hmSD沉默对PC-3细胞增殖迁移影响的体外研究
摘要
目的:构建表达pGCsi-hmSD质粒,转染前列腺癌PC-3细胞,观察沉默hmSD基因对PC-3细胞增殖,迁移能力及MMP-2 mRNA表达的影响,为前列腺癌的基因治疗提供靶向参考。
方法:RT-PCR检测hmSD mRNA在PC-3细胞系的表达;以基因重组技术构建表达pGCsi-hmSD质粒,应用真核细胞转染技术转染前列腺癌PC-3细胞并检测转染效率;PC-3细胞转染pGCsi-hmSD质粒后,RT-PCR检测hmSD mRNA表达的变化,倒置显微镜观察细胞形态的变化,MTT检测其对细胞增殖的影响,流式细胞术检测细胞凋亡的改变;划痕实验和Transwell实验检测细胞迁移的变化,RT-PCR检测转染pGCsi-hmSD质粒PC-3细胞MMP-2 mRNA表达的改变。
结果:1.经限制性酶切鉴定及DNA序列测定证实,成功构建了pGCsi-hmSD质粒;2.PC-3细胞转染效率各组均大于50%,且各组转染效率没有差异。3. PC-3细胞转染pGCsi-hmSD质粒后, RT-PCR检测hmSDmRNA表达水平降低,倒置显微镜下观察细胞数目减少,皱缩,MTT实验结果表明细胞增殖受抑制,流式细胞术结果表明其促进细胞凋亡。4.划痕实验和Transwell实验结果为PC-3细胞转染pGCsi-hmSD质粒后细胞迁移能力降低,RT-PCR结果显示MMP-2 mRNA表达下降。
结论: 1.成功构建了pGCsi-hmSD质粒,可特异性沉默PC-3细胞的hmSD表达。2.hmSD能够促进PC-3细胞的增殖,抑制肿瘤细胞凋亡。3. hmSD-RNAi可降低PC-3细胞的迁移能力,可能与抑制MMP-2 mRNA表达有关。
Prostate cancer is a leading cause of cancer-related deaths in the Western countries. in China, the incidence and mortality rates of prostate cancer have increased markedly, because of the chang of Life style and diet structure.because the effect of traditional chemotherapy, radiotherapy and surgical treatment are not very ideal , a new treatment of tumor gene therapy is thought highly of increasingly.
Sialidase is a kind of glycosidase,which can hydrolyzing sialic acid from non-deoxidized end glycogen and glycolipid.Human sialidases are overexpressed in mang tumors.human membrane-associated membrane (Neu3) is one of sialidases .In some tumors ,With the adjacent non-tumor tissue compared to the expressional level of Neu3 mRNA and protein in the tumor tissue significantly increased. the neu3 expression is higher, the tumor malignancy is higher. Therefore,Targeted silencing of hmSDis a promising approach in cancer control.
Small RNA (siRNAs) are short double-stranded RNA molecules that can target complementary mRNAs for degradation via a cellular process termed RNA interference (RNAi). Tumor is due to the proliferation and differentiation of cells control disorders and the surrounding tissue disorder, including the activation of oncogenes and tumor suppressor gene inactive- ation and apoptosis-related gene expression abnormalities in the process of longevity to promote the growth of tumor growth factor.Therefore, the use of RNAi technology without affecting the normal gene function under the premise inhibit gene mutation or overexpression of expression, so as to achieve the purpose of gene therapy.
Objective:
To investigate the efficacy of hmSD gene silencing in growth inhibition of PC-3 cell and to elucidate the underlying mechanisms. Methods:
1. Plasmid construction
According to the design principles, three suitable target sites against hmSD were selected and the specificities were determined by BLAST. The complementary oligonucleotides were annealed and ligated into the lineared pGCsi-U6/Neo/GFP siRNA expression vector, to construct recombinant plasmids pGCsi-hmSD1 , pGCsi-hmSD2and pGCsi-hmSD3 . Construction of good will have three recombinant plasmids were transformed into JM109 competent bacteria, positive clones selected strains, extracted plasmid, identified by restriction enzyme digestion, and sequenced.
