lncRNA Xist/miR-215-5p下调LPAR4表达对乳腺癌细胞增殖与转移的影响
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摘要
目的:探讨溶血磷脂酸受体4(LPAR4)在乳腺癌组织中的表达水平及与乳腺癌发生发展的关系,探究lncRNA Xist/miR-215-5p对LPAR4的调控作用。方法:通过Targetscan和Starbase预测LPAR4的上游通路lncRNA Xist/miR-215-5p。使用慢病毒转染LPAR4和miR-215-5p;使用质粒转染lncRNA Xist。在MDA-MB-231细胞系中,通过MTT和划痕实验检测细胞的增殖和转移能力。结果:LPAR4在乳腺癌细胞系中高表达,miR-215-5p和lncRNA Xist为低表达。Western blot显示LPAR4在乳腺癌细胞系中高表达。MTT和划痕实验提示上调LPAR4可以促进MDA-MB-231细胞的增殖和转移能力。进一步实验发现lncRNA Xist可以上调miR-215-5p,并且负性调控LPAR4;且lncRNA Xist可以抑制MDA-MB-231细胞的增殖能力。除此之外,在lncRNA Xist下调细胞中,上调miR-215-5p可以抑制lncRNA Xist下调介导的细胞增殖能力增强。结论:lncRNA Xist/miR-215-5p可能通过调控LPAR4的表达,为治疗乳腺癌提供可能的新靶点。LPAR4在乳腺癌细胞中高表达,可能成为乳腺癌潜在的肿瘤标志物。
Objective: To evaluate the expression of LPAR4 in breast cancer tissues and its association with breast cancer progression,and the regulating role of lncRNA Xist/miR-215-5p for LPAR4. Methods: The expression of LPAR4,lncRNA Xist and miR-215-5p in breast cancer cell line was tested by qRT-PCR. LPAR4 and miR-215-5p lentivirus and Xist plasmid were transfected. In MDA-MB-231 cell line,MTT and wound healing tests were used to investigate the proliferation and migration ability. Results: qRT-PCR indicated that LPAR4 expressed higher in breast cancer cell line,and miR-215-5p and Xist were expressed lower. MTT and wound healing test indicated that up regulation of LPAR4 could promote the proliferation and migration of MDA-MB-231 cell line. Furthermore,Xist could up regulate miR-215-5p and down regulate LPAR4,and Xist could inhibit the proliferation of MDA-MB-231. In addition,in Xist down regulated cell,up regulation of miR-215-5p could inhibit the Xist-associated enhanced cell proliferation. Conclusion: LPAR4 was associated with poorer prognosis in breast cancer and had the potential to be a novel biomarker. lncRNA Xist/miR-215-5p could provide novel therapeutic targets for breast cancer by targeting LPAR4.
Objective: To evaluate the expression of LPAR4 in breast cancer tissues and its association with breast cancer progression,and the regulating role of lncRNA Xist/miR-215-5p for LPAR4. Methods: The expression of LPAR4,lncRNA Xist and miR-215-5p in breast cancer cell line was tested by qRT-PCR. LPAR4 and miR-215-5p lentivirus and Xist plasmid were transfected. In MDA-MB-231 cell line,MTT and wound healing tests were used to investigate the proliferation and migration ability. Results: qRT-PCR indicated that LPAR4 expressed higher in breast cancer cell line,and miR-215-5p and Xist were expressed lower. MTT and wound healing test indicated that up regulation of LPAR4 could promote the proliferation and migration of MDA-MB-231 cell line. Furthermore,Xist could up regulate miR-215-5p and down regulate LPAR4,and Xist could inhibit the proliferation of MDA-MB-231. In addition,in Xist down regulated cell,up regulation of miR-215-5p could inhibit the Xist-associated enhanced cell proliferation. Conclusion: LPAR4 was associated with poorer prognosis in breast cancer and had the potential to be a novel biomarker. lncRNA Xist/miR-215-5p could provide novel therapeutic targets for breast cancer by targeting LPAR4.
引文
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[3]Kitayama J,Shida D,Sako A,et al.Over-expression of lysophosphatidic acid receptor-2 in human invasive ductal carcinoma[J].Breast Cancer Res,2014,6(6):640-646.
[4]Popnikolov NK,Dalwadi BH,Thomas JD,et al.Association of autotaxin and lysophosphatidic acid receptor 3 with aggressiveness of human breast carcinoma[J].Tumour Biol,2012,33(6):2237-2243.
[5]Liu S,Umezu-Goto M,Murph M,et al.Expression of autotaxin and lysophosphatidic acid receptors increases mammary tumorigenesis,invasion,and metastases[J].Cancer Cell,2009,15(6):539-550.
