miR-375靶向调控YAP表达对肝癌细胞增殖和侵袭能力的影响
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摘要
目的:探讨miR-375靶向调控Yes相关蛋白(Yes-associated protein, YAP)的表达对肝癌细胞增殖和侵袭能力的影响。方法:收集2015年1月至2016年12月河南中医药大学第二附属医院普外二科行手术切除的70例肝细胞癌(hepatocellular carcinoma, HCC)患者的癌组织及对应的癌旁组织标本,以及肝癌细胞系SMMC-7721、Hb611、HepG2和BEL-7405,用qPCR法检测HCC癌组织和肝癌细胞中miR-375的表达水平。双荧光素酶报告基因检测miR-375与YAP的相互作用;分析miR-375和HCC患者的临床病理特征之间的关系,MTT法检测miR-375对肝癌细胞增殖能力的影响,Transwell小室法检测抑制miR-375表达后HCC细胞侵袭能力的变化;Western blotting检测HepG2细胞中YAP的表达。建立裸鼠肝癌皮下移植瘤模型,观察抑制miR-375表达后移植瘤体积和质量的变化,免疫组化法和Western blotting检测裸鼠移植瘤中YAP的表达情况。结果:HCC组织中miR-375和YAP表达水平明显高于癌旁组织(均P<0.05),肝癌HepG2细胞中miR-375的表达水平最高(P<0.05)。miR-375能与YAP的3’UTR特异性结合,调控YAP的表达活性。抑制miR-375的表达后,HepG2细胞增殖、侵袭的能力显著减弱(均P<0.05),裸鼠移植瘤体积和质量都明显减小(均P<0.05),移植瘤组织细胞中YAP的表达水平相应下调(P<0.05)。结论:miR-375在肝癌的发生发展过程中起重要作用,可以通过靶向调控YAP的表达影响肝癌细胞的恶性生物学行为。
Objective: To investigate the mechanisms of miR-375 affecting the proliferation and invasion of hepatoma cells via targeting YAP(Yes-associated protein). Methods: The cancerous tissues and corresponding para-cancerous tissues of 70 patients with hepatocellular carcinoma(HCC) who underwent surgical resection at the Second Affiliated Hospital of Henan University of Traditional Chinese Medicine from January 2015 to December 2016, as well as the hepatoma cell lines SMMC-7721, Hb611, HepG2 and BEL-7405 were collected for this study. qPCR method was used to detect the expression level of miR-375 in collected HCC tissues and different hepatoma cell lines; Dual luciferase reporter gene assay was used to verify the interaction between miR-375 and YAP; The relationship between miR-375 and clinicopathological features of HCC patients was also analyzed; MTT assay was used to detect the effect of miR-375 on the proliferation of hepatoma cells; Transwell invasion assay was used to detect the invasive ability of hepatoma cells after inhibiting the expression of miR-375; Western blotting was used to detect the expression of YAP in HepG2 cells. The nude mouse model of subcutaneously transplanted xenograft was established, and the tumor volume and mass of transplanted hepatoma cells were detected after inhibiting the expression of miR-375. The expression of YAP in xenograft of nude mice was detected by immunohistochemistry and Western blotting. Results: The expression of miR-375 and YAP in HCC tissues was significantly higher than that in para-cancerous tissues(all P<0.05). The expression of miR-375 in HepG2 cells was the highest(P<0.05). miR-375 could specifically bind to the 3' UTR of YAP and regulate the expression activity of YAP. After inhibiting the expression of miR-375, the proliferation and invasion abilities of HepG2 cells were reduced(all P<0.05); The tumor volume and mass of transplanted xenografts were significantly reduced(both P<0.05); The expression of YAP protein in the transplanted xenografts was down-regulated(P<0.05). Conclusion: miR-375 plays an important role in the occurrence and development of liver cancer, and can influence the malignant biological behaviors of hepatoma cells by targeting and regulating the expression of YAP.
Objective: To investigate the mechanisms of miR-375 affecting the proliferation and invasion of hepatoma cells via targeting YAP(Yes-associated protein). Methods: The cancerous tissues and corresponding para-cancerous tissues of 70 patients with hepatocellular carcinoma(HCC) who underwent surgical resection at the Second Affiliated Hospital of Henan University of Traditional Chinese Medicine from January 2015 to December 2016, as well as the hepatoma cell lines SMMC-7721, Hb611, HepG2 and BEL-7405 were collected for this study. qPCR method was used to detect the expression level of miR-375 in collected HCC tissues and different hepatoma cell lines; Dual luciferase reporter gene assay was used to verify the interaction between miR-375 and YAP; The relationship between miR-375 and clinicopathological features of HCC patients was also analyzed; MTT assay was used to detect the effect of miR-375 on the proliferation of hepatoma cells; Transwell invasion assay was used to detect the invasive ability of hepatoma cells after inhibiting the expression of miR-375; Western blotting was used to detect the expression of YAP in HepG2 cells. The nude mouse model of subcutaneously transplanted xenograft was established, and the tumor volume and mass of transplanted hepatoma cells were detected after inhibiting the expression of miR-375. The expression of YAP in xenograft of nude mice was detected by immunohistochemistry and Western blotting. Results: The expression of miR-375 and YAP in HCC tissues was significantly higher than that in para-cancerous tissues(all P<0.05). The expression of miR-375 in HepG2 cells was the highest(P<0.05). miR-375 could specifically bind to the 3' UTR of YAP and regulate the expression activity of YAP. After inhibiting the expression of miR-375, the proliferation and invasion abilities of HepG2 cells were reduced(all P<0.05); The tumor volume and mass of transplanted xenografts were significantly reduced(both P<0.05); The expression of YAP protein in the transplanted xenografts was down-regulated(P<0.05). Conclusion: miR-375 plays an important role in the occurrence and development of liver cancer, and can influence the malignant biological behaviors of hepatoma cells by targeting and regulating the expression of YAP.
