切换至 "中华医学电子期刊资源库"

中华肺部疾病杂志(电子版) ›› 2025, Vol. 18 ›› Issue (01) : 1 -7. doi: 10.3877/cma.j.issn.1674-6902.2025.01.001

论著

过表达KLF7 对非小细胞肺癌细胞增殖凋亡的影响
吕巧1, 闵迁1, 姜露1, 陈黔1, 彭锦1, 代黔1   
  1. 1. 400037 重庆,陆军军医大学第二附属医院临床医学研究中心
  • 收稿日期:2024-11-08 出版日期:2025-02-25
  • 通信作者: 代黔
  • 基金资助:
    重庆市自然科学基金面上项目(CSTB2023NSCQ-MSX0417)

Effect of overexpression of KLF7 on proliferation and apoptosis of human non-small cell lung cancer cells

Qiao Lv1, Qian Min1, Lu Jiang1, Qian Chen1, Jin Peng1, Qian Dai1   

  1. 1. Clinical Research Center,The Second Affiliated Hospital, Army Medical University, Chongqing 400037, China
  • Received:2024-11-08 Published:2025-02-25
  • Corresponding author: Qian Dai
引用本文:

吕巧, 闵迁, 姜露, 陈黔, 彭锦, 代黔. 过表达KLF7 对非小细胞肺癌细胞增殖凋亡的影响[J/OL]. 中华肺部疾病杂志(电子版), 2025, 18(01): 1-7.

Qiao Lv, Qian Min, Lu Jiang, Qian Chen, Jin Peng, Qian Dai. Effect of overexpression of KLF7 on proliferation and apoptosis of human non-small cell lung cancer cells[J/OL]. Chinese Journal of Lung Diseases(Electronic Edition), 2025, 18(01): 1-7.

目的

通过构建Krüppel 样转录因子7(krüppel-like factor 7,KLF7)真核表达载体,分析KLF7 对肺癌细胞增殖、凋亡的调控作用,为发现非小细胞肺癌(non-small-cell lung cancer,NSCLC)治疗靶点提供新的线索。

方法

通过聚合酶链反应(polymerase chain reaction,PCR)方法克隆KLF7 完整序列,酶切、连接、测序等试验方法构建过表达载体并验证表达效率。 将A594 细胞随机分空载实验为对照组和KLF7 过表达为KLF7 组,采用流式细胞术检测A594 细胞周期、凋亡和活性氧变化,EdU 检测A594细胞增殖情况,实时荧光定量PCR 及蛋白质印迹法检测增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、G1/S-特异性周期蛋白-D1(cyclin D1)、B-细胞淋巴瘤-2(B-cell lymphoma-2,Bcl-2) 、Bcl-2 相关X 蛋白(Bcl-2 -associated X protein,Bax) mRNA 和蛋白表达水平。

结果

克隆KLF7 CDS 区全长693bp,并连接在pCDNA3.1 载体上,构建KLF7 过表达载体,提高HEK293T 及A594 细胞中KLF7 的表达水平(P<0.01)。 与对照组相比,KLF7 组在G1 期细胞百分比降低,G2 期细胞百分比升高,增殖指数(proliferation index,PI)明显升高,同时EdU 阳性细胞比例升高(P<0.01)。 与对照组比较,KLF7 组活性氧水平受到抑制,细胞凋亡率降低(P<0.01)。 与对照组比较,KLF7 组PCNA、CyclinD1、Bcl-2 mRNA相对表达和蛋白表达水平升高,Bax 的mRNA 相对表达和蛋白表达水平降低(P<0.01)。

结论

本文成功构建KLF7 过表达载体,肺癌细胞中,KLF7 过表达能促进细胞增殖并抑制细胞凋亡,参与肺癌发生,有望成为新的NSCLC 生物标志物。

Objective

To investigate the regulation of KLF7 on the proliferation and apoptosis of lung cancer cells by constructing the KLF7 eukaryotic expression vector,thereby providing novel clues for the discovery of therapeutic targets for non-small cell lung cancer(NSCLC).

Methods

The entire sequence of KLF7 was cloned through PCR,and the overexpression vector was constructed by means of enzyme digestion,ligation,and sequencing,with the expression efficiency verified.A594 cells were randomly divided into the experimental control group (control group) and the KLF7 overexpression group (KLF7 group).Changes in the cell cycle,apoptosis,and reactive oxygen species of A594 cells were detected using flow cytometry,and the proliferation of A594 cells was examined by EdU.The expression levels of Proliferating Cell Nuclear Antigen(PCNA),G1/S-specific cyclin-D1 (CyclinD1),B-cell lymphoma-2 (Bcl-2,Bcl-2),Bcl-2-associated X protein (Bax) mRNA and protein were detected by real-time quantitative PCR and Western blotting.

