人胰腺癌组织中微RNA-134-5p的异常表达及其作用研究
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摘要
目的:研究微RNA-134-5p(microRNA-134-5p,miR-134-5p)在人胰腺癌组织中的异常表达及其对癌细胞生物学行为、RAN基因表达的影响。方法:于我院消化科、普外科收集30例确诊胰腺癌患者,以癌旁正常组织为对照,采用TaqMan实时荧光定量聚合酶链反应(quantitative real-time polymerase chain reaction,q RT-PCR)方法检测胰腺癌组织中miR-134-5p的异常表达情况;体外实验采用蛋白质印迹、双荧光素酶实验证实RAN基因是miR-134-5p的直接靶基因,流式细胞仪检测miR-134-5p对人胰腺癌细胞凋亡的影响,细胞计数试剂盒(cell counting kit-8,CCK8)法和Transwell小室试验检测miR-134-5p对人胰腺癌细胞增殖、迁移能力的影响。结果:与癌旁正常胰腺组织相比,人胰腺癌组织中miR-134-5p的表达水平明显降低; RAN基因为miR-134-5p的直接靶基因;瞬时转染增高miR-134-5p的表达可促进人胰腺癌细胞的凋亡,抑制其增殖和迁移能力;瞬时转染降低miR-134-5p的表达则获得相反的效果。结论:人胰腺癌组织中miR-134-5p的表达水平较正常胰腺组织明显降低,miR-134-5p可促进胰腺癌细胞的凋亡、抑制其增殖和迁移、抑制RAN的表达从而发挥抗肿瘤作用。
Objective To study the expression of microRNA-134-5 p(miR-134-5 p) in pancreatic cancer tissue and its effect on biological behaviors of pancreatic cancer cells. Methods Thirty patients with pancreatic cancer were enrolled.TaqMan quantitative real-time polymerase chain reaction(qRT-PCR) was used to detect the expression of miR-134-5 p in pancreatic cancer tissues as compared with that in normal pancreatic tissue. RAN was detected to be the direct target of miR-134-5 p by Targetscan. Luciferase reporter assay was employed to testify the detection. Western-blot was employed to detect the effect of miR-134-5 p on the expression of RAN in pancreatic cancer cells. The effects of miR-134-5 p on pancreatic cancer cells apoptosis, growth, migration and invasion abilities were examined by flow cytometry, cell counting kit-8(CCK8) assay and Matrigel migration assay, respectively. Results Compared with controls, the expression of miR-134-5 p was significantly down-regulated in pancreatic cancer tissues; RAN was detected and proved to be a direct target of miR-134-5 p. The apoptosis of pancreatic cancer cells was significantly enhanced and the proliferation and migration were down-regulated by increased expression of miR-134-5 p, while inhibition of miR-134-5 p obtained the opposite effects.Conclusions MiR-134-5 p was significantly down-regulated in pancreatic cancer tissue; miR-134-5 p might function as a tumor-suppressor by inhibiting the cell proliferation and migration, enhancing apoptosis, and decreasing expression of RAN in pancreatic cancer cells.
Objective To study the expression of microRNA-134-5 p(miR-134-5 p) in pancreatic cancer tissue and its effect on biological behaviors of pancreatic cancer cells. Methods Thirty patients with pancreatic cancer were enrolled.TaqMan quantitative real-time polymerase chain reaction(qRT-PCR) was used to detect the expression of miR-134-5 p in pancreatic cancer tissues as compared with that in normal pancreatic tissue. RAN was detected to be the direct target of miR-134-5 p by Targetscan. Luciferase reporter assay was employed to testify the detection. Western-blot was employed to detect the effect of miR-134-5 p on the expression of RAN in pancreatic cancer cells. The effects of miR-134-5 p on pancreatic cancer cells apoptosis, growth, migration and invasion abilities were examined by flow cytometry, cell counting kit-8(CCK8) assay and Matrigel migration assay, respectively. Results Compared with controls, the expression of miR-134-5 p was significantly down-regulated in pancreatic cancer tissues; RAN was detected and proved to be a direct target of miR-134-5 p. The apoptosis of pancreatic cancer cells was significantly enhanced and the proliferation and migration were down-regulated by increased expression of miR-134-5 p, while inhibition of miR-134-5 p obtained the opposite effects.Conclusions MiR-134-5 p was significantly down-regulated in pancreatic cancer tissue; miR-134-5 p might function as a tumor-suppressor by inhibiting the cell proliferation and migration, enhancing apoptosis, and decreasing expression of RAN in pancreatic cancer cells.
引文
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[30]Ly TK,Wang J,Pereira R,et al.Activation of the RANGTPase is subject to growth factor regulation and can give rise to cellular transformation[J].J Biol Chem,2010,285(8):5815-5826.
[2]Zhong X,Zhang D,Xiong M,et al.Noncoding RNA for cancer gene therapy[J].Recent Results Cancer Res,2016,209:51-60.
[3]Bhattacharyya NP,Das E,Bucha S,et al.Regulation of cell cycle associated genes by microRNA and transcription factor[J].Microrna,2016,5(3):180-200.
[4]Ivey KN,Srivastava D.MicroRNAs as regulators of differentiation and cell fate decisions[J].Cell Stem Cell,2010,7(1):36-41.
[5]Mei Q,Xue G,Li X,et al.Methylation-induced loss of miR-484 in microsatellite-unstable colorectal cancer promotes both viability and IL-8 production via CD137L[J].J Pathol,2015,236(2):165-174.
[6]Chen Y,Gao DY,Huang L.In vivo delivery of miRNAs for cancer therapy:challenges and strategies[J].Adv Drug Deliv Rev,2015,81:128-141.
