靶向调控miR-21-5p上调TIMP3抑制膀胱癌SCSP-571细胞ECM和EMT的机制研究
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摘要
目的探讨靶向调控miR-21-5p上调金属蛋白酶组织抑制因子(TIMP3)抑制膀胱癌细胞外基质降解(ECM)和上皮间质转化(EMT)的作用效果与机制。方法 96孔板培养膀胱癌SCSP-571细胞,分对照组、空载质粒组、miR-21-5p沉默组、TIMP3沉默组、miR-21-5p+TIMP3沉默组。对照组常规培养,空载质粒组加空载质粒共培养,miR-21-5p沉默组加入miR-21-5p inhibitor;TIMP3沉默组加入TIMP3-shRNA慢病毒感染质粒;miR-21-5p+TIMP3沉默组同时加入miR-21-5p inhibitor和TIMP3-shRNA慢病毒感染质粒。培养72 h,采用荧光定量PCR实验测定各组miR-21-5p和TIMP3的mRNA表达,WB实验测定各组ECM和EMT标志物的表达,Transwell小室实验测定各组细胞迁移能力和侵袭能力。结果与对照组相比,miR-21-5p沉默组E钙粘蛋白和TIMP3蛋白表达水平升高,同时N钙粘蛋白、纤维蛋白、MMP-2、MMP-7、MMP-9蛋白表达水平,细胞迁移与侵袭能力降低;TIMP3沉默组E钙粘蛋白和TIMP3蛋白表达水平降低,同时N钙粘蛋白、纤维蛋白、MMP-2、MMP-7、MMP-9蛋白表达水平,细胞迁移与侵袭能力升高,差异有统计学意义(均P<0.05)。miR-21-5p+TIMP3沉默组的各项指标介于miR-21-5p沉默组与TIMP3沉默组之间,差异有统计学意义(P<0.05)。结论靶向调控miR-21-5p能够起到抑制膀胱癌细胞侵袭与转移的效果,作用机制可能与提高了TIMP3表达,从而抑制了MMPs介导的ECM与EMT有关。
Objective To investigate the effect and mechanism of up-regulating metalloproteinase tissue inhibitors(TIMP3) by targeted regulation of miR-21-5 p to suppress matrix degradation(ECM) and epithelial mesenchymal transformation(EMT) in bladder cancer cells. Methods Bladder cancer SCSP-571 cells were cultured on 96-well plates and divided into Control group, Plasmid group, miR-21-5 p silencing group, TIMP3 silencing group and miR-21-5 p+TIMP3 silencing group. The cells in Control group were cultured as normal, cells in Plasmid group were co-cultured with plasma, cells in miR-21-5 p silencing group were co-cultured with miR-21-5 p inhibitor, cells in TIMP3 silencing group were co-cultured with TIMP3-shRNA plasma, and cells in miR-21-5 p+TIMP3 silencing group were co-cultured with both miR-21-5 p inhibitor and TIMP3-shRNA plasma. When cells were cultured for 72 h, the mRNA expression of miR-21-5 p and TIMP3 in each group was determined by fluorescence quantitative PCR and the expression of ECM and EMT markers in each group was determined by WB experiment. Cell migration and invasion were measured by Transwell assay. Results Compared with Control group and Plasmid group, the expression levels of E cadherin and TIMP3 in miR-21-5 p silencing group were increased, while the expression levels of N cadherin, fibrin, MMP-2, MMP-7 and MMP-9 were decreased, and the cell migration and invasion count were decreased. The expression levels of E cadherin and TIMP3 in TIMP3 silencing group were decreased, while the expression levels of N cadherin, fibrin, MMP-2, MMP-7 and MMP-9 were decreased, and cell migration and invasion were increased. The difference had statistical significance(P<0.05). The indexes of miR-21-5 p+TIMP3 silencing group were between miR-21-5 p silencing group and TIMP3 silencing group, and the differences were statistically significant(P<0.05).Conclusion The targeted regulation of miR-21-5 p could inhibit the invasion and metastasis of SCSP-571 cells, and the mechanism may be related to the improvement of TIMP3 expression, thereby inhibiting the MMPs mediated ECM and EMT.
Objective To investigate the effect and mechanism of up-regulating metalloproteinase tissue inhibitors(TIMP3) by targeted regulation of miR-21-5 p to suppress matrix degradation(ECM) and epithelial mesenchymal transformation(EMT) in bladder cancer cells. Methods Bladder cancer SCSP-571 cells were cultured on 96-well plates and divided into Control group, Plasmid group, miR-21-5 p silencing group, TIMP3 silencing group and miR-21-5 p+TIMP3 silencing group. The cells in Control group were cultured as normal, cells in Plasmid group were co-cultured with plasma, cells in miR-21-5 p silencing group were co-cultured with miR-21-5 p inhibitor, cells in TIMP3 silencing group were co-cultured with TIMP3-shRNA plasma, and cells in miR-21-5 p+TIMP3 silencing group were co-cultured with both miR-21-5 p inhibitor and TIMP3-shRNA plasma. When cells were cultured for 72 h, the mRNA expression of miR-21-5 p and TIMP3 in each group was determined by fluorescence quantitative PCR and the expression of ECM and EMT markers in each group was determined by WB experiment. Cell migration and invasion were measured by Transwell assay. Results Compared with Control group and Plasmid group, the expression levels of E cadherin and TIMP3 in miR-21-5 p silencing group were increased, while the expression levels of N cadherin, fibrin, MMP-2, MMP-7 and MMP-9 were decreased, and the cell migration and invasion count were decreased. The expression levels of E cadherin and TIMP3 in TIMP3 silencing group were decreased, while the expression levels of N cadherin, fibrin, MMP-2, MMP-7 and MMP-9 were decreased, and cell migration and invasion were increased. The difference had statistical significance(P<0.05). The indexes of miR-21-5 p+TIMP3 silencing group were between miR-21-5 p silencing group and TIMP3 silencing group, and the differences were statistically significant(P<0.05).Conclusion The targeted regulation of miR-21-5 p could inhibit the invasion and metastasis of SCSP-571 cells, and the mechanism may be related to the improvement of TIMP3 expression, thereby inhibiting the MMPs mediated ECM and EMT.
