带Flag标签的Sirt4真核表达载体的构建及功能验证
|
摘要
目的构建带Flag标签pcDNA3.0载体的Sirt4真核表达载体,同时验证该基因与肝癌细胞HepG2生长之间的关系。方法以人卵巢文库为模板,采用PCR技术扩增出Sirt4的CDS区编码序列,将PCR产物插入到带Flag标签的pcDNA3.0载体上,转化大肠杆菌DH5α感受态细胞提取质粒,测序正确后将pcDNA3.0-Flag和pcDNA3.0-Flag-Sirt4分别转染肝癌细胞HepG2,Western印迹实验鉴定目的基因蛋白表达,CCK-8法、克隆形成实验测定其对肝癌细胞HepG2生长的影响,Transwell证明Sirt4对肝癌细胞HepG2体外侵袭能力的影响。结果双酶切结果显示,pcDNA3.0-Flag-Sirt4真核表达载体构建成功;Western印迹实验验证该基因蛋白成功表达;CCK-8法和克隆形成实验证明过表达Sirt4基因抑制HepG2细胞增殖,Transwell证明过表达Sirt4明显抑制HepG2细胞体外侵袭能力。结论pcDNA3.0-Flag-Sirt4真核表达载体构建成功,过表达Sirt4基因可抑制肝癌细胞增殖及体外侵袭能力,为进一步研究Sirt4在肝癌中的发生与发展奠定了基础。
Objective To construct the eukaryotic expression vector of Flag tagged pcDNA3.0 vector Sirt4 while verify-ing the effect of overexpression of the gene on proliferation in hepatocellular carcinoma HepG2 cells. Methods The CDSregion coding sequence of Sirt4 was amplified from the human ovarian library using polymerase chain reaction(PCR) technique. Flag tagged pcDNA3.0 vector was inserted into the PCR product by double digestion. The plasmid was transformedinto DH-5 alpha for extraction. After sequencing was correct,pcDNA3.0-Flag and pcDNA3.0-Flag-Sirt4 were transfectedinto HepG2 cells respectively. Western blotting was used to identify the expression of the target gene protein. CCK-8 assay andclony formation assay were used to determine the effect of Sirt4 on the growth of HepG2 cells. Transwell demonstratedthe effect of Sirt4 on the invasion ability of HepG2 cells in vitro. Results The results of double digestion showedthat pcDNA3.0-Flag-Sirt4 eukaryotic expression vector was successfully constructed.Western blotting confirmed thesuccessful expression of the gene protein.CCK-8 method and clone formation experiments proved that overexpression ofpcDNA3.0-Flag-Sirt4 inhibited the proliferation of HepG2 cells.Transwell suggested that overexpression of pcDNA3.0-Flag-Sirt4 suppressed the invasion ability of HepG2 cells in vitro. Conclusion The successful construction of pcDNA3.0-Flag-Sirt4 eukaryotic expression vector proves that it can promote the proliferation of hepatocellular carcinoma(HCC)cells,which can contribute to further study of the occurrence and development of Sirt4 in HCC.
Objective To construct the eukaryotic expression vector of Flag tagged pcDNA3.0 vector Sirt4 while verify-ing the effect of overexpression of the gene on proliferation in hepatocellular carcinoma HepG2 cells. Methods The CDSregion coding sequence of Sirt4 was amplified from the human ovarian library using polymerase chain reaction(PCR) technique. Flag tagged pcDNA3.0 vector was inserted into the PCR product by double digestion. The plasmid was transformedinto DH-5 alpha for extraction. After sequencing was correct,pcDNA3.0-Flag and pcDNA3.0-Flag-Sirt4 were transfectedinto HepG2 cells respectively. Western blotting was used to identify the expression of the target gene protein. CCK-8 assay andclony formation assay were used to determine the effect of Sirt4 on the growth of HepG2 cells. Transwell demonstratedthe effect of Sirt4 on the invasion ability of HepG2 cells in vitro. Results The results of double digestion showedthat pcDNA3.0-Flag-Sirt4 eukaryotic expression vector was successfully constructed.Western blotting confirmed thesuccessful expression of the gene protein.CCK-8 method and clone formation experiments proved that overexpression ofpcDNA3.0-Flag-Sirt4 inhibited the proliferation of HepG2 cells.Transwell suggested that overexpression of pcDNA3.0-Flag-Sirt4 suppressed the invasion ability of HepG2 cells in vitro. Conclusion The successful construction of pcDNA3.0-Flag-Sirt4 eukaryotic expression vector proves that it can promote the proliferation of hepatocellular carcinoma(HCC)cells,which can contribute to further study of the occurrence and development of Sirt4 in HCC.
引文
[1] Mouchiroud L,Houtkooper R,Moullan N,et al. The NAD(+)/sirtuin pathway modulates longevity through activation of mito-chondrial UPR and FOXO signaling[J]. Cell,2013,154(2):430-441.
[2] Carafa V,Rotili D,Forgione M,et al. Sirtuin functions and modulation:from chemistry to the clinic[J]. Clin Epigenet,2016,8:61.
[3]宋春丽,任吉华,冉龙宽,等.SIRT2沉默对肝癌细胞的迁移和侵袭能力的影响[J].第三军医大学学报,2013,35(24):2634-2638.