[7]Fest J,Ruiter R,van Rooij FJ,et al.Underestimation of pancreatic cancer in the national cancer registry-reconsidering the incidence and survival rates[J].Eur J Cancer,2017,72:186-191.
[8]Bouvier AM,Bossard N,Colonna M,et al.Trends in net survival from pancreatic cancer in six European Latin countries:results from the SUDCAN population-based study[J].Eur J Cancer Prev,2017,26:S63-S69.
[9]Chen Y,Meng L,Yu Q,et al.The miR-134 attenuates the expression of transcription factor FOXM1 during pluripotent NT2/D1 embryonal carcinoma cell differentiation[J].Exp Cell Res,2015,330(2):442-450.
[10]Liu CJ,Shen WG,Peng SY,et al.miR-134 induces oncogenicity and metastasis in head and neck carcinoma through targeting WWOX gene[J].Int J Cancer,2014,134(4):811-821.
[11]Zha R,Guo W,Zhang Z,et al.Genome-wide screening identified that miR-134 acts as a metastasis suppressor by targeting integrinβ1 in hepatocellular carcinoma[J].PLoSOne,2014,9(2):e87665.
[12]Chen T,Gao F,Feng S,et al.MicroRNA-134 regulates lung cancer cell H69 growth and apoptosis by targeting WWOX gene and suppressing the ERK1/2 signaling pathway[J].Biochem Biophys Res Commun,2015,464(3):748-754.
[13]Liu Y,Zhang M,Qian J,et al.miR-134 functions as a tumor suppressor in cell proliferation and epithelial-tomesenchymal transition by targeting KRAS in renal cell carcinoma cells[J].DNA Cell Biol,2015,34(6):429-436.
[14]Wu Z,Zhou L,Ding G,et al.Overexpressions of miR-212 are associated with poor prognosis of patients with pancreatic ductal adenocarcinoma[J].Cancer Biomark,2017,18(1):35-39.
[15]Chang W,Liu M,Xu J,et al.MiR-377 inhibits the proliferation of pancreatic cancer by targeting Pim-3[J].Tumour Biol,2016,37(11):14813-14824.
[16]Zhang R,Leng H,Huang J,et al.miR-337 regulates the proliferation and invasion in pancreatic ductal adenocarcinoma by targeting HOXB7[J].Diagn Pathol,2014,9:171.
[17]Yin C,Wang PQ,Xu WP,et al.Hepatocyte nuclear factor-4αreverses malignancy of hepatocellular carcinoma through regulating miR-134 in the DLK1-DIO3 region[J].Hepatology,2013,58(6):1964-1976.
[18]Niu CS,Yang Y,Cheng CD.MiR-134 regulates the proliferation and invasion of glioblastoma cells by reducing Nanog expression[J].Int J Oncol,2013,42(5):1533-1540.
[19]Zhang Y,Kim J,Mueller AC,et al.Multiple receptor tyrosine kinases converge on microRNA-134 to control KRAS,STAT5B,and glioblastoma[J].Cell Death Differ,2014,21(5):720-734.
[20]Zhang X,Wang H,Zhang S,et al.MiR-134 functions as a regulator of cell proliferation,apoptosis,and migration involving lung septation[J].In Vitro Cell Dev Biol Anim,2012,48(2):131-136.
[21]O’Brien K,Lowry MC,Corcoran C,et al.miR-134 in extracellular vesicles reduces triple-negative breast cancer aggression and increases drug sensitivity[J].Oncotarget,2015,6(32):32774-32789.
[22]Liu CJ,Shen WG,Peng SY,et al.miR-134 induces oncogenicity and metastasis in head and neck carcinoma through targeting WWOX gene[J].Int J Cancer,2014,134(4):811-821.
[23]Yuan SM,Li H,Yang M,et al.High intensity focused ultrasound enhances anti-tumor immunity by inhibiting the negative regulatory effect of miR-134 on CD86 in a murine melanoma model[J].Oncotarget,2015,6(35):37626-37637.
[24]Lu L,Ju F,Zhao H,et al.MicroRNA-134 modulates resistance to doxorubicin in human breast cancer cells by downregulating ABCC1[J].Biotechnol Lett,2015,37(12):2387-2394.
[25]Chen T,Gao F,Feng S,et al.MicroRNA-134 regulates lung cancer cell H69 growth and apoptosis by targeting WWOX gene and suppressing the ERK1/2 signaling pathway[J].Biochem Biophys Res Commun,2015,464(3):748-754.
[26]Kitamura K,Seike M,Okano T,et al.MiR-134/487b/655cluster regulates TGF-β-induced epithelial-mesenchymal transition and drug resistance to gefitinib by targeting MAGI2 in lung adenocarcinoma cells[J].Mol Cancer Ther,2014,13(2):444-453.
[27]Rensen WM,Mangiacasale R,Ciciarello M,et al.The GTPase RAN:regulation of cell life and potential roles in cell transformation[J].Front Biosci,2008,13:4097-4121.
[28]Kurisetty VV,Johnston PG,Johnston N,et al.RANGTPase is an effector of the invasive/metastatic phenotype induced by osteopontin[J].Oncogene,2008,27(57):7139-7149.
[29]Muniyappa MK,Dowling P,Henry M,et al.MiRNA-29a regulates the expression of numerous proteins and reduces the invasiveness and proliferation of human carcinoma cell lines[J].Eur J Cancer,2009,45(17):3104-3118.
[30]Ly TK,Wang J,Pereira R,et al.Activation of the RANGTPase is subject to growth factor regulation and can give rise to cellular transformation[J].J Biol Chem,2010,285(8):5815-5826.