2. In vitro study
(1)The prostate cancer cell line PC-3 were transfected with plasmids pGCsi-hmSD1, pGCsi-hmSD2 and pGCsi-hmSD3 and the efficiency of transfection is Appraiseed. To determine the expression levels of hmSD, the semi-quantitative RT-PCR analysis with the samples extracted from treated and control cells were performed. cell morphology observed by Inverted microscope cell morphology. The cells were also analyzed for cell cycle phase distribution and apoptosis rate by flow cytometry, and detected for the inhibition of cell proliferation by MTT. Cell migration was assayed by Scratch test.besides, Cell migration was assayed using a modified Boydenchamber containing a polycarbonate Transwell membranefilter (Corning Costar, Cambridge, MA, USA) coated with collagen type I. Cells were deposited on the upper chamber in IMDM containing 1% fetal bovine serum (FBS). The lower chamber contained IMDM with 10% (control) FBS. Cells were incubated for 6 h at 37°Cin 5% CO2/95% air. After scraping non-migrated cells from the upper surface of the membrane with a cotton swab, the migrated cells remaining on the bottom surface were stained with 0.5% crystal violet. The stained insert waswashed thoroughly, dissolved with 1% Triton X-100, andabsorbance (O.D.) at 595 nm was measured. To determine the expression levels of MMP-2, the semi-quantitative RT-PCR analysis with the samples extracted from treated and control cells were performed
Results:
1.The plasmids pGCsi-hmSD containing siRNA-hmSD were constr- ucted, which have been confirmed by restriction enzyme digest and sequence analysis.
2. The results from semi-quantitative RT-PCR analysis for the PC-3 cell samples in pGCsi-hmSD1,pGCsi-hmSD2, pGCsi-hmSD3 and pGCsi-scramble groups demonstrated that hmSD siRNA could specifically reduce hmSD mRNA expression. the number of cells are reduced ,Using an inverted microscope observation. The results of MTT demonstrated that inhibition of cell proliferation were present in treated groups. Significant hypodiploidy peaks before G1 phase were present in both treated cell samples in FCM analysis. The results of Scratch test and Transwell migration assay demonstrated that inhibition of cell migration. The results from semi-quantitative RT-PCR analysis demonstrated that the level of MMP-2 mRNA expression reduce in transfect pGCsi-hmSD groups compare withpGCsi-scramble group
Conclusions:
In our studies, hmSD knockdown showed that the expression levels of hmSD is reduced and cell proliferation is inhibited.It maybe the underlying mechanisms that the expression levels of hmSD, Cell migration is inhibited It maybe the underlying mechanisms that the pathway of MMP-2.
方法:RT-PCR检测hmSD mRNA在PC-3细胞系的表达;以基因重组技术构建表达pGCsi-hmSD质粒,应用真核细胞转染技术转染前列腺癌PC-3细胞并检测转染效率;PC-3细胞转染pGCsi-hmSD质粒后,RT-PCR检测hmSD mRNA表达的变化,倒置显微镜观察细胞形态的变化,MTT检测其对细胞增殖的影响,流式细胞术检测细胞凋亡的改变;划痕实验和Transwell实验检测细胞迁移的变化,RT-PCR检测转染pGCsi-hmSD质粒PC-3细胞MMP-2 mRNA表达的改变。
结果:1.经限制性酶切鉴定及DNA序列测定证实,成功构建了pGCsi-hmSD质粒;2.PC-3细胞转染效率各组均大于50%,且各组转染效率没有差异。3. PC-3细胞转染pGCsi-hmSD质粒后, RT-PCR检测hmSDmRNA表达水平降低,倒置显微镜下观察细胞数目减少,皱缩,MTT实验结果表明细胞增殖受抑制,流式细胞术结果表明其促进细胞凋亡。4.划痕实验和Transwell实验结果为PC-3细胞转染pGCsi-hmSD质粒后细胞迁移能力降低,RT-PCR结果显示MMP-2 mRNA表达下降。
结论: 1.成功构建了pGCsi-hmSD质粒,可特异性沉默PC-3细胞的hmSD表达。2.hmSD能够促进PC-3细胞的增殖,抑制肿瘤细胞凋亡。3. hmSD-RNAi可降低PC-3细胞的迁移能力,可能与抑制MMP-2 mRNA表达有关。
Prostate cancer is a leading cause of cancer-related deaths in the Western countries. in China, the incidence and mortality rates of prostate cancer have increased markedly, because of the chang of Life style and diet structure.because the effect of traditional chemotherapy, radiotherapy and surgical treatment are not very ideal , a new treatment of tumor gene therapy is thought highly of increasingly.
