干扰miR-21调控人前列腺癌细胞株PC-3增殖、迁移及侵袭的实验研究
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摘要
目的观察干扰微小RNA-21(miR-21)对人前列腺癌细胞株PC-3增殖、迁移及侵袭的影响,并初步探讨其调控机制。方法收集对数期生长PC-3细胞株及人前列腺正常上皮细胞株RWPE-1,采用实时荧光定量聚合酶链反应(RT-PCR)法检测并对比其miR-21基因表达情况。取对数期生长PC-3细胞株,采用脂质体转染法将miR-21抑制剂(inhibitor)及NC inhibitor质粒转染至PC-3细胞,分别设为干扰组和空载组,另取不做处理PC-3细胞为空白组。采用倒置荧光显微镜检测转染效率;采用MTT法检测并对比各组不同时刻吸光度(OD值);采用划痕试验及Transwell试验检测并对比三组穿膜细胞数及迁移率;采用RT-PCR法检测并对比三组miR-21、基质金属蛋白酶组织抑制因子3(TIMP3)、基质金属蛋白酶-9(MMP-9)基因表达水平;采用蛋白免疫印迹法检测并对比三组TIMP3、MMP-9蛋白表达水平。结果 PC-3细胞miR-21相对表达量显著低于RWPE-1细胞(P <0. 05);倒置显微镜下检测转染24 h后干扰组和空载组转染效率分别为(82. 16±8. 20)%、(80. 23±8. 69)%;干扰组MTT实验不同时刻OD值均显著低于空白组和空载组(P <0. 05),空白组与空载组比较,差异均无统计学意义(P> 0. 05),三组MTT实验OD值均随时间延长呈显著升高趋势(P <0. 05);干扰组12 h、24 h迁移率均显著低于空白组和空载组(P <0. 05),空白组与空载组比较,差异均无统计学意义(P> 0. 05),三组24 h迁移率显著高于12 h(P <0. 05);干扰组穿膜细胞数显著少于空白组和空载组(P <0. 05),空白组与空载组比较,差异均无统计学意义(P> 0. 05);干扰组miR-21相对表达量、MMP-9 mRNA和蛋白相对表达量显著低于空白组和空载组(P <0. 05),空白组与空载组比较,差异均无统计学意义(P> 0. 05);干扰组TIMP3 mRNA和蛋白相对表达量显著高于空白组和空载组(P <0. 05),空白组与空载组比较,差异均无统计学意义(P> 0. 05)。结论前列腺癌细胞miR-21异常高表达,干扰miR-21可抑制人前列腺癌细胞株PC-3增殖、迁移及侵袭能力,可能与上调TIMP3 mRNA和蛋白、下调MMP-9 mRNA和蛋白有关。
Objective To observe the effect of microRNA-21( miR-21) on the proliferation,migration and invasion of human prostate cancer cell line PC-3,and to explore its regulatory mechanism. Methods The logarithmic growth of PC-3 cell line and human prostate normal epithelial cell line RWPE-1 were collected,and the expression of miR-21 gene was detected by real-time fluorescent quantitative polymerase chain reaction( RT-PCR). PC-3 cell line was grown in log phase,and the plasmid of carrying miR-21 inhibitor and NC inhibitor were transfected into PC-3 cells by lipofection,and they were set as interference group and empty group,respectively. The PC-3 cells were treated as blank group. Transfection efficiency was detected by inverted fluorescence microscope. The absorbance( OD value) of each group at different moments were detected and compared by MTT method. The number of transmembrane cells and the mobility of the 3 groups were tested by scratch test and Transwell test. The expressions of miR-21,tissue inhibitor of metalloproteinase-3( TIMP3) and matrix metalloproteinase-9( MMP-9)in the 3 groups were detected by RT-PCR and compared. The expressions of TIMP3 and MMP-9 proteins in the 3 groups were detected by Western blotting and compared. Results The relative expression of miR-21 in PC-3 cells was significantly lower than that in RWPE-1 cells( P< 0. 05). The transfection efficiency of the interference group and the empty group was( 82. 16 ± 8. 20) % and( 80. 23 ± 8. 69) % after transfection under inverted microscope for 24 h. The OD values of the MTT assay at different moments in the interference group were significantly lower than those in the blank and empty groups( P < 0. 05),while there was no significant difference between the blank group and the empty group( P > 0. 05). The OD values of the MTT assays increased significantly with time( P < 0. 05). The mobility of the interference group at 12 h and24 h were significantly lower than those of the blank group and the empty group( P < 0. 05),while there was no significant difference between the blank group and the empty group( P > 0. 05),and the mobility of the 3 groups at 24 h was significantly higher than that at 12 h( P < 0. 05).The number of transmembrane cells in the interference group was significantly lower than that in the blank group and the empty group( P < 0. 05).There was no significant difference between the blank group and the empty group( P > 0. 05). The relative expressions of miR-21 and MMP-9 mRNA and protein in the interference group were significantly lower than those in the blank group and the empty group( P < 0. 05). There was no significant difference between the blank group and the empty group( P > 0. 05). The relative expressions of TIMP3 mRNA and protein in the interference group were significantly higher than those in the blank group and the empty group( P < 0. 05). There was no significant difference between the blank group and the empty group( P > 0. 05). Conclusion Abnormally high expression of miR-21 in prostate cancer cells,interfering with miR-21 can inhibit the proliferation,migration and invasion of human prostate cancer cell line PC-3,which may be related to up-regulation of TIMP3 mRNA and protein and down-regulation of MMP-9 mRNA and protein.
