PHLPP2基因小干扰RNA慢病毒载体的构建及对舌癌细胞Tca8113增殖的影响
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摘要
目的构建稳定表达PHLPP2小发夹RNA(shRNA)的Tca8113细胞系,研究敲低PHLPP2对Akt Ser473磷酸化水平和Tca8113细胞生长的影响。方法利用RNA干扰(RNAi)技术,设计并合成针对PHLPP2基因的shRNA,并将其克隆到shRNA表达载体PSIH-H1上。经测序成功后,包装病毒并感染舌癌细胞Tca8113,建立敲低PHLPP2的稳定细胞株。通过实时定量PCR(qRT-PCR)和Western印迹实验检测RNAi的干扰效果。利用Western印迹实验和细胞生长曲线检测敲低PHLPP2对Akt Ser473磷酸化及细胞生长的影响。结果经测序证明,成功构建PHLPP2 shRNA真核表达载体。qRT-PCR和Western印迹实验证明,构建的shRNA能有效抑制PHLPP2的表达,并构建敲低PHLPP2基因的Tca8113稳定细胞系。实验表明,敲低PHLPP2可促进细胞的生长并升高Akt Ser473的磷酸化水平。结论成功构建PHLPP2基因的shRNA真核表达载体,转染细胞后能有效抑制PHLPP2基因的表达,且能升高Akt Ser473的磷酸化水平并促进细胞的生长,为进一步研究PHLPP2在舌癌中的功能及机制奠定了基础。
Objective To construct the lentiviral vector for PHLPP2 small-hairpin RNA( shRNA) and to detect its effect on tongue cancer Tca8113 cell growth and the phosphorylation level of Akt Ser473. Methods PHLPP2 shRNA was designed and constructed for the PSIH-H1 lentiviral vector. After sequencing,PSIH-H1-PHLPP2 was packaged to lentivirus in 293 T cells. After being infected with lentivirus and selected by puromycin,mixed Tca8113 colonies stably expressing PHLPP2 shRNA were obtained and the fusion PHLPP2 expression was detected by quantitative real-time PCR( qRT-PCR) and Western blotting. Finally,the effect of PHLPP2 shRNA on Tca8113 growth was determined by cell growth assay. The effect of knock-down of PHLPP2 on the phosphorylation of Akt Ser473 was determined by Western blotting. Results qRT-PCR and Western blotting showed that shRNA could suppress the PHLPP2 gene expression,which,in turn,could promote the growth of Tca8113 cells. Knock-down of PHLPP2 could markedly promote the phosphorylation of Akt Ser473. Conclusion The lentivirus-mediated PHLPP2 shRNA is obtained, which will contribute to further research on the function and mechanism of PHLPP2 in tongue cancer.
Objective To construct the lentiviral vector for PHLPP2 small-hairpin RNA( shRNA) and to detect its effect on tongue cancer Tca8113 cell growth and the phosphorylation level of Akt Ser473. Methods PHLPP2 shRNA was designed and constructed for the PSIH-H1 lentiviral vector. After sequencing,PSIH-H1-PHLPP2 was packaged to lentivirus in 293 T cells. After being infected with lentivirus and selected by puromycin,mixed Tca8113 colonies stably expressing PHLPP2 shRNA were obtained and the fusion PHLPP2 expression was detected by quantitative real-time PCR( qRT-PCR) and Western blotting. Finally,the effect of PHLPP2 shRNA on Tca8113 growth was determined by cell growth assay. The effect of knock-down of PHLPP2 on the phosphorylation of Akt Ser473 was determined by Western blotting. Results qRT-PCR and Western blotting showed that shRNA could suppress the PHLPP2 gene expression,which,in turn,could promote the growth of Tca8113 cells. Knock-down of PHLPP2 could markedly promote the phosphorylation of Akt Ser473. Conclusion The lentivirus-mediated PHLPP2 shRNA is obtained, which will contribute to further research on the function and mechanism of PHLPP2 in tongue cancer.
引文
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[10]衣素琴,庄园,国峰.PHLPP1与肿瘤的关系及研究[J].中华临床医师杂志,2013,7(5):95-98.
[11]Qiao M,Iglehart JD,Pardee AB.Metastatic potential of 21T human breast cancer cells depends on Akt/protein kinase B activation[J].Cancer Res,2007,67(11):5293-5299.
[12]Park WS,Heo WD,Whalen JH,et al.Comprehensive identification of PIP3-regulated PH domains from C.elegans to H.sapiens by model prediction and live imaging[J].Mol Cell,2008,30(3):381-392.
[2]Wade J,Smith H,Hankins M,et al.Conducting oral examinations for cancer in general practice:what are the barriers[J]?Fam Pract,2010,27(1):77-84.
[3]任振虎.舌癌手术治疗的进展[J].实用口腔医学杂志,2014,30(1):110-114.
[4]朱海艳.舌癌患者术后语音康复训练的护理[J].口腔医学,2013,(3):207-208.
[5]Gao T,Furnari F,Newton AC,et al.PHLPP:A phosphatase that directly dephosphorylates Akt,promotes apoptosis,and suppresses tumor growth[J].Mol Cell,2005,18(1):13-24.
[6]Brognard J,Sierecki E,Gao T,et al.PHLPP And a second isoform,PHLLPP2,differentially attenuate the amplitude of Akt signaling by regulating distinct Akt isoforms[J].Mol Cell,2007,25(6):917-931.
[7]Wang Z,Shu H,Wang Z,et al.Loss expression of PHLLPP1 correlates with lymph node metastasis and exhibits a poor prognosis in patients with gastric cancer[J].J Surg Onlol,2013,108(7):427-432.
[8]Liu J,Weiss HL,Rychahou P,et al.Loss of PHLPP expression in colon cancer:role in proliferation and tumorigenesis[J].Oncogene,2009,28(7):994-1004.
[9]Qiao M,Wang Y,Xu X,et al.Mstl Is an interacting protein that mediates PHLPPs'induced apoptosis[J].Mol Cell,2010,38(4):512-523.
[10]衣素琴,庄园,国峰.PHLPP1与肿瘤的关系及研究[J].中华临床医师杂志,2013,7(5):95-98.
[11]Qiao M,Iglehart JD,Pardee AB.Metastatic potential of 21T human breast cancer cells depends on Akt/protein kinase B activation[J].Cancer Res,2007,67(11):5293-5299.
[12]Park WS,Heo WD,Whalen JH,et al.Comprehensive identification of PIP3-regulated PH domains from C.elegans to H.sapiens by model prediction and live imaging[J].Mol Cell,2008,30(3):381-392.