上调miR-34b-5p通过抑制胰岛素样生长因子1受体干扰肾癌Caki-1细胞的增殖和侵袭
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摘要
目的观察微小RNA-34b-5p(miR-34b-5p)对人肾癌细胞增殖和侵袭的影响及分子机制。方法采用实时荧光定量PCR(real-time quantitative PCR, qRT-PCR)检测人肾癌细胞ACHN、Caki-1、OS-RC-2、786-O、A498和人正常肾小管上皮细胞HK-2中miR-34b-5p的表达量。以miR-34b-5p表达量最低的Caki-1细胞为转染对象,应用脂质体转染miR-34b-5p或微小RNA(miR-NC)。采用qRT-PCR检测各组细胞中miR-34b-5p的表达水平。MTT法和Transwell侵袭实验检测肾癌细胞的增殖和侵袭能力。生物信息学预测并采用双荧光素酶报告基因验证miR-34b-5p的潜在靶基因。qRT-PCR和Western blot法分别检测miR-34b-5p潜在靶基因及下游通路蛋白的表达。结果肾癌细胞中miR-34b-5p的表达量低于正常肾小管上皮细胞。用脂质体转染miR-34b-5p后Caki-1细胞中miR-34b-5p的表达量明显高于miR-NC组,提示过表达成功。上调miR-34b-5p可抑制肾癌细胞的增殖能力和侵袭能力。生物信息学预测和双荧光素酶报告基因证实胰岛素样生长因子1受体(insulin like growth factor 1 receptor, IGF1R)为miR-34b-5p的靶基因。上调miR-34b-5p可降低肾癌Caki-1细胞中IGF1R基因及PI3K/Akt信号通路蛋白的表达量。结论上调miR-34b-5p可抑制人肾癌Caki-1细胞的增殖和侵袭,其可能的分子机制为降低IGF1R基因及下游PI3K/Akt信号通路蛋白的表达。
Purpose To investigate the effect of microRNA-34 b-5 p(miR-34 b-5 p) on the proliferation and invasion of human renal cell carcinoma and its molecular mechanism. Methods Real-time quantitative PCR was used to detect the expression of miR-34 b-5 p in ACHN, Caki-1, OS-RC-2, 786-O, A498 human renal cell carcinoma and HK-2 human normal renal tubular epithelial cells. miR-34 b-5 p or miR-NC was transfected into Caki-1 cells with the lowest expression of miR-34 b-5 p using liposomes. Real-time quantitative PCR was used to detect the expression of miR-34 b-5 p in each group. MTT assay and Transwell invasion assay were used to detect the proliferation and invasion ability of renal carcinoma cells. Bioinformatics and dual-luciferase reporter assays were used to predict and validate miR-34 b-5 p potential target genes. Real-time quantitative PCR and Western blot were used to detect the expression of potential target genes of miR-34 b-5 p and downstream pathway proteins. Results The expression of miR-34 b-5 p in renal cell carcinoma cells was lower than that in normal renal tubular epithelial cells. The expression of miR-34 b-5 p in Caki-1 cells transfected with miR-34 b-5 p was significantly higher than that in miR-NC group, suggesting successful transfection. Up-regulation of miR-34 b-5 p can inhibit the proliferation and invasion of renal cell carcinoma cells. Bioinformatics prediction and dual luciferase reporter gene confirmed that insulin-like growth factor 1 receptor(IGF1 R) is the target gene of miR-34 b-5 p. Upregulation of miR-34 b-5 p can reduce the expression of IGF1 R gene and PI3 K/Akt signaling pathway proteins in Caki-1 cells. Conclusion Up-regulation of miR-34 b-5 p inhibits the proliferation and invasion of human renal carcinoma Caki-1 cells by decreasing the expression of IGF1 R gene and PI3 K/Akt signaling pathway proteins.
Purpose To investigate the effect of microRNA-34 b-5 p(miR-34 b-5 p) on the proliferation and invasion of human renal cell carcinoma and its molecular mechanism. Methods Real-time quantitative PCR was used to detect the expression of miR-34 b-5 p in ACHN, Caki-1, OS-RC-2, 786-O, A498 human renal cell carcinoma and HK-2 human normal renal tubular epithelial cells. miR-34 b-5 p or miR-NC was transfected into Caki-1 cells with the lowest expression of miR-34 b-5 p using liposomes. Real-time quantitative PCR was used to detect the expression of miR-34 b-5 p in each group. MTT assay and Transwell invasion assay were used to detect the proliferation and invasion ability of renal carcinoma cells. Bioinformatics and dual-luciferase reporter assays were used to predict and validate miR-34 b-5 p potential target genes. Real-time quantitative PCR and Western blot were used to detect the expression of potential target genes of miR-34 b-5 p and downstream pathway proteins. Results The expression of miR-34 b-5 p in renal cell carcinoma cells was lower than that in normal renal tubular epithelial cells. The expression of miR-34 b-5 p in Caki-1 cells transfected with miR-34 b-5 p was significantly higher than that in miR-NC group, suggesting successful transfection. Up-regulation of miR-34 b-5 p can inhibit the proliferation and invasion of renal cell carcinoma cells. Bioinformatics prediction and dual luciferase reporter gene confirmed that insulin-like growth factor 1 receptor(IGF1 R) is the target gene of miR-34 b-5 p. Upregulation of miR-34 b-5 p can reduce the expression of IGF1 R gene and PI3 K/Akt signaling pathway proteins in Caki-1 cells. Conclusion Up-regulation of miR-34 b-5 p inhibits the proliferation and invasion of human renal carcinoma Caki-1 cells by decreasing the expression of IGF1 R gene and PI3 K/Akt signaling pathway proteins.
