微小RNA-99a-5p靶向调控IGF1R表达及对肝癌HepG2细胞侵袭迁移的影响
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摘要
目的探讨微小RNA-99a-5p(miR-99a-5p)对肝癌细胞HepG2侵袭和迁移的影响及对胰岛素样生长因子1受体(IGF1R)的靶向调控作用。方法收集本院2014年1月至2017年3月经手术切除的90对癌组织及癌旁组织,采用实时定量PCR(QPCR)检测以上组织中miR-99a-5p及IGF1R mRNA水平;采用脂质体法向HepG2细胞分别转染miR-99a-5p模拟物(过表达组)和miR-99a-5p阴性对照(NC组),采用QPCR法检测转染48 h后各组的miR-99a-5p水平以评价转染效率;分别采用Transwell和划痕实验比较各组的侵袭和迁移情况;QPCR和Western blotting检测各组的IGF1R mRNA和蛋白水平;双荧光素酶报告基因实验验证miR-99a-5p对IGF1R的靶向作用。结果 QPCR检测结果显示,癌组织的miR-99a-5p水平为0.823±0.039,低于癌旁组织的1.016±0.013,而癌组织的IGF1R mRNA水平为1.329±0.024,高于癌旁组织的1.035±0.026,差异均有统计学意义(P<0.05)。过表达组转染48 h后的miR-99a-5p水平为1.487±0.027,均高于对照组的1.021±0.013和NC组的1.019±0.025,差异有统计学意义(P<0.05)。对照组、NC组和过表达组的愈合率分别为(60.5±2.4)%、(58.4±3.3)%和(42.1±2.9)%,穿膜细胞数分别为(92.1±3.7)个、(95.2±4.9)个和(36.7±4.5)个,过表达组的愈合率和穿膜细胞数均少于其余两组(P<0.05);过表达组的IGF1R mRNA和蛋白水平均低于其余两组(P<0.05)。双荧光素酶报告基因试验结果显示,miR-99a-5p显著抑制野生型IGF1R-3’-UTR质粒转染细胞的荧光素酶活性(P<0.01),但是对突变型IGF1R-3’-UTR荧光素酶活性无显著影响。结论 miR-99a-5p在肝癌中表达降低,且可抑制肝癌细胞的侵袭迁移过程,可能是通过靶向调控IGF1R来实现,可作为肝癌的潜在治疗靶点。
Objective To investigate the effect of microRNA-99 a-5 p( miR-99 a-5 p) on the invasion and migration of hepatoma cells HepG2 and its regulatory role on insulin-like growth factor 1 receptor( IGF1 R). Methods Ninety pairs of cancerous and paracancerous tissues surgically removed in our hospital were collected from January 2014 to March 2017. Real-time quantitative PCR( QPCR) was used to detect the level of miR-99 a-5 p and IGF1 R mRNA in the above tissues. HepG2 cells were transfected with miR-99 a-5 p mimics( overexpression group) and miR-99 a-5 p negative control( NC group) by liposome method. QPCR method was used to detect the miR-99 a-5 p level of each group after transfection for 48 h to evaluate the transfection efficiency. The invasion and migration of each group were evaluated by Transwell and scratch test,respectively. The mRNA and protein levels of IGF1 R were detected by QPCR and Western blotting,respectively. The dual luciferase reporter gene test was applied to verify the targeting effect of miR-99 a-5 p on IGF1 R. Results The results of QPCR showed that the level of miR-99 a-5 p in the cancer tissues was 0. 823 ± 0. 039,lower than1. 016 ± 0. 013 in normal paracancerous tissues,while the level of IGF1 R mRNA in the cancer tissues was 1. 329 ± 0. 024,higher than1. 035 ± 0. 026 in normal paracancerous tissues( P < 0. 05). The miR-99 a-5 p level in the overexpression group was 1. 487 ± 0. 027,higher than 1. 021 ± 0. 013 of control group and 1. 019 ± 0. 025 of NC group( P < 0. 05). The healing rates of control group,NC group and overexpression group were( 60. 5 ± 2. 4) %,( 58. 4 ± 3. 3) % and( 42. 1 ± 2. 9) %,and the numbers of membrane cells were92. 1 ± 3. 7,95. 2 ± 4. 9 and 36. 7 ± 4. 5,respectively. Compared with other two groups,the healing rates and numbers of membrane cells of overexpression group decreased( P < 0. 05). The mRNA and protein levels of IGF1 R in overexpression group were lower than those in other two groups( P < 0. 05). The results of double fluorescence report showed that miR-99 a-5 p significantly inhibited the luciferase activity of cells transfected with the wild type IGF1 R-3'-UTR plasmid( P < 0. 01),but had no significant effect on the activity of the mutant IGF1 R-3'-UTR luciferase activity. Conclusion The expression of miR-99 a-5 p is reduced in hepatoma cancer. MiR-99 a-5 p can inhibit the invasion and migration of hepatoma cells. It may be realized by targeting IGF1 R,which can be used as a potential target for the treatment of hepatoma cancer.