Objective To observe the effect of microRNA-21( miR-21) on the proliferation,migration and invasion of human prostate cancer cell line PC-3,and to explore its regulatory mechanism. Methods The logarithmic growth of PC-3 cell line and human prostate normal epithelial cell line RWPE-1 were collected,and the expression of miR-21 gene was detected by real-time fluorescent quantitative polymerase chain reaction( RT-PCR). PC-3 cell line was grown in log phase,and the plasmid of carrying miR-21 inhibitor and NC inhibitor were transfected into PC-3 cells by lipofection,and they were set as interference group and empty group,respectively. The PC-3 cells were treated as blank group. Transfection efficiency was detected by inverted fluorescence microscope. The absorbance( OD value) of each group at different moments were detected and compared by MTT method. The number of transmembrane cells and the mobility of the 3 groups were tested by scratch test and Transwell test. The expressions of miR-21,tissue inhibitor of metalloproteinase-3( TIMP3) and matrix metalloproteinase-9( MMP-9)in the 3 groups were detected by RT-PCR and compared. The expressions of TIMP3 and MMP-9 proteins in the 3 groups were detected by Western blotting and compared. Results The relative expression of miR-21 in PC-3 cells was significantly lower than that in RWPE-1 cells( P< 0. 05). The transfection efficiency of the interference group and the empty group was( 82. 16 ± 8. 20) % and( 80. 23 ± 8. 69) % after transfection under inverted microscope for 24 h. The OD values of the MTT assay at different moments in the interference group were significantly lower than those in the blank and empty groups( P < 0. 05),while there was no significant difference between the blank group and the empty group( P > 0. 05). The OD values of the MTT assays increased significantly with time( P < 0. 05). The mobility of the interference group at 12 h and24 h were significantly lower than those of the blank group and the empty group( P < 0. 05),while there was no significant difference between the blank group and the empty group( P > 0. 05),and the mobility of the 3 groups at 24 h was significantly higher than that at 12 h( P < 0. 05).The number of transmembrane cells in the interference group was significantly lower than that in the blank group and the empty group( P < 0. 05).There was no significant difference between the blank group and the empty group( P > 0. 05). The relative expressions of miR-21 and MMP-9 mRNA and protein in the interference group were significantly lower than those in the blank group and the empty group( P < 0. 05). There was no significant difference between the blank group and the empty group( P > 0. 05). The relative expressions of TIMP3 mRNA and protein in the interference group were significantly higher than those in the blank group and the empty group( P < 0. 05). There was no significant difference between the blank group and the empty group( P > 0. 05). Conclusion Abnormally high expression of miR-21 in prostate cancer cells,interfering with miR-21 can inhibit the proliferation,migration and invasion of human prostate cancer cell line PC-3,which may be related to up-regulation of TIMP3 mRNA and protein and down-regulation of MMP-9 mRNA and protein.
引文
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[14]李振辉,韩俊岭,师磊,等.人膀胱癌5637细胞中miR-21表达及其对细胞增殖和侵袭的影响[J].山东医药,2016,56(37):8-10.
[15]Huang X,Shen W,Xi H,et al.Prognostic role of extracellular matrix metalloproteinase inducer/CD147 in gastrointestinal cancer:a meta-analysis of related studies[J].Oncotarget,2016,7(49):81003-81011.
[16]Hu J,Ni S,Cao Y,et al.The Angiogenic Effect of microRNA-21Targeting TIMP3 through the Regulation of MMP2 and MMP9[J].PLo SOne,2016,11(2):149-153.