Results

The total length of the CDS region of KLF7 was cloned to 693 bp and linked to the pCDNA3.1 vector to construct the KLF7 overexpression vector,which could significantly enhance the expression level of KLF7 in HEK293T and A594 cells (P<0.01).Compared with the control group,the percentage of cells in the G1 phase decreased,the percentage of cells in the G2 phase increased,the proliferation index (PI) significantly increased,and the percentage of EdU-positive cells significantly increased in the KLF7 overexpression group(P<0.01).Compared with the control group,the ROS level was inhibited and the apoptosis rate decreased in the KLF7 overexpression group (P<0.01).Compared with the control group,the relative expression levels of PCNA,CyclinD1,and Bcl-2 mRNA and protein increased in the KLF7 overexpression group,while the relative expression levels of Bax mRNA and protein decreased (P <0.01).

Conclusion

In this study,the KLF7 overexpression vector was successfully constructed.In lung cancer cells,KLF7 overexpression can significantly promote cell proliferation and inhibit cell apoptosis,and participate in the occurrence of lung cancer,which may serve as a new biomarker of NSCLC.

表1 引物信息
图1 KLF7 克隆、载体构建与表达效率印证 A:KLF7 过表达载体构建;B:KLF7 在293T 细胞过表达效率检测;C:KLF7在A594 细胞过表达效率检测 注:M1 为DL2000 DNA Marker; 1 为KLF7 PCR 扩增产物;M2 为DL15000 DNA Marker;M3 为Supercoiled DNA Ladder Marker;2 为pcDNA3.1 酶切产物;3 为pcDNA3.1;4 为KLF7过表达载体。 *与对照组相比,差异显著(P<0.05);**为差异极显著(P<0.01)
图2 KLF7 对A594 细胞增殖的影响 A:流式细胞仪测定细胞周期的分布及细胞增殖指数(S+G2/G1+G2+S);B:EdU 检测细胞增殖比例 注:KLF7 促进A594 细胞增殖。 分别用空载与KLF7 转染A594 细胞
图3 KLF7 对A594 细胞周期的影响 qPCR 或Western blot方法检测细胞增殖相关基因(PCNA 和CyclinD1)mRNA(A)和蛋白(B)的表达 注:KLF7 抑制A594 细胞凋亡。 分别用空载与KLF7 转染A594 细胞
图4 KLF7 对A594 细胞凋亡的影响 流式细胞仪检测细胞活性氧(A)与凋亡(B) 注:分别用空载与KLF7 转染A594 细胞
图5 KLF7 对A594 细胞凋亡的影响 qPCR 与Western blot检测细胞促凋亡基因(BAX)和抑凋亡基因(Bcl-2) mRNA(A)与蛋白(B)的表达 注:分别用空载与KLF7 转染A594 细胞
1
Bray F,Ferlay J,Soerjomataram I,et al.Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J].CA Cancer J Clin,2018,68(6): 394-424.
2
Bray F,Laversanne M,Sung H,et al.Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J].CA Cancer J Clin,2024,74(3): 229-263.
3
Julian RM,Ping Y,Stephen DC,et al.Non-small cell lung cancer:epidemiology,risk factors,treatment,and survivorship[J].Mayo Clin Proc,2008,83(5): 584-594.
4
Chu QS.Targeting non-small cell lung cancer:driver mutation beyond epidermal growth factor mutation and anaplastic lymphoma kinase fusion[J].Ther Adv Med Oncol,2020,12: 1758835919895756.
5
Melosky B,Wheatley-Price P,Juergens RA,et al.The rapidly evolving landscape of novel targeted therapies in advanced non-small cell lung cancer[J].Lung Cancer,2021,160: 136-151.
6
Fan YB,Lu HC,Liang WY ,et al.Krüppel-like factors and vascular wall homeostasis[J].J Mol Cell Biol,2017,9(5): 352-363.
7
McConnell BB,Yang VW.Mammalian Krüppel-like factors in health and diseases[J].Physiol Rev,2010,90(4): 1337-1381.
8
Yang M,Kim HS,Cho MY.Different methylation profiles between intestinal and diffuse sporadic gastric carcinogenesis[J].Clin Res Hepatol Gastroenterol,2014,38(5): 613-620.
9
