miR-148b-3p在前列腺癌细胞增殖和迁移中的作用机制
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摘要
目的探讨微小RNA(miRNA,miR)-148b-3p表达对前列腺癌细胞增殖和迁移的影响机制。方法采用实时定量聚合酶链反应(qPCR)检测前列腺癌细胞株LNCap、22RV1、DU-145、PC-3和人正常前列腺上皮细胞RWPE-1中miR-148b-3p的表达。采用Lipofectamine 2000分别将miR-148b-3p inhibitor模拟物(实验组)和miR-NC(对照组)转染入PC-3细胞。qPCR检测PC-3细胞中miR-148b-3p的表达变化。CCK8法检测细胞增殖能力;Transwell实验检测细胞迁移能力。采用生物信息学技术和双荧光素酶报告基因检测实验分别预测和验证miR-148b-3p的潜在靶基因。qPCR和Western blot检测PTEN基因及CDK6、Cyclin D2、Claudin-1和Vimentin蛋白表达。结果前列腺癌细胞株LNCap、22RV1、DU-145、PC-3和人正常前列腺上皮细胞RWPE-1中miR-148b-3p的表达分别为0.18±0.08、0.60±0.10、0.44±0.07、0.55±0.13和1.02±0.24,前列腺癌细胞中miR-148b-3p表达明显降低(P<0.01)。对照组和实验组PC-3细胞中miR-148b-3p的表达量分别为1.09±0.57和72.27±13.78(P<0.001,t=10.320)。与对照组相比,细胞增殖能力和迁移能力均降低(P<0.01)。通过生物信息学技术和双荧光素酶报告基因预测并验证了PTEN为miR-148b-3p的潜在靶基因。对照组和实验组PTEN mRNA的表达量分别为1.03±0.29和4.10±1.15(P=0.002,t=5.179)。与对照组相比,实验组中PTEN和Claudin-1蛋白表达升高,CDK6、Cyclin D2和Vimentin蛋白表达降低。结论 miR-148b-3p可通过上调PTEN基因的表达抑制PC-3细胞的增殖和迁移,延缓前列腺癌的进展。
Objective To investigate the effect of miR-148b-3p low expression on the proliferation and migration of prostate cancer and its mechanism. Methods The expressions of miR-148b-3p in prostate cancer cell lines LNCap,22 RV1,DU-145,PC-3 and human normal epithelial cells RWPE-1 were detected by real-time quantitative polymerase chain reaction(qPCR). miR-148b-3p inhibitor(experimental group) and miR-NC(control group) were transfected into PC-3 cells using Lipofectamine 2000 respectively. qPCR was used to detect the expression of miR-148b-3p in PC-3 cells. CCK8 method was used to detect cell proliferation ability. Transwell assay was used to detect cell migration ability. Bioinformatics and dual luciferase reporter assay were used to predict and validate the potential target genes of miR-148b-3p respectively. The expression of PTEN gene and CDK6,Cyclin D2,Claudin-1 and Vimentin protein levels were detected by qPCR and Western blot. Results The expressions of miR-148b-3p in the prostate cancer cell lines LNCap,22 RV1,DU-145,PC-3 and human normal prostate epithelial cells RWPE-1 were 0. 18 ± 0. 08,0. 60 ± 0. 10,0. 44 ± 0. 07,0. 55 ±0. 13 and 1. 02 ± 0. 24,respectively. The expression of miR-148b-3p in prostate cancer cells was significantly increased(P < 0. 01). The expression of miR-148b-3p in control and experimental groups was 1. 09 ± 0. 57 and 72. 27 ± 13. 78,respectively(P < 0. 001,t = 10. 320). Compared with the control group,the proliferation and migration ability of cells in the experimental group decreased(P < 0. 01). Bioinformatics and dual luciferase reporter genes predicted and validated PTEN as a potential target gene for miR-148b-3p. The expression of PTEN mRNA in control group and experimental group were 1. 03 ± 0. 29 and 4. 10 ± 1. 15,respectively(P =0. 002,t = 5. 179). Compared with control group,the expression of PTEN and Claudin-1 proteins were increased in experimental group,the expression of CDK6,Cyclin D2,and Vimentin proteins were decreased. Conclusion Low expression of miR-148b-3p can inhibit the proliferation and migration of PC-3 cells and prevent the progress of prostate cancer by up-regulating the expression of PTEN gene.