[17]梁亮,张寅斌,张扬,等.miR-21影响宫颈癌He La细胞生长和凋亡的机制探讨[J].现代肿瘤医学,2016,24(19):3005-3009.
[18]Petrovi!c N.miR-21 Might be Involved in Breast Cancer Promotion and Invasion Rather than in Initial Events of Breast Cancer Development[J].Mol Diagn Ther,2016,20(2):97-110.
[19]Wu ZH,Tao ZH,Zhang J,et al.MiRNA-21 induces epithelial to mesenchymal transition and gemcitabine resistance via the PTEN/AKTpathway in breast cancer[J].Tumour Biol,2016,37(6):7245-7254.
[2]梁朝朝,周骏,邰胜,等.达芬奇机器人辅助腹腔镜前列腺癌根治术69例报告[J].临床泌尿外科杂志,2016,31(1):23-25.
[3]Shi J.Considering Exosomal miR-21 as a Biomarker for Cancer[J].JClin Med,2016,5(4):152-159.
[4]Ohno R,Uozaki H,Kikuchi Y,et al.Both cancerous miR-21 and stromal miR-21 in urothelial carcinoma are related to tumour progression[J].Histopathology,2016,69(6):993-999.
[5]Kuang Y,Nie YJ.Exploration of the regulatory effect of miR-21 on breast cancer cell line proliferation and invasion as well as the downstream target genes[J].Asian Pac J Trop Med,2016,9(5):470-473.
[6]Eeles R,Goh C,Castro E,et al.The genetic epidemiology of prostate cancer and its clinical implications[J].Nat Rev Urol,2014,11(1):18-31.
[7]赵强,杨勇,李莉,等.局部治疗对激素敏感性转移性前列腺癌预后影响的Meta分析[J].中华泌尿外科杂志,2017,38(10):54-58.
[8]Backes C,Meese E,Keller A.Specific miRNA Disease Biomarkers in Blood,Serum and Plasma:Challenges and Prospects[J].Mol Diagn T-her,2016,20(6):509-518.
[9]Zhou B,Wang J,Zheng G,et al.Methylated urolithin A,the modified ellagitannin-derived metabolite,suppresses cell viability of DU145 human prostate cancer cells via targeting miR-21[J].Food Chem Toxicol,2016,97(1):375-384.
[10]吕光耀,付启忠,刘颖,等.miR-21在前列腺癌中的表达及抑制其表达对前列腺癌细胞增殖凋亡的影响[J].临床和实验医学杂志,2018,17(3):280-284.
[11]Xue X,Liu Y,Wang Y,et al.MiR-21 and MiR-155 promote non-small cell lung cancer progression by downregulating SOCS1,SOCS6,and PTEN[J].Oncotarget,2016,7(51):84508-84519.
[12]Bruhn O,Drerup K,Kaehler M,et al.Length variants of the ABCB1 3'-UTR and loss of miRNA binding sites:possible consequences in regulation and pharmacotherapy resistance[J].Pharmacogenomics,2016,17(4):327-340.
[13]杨朝晖,章辉,顾华敏,等.宫颈癌组织中miR-21和Stat3的表达及相关性研究[J].浙江医学,2017,39(2):93-96.
[14]李振辉,韩俊岭,师磊,等.人膀胱癌5637细胞中miR-21表达及其对细胞增殖和侵袭的影响[J].山东医药,2016,56(37):8-10.
[15]Huang X,Shen W,Xi H,et al.Prognostic role of extracellular matrix metalloproteinase inducer/CD147 in gastrointestinal cancer:a meta-analysis of related studies[J].Oncotarget,2016,7(49):81003-81011.
[16]Hu J,Ni S,Cao Y,et al.The Angiogenic Effect of microRNA-21Targeting TIMP3 through the Regulation of MMP2 and MMP9[J].PLo SOne,2016,11(2):149-153.
[17]梁亮,张寅斌,张扬,等.miR-21影响宫颈癌He La细胞生长和凋亡的机制探讨[J].现代肿瘤医学,2016,24(19):3005-3009.
[18]Petrovi!c N.miR-21 Might be Involved in Breast Cancer Promotion and Invasion Rather than in Initial Events of Breast Cancer Development[J].Mol Diagn Ther,2016,20(2):97-110.
[19]Wu ZH,Tao ZH,Zhang J,et al.MiRNA-21 induces epithelial to mesenchymal transition and gemcitabine resistance via the PTEN/AKTpathway in breast cancer[J].Tumour Biol,2016,37(6):7245-7254.