Mattei AL,Bailly N,Meissner A.DNA methylation: a historical perspective[J].Trends Genet,2022,38(7): 676-707.
10
Wu ZH,Zhong Y,Zhou T,et al.miRNA biomarkers for predicting overall survival outcomes for head and neck squamous cell carcinoma[J].Genomics,2021,113(1 Pt 1): 135-141.
11
Ding X,Wang X,Gong Y,et al.KLF7 overexpression in human oral squamous cell carcinoma promotes migration and epithelialmesenchymal transition[J].Oncol Lett,2017,13(4):2281-2289.
12
Jiang Z,Yu T,Fan Z,et al.Krüppel-Like Factor 7 is a Marker of Aggressive Gastric Cancer and Poor Prognosis[J].Cell Physiol Biochem,2017,43(3): 1090-1099.
13
Yang J,Xie K,Wang Z,et al.Elevated KLF7 levels may serve as a prognostic signature and might contribute to progression of squamous carcinoma[J].FEBS Open Bio,2020,10(8): 1577-1586.
14
Niu RG,Tang Yl,Xi YF,et al.High Expression of Krüppel-like Factor 7 Indicates Unfavorable Clinical Outcomes in Patients with Lung Adenocarcinoma[J].J Sur Res,2020,250: 216-223.
15
Narjust D Rafael SD,Molina JR.Non-small cell lung cancer epidemiology,risk factors,treatment,and survivorship[J].Mayo Clin Pro,2019,94(8): 1623-1640.
16
吴国明,钱桂生.非小细胞肺癌靶向治疗研究进展及新理念[J/CD].中华肺部疾病杂志(电子版),2019,12(4): 405-408.
17
汪 艳,孙美玲,闵凌峰.基于TCGA 数据库肺腺癌铁死亡相关基因CA9 的鉴定[J/CD].中华肺部疾病杂志(电子版),2024,17(6): 888-894.
18
Kelsey CR,Marks LB,Hollis D,et al.Local recurrence after surgery for early stage lung cancer[J].Cancer,2009,115(22):5218-5227.
19
Wang X,Wang X,Hui B,et al.Efficacy of neoadjuvant chemoimmunotherapy in non-small cell lung cancer: a real-world,multicenter,retrospective study [J].Transl Lung Cancer Res,2024,13(4): 849-860.
20
Gourcerol D,Scherpereel A,Debeugny S,et al.Relevance of an extensive follow-up after surgery for nonsmall cell lung cancer[J].Eur Respir J,2013,42(5): 1357-1364.
21
The Lancet.GLOBOCAN 2018: counting the toll of cancer[J].The Lancet,2018,392(10152):985.
22
Joanna K,COOK T,Urrutia R.Sp1- and Kruppel-like transcription[J].Genome Biol,2003,53(2): 149-159.
23
Zhang Z,Wu C,Lin T,et al.Loss of the third CH zinc finger of chicken KLF7 affects its transcriptional regulation activities in adipose tissue[J].Acta Biochim Biophy Sin (Shanghai),2019,52(1): 84-90.
24
Tripathi R,Rath G,Jawanjal P,et al.Cyclin D1 protein affecting global women’s health by regulating HPV mediated adenocarcinoma of the uterine cervix[J].Sci Rep,2019,9(1): 5019.
25
Wangefjord S,Manjer J,Gaber A,et al.Cyclin D1 expression in colorectal cancer is a favorable prognostic factor in men but not in women in a prospective,population-based cohort study[J].Biol Sex Differ,2011,2: 10.
26
Garces S,Medeiros LJ,Marques-Piubelli ML,et al.Cyclin D1 expression in Rosai-Dorfman disease: a near-constant finding that is not invariably associated with mitogen-activated protein kinase/extracellular signal-regulated kinase pathway activation[J].Hum Pathol,2022,121: 36-45.
27
GonzÁlez-MagaÑa A,Blanco FJ.Human PCNA Structure,Function and Interactions[J].Biomolecules,2020,10(4): 570.
28
Cersosimo RJ.Cyclin-dependent kinase 4/6 inhibitors for the management of advanced or metastatic breast cancer in women[J].Am J Health Syst Pharm,2019,76(16): 1183-1202.
29