引文
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[3]CALLEGARI E,GRAMANTIERI L,DOMENICALI M,et al.MicroRNAs in liver cancer:a model for investigating pathogenesis and novel therapeutic approaches[J].Cell Death Differ,2015,22(1):46-57.DOI:10.1038/cdd.2014.136.
[4]SHEN H,ZHU F,LIU J,et al.Alteration in miR-21/PTEN expression modulates gefitinib resistance in non-small cell lung cancer[J/OL].PLoS One,2014,9(7):e103305[2018-04-16].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4110008/.DOI:10.1371/journal.pone.0103305.
[5]MOHANTY V,SHAH A,ALLENDER E,et al.Folate receptor alpha upregulates Oct4,Sox2 and Klf4 and downregulates miR-138and miR-let-7 in cranial neural crest cells[J].Stem Cell,2016,34(11):2721-2732.DOI:10.1002/stem.2421.
[6]JOHNSON R,HALDER G.The two faces of Hippo:targeting the Hippo pathway for regenerative medicine and cancer treatment[J].Nat Rev Drug Discov,2014,13(1):63-79.DOI:10.1038/nrd4161.
[7]蔡芸芸,高嵩,刘鲁明,等.半枝莲提取物通过Hippo/YAP通路抑制胰腺癌PANC-1细胞的增殖,侵袭转移和成瘤能力的研究[J].中华中医药杂志,2017,2017(7):2947-2951.
[8]WANG J,WANG H,ZHANG Y,et al.Mutual inhibition between YAP and SRSF1 maintains long non-coding RNA,Malat1-induced tumourigenesis in liver cancer[J].Cell Signal,2014,26(5):1048-1059.DOI:10.1016/j.cellsig.2014.01.022.
[9]陈彭,刘利.miR-21作用于PTEN与肿瘤耐药关系的研究进展[J].现代肿瘤医学,2015,23(13):1917-1920.DOI:10.3969/j.issn.1672-4992.2015.13.42.
[10]JIMENEZ L,SHARMA V P,LIM J,et al.MicroRNA-375 impairs head and neck squamous cell carcinoma invasion by suppressing invadopodia activity[J].Arch Pathol Lab Med,2015,139(11):1349-1361.DOI:10.5858/arpa.2014-0471-OA.
[11]MIAO L,LIU K,XIE M,et al.miR-375 inhibits Helicobacter pylori-induced gastric carcinogenesis by blocking JAK2-STAT3 signaling[J].Cancer Immunol Immunother,2014,63(7):699-711.DOI:10.1007/s00262-014-1550-y.
[12]MUSSNICH P,ROSA R,BIANCO R,et al.MiR-199a-5p and miR-375 affect colon cancer cell sensitivity to cetuximab by targeting PHLPP1[J].Expert Opin Ther Targets,2015,19(8):1017-1026.DOI:10.1517/14728222.2015.1057569.
[13]SELTH L A,DAS R,TOWNLEY S L,et al.A ZEB1-miR-375-YAP1 pathway regulates epithelial plasticity in prostate cancer[J].Oncogene,2017,36(1):24-34.DOI:10.1038/onc.2016.185.
[14]HAN X,YAN S,WEIJIE Z,et al.Critical role of miR-10b in transforming growth factor-β1-induced epithelial-mesenchymal transition in breast cancer[J].Cancer Gene Ther,2014,21(2):60-67.DOI:10.1038/cgt.2013.82.
[15]ZHOU J,SONG S,HE S,et al.MicroRNA-375 targets PDK1 in pancreatic carcinoma and suppresses cell growth through the Akt signaling pathway[J].Int J Mol Med,2014,33(4):950-956.DOI:10.3892/ijmm.2014.1638.
[16]HU S,ZHANG M,SUN F,et al.miR-375 controls porcine pancreatic stem cell fate by targeting 3-phosphoinositide-dependent protein kinase-1(Pdk1)[J].Cell Prolif,2016,49(3):395-406.DOI:10.1111/cpr.12263.
[17]杨进,魏蕊,洪天配,等.miR-375在人胚胎干细胞定向分化为胰岛素分泌细胞过程中对肝细胞核因子1β表达的调控作用[J].中华糖尿病杂志,2015,7(12):730-734.DOI:10.3760/cma.j.issn.1674-5809.2015.12.004.
[18]MOROISHI T,HANSEN C G,GUAN K L.The emerging roles of YAP and TAZ in cancer[J].Nat Rev Cancer,2015,15(2):73-79.DOI:10.1038/nrc3876.
[19]LIN L,SABNIS A J,CHAN E,et al.The Hippo effector YAP promotes resistance to RAF-and MEK-targeted cancer therapies[J].Nat Genet,2015,47(3):250-256.DOI:10.1038/ng.3218.
[20]THONGON N,CASTIGLIONI I,ZUCAL C,et al.The GSK3βinhibitor BIS I reverts YAP-dependent EMT signature in PDAC cell lines by decreasing SMADs expression level[J].Oncotarget,2016,7(18):26551-26566.DOI:10.18632/oncotarget.8437.
[21]DI BENEDETTO A,MOTTOLESE M,SPERATI F,et al.The Hippo transducers TAZ/YAP and their target CTGF in male breast cancer[J].Oncotarget,2016,7(28):43188-43198.DOI:10.18632/oncotarget.9668.