miR-105-5p在胃癌中的表达及其意义与生物学功能
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摘要
目的:探讨miR-105-5p在胃癌(GC)中的表达情况、临床意义及生物学功能及其潜在的作用机制。方法:应用real-time PCR检测miR-105-5p在GC组织与癌旁组织以及不同GC细胞(MGC-803,MKN-1,SGC-7901,BGC-823和AGS)与正常胃黏膜细胞(GES-1)中的表达;分析miR-105-5p的表达与GC临床病理特征及患者预后的关系;采用Transwell迁移和侵袭实验以及MTT实验检测miR-105-5p对GC细胞迁移与侵袭能力以及增殖能力的影响;应用生物信息学工具预测miR-105-5p的下游靶点,并应用荧光素酶报告实验以及Western blot分析miR-105-5p对靶点的调节作用。结果:miR-105-5p在GC组织的表达明显高于癌旁组织,在各GC细胞中的表达均明显高于正常胃黏膜细胞(均P<0.05)。miR-105-5p的表达水平与肿瘤大小(P=0.020)和远处转移(P=0.004)明显有关;miR-105-5p低表达GC患者的总体生存率明显高于miR-105-5p高表达组的患者(P=0.001 8)。选择miR-105-5p表达量相对较低的BGC-823细胞和相对较高的MKN-1细胞,分别转染miR-105-5p模拟物和miR-105-5p抑制物,转染后结果显示,BGC-823细胞的迁移、侵袭和增殖能力明显增强,而MKN-1细胞的迁移、侵袭和增殖能力明显抑制(均P<0.05)。生物信息学分析发现,DIRAS家族GTP结合RAS样3(DIRAS3)可能是miR-105-5p的作用靶点;荧光素酶报告实验显示,miR-105-5p能够负向调节DIRAS3-3'UTR的荧光素酶活性;Western blot显示,转染miR-105-5p模拟物的BGC-823细胞中DIRAS3的表达明显下调,转染miR-105-5p抑制物的MKN-1细胞DIRAS3的表达明显上调(均P<0.05)。结论:miR-105-5p在GC中表达升高,其可能通过靶向作用于DIRAS3增强GC细胞的迁移、侵袭及增殖,进而促进GC的发生发展。
Objective: To investigate the expression, clinical signifi cance and biological function of miR-105-5 p in gastric cancer(GC), as well as its potential action mechanism.Methods: The expressions of miR-105-5 p in GC and tumor adjacent tissues as well as in different GC cell lines(MGC 803, MKN 1, SGC 7901, BGC 823 and AGS) and normal gastric mucosal cell line(GES-1) were determined by real-time PCR. Th e relations of miR-105-5 p expression with the clinicopathologic features and prognosis of GC patients were analyzed. Th e eff ects of miR-105-5 p on migration, invasion and proliferation of GC cells were examined by Transwell migration and invasion assay and MTT assay, respectively. Th e potential target gene for miR-105-5 p was predicted by using Bioinformatics tools, and then, the correlation between miR-105-5 p and its target gene was verifi ed by Luciferase reporter assay and Western blot, respectively. Results: Th e expression of miR-105-5 p in GC tissues was signifi cantly higher than that in tumor adjacent tissue, and in each studied GC cell line was signifi cantly higher than that in normal gastric mucosal cell line(all P<0.05). The expression of miR-105-5 p was significantly associated with tumor size(P=0.020) and distant metastasis(P=0.004); the overall survival rate in GC patients with low miR-105-5 p expression was significantly higher than that in GC patients with high miR-105-5 p expression(P=0.001 8). The BGC-823 cells with a relatively low miR-105-5 p expression and the MKN 1 cells with a relatively high miR-105-5 p expression were transfected with miR-105-5 p mimics and miR-105-5 p inhibitors respectively, and the results aft er transfection showed that the migration, invasion and proliferative abilities in BGC-823 cells were significantly increased, while were significantly decreased in MKN 1 cells(all P<0.05). Bioinformatics analysis suggested that DIRAS family GTPase 3(DIRAS3) was possibly be the target of miR-105-5 p; luciferase reporter assay indicated that the luciferase activity of DIRAS3-3'-UTR was negatively regulated by miR-105-5 p; Western blot demonstrated that the DIRAS3 expression was significantly down-regulated in BGC-823 after transfection with miR-105-5 p mimics, and was significantly up-regulated in MKN 1 cells after transfection with miR-105-5 p inhibitors(both P<0.05).Conclusion: The expression of miR-105-5 p is elevated in GC, promotes migration, which can enhance the migration, invasion and proliferative abilities of GC cells probably through targeting DIRAS3, and thereby promote the occurrence and development of GC.
