乳腺癌组织和细胞中低表达的LZTS2通过PI3K/AKT信号通路抑制癌细胞增殖、迁移和EMT
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摘要
目的:探讨亮氨酸拉链肿瘤抑制因子2(leucine zipper tumor suppressor 2, LZTS2)基因在人乳腺癌组织和细胞系中的表达及其对乳腺癌细胞增殖、迁移和EMT的影响及其作用机制。方法:收集2016年1月至2016年12月开封中心医院乳腺外科收治的50例女性乳腺癌患者的癌及癌旁组织标本和乳腺癌细胞系MCF-7、MDA-MB-231、MDA-MB-468以及正常人乳腺上皮细胞株HBL-100,用qPCR和Western blotting检测乳腺癌组织和细胞中LZTS2 mRNA和蛋白表达水平。构建pcDNA-LZTS2真核表达载体并采用脂质体转染MCF-7细胞,同时转染pcDNA3.1作为阴性对照。用Western blotting检测转染48～72 h后MCF-7细胞中LZTS2蛋白表达水平;用MTT法、Transwell小室法检测LZTS2过表达对细胞增殖、迁移和侵袭的影响,同时用Western blotting检测细胞中EMT相关蛋白Cyclin D1、波形蛋白、神经钙黏蛋白、上皮钙黏蛋白以及PI3K/AKT信号通路中相关蛋白的表达。结果:人乳腺癌组织中LZTS2 m RNA和蛋白表达水平均明显低于癌旁组织(P<0.05或P<0.01);乳腺癌MCF-7、MDA-MB-231和MDA-MB-468细胞中LZTS2 m RNA和蛋白表达水平显著低于乳腺上皮细胞HBL-100(P<0.05或P<0.01)。与空白对照组和pcDNA3.1组相比,pcDNA-LZTS2组MCF-7细胞中LZTS2蛋白表达水平明显上调(P<0.01),细胞增殖、迁移和侵袭能力显著受到抑制(P<0.05或P<0.01),同时过表达LZTS2细胞中Cyclin D1、波形蛋白和神经钙黏蛋白表达水平均明显降低(P<0.05或P<0.01)、上皮钙黏蛋白表达水平明显升高(均P<0.01),显示LZTS2过表达通过降低p-PI3K和p-AKT表达而抑制PI3K/AKT信号通路。结论:LZTS2在乳腺癌中低表达,过表达LZTS2能够抑制乳腺癌细胞的增殖、迁移和侵袭能力,可能与抑制细胞EMT过程的PI3K/AKT信号通路有关。
Objective: To evaluate the expression of leucine zipper tumor suppressor 2(LZTS2) in human breast cancer tissues and cell lines, and to investigate the effects and mechanisms of LZTS2 over-expression on proliferation, invasion and epithelial-mesenchymal transition(EMT) of breast cancer cells. Methods: Fifty pairs of cancerous tissues and para-cancerous tissues resected from breast cancer patients in Department of Breast Surgery of Kaifeng Central Hospital from January, 2016 to December, 2016, as well as breast cancer cell lines(MCF-7, MDA-MB-231, MDA-MB-468) and normal mammary epithelial HBL-100 cells were collected for this study;and Real-time quantitative PCR(qPCR) and Western blotting were used to determine the mRNA and protein expressions of LZTS2 in collected tissues and cell lines. MCF-7 cells were transfected with pcDNA-LZTS2 or pcDNA3.1(negative control) using lipofectamineTM2000, and the protein expression of LZTS2 at 49-72 h after transfection was measured by Western blotting; Then, the effects of LZTS2 over-expression on proliferation, migration and invasion of MCF-7 cells were detected by MTT assay and Transwell assay, respectively; Furthermore, Western blotting was performed to detect the expressions of EMT associated proteins(Cyclin D1, Vimentin, Ncadherin, E-cadherin) and PI3 K/AKT signaling pathways-related molecules. Results: The mRNA and protein expressions of LZTS2 were down-regulated in breast cancerous tissues and cell lines(MCF-7, MDA-MB-468 and MDA-MB-231) as compared with paired para-cancerous tissues or normal mammary epithelial HBL-100 cells(P<0.05 or P<0.01). Compared with and blank control or pcDNA3.1 group, the protein expression of LZTS2 in MCF-7 cells of pcDNA-LZTS2 group significantly increased(P<0.01), while the proliferation, migration and invasion of MCF-7 cells significantly reduced(P<0.05 or P<0.01). In addition, forced expression of LZTS2significantly down-regulated the protein expressions of Cyclin D1, Vimentin and N-cadherin(P<0.05 or P<0.01) but up-regulated the expression of E-cadherin in MCF-7 cells(P<0.01), indicating LZTS2 over-expression suppressed PI3 K/AKT signaling pathway through inhibiting the expression p-PI3 K and p-AKT. Conclusion: The findings collectively demonstrated that the expression of LZTS2 was decreased in breast cancer, and over-expression of LZTS2 efficiently inhibited the proliferation, migration and invasion of breast cancer cells, which might be related with the suppression of PI3 K/AKT signaling pathway involved in EMT.
