|Table of Contents|

The effects of silencing of POLE2 gene expression on proliferation of non-small cell lung cancer cells

Journal Of Modern Oncology[ISSN:1672-4992/CN:61-1415/R]

Issue:
2019 01
Page:
35-40
Research Field:
Publishing date:

Info

Title:
The effects of silencing of POLE2 gene expression on proliferation of non-small cell lung cancer cells
Author(s):
Li Namiao1; 2; Zhang Yingying1; 2; Fan Yali1; 2; Zhao Yuling1; 2; Dong Ya1; 2; Bai Jie1; Li Jianying1
1.Department of Respairtory Disease，Xi'an Central Hospital Affiliated to Medical Colleage of Xi'an Jiaotong University，Shaanxi Xi'an 710003，China；2.Medical College of Yan'an University，Shaanxi Yan'an 716000，China.
Keywords:
lentivirus infections; genes; non-small cell lung cancer; RNA interference（RNAi）
PACS:
R734.2
DOI:
10.3969/j.issn.1672-4992.2019.01.009
Abstract:
Objective：To study the function of DNA polymerase εB subunit(POLE2) in non-small cell lung cancer(NSCLC).Methods：RT-PCR technique was used to detect the expression of POLE2 gene in A549，NCI-H1975 and NCI-H1299 cells.First，a single strand of oligonucleotides for POLE2 siRNA target sequences was designed and synthesized，and two stranded DNA was formed by annealing，which produces GV115-shPOLE2 lentivirus vector with the GV115 vector after AgeI and EcoRI enzyme digestion.The vector contains green fluorescent protein(GFR)，and the positive clones are screened and sequenced by polymerase chain reaction(PCR).It was cotransfected by GV115-shPOLE2 along with Helper1.0 and Helper2.0 into 293T cells to package lentivirus particles.At the same time，the packed virus infected non-small cell lung cancer cell(A549)，the level of FIAG protein on 5 d after infection Wag detected by Western blot to screen the target of POLE2.The cell function experiment was carried out：The effect of POLE2 knockout on cell proliferation ability was detected by CCK-8 method.The effect of POLE2 gene knockout on cell clone formation ability was detected by clone formation assay.The effect of POLE2 gene knockout on apoptosis was detected by Casepase3/7 assay.Results：RT-PCR results showed that the expression abundance of POLE2 gene in three cell lines was highly expressed.The results of PCR and sequencing show that the GV115-shPOLE2 lentivirus vector was constructed correctly.The Western blot method proved that POLE2 siRNA had obvious knockout effect on the heterogenous expression of POLE2，which was an effective target.The proliferation of A549 cells was significantly slowed down，the number of cell clones decreased，and the number of apoptotic cells increased.Compared with those without A549 infection，the difference was statistically significant(P<0.05).Conclusion：Lentiviral vector-mediated siRNA technique effectively inhibits the expression of POLE2，induces the NSCLC cell apoptosis and inhibits the tumor growth.Elucidation of the precise role of POLE2 in these pathways may lead to new targeted therapies for non-small eell lung cancer.

References:

[1]Chen WQ，Zheng RS，Baade PD，et al.Cancer statistics in China，2015［J］.CA Cancer J Clin，2016，66(2)：115-132.
[2]Huang Q，Zhang S.Role of POLE mutations in the occurrence and development of tumors［J］.J Clin Pathol Res，2015，35（12）：2173-2179.［黄茜，张声.POLE突变在肿瘤发生发展中的作用［J］.临床与病理杂志，2015，35（12）：2173-2179.］
[3]Briggs S，Tomlinson I.Germline and somatic polymerase μ and ' mutations define a new class of hypermutated colorectal and endometrial cancers［J］.Journal of Pathology，2013，230(2)：148-153.
[4]Frugoni F，Dobbs K，Felgentreff K，et al.A novel mutation in the POLE2 gene causing combined immunodeficiency［J］.J Allergy Clin Immunol，2016，137(2)：635-638.
[5]Daniel C，Peter B，Dobbins SE，et al.Rare disruptive mutations and their contribution to the heritable risk of colorectal cancer［J］.Nature Communications，2016，7：11883.
[6]Spier I，Holzapfel S，Altmüller J，et al.Frequency and phenotypic spectrum of germline mutations in POLE and seven other polymerase genes in 266 patients with colorectal adenomas and carcinomas［J］.International Journal of Cancer Journal International Du Cancer，2015，137(2)：320-331.
[7]Zekri AR，Hassan ZK，Bahnassy AA，et al.Differentially expressed genes in metastatic advanced egyptian bladder cancer［J］.Asian Pacific Journal of Cancer Prevention，2015，16(8)：3543-3549.
[8]Tiscornia G.Design and cloning of lentiviral vectors expressing small interfering RNAs［J］.Nature Protocols，2006，1(1)：234-240.
[9]Stewart SA，Dykxhnom DM,Palliser D，et al.Lentivirus-delivered stable gene silencing by RNAi in primary ceUs［J］.RNA，2003，9：493-501.
[10]Pi W，Yu CH，Liu ZB.The effect of lentiviral expression system-mediated RNAi technology targeting silencing Bi-1 gene on biological characteristics of nasopharyngeal carcinoma cells［J］.Chinese Gerontology，2017，37(3)：555-557.［皮文，于长华，刘志标.慢病毒表达系统介导的RNAi技术靶向沉默Bi-1基因对鼻咽癌细胞生物学特性的影响［J］.中国老年学，2017，37(3)：555-557.］
[11]Meng FR，Cheng C，Wan HS，et al.Lentiviral vector and its research progress［J］.Chinese Journal of Lung Cancer，2014，17(12)：870-876.［孟凡荣，陈琛，万海粟，等.慢病毒载体及其研究进展［J］.中国肺癌杂志，2014，17(12)：870-876.］
[12]Liu D，Zhang XX，Xi BX，et al.Sine oculis homeobox homolog 1 promotes DNA replication and cell proliferation in cervical cancer［J］.International Journal of Oncology，2014，45(3)：1232.
[13]Hartmann E，Fernàndez V，Moreno V，et al.Five-gene model to predict survival in mantle-cell lymphoma using frozen or formalin-fixed，paraffin-embedded tissue［J］.Journal of Clinical Oncology Official Journal of the American Society of Clinical Oncology，2008，26(30)：4966-4972.
[14]Jokela M，Mkiniemi M，Lehtonen S，et al.The small subunits of human and mouse DNA polymerase ε are homologous to the second largest subunit of the yeast Saccharomyces cerevisiae DNA polymerase ε［J］.Nucleic Acids Research，1998，26(3)：730-734.
[15]Huang D，Jokela M，Tuusa J，et al.E2F mediates induction of the Sp1-controlled promoter of the human DNA polymerase ε B-subunit gene POLE2［J］.Nucleic Acids Research，2001，29(13)：2810-2821.

Memo

Memo:
陕西省中医药管理局面上项目（编号：JCMS062）；西安市科技攻关项目［编号：2016047SF/YX03（3）］
Last Update: 2018-11-30