Objective: To investigate the expression, clinical signifi cance and biological function of miR-105-5 p in gastric cancer(GC), as well as its potential action mechanism.Methods: The expressions of miR-105-5 p in GC and tumor adjacent tissues as well as in different GC cell lines(MGC 803, MKN 1, SGC 7901, BGC 823 and AGS) and normal gastric mucosal cell line(GES-1) were determined by real-time PCR. Th e relations of miR-105-5 p expression with the clinicopathologic features and prognosis of GC patients were analyzed. Th e eff ects of miR-105-5 p on migration, invasion and proliferation of GC cells were examined by Transwell migration and invasion assay and MTT assay, respectively. Th e potential target gene for miR-105-5 p was predicted by using Bioinformatics tools, and then, the correlation between miR-105-5 p and its target gene was verifi ed by Luciferase reporter assay and Western blot, respectively. Results: Th e expression of miR-105-5 p in GC tissues was signifi cantly higher than that in tumor adjacent tissue, and in each studied GC cell line was signifi cantly higher than that in normal gastric mucosal cell line(all P<0.05). The expression of miR-105-5 p was significantly associated with tumor size(P=0.020) and distant metastasis(P=0.004); the overall survival rate in GC patients with low miR-105-5 p expression was significantly higher than that in GC patients with high miR-105-5 p expression(P=0.001 8). The BGC-823 cells with a relatively low miR-105-5 p expression and the MKN 1 cells with a relatively high miR-105-5 p expression were transfected with miR-105-5 p mimics and miR-105-5 p inhibitors respectively, and the results aft er transfection showed that the migration, invasion and proliferative abilities in BGC-823 cells were significantly increased, while were significantly decreased in MKN 1 cells(all P<0.05). Bioinformatics analysis suggested that DIRAS family GTPase 3(DIRAS3) was possibly be the target of miR-105-5 p; luciferase reporter assay indicated that the luciferase activity of DIRAS3-3'-UTR was negatively regulated by miR-105-5 p; Western blot demonstrated that the DIRAS3 expression was significantly down-regulated in BGC-823 after transfection with miR-105-5 p mimics, and was significantly up-regulated in MKN 1 cells after transfection with miR-105-5 p inhibitors(both P<0.05).Conclusion: The expression of miR-105-5 p is elevated in GC, promotes migration, which can enhance the migration, invasion and proliferative abilities of GC cells probably through targeting DIRAS3, and thereby promote the occurrence and development of GC.
引文
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[17]Chen S,Wu J,Jiao K,et al.MicroRNA-495-3p inhibits multidrug resistance by modulating autophagy through GRP78/mTOR axis in gastric cancer[J].Cell Death Dis,2018,9(11):1070.doi:10.1038/s41419-018-0950-x.
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[21]Hu Y,Yang H,Lu XQ,et al.ARHI suppresses pancreatic cancer by regulating MAPK/ERK 1/2 pathway[J].Pancreas,2015,44(2):342-343.doi:10.1097/MPA.0000000000000274.
[22]Lu Z,Yang H,Sutton MN,et al.ARHI(DIRAS3)induces autophagy in ovarian cancer cells by downregulating the epidermal growth factor receptor,inhibiting PI3K and Ras/MAP signaling and activating the FOXo3a-mediated induction of Rab7[J].Cell Death Differ,2014,21(8):1275-1289.doi:10.1038/cdd.2014.48.