[6]García DM,Baek D,Shin C,et al.Weak seed-pairing stability and high target-site abundance decrease the proficiency of lsy-6 and other miRNAs[J].Nat Struct Mol Biol,2011,18(3):1139-1146.
[7]Gyvyte U,Juzenas S,Salteniene V,et al.MiRNA profiling of gastrointestinal stromal tumors by next-generation sequencing[J].Oncotarget,2017,8(3):202-209.
[8]Vychytilovafaltejskova P,Merhautova J,Machackova T,et al.MiR-215-5p is a tumor suppressor in colorectal cancer targeting EG-FR ligand epiregulin and its transcriptional inducer HOXB9[J].Oncogenesis,2017,6(11):399-406.
[9]Li J H,Liu S,Zhou H,et al.Starbase v2.0:Decoding miRNA-ceRNA,miRNA-ncRNA and protein-RNA interaction networks from large-scale CLIP-Seq data[J].Nucleic Acids Research,2014(42):D92.
[10]Engreitz J M,Pandyajones A,Mcdonel P,et al.The Xist lncRNAexploits three-dimensional genome architecture to spread across the X-chromosome[J].Science,2013,341(6147):767-771.
[11]WEI Wei,YANG Bo,TANG Ling,et al.Expression of long noncoding RNA XIST in pancreatic cancer and its significance[J].Chinese Journal of General Surgery,2017,26(3):304-310.[魏伟,杨波,唐翎,等.长链非编码RNA XIST在胰腺癌中的表达及意义[J].中国普通外科杂志,2017,26(3):304-310.]
[12]Rui Z,Tian X.Long non-coding RNA XIST regulates PDCD4expression by interacting with miR-21-5p and inhibits osteosarcoma cell growth and metastasis[J].Int J Oncol,2017,51(5):1460-1470.
[13]TANG Ping,WEI Bing,David G Hicks,et al.Molecular phenotypes of breast cancer and their clinical application[J].Chinese JPathol,2009,38(1):13-17.[唐平,魏兵,David G Hicks,等.乳腺癌的分子分类及其临床应用[J].中华病理学杂志,2009,38(1):13-17.]
[14]Voduc D,Nielsen TO.Basal and triple-negative breast cancers:Impact on clinical decision-making and novel therapeutic options[J].Clinical Breast Cancer,2008,8(6):171-178.
[15]Hartman ZC,Poage GM,den Hollander P,et al.Growth of triplenegative breast cancer cells relies upon coordinate autocrine expression of the proinflammatory cytokines IL-6 and IL-8[J].Cancer Res,2013,73(11):3470-3480.
[16]Yu FX,Zhao B,Panupinthu N,et al.Regulation of the HippoYAP pathway by G-protein-coupled receptor signaling[J].Cell,2012,150(4):780-791.
[17]Wu J,Mukherjee A,Lebman DA,et al.Lysophosphatidic acidinduced p21Waf1 expression mediates the cytostatic response of breast and ovarian cancer cells to TGFbeta[J].Mol Cancer Res,2011,9(11):1562-1570.
[18]Lai YJ,Lin WC,Lin FT,et al.PTPL1/FAP-1 negatively regulates TRIP6 function in lysophosphatidic acid-induced cell migration[J].J Biol Chem,2007,282(33):24381-24387.
[19]Li TT,Alemayehu M,Aziziyeh AI,et al.Beta-arrestin/Ral signaling regulates lysophosphatidic acid-mediated migration and invasion of human breast tumor cells[J].Mol Cancer Res,2009,7(7):1064-1077.
[20]Boucharaba A,Serre CM,Gres S,et al.Platelet-derived lysophosphatidic acid supports the progression of osteolytic bone metastases in breast cancer[J].The Journal of Clinical Investigation,2014,7(8):123-131.
[21]Hu S,Chang J,Li Y,et al.Long non-coding RNA XIST as a potential prognostic biomarker in human cancers:A Meta-analysis[J].Oncotarget,2018,9(17):13911-13919.
[22]Zheng R,Lin S,Guan L,et al.Long non-coding RNA XIST inhibited breast cancer cell growth,migration,and invasion via miR-155/CDX1 axis[J].Biochemical&Biophysical Research Communications,2018,3(4):1253-1259.
[23]Huang YS,Chang CC,Lee SS,et al.Xist reduction in breast cancer upregulates AKT phosphorylation via HDAC3-mediated repression of PHLPP1 expression[J].Oncotarget,2016,7(28):43256-43266.
[24]Zhao J,Xu J,Zhang R.SRPX2 regulates colon cancer cell metabolism by miR-192/215 via PI3K-Akt[J].American Journal of Translational Research,2018,10(2):483-490.
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