miR-1271-5p在肾细胞癌组织和细胞中的表达及其对A-498细胞增殖及凋亡的影响
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摘要
目的:探讨miR-1271-5p在肾细胞癌(renal cell carcinoma,RCC)组织和细胞系中的表达及其对RCC细胞株A-498增殖及凋亡的影响。方法:用实时荧光定量PCR(q PCR)检测手术切除并经病理确诊为RCC组织和癌旁组织,以及RCC细胞系ACHN、A498、HK-2、786-O、Ca Ki-1和人胚肾细胞株HEK293中miR-1271-5p的表达水平。用miR-1271-5p(实验组)和miR-NC(对照组)分别转染A-498细胞。通过生物信息学预测鸟嘌呤交换因子DOCK1为miR-1271-5p可能的靶基因,构建DOCK1基因的3’UTR野生型及突变体序列双荧光素酶报告基因载体并进行荧光素酶活性检测,q PCR检测两组细胞中DOCK1 m RNA表达水平,Western blotting检测两组细胞中DOCK1、p-ERK、p-AKT、Bcl-2和Bax蛋白的表达情况,MTS法、集落形成实验和流式细胞术检测A-489细胞增殖、集落形成数目和凋亡情况。结果:RCC组织和细胞系中miR-1271-5p表达水平显著低于癌旁组织和人胚肾HEK293细胞(均P<0.01)。双荧光素酶报告基因系统结果显示DOCK1是miR-1271-5p的靶基因(P<0.01)。与miR-NC组细胞相比,miR-1271-5p组A-498细胞中DOCK1 m RNA的表达水平显著下降(P<0.01);DOCK1、p-ERK、p-AKT、Bcl-2蛋白表达水平显著下调(P<0.05),Bax蛋白明显上调(P<0.05);A-498细胞增殖活力显著降低(P<0.01);集落形成数显著减少(P<0.05);细胞凋亡率显著增高(P<0.01)。结论:RCC组织和细胞系中miR-1271-5p表达下调,通过干扰DOCK1基因表达能明显抑制A-489细胞的增殖及诱导其凋亡,miR-1271-5p可能成为未来RCC治疗的分子靶标。
Objective:To investigate the expression of miR-1271-5 p in renal cell carcinoma(RCC)tissues and cell lines and its effect on the proliferation and apoptosis of RCC A-498 cell line.Methods:Pathologically confirmed RCC tissues and para-cancerous tissues were collected.Real-time fluorescent quantitative PCR(q PCR)was used to analyze the expression of miR-1271-5 p in collected RCC tissues and RCC cell lines(ACHN,A498,HK-2,786-O,Ca Ki-1) as well as human embryonic kidney cell line HEK293.miR-1271-5 p(experiment group) and miR-NC(control group) were transfected into A-498 cells,respectively.Bioinformatics predicts that DOCK1 is a possible target gene for miR-1271-5 p.Double luciferase reporter gene vector of DOCK1 gene 3 'UTR(both wild and mutant type)were constructed and the luciferase activity was detected.The expression of DOCK1 m RNA was detected by q PCR.Western blotting was used to analyze the protein expressions of DOCK1,p-ERK,p-AKT,Bcl-2 and Bax in two groups of cells.MTS assay and colony formation assay were performed to detect cell viability and proliferation.Cell apoptosis was analyzed by flow cytometry.Results:Compared with para-cancerous tissue and human embryonic kidney cells,the expression of miR-1271-5 p was significantly decreased in RCC tissues and cell lines(P<0.01).Double luciferase reporter gene system showed that DOCK1 is a target gene of miR-1271-5 p(P<0.01).Compared with miR-NC transfected cells,the expression of DOCK1 m RNA in A-498 cells transfected with miR-1271-5 p was significantly decreased(P<0.01);the protein expressions of DOCK1,p-ERK,p-AKT and Bcl-2 were significantly down-regulated(P<0.05)while the expression of Bax protein was significantly up-regulated(P<0.05);The viability and colony formation of A-498 cells was significantly decreased(all P<0.01),while apoptosis was significantly increased(P<0.01).Conclusion:miR-1271-5 p was downregulated in RCC tissues and cell lines.miR-1271-5 p significantly inhibited the proliferation and induced apoptosis of RCC A-489 cell by interfering with the expression of DOCK1 gene.This provides a theoretical basis for miR-1271-5 p as a promise molecular target in RCC treatment.