引文
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[16] XING S,YU W,ZHANG X,et al.Isoviolanthin Extracted from Dendrobium officinale Reverses TGF-beta1-Mediated Epithelial(-)Mesenchymal Transition in Hepatocellular Carcinoma Cells via Deactivating the TGF-beta/Smad and PI3K/Akt/mTOR Signaling Pathways[J].Int J Mol Sci,2018,19(6):E1556.
[17] 杨玉帛,冯德超,王晓明,等.膀胱癌上皮间质转化的研究进展[J].临床泌尿外科杂志,2018,(11):919-924,928.
[2] 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.
[3] KRIEBEL S,SCHMIDT D,HOLDENRIEDER S,et al.Analysis of tissue and serum microRNA expression in patients with upper urinary tract urothelial cancer[J].PLoS One,2015,10(1):e0117284.
[4] SMITH D A,NEWBURY L J,DRAGO G,et al.Electrochemical detection of urinary microRNAs via sulfonamide-bound antisense hybridisation[J].Sens Actuators B,Chem,2017,253(1):335-341.
[5] GHORBANMEHR N,GHARBI S,KORSCHING E,et al.miR-21-5p,miR-141-3p,and miR-205-5p levels in urine-promising biomarkers for the identification of prostate and bladder cancer[J].Prostate,2019,79(1):88-95.
[6] 王婵娟,朱熹.EMT介导膀胱癌耐药的机制及相关分子[J].肿瘤学杂志,2018,24(8):813-817.
[7] STRZELCZYK J K,KRAKOWCZYK L,OWCZAREK A J.Aberrant DNA methylation of the p16,APC,MGMT,TIMP3 and CDH1 gene promoters in tumours and the surgical margins of patients with oral cavity cancer[J].J Cancer,2018,9(11):1896-1904.
[8] WANG K,ZHAO S,LIU B,et al.Perturbations of BMP/TGF-beta and VEGF/VEGFR signalling pathways in non-syndromic sporadic brain arteriovenous malformations (BAVM)[J].J Med Genet,2018,55(10):675-684.
[9] MALEVA KOSTOVSKA I,JAKIMOVSKA M,POPOVSKA-JANKOVIC K,et al.TIMP3 Promoter Methylation Represents an Epigenetic Marker of BRCA1ness Breast Cancer Tumours[J].Pathol Oncol Res,2018,24(4):937-940.
[10] TONG Z,LIU Y,CHEN B,et al.Association between MMP3 and TIMP3 polymorphisms and risk of osteoarthritis[J].Oncotarget,2017,8(48):83563-83569.
[11] 王刚,杨涛,彭克楠,等.Twist对膀胱癌细胞迁移、侵袭及MMP-2、MMP-9表达的影响[J].中国老年学杂志,2018,38(10):2439-2442.
[12] 徐海波,熊异平,余恒勇.膀胱癌组织中MMP-2、MMP-9、Survivin、Livin和VEGF表达的临床意义分析[J].标记免疫分析与临床,2017,24(1):51-54.
[13] 钟鹏,杨川.血清P53、MMP-7、MMP-9在膀胱癌患者中的表达及意义[J].国际检验医学杂志,2018,39(9):1052-1055.
[14] LI Q,WANG C,WANG Y,et al.HSCs-derived COMP drives hepatocellular carcinoma progression by activating MEK/ERK and PI3K/AKT signaling pathways[J].J Exp Clin Cancer Res,2018,37(1):231.
[15] WANG X,WANG B,XIE J,et al.Melatonin inhibits epithelialtomesenchymal transition in gastric cancer cells via attenuation of IL1beta/NFkappaB/MMP2/MMP9 signaling[J].Int J Mol Med,2018,42(4):2221-2228.
[16] XING S,YU W,ZHANG X,et al.Isoviolanthin Extracted from Dendrobium officinale Reverses TGF-beta1-Mediated Epithelial(-)Mesenchymal Transition in Hepatocellular Carcinoma Cells via Deactivating the TGF-beta/Smad and PI3K/Akt/mTOR Signaling Pathways[J].Int J Mol Sci,2018,19(6):E1556.
[17] 杨玉帛,冯德超,王晓明,等.膀胱癌上皮间质转化的研究进展[J].临床泌尿外科杂志,2018,(11):919-924,928.