引文
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[2] Petrozza V,Pastore A L,Palleschi G,et al.Secreted miR-210-3p as non-invasive biomarker in clear cell renal cell carcinoma[J].Oncotarget,2017,8(41):69551-69558.
[3] Zhang X,Wang C,Li H,et al.miR-338-3p inhibits the invasion of renal cell carcinoma by downregulation of ALK5[J].Oncotarget,2017,8(38):64106-64113.
[4] 曾小芳,熊艳杰,乌日娜,等.抑制miRNA-301a表达对卵巢癌中SKOV3细胞增殖、迁移及侵袭的影响[J].临床与实验病理学杂志,2018,34(1):92-94.
[5] Ding D,Zhang Y,Wen L,et al.MiR-367 regulates cell proliferation and metastasis by targeting metastasis-associated protein 3 (MTA3) in clear-cell renal cell carcinoma[J].Oncotarget,2017,8(38):63084-63095.
[6] Fang L L,Sun B F,Huang L R,et al.Potent inhibition of miR-34b on migration and invasion in metastatic prostate cancer cells by regulating the TGF-beta pathway[J].Int J Mol Sci,2017,18(12):2762.doi:10.3390/ijms18122762.
[7] Maroof H,Islam F,Ariana A,et al.The roles of microRNA-34b-5p in angiogenesis of thyroid carcinoma[J].Endocrine,2017,58(1):153-166.
[8] Xiao J,Li Y,Zhang W,et al.miR-34b inhibits nasopharyngeal carcinoma cell proliferation by targeting ubiquitin-specific peptidase 22[J].Onco Targets Ther,2016,9:1525-1534.
[9] Quan J,Liu S,Dai K,et al.MicroRNA-23a/24-2/27a as a potential diagnostic biomarker for cancer:a systematic review and meta-analysis[J].Mol Clin Oncol,2018,8(1):159-169.
[10] Yoshino H,Yonezawa T,Yonemori M,et al.Downregulation of microRNA-1274a induces cell apoptosis through regulation of BMPR1B in clear cell renal cell carcinoma[J].Oncol Rep,2018,39(1):173-181.
[11] 闫玉,王瑞芬,乔萌,等.胃癌相关纤维母细胞中miRNAs的表达及对癌细胞迁移和侵袭的影响[J].临床与实验病理学杂志,2017,33(6):601-605.
[12] Fan Y,Ma X,Li H,et al.miR-122 promotes metastasis of clear-cell renal cell carcinoma by downregulating dicer[J].Int J Cancer,2018,142(3):547-560.
[13] He T,Chen P,Jin L,et al.miR6605p is associated with cell migration,invasion,proliferation and apoptosis in renal cell carcinoma[J].Mol Med Rep,2018,17(1):2051-2060.
[14] Hu B,Wang J,Jin X.MicroRNA-138 suppresses cell proliferation and invasion of renal cell carcinoma by directly targeting SOX9[J].Oncol Lett,2017,14(6):7583-7588.
[15] Gu C,Wang Z,Jin Z,et al.MicroRNA-212 inhibits the proliferation,migration and invasion of renal cell carcinoma by targeting X-linked inhibitor of apoptosis protein (XIAP)[J].Oncotarget,2017,8(54):92119-92133.
[16] Jin L,Li Y,Nie L,et al.MicroRNA242 is associated with cell proliferation,invasion,migration and apoptosis in renal cell carcinoma[J].Mol Med Rep,2017,16(6):9157-9164.
[17] Wang X,Li H,Cui L,et al.MicroRNA-182 suppresses clear cell renal cell carcinoma migration and invasion by targeting IGF1R[J].Neoplasma,2016,63(5):717-725.
[18] Selfe J,Goddard N C,Mcintyre A,et al.IGF1R signalling in testicular germ cell tumour cells impacts on cell survival and acquired cisplatin resistance[J].J Pathol,2018,244(2):242-253.
[19] Yoon S O,Shin S,Karreth F A,et al.Focal adhesion- and IGF1R-dependent survival and migratory pathways mediate tumor resistance to mTORC1/2 inhibition[J].Mol Cell,2017,67(3):512-527.
[20] May C D,Landers S M,Bolshakov S,et al.Co-targeting PI3K,mTOR,and IGF1R with small molecule inhibitors for treating undifferentiated pleomorphic sarcoma[J].Cancer Biol Ther,2017,18(10):816-826.
[21] Zhang L,Qi M,Feng T,et al.IDH1R132H promotes malignant transformation of benign prostatic epithelium by dysregulating microRNAs:involvement of IGF1R-AKT/STAT3 signaling pathway[J].Neoplasia,2018,20(2):207-217.
[22] Zhou Y,Geng P,Liu Y,et al.Rotavirus-encoded virus-like small RNA triggers autophagy by targeting IGF1R via the PI3K/Akt/mTOR pathway[J].Biochim Biophys Acta,2018,1864(1):60-68.
[23] Das F,Dey N,Bera A,et al.MicroRNA-214 reduces insulin-like growth factor-1 (IGF-1) receptor expression and downstream mTORC1 signaling in renal carcinoma cells[J].J Biol Chem,2016,291(28):14662-14676.