Objective To investigate the effect of miR-148b-3p low expression on the proliferation and migration of prostate cancer and its mechanism. Methods The expressions of miR-148b-3p in prostate cancer cell lines LNCap,22 RV1,DU-145,PC-3 and human normal epithelial cells RWPE-1 were detected by real-time quantitative polymerase chain reaction(qPCR). miR-148b-3p inhibitor(experimental group) and miR-NC(control group) were transfected into PC-3 cells using Lipofectamine 2000 respectively. qPCR was used to detect the expression of miR-148b-3p in PC-3 cells. CCK8 method was used to detect cell proliferation ability. Transwell assay was used to detect cell migration ability. Bioinformatics and dual luciferase reporter assay were used to predict and validate the potential target genes of miR-148b-3p respectively. The expression of PTEN gene and CDK6,Cyclin D2,Claudin-1 and Vimentin protein levels were detected by qPCR and Western blot. Results The expressions of miR-148b-3p in the prostate cancer cell lines LNCap,22 RV1,DU-145,PC-3 and human normal prostate epithelial cells RWPE-1 were 0. 18 ± 0. 08,0. 60 ± 0. 10,0. 44 ± 0. 07,0. 55 ±0. 13 and 1. 02 ± 0. 24,respectively. The expression of miR-148b-3p in prostate cancer cells was significantly increased(P < 0. 01). The expression of miR-148b-3p in control and experimental groups was 1. 09 ± 0. 57 and 72. 27 ± 13. 78,respectively(P < 0. 001,t = 10. 320). Compared with the control group,the proliferation and migration ability of cells in the experimental group decreased(P < 0. 01). Bioinformatics and dual luciferase reporter genes predicted and validated PTEN as a potential target gene for miR-148b-3p. The expression of PTEN mRNA in control group and experimental group were 1. 03 ± 0. 29 and 4. 10 ± 1. 15,respectively(P =0. 002,t = 5. 179). Compared with control group,the expression of PTEN and Claudin-1 proteins were increased in experimental group,the expression of CDK6,Cyclin D2,and Vimentin proteins were decreased. Conclusion Low expression of miR-148b-3p can inhibit the proliferation and migration of PC-3 cells and prevent the progress of prostate cancer by up-regulating the expression of PTEN gene.
引文
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[23]Sekhon K,Bucay N,Majid S,et al.MicroRNAs and epithelial-mesenchymal transition in prostate cancer[J].Oncotarget,2016,7(41):67597-67611.
[2]Dai Y,Ren D,Yang Q,et al.The TGF-beta signalling negative regulator PICK1 represses prostate cancer metastasis to bone[J].Br J Cancer,2017,117(5):685-694.
[3]Chen W Y,Tsai Y C,Siu M K,et al.Inhibition of the androgen receptor induces a novel tumor promoter,ZBTB46,for prostate cancer metastasis[J].Oncogene,2017,36(45):6213-6224.
[4]Qiao W,Cao N,Yang L.MicroRNA-154 inhibits the growth and metastasis of gastric cancer cells by directly targeting MTDH[J].Oncol Lett,2017,14(3):3268-3274.
[5]王燕,张金莉,彭莉萍,等.结肠癌转移相关潜在微小RNA(miRNA)的分离与鉴定[J].遵义医学院学报,2015,38(5):479-487.
[6]Ding X,Liu J,Liu T,et al.miR-148b inhibits glycolysis in gastric cancer through targeting SLC2A1[J].Cancer Med,2017,6(6):1301-1310.