Objective:To investigate the expression of miR-1271-5 p in renal cell carcinoma(RCC)tissues and cell lines and its effect on the proliferation and apoptosis of RCC A-498 cell line.Methods:Pathologically confirmed RCC tissues and para-cancerous tissues were collected.Real-time fluorescent quantitative PCR(q PCR)was used to analyze the expression of miR-1271-5 p in collected RCC tissues and RCC cell lines(ACHN,A498,HK-2,786-O,Ca Ki-1) as well as human embryonic kidney cell line HEK293.miR-1271-5 p(experiment group) and miR-NC(control group) were transfected into A-498 cells,respectively.Bioinformatics predicts that DOCK1 is a possible target gene for miR-1271-5 p.Double luciferase reporter gene vector of DOCK1 gene 3 'UTR(both wild and mutant type)were constructed and the luciferase activity was detected.The expression of DOCK1 m RNA was detected by q PCR.Western blotting was used to analyze the protein expressions of DOCK1,p-ERK,p-AKT,Bcl-2 and Bax in two groups of cells.MTS assay and colony formation assay were performed to detect cell viability and proliferation.Cell apoptosis was analyzed by flow cytometry.Results:Compared with para-cancerous tissue and human embryonic kidney cells,the expression of miR-1271-5 p was significantly decreased in RCC tissues and cell lines(P<0.01).Double luciferase reporter gene system showed that DOCK1 is a target gene of miR-1271-5 p(P<0.01).Compared with miR-NC transfected cells,the expression of DOCK1 m RNA in A-498 cells transfected with miR-1271-5 p was significantly decreased(P<0.01);the protein expressions of DOCK1,p-ERK,p-AKT and Bcl-2 were significantly down-regulated(P<0.05)while the expression of Bax protein was significantly up-regulated(P<0.05);The viability and colony formation of A-498 cells was significantly decreased(all P<0.01),while apoptosis was significantly increased(P<0.01).Conclusion:miR-1271-5 p was downregulated in RCC tissues and cell lines.miR-1271-5 p significantly inhibited the proliferation and induced apoptosis of RCC A-489 cell by interfering with the expression of DOCK1 gene.This provides a theoretical basis for miR-1271-5 p as a promise molecular target in RCC treatment.
引文
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[3]WANG J,SONG C,TANG H,et al.mi R-629-3p may serve as a novel biomarker and potential therapeutic target for lung metastases of triplenegative breast cancer[J/OL].Breast Cancer Res,2017,19(1):72[2017-09-01].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477310/.DOI:10.1186/s13058-017-0865-y.
[4]LIU H,WANG H,LIU X,et al.mi R-1271 inhibits migration,invasion and epithelial-mesenchymal transition by targeting ZEB1 and TWIST1 in pancreatic cancer cells[J].Biochem Biophys Res Commun,2016,472(2):346-352.DOI:10.1016/j.bbrc.2016.02.096.
[5]LIU X,MA L,RAO Q,et al.Mi R-1271 Inhibits ovarian cancer growth by targeting cyclin G1[J/OL].Med Sci Monit,2015,21:3152-3158[2017-09-01].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4617187/.
[6]XIANG X J,DENG J,LIU Y W,et al.Mi R-1271 inhibits cell proliferation,invasion and EMT in gastric cancer by targeting FOXQ1[J].Cell Physiol Biochem,2015,36(4):1382-1394.DOI:10.1159/000430304.
[7]KONG D,ZHANG G,MA H,et al.mi R-1271 inhibits OSCC cell growth and metastasis by targeting ALK[J].Neoplasma,2015,62(4):559-566.DOI:10.4149/neo_2015_067.
[8]HAN X,WANG X,ZHAO B,et al.Micro RNA-187 inhibits tumor growth and metastasis via targeting of IGF-1R in hepatocellular carcinoma[J].Mol Med Rep,2017,16(2):2241-2246.DOI:10.3892/mmr.2017.6788.
[9]LIN M F,YANG Y F,PENG Z P,et al.FOXK2,regulted by mi R-1271-5p,promotes cell growth and indicates unfavorable prognosis in hepatocellular carcinoma[J/OL].Int J Biochem Cell Biol,2017,88:155-161[2017-09-01].http://sciencedirect.com/science/journal/13572725.DOI:10.1016/j.biocel.2017.05.019.
[10]LI L,QU Y W,LI Y P.Over-expression of mi R-1271 inhibits endometrial cancer cells proliferation and induces cell apoptosis by targeting CDK1[J/OL].Eur Rev Med Pharmacol Sci,2017,21(12):2816-2822[2017-09-01].http://www.europeanreview.org/article/12939.
[11]WANG M,GAO W,LU D,et al.Mi R-1271 inhibits cell growth in prostate cancer by targeting ERG[J/OL].Pathol Oncol Res,2017,2017[2017-09-01].https://link.springer.com/article/10.1007%2Fs12253-017-0254-y.DOI:10.1007/s12253-017-0254-y.
[12]WANG Y,XU L,JIANG L.mi R-1271 promotes non-small-cell lung cancer cell proliferation and invasion via targeting HOXA5[J].Biochem Biophys Res Commun,2015,458(3):714-719.https://doi.org/10.1016/j.bbrc.2015.02.033.DOI:10.1016/j.bbrc.2015.02.033.
[13]ZHANG B,LI H,YIN C,et al.Dock1 promotes the mesenchymal transition of glioma and is modulated by Mi R-31[J].Neuropathol Appl Neurobiol,2017,43(5):419-432.DOI:10.1111/nan.12321.
[14]LIU F,JIANG Y J,ZHAO H J,et al.Electroacupuncture ameliorates cognitive impairment and regulates the expression of apoptosis-related genes Bcl-2 and Bax in rats with cerebral ischaemia-reperfusion injury[J/OL].Acupunct Med,2015,33(6):478-484[2017-09-01].http://aim.bmj.com/content/33/6/478.long.DOI:10.1136/acupmed-2014-010728.
[15]WU C,GENG X,WAN S,et al.Cecropin-P17,an analog of cecropin B,inhibits human hepatocellular carcinoma cell Hep G-2 proliferation via regulation of ROS,Caspase,Bax,and Bcl-2[J].J Pept Sci,2015,21(8):661-668.DOI:10.1002/psc.2786.