[23]Qiu J,Li X,He Y,et al.Distinct subgroup of the Ras family member3(DIRAS3)expression impairs metastasis and induces autophagy of gastric cancer cells in mice[J].J Cancer Res Clin Oncol,2018,144(10):1869-1886.doi:10.1007/s00432-018-2708-3.
[24]Tang HL,Hu YQ,Qin XP,et al.Aplasia ras homolog member Iis downregulated in gastric cancer and silencing its expression promotes cell growth in vitro[J].J Gastroenterol Hepatol,2012,27(8):1395-1404.doi:10.1111/j.1440-1746.2012.07146.x.
[25]Wang W,Bu XM,Wang J,et al.The expression of ARHI in pT2a and pT2b stage gastric cancer and its clinical signifi cance[J].Oncol Rep,2012,27(6):1953-1959.doi:10.3892/or.2012.1727.
[2]Einama T,Abe H,Shichi S,et al.Long-term survival and prognosis associated with conversion surgery in patients with metastatic gastric cancer[J].Mol Clin Oncol,2017,6(2):163-166.doi:10.3892/mco.2017.1128.
[3]吴永丰,刘兴洲,柳东,等.血管导向淋巴清扫术在腹腔镜胃癌根治术中的应用价值[J].中国普通外科杂志,2018,27(12):1589-1596.doi:10.7659/j.issn.1005-6947.2018.12.015.Wu YF,Liu XZ,Liu D,et al.Application value of vessel-guided lymph node dissection in laparoscopic radical gastrectomy for gastric cancer[J].Chinese Journal of General Surgery,2018,27(12):1589-1596.doi:10.7659/j.issn.1005-6947.2018.12.015.
[4]殷凯,瞿建国,陈吉祥,等.胃癌神经侵犯的相关临床病理因素:单中心509例分析[J].中国普通外科杂志,2018,27(4):396-400.doi:10.3978/j.issn.1005-6947.2018.04.002.Yin K,Qu JG,Chen JX,et al.Clinicopathologic factors related to perineural invasion of gastric cancer:analysis of 509 cases in a single center[J].Chinese Journal of General Surgery,2018,27(4):396-400.doi:10.3978/j.issn.1005-6947.2018.04.002.
[5]Beom SH,Choi YY,Baek SE,et al.Multidisciplinary treatment for patients with stage IV gastric cancer:the role of conversion surgery following chemotherapy[J].BMC Cancer,2018,18(1):1116.doi:10.1186/s12885-018-4998-x.
[6]Lochman P,Koci J,Paral J.Quality of life after proximal gastrectomy a review[J].Rozhl Chir,2018,97(8):368-372.
[7]Haider MT,Taipaleenmaki H.Targeting the Metastatic Bone Microenvironment by MicroRNAs[J].Front Endocrinol(Lausanne),2018,9:202.doi:10.3389/fendo.2018.00202.
[8]Yuan HL,Wang T,Zhang KH.MicroRNAs as potential biomarkers for diagnosis,therapy and prognosis of gastric cancer[J].Onco Targets Ther,2018,11:3891-3900.doi:10.2147/OTT.S156921.
[9]房锋,宋天强.微小RNA在肝细胞癌中的相关研究进展[J].中国普通外科杂志,2018,27(7):899-909.doi:10.3978/j.issn.1005-6947.2018.07.015.Fang F,Song TQ.Research progress associated with microRNAs in hepatocellular carcinoma[J].Chinese Journal of General Surgery,2018,27(7):899-909.doi:10.3978/j.issn.1005-6947.2018.07.015.
[10]Anfossi S,Fu X,Nagvekar R,et al.MicroRNAs,Regulatory Messengers Inside and Outside Cancer Cells[J].Adv Exp Med Biol,2018,1056:87-108.doi:10.1007/978-3-319-74470-4_6.
[11]王宇锋,刘志奎,姚博文,等.肝癌细胞中miR-376c的表达及其与高迁移率族蛋白A2的关系[J].中国普通外科杂志,2017,26(7):861-869.doi:10.3978/j.issn.1005-6947.2017.07.008.Wang YF,Liu ZK,Yao BW,et al.Expression of miR-376c in hepatocellular carcinoma cells and its relation with high mobility group A2[J].Chinese Journal of General Surgery,2017,26(7):861-869.doi:10.3978/j.issn.1005-6947.2017.07.008.