Objective: To evaluate the expression of leucine zipper tumor suppressor 2(LZTS2) in human breast cancer tissues and cell lines, and to investigate the effects and mechanisms of LZTS2 over-expression on proliferation, invasion and epithelial-mesenchymal transition(EMT) of breast cancer cells. Methods: Fifty pairs of cancerous tissues and para-cancerous tissues resected from breast cancer patients in Department of Breast Surgery of Kaifeng Central Hospital from January, 2016 to December, 2016, as well as breast cancer cell lines(MCF-7, MDA-MB-231, MDA-MB-468) and normal mammary epithelial HBL-100 cells were collected for this study;and Real-time quantitative PCR(qPCR) and Western blotting were used to determine the mRNA and protein expressions of LZTS2 in collected tissues and cell lines. MCF-7 cells were transfected with pcDNA-LZTS2 or pcDNA3.1(negative control) using lipofectamineTM2000, and the protein expression of LZTS2 at 49-72 h after transfection was measured by Western blotting; Then, the effects of LZTS2 over-expression on proliferation, migration and invasion of MCF-7 cells were detected by MTT assay and Transwell assay, respectively; Furthermore, Western blotting was performed to detect the expressions of EMT associated proteins(Cyclin D1, Vimentin, Ncadherin, E-cadherin) and PI3 K/AKT signaling pathways-related molecules. Results: The mRNA and protein expressions of LZTS2 were down-regulated in breast cancerous tissues and cell lines(MCF-7, MDA-MB-468 and MDA-MB-231) as compared with paired para-cancerous tissues or normal mammary epithelial HBL-100 cells(P<0.05 or P<0.01). Compared with and blank control or pcDNA3.1 group, the protein expression of LZTS2 in MCF-7 cells of pcDNA-LZTS2 group significantly increased(P<0.01), while the proliferation, migration and invasion of MCF-7 cells significantly reduced(P<0.05 or P<0.01). In addition, forced expression of LZTS2significantly down-regulated the protein expressions of Cyclin D1, Vimentin and N-cadherin(P<0.05 or P<0.01) but up-regulated the expression of E-cadherin in MCF-7 cells(P<0.01), indicating LZTS2 over-expression suppressed PI3 K/AKT signaling pathway through inhibiting the expression p-PI3 K and p-AKT. Conclusion: The findings collectively demonstrated that the expression of LZTS2 was decreased in breast cancer, and over-expression of LZTS2 efficiently inhibited the proliferation, migration and invasion of breast cancer cells, which might be related with the suppression of PI3 K/AKT signaling pathway involved in EMT.
引文
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[16]QIU X Y,HU D X,CHEN W Q,et al.PD-L1 confers glioblastoma multiforme malignancy via Ras binding and Ras/Erk/EMT activation[J/OL].Biochim Biophys Acta,2018,1864(5 Pt A):1754-1769[2018-07-05].http://www.sciencedirect.com/science/journal/09254439.DOI:10.1016/j.bbadis.2018.03.002.
[17]赵家义,韩一平.PI3K-AKT-mTOR信号通路抑制剂与肿瘤免疫治疗[J].中国肿瘤生物治疗杂志,2017,24(12):1424-1430.DOI:10.3872/j.issn.1007-385x.2017.12.05.
[18]LI Z,XU X,LI Y,et al.Synergistic antitumor effect of BKM120 with prima-1Met via inhibiting PI3K/AKT/mTOR and CPSF4/hTERTsignaling and reactivating mutant P53[J].Cell Physiol Biochem,2018,45(5):1772-1786.DOI:10.1159/000487786.
[2]JIA X P,MENG L L,FANG J C,et al.Aberrant expression of miR-142-3p and its target gene HMGA1 and FZD7 in breast cancer and its clinical significance[J].Clin Lab,2018,64(6):915-921.DOI:10.7754/Clin.Lab.2017.171114.
[3]梅虹,李常恩,杨梁,等.长链非编码RNA LINC01001在乳腺癌中的表达及其对MCF-7细胞增殖的影响[J].中国肿瘤生物治疗杂志,2018,25(2):158-162.DOI:10.3872/j.issn.1007-385x.2018.02.009.