[4] Jemal A,Bray F,Center M,et al. Global cancer statistics[J].CA Cancer J Clin,2011,61(2):69-90.
[5] Yuan H,Su L,Chen W. The emerging and diverse roles of sirtuins in cancer:a clinical perspective[J]. Onco Targets Ther,2013,6:1399-1416.
[6] Chae J,Nohs J,Kwon K,et al. Expression of DBC1 and SIRT1 is associated with poor prognosis of gastric carcinoma[J]. Clin Cancer Res,2009,15(13):4453-4459.
[7] Stunkel W,Peh B,Tan Y,et al. Function of the SIRT1 protein deacetylase in cancer[J]. Biotechnol J,2007,2(11):1360-1368.
[8] Huffman D,Grizzle W,Bamman M,et al. SIRT1 is significantly elevated in mouse and human prostate cancer[J]. Cancer Res,2007,67(14):6612-6618.
[9] Hida Y,Kubo Y,Murao K,et al. Strong expression of a longevity-related protein,SIRT1,in Bowen′s disease[J]. Arch Dermatol Res,2007,299(2):103-106.
[10] Wang R,Sengupta K,Li C,et al. Impaired DNA damage response,genome instability,and tumorigenesis in SIRT1 mutant mice[J]. Cancer Cell,2008,14(4):312-323.
[11] Zhu Y,Wang G,Li X,et al. Knockout of SIRT4 decreases chemosensitivity to 5-FU in colorectal cancer cells[J]. Oncol Lett,2018,16(2):1675-1681.
[12] Wang Y,Guo Y,Gao J,et al. Tumor-suppressive function of SIRT4 in neuroblastoma through mitochondrial damage[J].Cancer Manag Res,2018,9(10):5591-5603.
[13] Sun H,Huang D,Liu G,et al. SIRT4 acts as a tumor suppressor in gastric cancer by inhibiting cell proliferation,migration,and invasion[J]. Onco Targets Ther,2018,10(11):3959-3968.
[14] Fu L,Dong Q,He J,et al. SIRT4 inhibits malignancy progression of NSCLCs,through mitochondrial dynamics mediated by the ERK-Drp1 pathway[J]. Oncogene,2017,36(19):2724-2736.
[15] Huang G,Zhu G. Sirtuin-4(SIRT4),a therapeutic target with oncogenic and tumor-suppressive activity in cancer[J]. Onco Targets Ther,2018,11(11):3395-3400.
[2] Carafa V,Rotili D,Forgione M,et al. Sirtuin functions and modulation:from chemistry to the clinic[J]. Clin Epigenet,2016,8:61.
[3]宋春丽,任吉华,冉龙宽,等.SIRT2沉默对肝癌细胞的迁移和侵袭能力的影响[J].第三军医大学学报,2013,35(24):2634-2638.
[4] Jemal A,Bray F,Center M,et al. Global cancer statistics[J].CA Cancer J Clin,2011,61(2):69-90.
[5] Yuan H,Su L,Chen W. The emerging and diverse roles of sirtuins in cancer:a clinical perspective[J]. Onco Targets Ther,2013,6:1399-1416.
[6] Chae J,Nohs J,Kwon K,et al. Expression of DBC1 and SIRT1 is associated with poor prognosis of gastric carcinoma[J]. Clin Cancer Res,2009,15(13):4453-4459.
[7] Stunkel W,Peh B,Tan Y,et al. Function of the SIRT1 protein deacetylase in cancer[J]. Biotechnol J,2007,2(11):1360-1368.
[8] Huffman D,Grizzle W,Bamman M,et al. SIRT1 is significantly elevated in mouse and human prostate cancer[J]. Cancer Res,2007,67(14):6612-6618.
[9] Hida Y,Kubo Y,Murao K,et al. Strong expression of a longevity-related protein,SIRT1,in Bowen′s disease[J]. Arch Dermatol Res,2007,299(2):103-106.
[10] Wang R,Sengupta K,Li C,et al. Impaired DNA damage response,genome instability,and tumorigenesis in SIRT1 mutant mice[J]. Cancer Cell,2008,14(4):312-323.
[11] Zhu Y,Wang G,Li X,et al. Knockout of SIRT4 decreases chemosensitivity to 5-FU in colorectal cancer cells[J]. Oncol Lett,2018,16(2):1675-1681.
[12] Wang Y,Guo Y,Gao J,et al. Tumor-suppressive function of SIRT4 in neuroblastoma through mitochondrial damage[J].Cancer Manag Res,2018,9(10):5591-5603.
[13] Sun H,Huang D,Liu G,et al. SIRT4 acts as a tumor suppressor in gastric cancer by inhibiting cell proliferation,migration,and invasion[J]. Onco Targets Ther,2018,10(11):3959-3968.
[14] Fu L,Dong Q,He J,et al. SIRT4 inhibits malignancy progression of NSCLCs,through mitochondrial dynamics mediated by the ERK-Drp1 pathway[J]. Oncogene,2017,36(19):2724-2736.
[15] Huang G,Zhu G. Sirtuin-4(SIRT4),a therapeutic target with oncogenic and tumor-suppressive activity in cancer[J]. Onco Targets Ther,2018,11(11):3395-3400.