Sialidase is a kind of glycosidase,which can hydrolyzing sialic acid from non-deoxidized end glycogen and glycolipid.Human sialidases are overexpressed in mang tumors.human membrane-associated membrane (Neu3) is one of sialidases .In some tumors ,With the adjacent non-tumor tissue compared to the expressional level of Neu3 mRNA and protein in the tumor tissue significantly increased. the neu3 expression is higher, the tumor malignancy is higher. Therefore,Targeted silencing of hmSDis a promising approach in cancer control.
Small RNA (siRNAs) are short double-stranded RNA molecules that can target complementary mRNAs for degradation via a cellular process termed RNA interference (RNAi). Tumor is due to the proliferation and differentiation of cells control disorders and the surrounding tissue disorder, including the activation of oncogenes and tumor suppressor gene inactive- ation and apoptosis-related gene expression abnormalities in the process of longevity to promote the growth of tumor growth factor.Therefore, the use of RNAi technology without affecting the normal gene function under the premise inhibit gene mutation or overexpression of expression, so as to achieve the purpose of gene therapy.
Objective:
To investigate the efficacy of hmSD gene silencing in growth inhibition of PC-3 cell and to elucidate the underlying mechanisms. Methods:
1. Plasmid construction
According to the design principles, three suitable target sites against hmSD were selected and the specificities were determined by BLAST. The complementary oligonucleotides were annealed and ligated into the lineared pGCsi-U6/Neo/GFP siRNA expression vector, to construct recombinant plasmids pGCsi-hmSD1 , pGCsi-hmSD2and pGCsi-hmSD3 . Construction of good will have three recombinant plasmids were transformed into JM109 competent bacteria, positive clones selected strains, extracted plasmid, identified by restriction enzyme digestion, and sequenced.
2. In vitro study
(1)The prostate cancer cell line PC-3 were transfected with plasmids pGCsi-hmSD1, pGCsi-hmSD2 and pGCsi-hmSD3 and the efficiency of transfection is Appraiseed. To determine the expression levels of hmSD, the semi-quantitative RT-PCR analysis with the samples extracted from treated and control cells were performed. cell morphology observed by Inverted microscope cell morphology. The cells were also analyzed for cell cycle phase distribution and apoptosis rate by flow cytometry, and detected for the inhibition of cell proliferation by MTT. Cell migration was assayed by Scratch test.besides, Cell migration was assayed using a modified Boydenchamber containing a polycarbonate Transwell membranefilter (Corning Costar, Cambridge, MA, USA) coated with collagen type I. Cells were deposited on the upper chamber in IMDM containing 1% fetal bovine serum (FBS). The lower chamber contained IMDM with 10% (control) FBS. Cells were incubated for 6 h at 37°Cin 5% CO2/95% air. After scraping non-migrated cells from the upper surface of the membrane with a cotton swab, the migrated cells remaining on the bottom surface were stained with 0.5% crystal violet. The stained insert waswashed thoroughly, dissolved with 1% Triton X-100, andabsorbance (O.D.) at 595 nm was measured. To determine the expression levels of MMP-2, the semi-quantitative RT-PCR analysis with the samples extracted from treated and control cells were performed
Results:
1.The plasmids pGCsi-hmSD containing siRNA-hmSD were constr- ucted, which have been confirmed by restriction enzyme digest and sequence analysis.
2. The results from semi-quantitative RT-PCR analysis for the PC-3 cell samples in pGCsi-hmSD1,pGCsi-hmSD2, pGCsi-hmSD3 and pGCsi-scramble groups demonstrated that hmSD siRNA could specifically reduce hmSD mRNA expression. the number of cells are reduced ,Using an inverted microscope observation. The results of MTT demonstrated that inhibition of cell proliferation were present in treated groups. Significant hypodiploidy peaks before G1 phase were present in both treated cell samples in FCM analysis. The results of Scratch test and Transwell migration assay demonstrated that inhibition of cell migration. The results from semi-quantitative RT-PCR analysis demonstrated that the level of MMP-2 mRNA expression reduce in transfect pGCsi-hmSD groups compare withpGCsi-scramble group
Conclusions:
In our studies, hmSD knockdown showed that the expression levels of hmSD is reduced and cell proliferation is inhibited.It maybe the underlying mechanisms that the expression levels of hmSD, Cell migration is inhibited It maybe the underlying mechanisms that the pathway of MMP-2.