Al-Maghrabi J,Mufti S,Gomaa W,et al.Immunoexpression of cyclin D1 in colorectal carcinomas is not correlated with survival outcome[J].J Microsc Ultrastruct,2015,3(2): 62-67.
30
Tchakarska G,Sola B.The double dealing of cyclin D1[J].Cell Cycle,2019,19(2): 163-178.
31
RouÉ G,Pichereau V,Lincet H,et al.Cyclin D1 mediates resistance to apoptosis through upregulation of molecular chaperones and consequent redistribution of cell death regulators[J].Oncogene,2008,27(36): 4909-4920.
32
Beltran E,Fresquet V,Martinez-Useros J,et al.A cyclin-D1 interaction with BAX underlies its oncogenic role and potential as a therapeutic target in mantle cell lymphoma[J].Pro Natl Acad Sci USA,2011,108(30): 12461-12466.
33
De Donato M,Babini G,Mozzetti S,et al.KLF7: a new candidate biomarker and therapeutic target for high-grade serous ovarian cancer[J].J Exp Clin Cancer Res,2020,39(1): 265.
34
Guo Y,Chai B,Jia J,et al.KLF7/VPS35 axis contributes to hepatocellular carcinoma progression through CCDC85C-activated βcatenin pathway[J].Cell Biosc,2021,11(1): 73.
35
Wang X,Shen QW,Wang J,et al.KLF7 Regulates Satellite Cell Quiescence in Response to Extracellular Signaling[J].Stem Cells,2016,34(5): 1310-1320.
36
Feng W,Chen J,Huang W,et al.HMGB1-mediated elevation of KLF7 facilitates hepatocellular carcinoma progression and metastasis through upregulating TLR4 and PTK2[J].Theranostics,2023,13(12): 4042-4058.
37
Zhao L,Zhang Y,Liu J,et al.miR-185 inhibits the proliferation and invasion of non-small cell lung cancer by targeting KLF7[J].Oncol Res ,2019,27(9): 1015-1023.
38
AN YX,Shang YJ,Xu ZW,et al.STAT3-induced long noncoding RNA LINC00668 promotes migration and invasion of non-small cell lung cancer via the miR-193a/KLF7 axis [ J ].Biomed Pharmacother,2019,116: 109023.
39
Li K,Yuan C.MicroRNA-103 modulates tumor progression by targeting KLF7 in non-small cell lung cancer[J].Int J Mol Med,2020,46(3): 1013-1028.
40
Mao Y,Chen YC,Zhang ZW.Molecular function of Krüppel-like factor 7 in biology[J].Acta Biochim Biophys Sin(Shanghai),2023,55(5): 713-725.
41
Zhou R,Al-Henhena N,Ying RPY,et al.Chemopreventive efficacy of andrographis paniculata on azoxymethane-induced aberrant colon crypt foci in vivo[J].PLoS ONE,2014,9(11): e111118.
42
Huang Q,Li S,Cheng P,et al.High expression of anti-apoptotic protein Bcl-2 is a good prognostic factor in colorectal cancer: Result of a meta-analysis[J].World J Gastroenterol,2017,23(27):5018-5033.
43
Jacob MW,Tarun Y,Rutter J.Stressed to death Mitochondrial stress responses connect respiration and apoptosis in cancer[J].Mol Cell,2022,82(18): 3321-3332.
44
Kaloni D,Diepstraten ST,Strasser A,et al.BCL-2 protein family:attractive targets for cancer therapy[J].Apoptosis,2022,28(1-2):20-38.
45
Lee YG,Guruprasad P,Ghilardi G,et al.Modulation of BCL-2 in both T cells and tumor cells to enhance chimeric antigen receptor tcell Immunotherapy against cancer[J].Cancer Discov,2022,12(10): 2372-2391.
46
Singh P,Lim B.Targeting Apoptosis in Cancer[J].Curr Oncol Rep,2022,24(3): 273-284.
47
Yeretssian G,Correa RG,Doiron K,et al.Non-apoptotic role of BID in inflammation and innate immunity [J].Nature,2011,474(7349): 96-99.
48
