018,19(6):2320-2329.