引文
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[2] Morales A, Colell A, Mari, M, et al.Glycosphingolipids and mitochondria:Role in apoptosis and disease.GLYCOCONJUGATE,2003,20 (9):579-588.
[3] Lahdenne Pekka, Heikinheimo Markku.Clinical use of tumor markers in childhood malignancies.Annals of Medicine,2002,34(5):316-323.
[4]Dall'Olio Fabio, Chiricolo Mariella, et al. Sialyltransferases in cancer. Glycoconjugate Journal, 2001,18 (11-12):841-850.
[5] Wang Y,Yamaguchi K,Shimada Y,et al.Site-directed mutage-nesis of human membrane-associated ganglioside sialidase:identification of amino-acid residues contributing to substrate specificity. Eur.J. Bioche,2001,268:2201–2208.
[6] Miyagi T,Kato K,Ueno S,Wada T.Aberrant expression of sialidase cancer. Trends Glycosci.Glycotechnol,2004,16,371–381.
[7] Zanchetti G, Colombi P, Manzoni M, et al. Sialidase NEU3 is a peripheral membrane protein localized on the cell surface and in endosomal structures Biochem J,2007,408(20):211-9
[8] Yan W, Yamaguchi K, Wada T, at el. A Close Association of the Ganglioside- specific Sialidase Neu3 with Caveolin in Membrane Microdomains. The Journal of Biologigal chemistry,2002,277 (29):26252-26259.
[9] Kato K, Shiga K, Yamaguchi K, et al. Plasma-membrane-associated sialidase (NEU3) differentially regulates integrin-mediated cell proliferation through laminin-and fibronectin-derived signalling. Biochemical Journal, 2006 Mar 5, 394 (Pt3): 647-656
[10] Kakugawa Y, Wada T, Yamaguchi K, et al. Up-regulation of plasma membrane- associated ganglioside sialidase (Neu3) in human colon cancer and its involvement in apoptosis suppression.Proc. Natl. Acad. Sci. U.S.A, 2002,99:10718–10723.
[11] Ueno Seiji, Saito Seiichi, Wada Tadashi, et al. Plasma membrane-associated sialidase is up-regulated in renal cell carcinoma and promotes interleukin-6-induced apoptosis suppression and cell motility. J Biol Chem,2006,281 (12):7756-7764.
[12] Pshezhetsky AV, Richard C, Michaud L, et al. Cloning,expression and chromosomal mapping of human lysosomal sialidase and characterizeationation of mutations in sialidosis. Nat Genet,1997 Mar, 15(3):316-320.
[13] Napoli C, Lemieux C, Jorgensen R. Introduction of a chalconesynt hasintoPetunia results in reversible co-suppression of homoogous genes in trans. Plant Cell,1990,2 (4) :279-289.
[14] Romano N, Macino G. Quelling: transient inaction inactivation of gene expression in neurospora crassa by t ransformation with homologus sequences. Mol Microbiol,1992,6 (22) :3343-3353.
[15]Guo S, Kempheus KJ. Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmet rically dist ributed. Cell, 1995, 81 (4) :611-620.
[16] Fire A, Xu S, Montgomery MK,et al. Potent and specific genetic inference by double -stranded RNA in Caenorhabditis elegans. Nature, 1998,391 (6669) :806-811.
[17] Guru T. A silence that speaks volumes. Nature, 2000, 404 (3) :804-808.
[18] Elbashir SM, Harbrt HJ. Analysis of gene fanction in somatic mammalian cells using small interfering RNAS. Methods, 2002, 26 (2) :199-213.
[19] Zhou Hongxia, Xia Xu Gang, Xu Zuoshang. An RNA polymerase III constructsynthesizes short-hairpin RNA with aquantitative indicator and mediates highly efficientRNAi.Nucleic Acids Res, 2005, 33 (6) :e62-e64.
[20] Hannon GJ. RNA interference. Nature, 2002 , 418 ( 6894 ) : 244-251.
[21] Sui G, Soo hoo C, Affar ElB, et al. A DNA vector basedRNAi technology to supress gene expression in mammalian cells. ProcNatl Acad Sci, 2002,99 (8): 5515-5520.
[22] Yang S, Tutton S, Pierce E, et al. Specific double-stranded RNA interference in undifferentiated mouse embryonic stem cell. Mol Cell Biol, 2001,21 (22): 7807 -7817.
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