[12]Shen Z,Zhou R,Liu C,et al.MicroRNA-105 is involved in TNF-alpha-related tumor microenvironment enhanced colorectal cancer progression[J].Cell Death Dis,2017,8(12):3213.doi:10.1038/s41419-017-0048-x.
[13]Shen G,Rong X,Zhao J,et al.MicroRNA-105 suppresses cell proliferation and inhibits PI3K/AKT signaling in human hepatocellular carcinoma[J].Carcinogenesis,2014,35(12):2748-2755.doi:10.1093/carcin/bgu208.
[14]Liu X,Wang H,Zhu Z,et al.MicroRNA-105 targets SOX9 and inhibits human glioma cell progression[J].FEBS Lett,2016,590(23):4329-4342.doi:10.1002/1873-3468.12458.
[15]Honeywell DR,Cabrita MA,Zhao H,et al.miR-105 inhibits prostate tumour growth by suppressing CDK6 levels[J].PLoS One,2013,8(8):e70515.doi:10.1371/journal.pone.0070515.
[16]Guo H,Ji F,Zhao X,et al.MicroRNA-371a-3p promotes progression of gastric cancer by targeting TOB1[J].Cancer Lett,2019,443:179-188.doi:10.1016/j.canlet.2018.11.021.
[17]Chen S,Wu J,Jiao K,et al.MicroRNA-495-3p inhibits multidrug resistance by modulating autophagy through GRP78/mTOR axis in gastric cancer[J].Cell Death Dis,2018,9(11):1070.doi:10.1038/s41419-018-0950-x.
[18]Yang N,Zhu S,Lv X,et al.MicroRNAs:Pleiotropic Regulators in the Tumor Microenvironment[J].Front Immunol,2018,9:2491.doi:10.3389/fi mmu.2018.02491.
[19]Yu Y,Fujii S,Yuan J,et al.Epigenetic regulation of ARHI in breast and ovarian cancer cells[J].Ann N Y Acad Sci,2003,983:268-277.
[20]Ye K,Wang S,Yang Y,et al.Aplasia Ras homologue member overexpression inhibits tumor growth and induces apoptosis through inhibition of PI3K/Akt survival pathways in human osteosarcoma MG-63 cells in culture[J].Int J Mol Med,2015,36(3):776-782.doi:10.3892/ijmm.2015.2278.
[21]Hu Y,Yang H,Lu XQ,et al.ARHI suppresses pancreatic cancer by regulating MAPK/ERK 1/2 pathway[J].Pancreas,2015,44(2):342-343.doi:10.1097/MPA.0000000000000274.
[22]Lu Z,Yang H,Sutton MN,et al.ARHI(DIRAS3)induces autophagy in ovarian cancer cells by downregulating the epidermal growth factor receptor,inhibiting PI3K and Ras/MAP signaling and activating the FOXo3a-mediated induction of Rab7[J].Cell Death Differ,2014,21(8):1275-1289.doi:10.1038/cdd.2014.48.
[23]Qiu J,Li X,He Y,et al.Distinct subgroup of the Ras family member3(DIRAS3)expression impairs metastasis and induces autophagy of gastric cancer cells in mice[J].J Cancer Res Clin Oncol,2018,144(10):1869-1886.doi:10.1007/s00432-018-2708-3.
[24]Tang HL,Hu YQ,Qin XP,et al.Aplasia ras homolog member Iis downregulated in gastric cancer and silencing its expression promotes cell growth in vitro[J].J Gastroenterol Hepatol,2012,27(8):1395-1404.doi:10.1111/j.1440-1746.2012.07146.x.
[25]Wang W,Bu XM,Wang J,et al.The expression of ARHI in pT2a and pT2b stage gastric cancer and its clinical signifi cance[J].Oncol Rep,2012,27(6):1953-1959.doi:10.3892/or.2012.1727.