[4]YU E J,HOOKER E,JOHNSON D T,et al.LZTS2 and PTEN collaboratively regulateβ-catenin in prostatic tumorigenesis[J/OL].PLoS One,2017,12(3):e0174357[2018-07-05].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5360334/.DOI:10.1371/journal.pone.0174357.
[5]SHEN Z,LIN L,CAO B,et al.A potential biomarker for the diagnosis and prognosis of laryngeal squamous cell carcinoma[J/OL].World J Surg Oncol,2018,16(1):42[2018-07-05].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834860/.DOI:10.1186/s12957-018-1349-y.
[6]XU S,LI Y,LU Y,et al.LZTS2 inhibits PI3K/AKT activation and radioresistance in nasopharyngeal carcinoma by interacting with P85[J/OL].Cancer Lett,2018,420:38-48[2018-07-05].http://sciencedirect.com/science/journal/03043835.DOI:10.1016/j.canlet.2018.01.067.
[7]CUI Q Z,TANG Z P,ZHANG X P,et al.Leucine zipper tumor suppressor 2 inhibits cell proliferation and regulates Lef/Tcf-dependent transcription through Akt/GSK3βsignaling pathway in lung cancer[J].J Histochem Cytochem,2013,61(9):659-670.DOI:10.1369/0022155413495875.
[8]JOHNSON D T,LUONG R,LEE S H,et al.Deletion of leucine zipper tumor suppressor 2(LZTS2)increases susceptibility to tumor development[J].J Biol Chem,2013,288(6):3727-3738.DOI:10.1074/jbc.M112.417568.
[9]SUDO H,MARU Y.LAPSER1/LZTS2:a pluripotent tumor suppressor linked to the inhibition of katanin-mediated microtubule severing[J].Hum Mol Genet,2008,17(16):2524-2540.DOI:10.1093/hmg/ddn153.
[10]KIM J M,SONG J S,CHO H H,et al.Effect of the modulation of leucine zipper tumor suppressor 2 expression on proliferation of various cancer cells functions as a tumor suppressor[J].Mol Cell Biochem,2011,346(1/2):125-136.DOI:10.1007/s11010-010-0599-y.
[11]EXPóSITO-VILLèN A,E ARáNEGA A,FRANCO D.Functional role of non-coding RNAs during epithelial-to-mesenchymal transition[J/OL].Noncoding RNA,2018,4(2).pii:E14[2018-07-05].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6027143/.DOI:10.3390/ncrna4020014.
[12]CYPRIAN F S,AL-FARSI H F,VRANIC S,et al.Epstein-Barr virus and human papillomaviruses interactions and their roles in the initiation of epithelial-mesenchymal transition and cancer progression[J/OL].Front Oncol,2018,8:111[2018-07-05].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5938391/.DOI:10.3389/fonc.2018.00111.
[13]YAO W F,LIU J W,HUANG D S.MiR-200a inhibits cell proliferation and EMT by down-regulating the ASPH expression levels and affecting ERK and PI3K/Akt pathways in human hepatoma cells[J/OL].Am J Transl Res,2018,10(4):1117-1130[2018-07-05].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5934571/.
[14]HUI L,LI H,LU G,et al.Low dose of bisphenol a modulates ovarian cancer gene expression profile and promotes epithelial to mesenchymal transition via canonical Wnt pathway[J].Toxicol Sci,2018,164(2):527-538.DOI:10.1093/toxsci/kfy107.
[15]HE K,DUAN G,LI Y.Dehydroeffusol inhibits viability and epithelial-mesenchymal transition through the hedgehog and Akt/mTORsignaling pathways in neuroblastoma cells[J/OL].Eur J Pharmacol,2018,829:93-101[2018-07-05].http://sciencedirect.com/science/journal/00142999.DOI:10.1016/j.ejphar.2018.04.012.
[16]QIU X Y,HU D X,CHEN W Q,et al.PD-L1 confers glioblastoma multiforme malignancy via Ras binding and Ras/Erk/EMT activation[J/OL].Biochim Biophys Acta,2018,1864(5 Pt A):1754-1769[2018-07-05].http://www.sciencedirect.com/science/journal/09254439.DOI:10.1016/j.bbadis.2018.03.002.
[17]赵家义,韩一平.PI3K-AKT-mTOR信号通路抑制剂与肿瘤免疫治疗[J].中国肿瘤生物治疗杂志,2017,24(12):1424-1430.DOI:10.3872/j.issn.1007-385x.2017.12.05.
[18]LI Z,XU X,LI Y,et al.Synergistic antitumor effect of BKM120 with prima-1Met via inhibiting PI3K/AKT/mTOR and CPSF4/hTERTsignaling and reactivating mutant P53[J].Cell Physiol Biochem,2018,45(5):1772-1786.DOI:10.1159/000487786.