[7]Yang Z G,Ma X D,He Z H,et al.miR-483-5p promotes prostate cancer cell proliferation and invasion by targeting RBM5[J].Int Braz J Urol,2017,43(6):1060-1067.
[8]赵娟娟,徐华林,陶弋婧,等.基于SYBR GreenⅠ的茎-环Real-time PCR定量检测miRNA-7方法的建立[J].遵义医学院学报,2015,38(6):636-641.
[9]郑文,赵娟娟,何志旭,等.微小RNA-21与NSCLC放化疗抵抗的研究新进展[J].遵义医学院学报,2017,40(2):214-217.
[10]Liu Y,Xu X,Xu X,et al.MicroRNA-193a-3p inhibits cell proliferation in prostate cancer by targeting cyclin D1[J].Oncol Lett,2017,14(5):5121-5128.
[11]Karatas O F,Wang J,Shao L,et al.miR-33a is a tumor suppressor microRNA that is decreased in prostate cancer[J].Oncotarget,2017,8(36):60243-60256.
[12]Epis M R,Giles K M,Beveridge D J,et al.miR-331-3p and Aurora Kinase inhibitor II co-treatment suppresses prostate cancer tumorigenesis and progression[J].Oncotarget,2017,8(33):55116-55134.
[13]Xu S,Ge J,Zhang Z,et al.MiR-129 inhibits cell proliferation and metastasis by targeting ETS1 via PI3K/AKT/m TOR pathway in prostate cancer[J].Biomed Pharmacother,2017,96:634-641.
[14]Ren D,Yang Q,Dai Y,et al.Oncogenic miR-210-3p promotes prostate cancer cell EMT and bone metastasis via NF-kappa B signaling pathway[J].Mol Cancer,2017,16(1):117.
[15]Guo J,Xiao Z,Yu X,et al.miR-20b promotes cellular proliferation and migration by directly regulating phosphatase and tensin homolog in prostate cancer[J].Oncol Lett,2017,14(6):6895-6900.
[16]Zhou W,Huang S,Jiang Q,et al.Suppression of miR-4735-3p in androgen receptor-expressing prostate cancer cells increases cell death during chemotherapy[J].Am J Transl Res,2017,9(8):3714-3722.
[17]Mou Z,Xu X,Dong M,et al.MicroRNA-148b acts as a tumor suppressor in cervical cancer by inducing G1/S-phase cell cycle arrest and apoptosis in a caspase-3-dependent manner[J].Med Sci Monit,2016,22:2809-2815.
[18]Huang M X.Down-expression of circulating micro ribonucleic acid(miRNA)-148/152 family in plasma samples of non-small cell lung cancer patients[J].J Cancer Res Ther,2016,12(2):671-675.
[19]Jefferies M T,Cox A C,Shorning B Y,et al.PTEN loss and activation of K-RAS and beta-catenin cooperate to accelerate prostate tumourigenesis[J].J Pathol,2017,243(4):442-456.
[20]Wozniak D J,Kajdacsy-Balla A,Macias V,et al.PTENis a protein phosphatase that targets active PTK6 and inhibits PTK6 oncogenic signaling in prostate cancer[J].Nat Commun,2017,8(1):1508.
[21]Li X,Gong X,Chen J,et al.miR-340 inhibits glioblastoma cell proliferation by suppressing CDK6,cyclin-D1and cyclin-D2[J].Biochem Biophys Res Commun,2015,460(3):670-677.
[22]Montanari M,Rossetti S,Cavaliere C,et al.Epithelialmesenchymal transition in prostate cancer:an overview[J].Oncotarget,2017,8(21):35376-35389.
[23]Sekhon K,Bucay N,Majid S,et al.MicroRNAs and epithelial-mesenchymal transition in prostate cancer[J].Oncotarget,2016,7(41):67597-67611.