Woods ML,Weiss A,Sokol AM,et al.Epigenetically silenced apoptosis-associated tyrosine kinase (AATK) facilitates a decreased expression of Cyclin D1 and WEE1,phosphorylates TP53 and reduces cell proliferation in a kinase-dependent manner[J].Cancer Gene Ther,2022,29(12): 1975-1987.
49
Cekanova M,Fernando RI,Siriwardhana N,et al.BCL-2 family protein,BAD is down-regulated in breast cancer and inhibits cell invasion[J].Exp Cell Res,2015,331(1): 1-10.
50
Alam S,Pal A,Kumar R,et al.Nexrutine inhibits azoxymethaneinduced colonic aberrant crypt formation in rat colon and induced apoptotic cell death in colon adenocarcinoma cells [ J].Mol Carcinog,2015,55(8): 1262-1274.
51
Ahmed KA ,Shareef SH,Faraj TA ,et al.Chemoprotective effect of arbutin on azoxymethane-induced aberrant crypt foci in rat colon via modulation of PCNA Bax protein[J].Braz J Med Biol Res,2024,57: e13306.
52
Wang J,Teng F,Chai H,et al.GNA14 stimulation of KLF7 promotes malignant growth of endometrial cancer through upregulation of HAS2[J].BMC Cancer,2021,21(1): 456.
53
Wang Y,Zhu XF,Li WY,et al.Krüppel-like factor 7 attenuates hippocampal neuronal injury after traumatic brain injury[J].Neural Regen Res,2022,17(3): 661-672.
[1] 钟雅雯, 王煜, 王海臻, 黄莉萍. 肌苷通过抑制线粒体通透性转换孔开放缓解缺氧/复氧诱导的人绒毛膜滋养层细胞凋亡[J/OL]. 中华妇幼临床医学杂志(电子版), 2024, 20(05): 525-533.
[2] 黄福, 王黔, 金相任, 唐云川. VEGFR2、miR-27a-5p在胃癌组织中的表达与临床病理参数及预后的关系研究[J/OL]. 中华普外科手术学杂志(电子版), 2024, 18(05): 558-561.
[3] 祝炜安, 林华慧, 吴建杰, 黄炯煅, 吴婷婷, 赖文杰. RDM1通过CDK4促进前列腺癌细胞进展的研究[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(06): 618-625.
[4] 郑俊, 吴杰英, 谭海波, 郑安全, 李腾成. EGFR-MEK-TZ三联合分子的构建及其对去势抵抗性前列腺癌细胞增殖与凋亡的影响[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(05): 503-508.
[5] 罗玉, 徐春园, 王振, 胡丹. 晚期非小细胞肺癌抗程序性细胞死亡蛋白-1治疗预后影响因素与研究进展[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(06): 1037-1041.
[6] 刘岩, 赵沛妍, 田琳, 崔贺然, 狄娜, 李慧, 程颖. 新型OX40 抗体激动剂对NSCLC 人源化小鼠模型的抑制作用及机制分析[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(06): 901-906.
[7] 谢鹏飞, 陈佳, 张晓东, 张春荣, 许峰. 肺叶气肿比值预测NSCLC 电视胸腔镜肺叶切除术后持续性漏气的临床分析[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(06): 907-912.
[8] 焦咪, 郭亚焕, 锻炼, 方芳. SCARA5 甲基化对可切除非小细胞肺癌辅助化疗的预后意义[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(06): 936-941.
[9] 张青, 宋明, 闫嘉, 王娇. 卡瑞利珠单抗联合克唑替尼治疗ALK 阳性NSCLC 疗效分析[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(06): 967-970.
[10] 张礼江, 沈玲佳, 施我大. 倾向性评分匹配分析奥希替尼对晚期NSCLC 预后的影响[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(05): 820-822.
[11] 杨慧, 郭丽娟, 冯晓丹, 李静, 黄成谋, 蔡兴锐, 覃英娇, 王远礼. 非小细胞肺癌铂类药物耐药mi RNA表达特征及预测分析[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(05): 719-724.
[12] 赖淼, 景鑫, 李桂珍, 李怡. 非小细胞肺癌EGFR 突变亚型的临床病理和预后意义[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(05): 731-737.
[13] 黄程鑫, 陈莉, 刘伊楚, 王水良, 赖晓凤. OPA1 在乳腺癌组织的表达特征及在ER阳性乳腺癌细胞中的生物学功能研究[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(05): 275-284.
[14] 杜霞, 马梦青, 曹长春. 造影剂诱导的急性肾损伤的发病机制及干预靶点研究进展[J/OL]. 中华肾病研究电子杂志, 2024, 13(05): 279-282.
[15] 江倩, 王红蕊, 朱玥荃, 李响, 耿晓坤, 李凤武. 药物诱导亚低温对缺血性脑卒中的神经保护作用及DRP-1 调控线粒体功能在其中的潜在分子机制[J/OL]. 中华脑血管病杂志(电子版), 2024, 18(06): 586-594.
阅读次数
全文
0
HTML PDF
最新录用 在线预览 正式出版 最新录用 在线预览 正式出版
0 0 0 0 0 0


摘要
39
最新录用 在线预览 正式出版
0 0 39
  来源 本网站 其他网站
  次数 26 13
  比例 67% 33%