Objective To investigate the effect of microRNA-99 a-5 p( miR-99 a-5 p) on the invasion and migration of hepatoma cells HepG2 and its regulatory role on insulin-like growth factor 1 receptor( IGF1 R). Methods Ninety pairs of cancerous and paracancerous tissues surgically removed in our hospital were collected from January 2014 to March 2017. Real-time quantitative PCR( QPCR) was used to detect the level of miR-99 a-5 p and IGF1 R mRNA in the above tissues. HepG2 cells were transfected with miR-99 a-5 p mimics( overexpression group) and miR-99 a-5 p negative control( NC group) by liposome method. QPCR method was used to detect the miR-99 a-5 p level of each group after transfection for 48 h to evaluate the transfection efficiency. The invasion and migration of each group were evaluated by Transwell and scratch test,respectively. The mRNA and protein levels of IGF1 R were detected by QPCR and Western blotting,respectively. The dual luciferase reporter gene test was applied to verify the targeting effect of miR-99 a-5 p on IGF1 R. Results The results of QPCR showed that the level of miR-99 a-5 p in the cancer tissues was 0. 823 ± 0. 039,lower than1. 016 ± 0. 013 in normal paracancerous tissues,while the level of IGF1 R mRNA in the cancer tissues was 1. 329 ± 0. 024,higher than1. 035 ± 0. 026 in normal paracancerous tissues( P < 0. 05). The miR-99 a-5 p level in the overexpression group was 1. 487 ± 0. 027,higher than 1. 021 ± 0. 013 of control group and 1. 019 ± 0. 025 of NC group( P < 0. 05). The healing rates of control group,NC group and overexpression group were( 60. 5 ± 2. 4) %,( 58. 4 ± 3. 3) % and( 42. 1 ± 2. 9) %,and the numbers of membrane cells were92. 1 ± 3. 7,95. 2 ± 4. 9 and 36. 7 ± 4. 5,respectively. Compared with other two groups,the healing rates and numbers of membrane cells of overexpression group decreased( P < 0. 05). The mRNA and protein levels of IGF1 R in overexpression group were lower than those in other two groups( P < 0. 05). The results of double fluorescence report showed that miR-99 a-5 p significantly inhibited the luciferase activity of cells transfected with the wild type IGF1 R-3'-UTR plasmid( P < 0. 01),but had no significant effect on the activity of the mutant IGF1 R-3'-UTR luciferase activity. Conclusion The expression of miR-99 a-5 p is reduced in hepatoma cancer. MiR-99 a-5 p can inhibit the invasion and migration of hepatoma cells. It may be realized by targeting IGF1 R,which can be used as a potential target for the treatment of hepatoma cancer.
引文
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[2]Song Y,Wang F,Huang Q,et al.MicroRNAs contribute to hepatocellular carcinoma[J].Mini Rev Med Chem,2015,15(6):459-466.
[3]Li ZB,Li ZZ,Li L,et al.MiR-21 and miR-183 can simultaneously target SOCS6 and modulate growth and invasion of hepatocellular carcinoma(HCC)cells[J].Eur Rev Med Pharmacol Sci,2015,19(17):3208-3217.
[4]Hua HW,Jiang F,Huang Q,et al.MicroRNA-153 promotes Wnt/β-catenin activation in hepatocellular carcinoma through suppression of WWOX[J].Oncotarget,2015,6(6):3840-3847.
[5]Zhang JJ,Wang CY,Hua L,et al.MiR-107 promotes hepatocellular carcinoma cell proliferation by targeting Axin2[J].Int J Clin Exp Pathol,2015,8(5):5168-5174.
[6]Chen JJ,Tang YS,Huang SF,et al.HBx protein-induced upregulation of microRNA-221 promotes aberrant proliferation in HBV related hepatocellular carcinoma by targeting estrogenreceptor-α[J].Oncol Rep,2015,33(2):792-798.
[7]Huang H,Zhu Y,Li S,et al.MicroRNA-122 mimic transfection contributes to apoptosis in Hep G2 cell[J].Mol Med Rep,2015,12(5):6918-6924.
[8]Chen L,Zheng J,Zhang Y,et al.Tumor-specific expression of microRNA-26a suppresses human hepatocellular carcinoma growth via cyclin-dependent and-independent pathways[J].Mol Ther,2011,19(8):1521-1528.
[9]Yan MD,Yao CJ,Chow JM,et al.Fucoidan elevates microRNA-29b to regulate DNMT3B-MTSS1 axis and inhibit EMT in human hepatocellular carcinoma cells[J].Mar Drugs,2015,13(10):6099-6116.
[10]Thurnherr T,Mah WC,Lei Z,et al.Differentially expressed miRNAs in hepatocellular carcinoma target genes in the genetic information processing and metabolism pathways[J/OL].Sci Rep,2016[2017-10-22].https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/26817861.
[11]Shen J,Siegel AB,Remotti H,et al.Identifying microRNA panels specifically associated with hepatocellular carcinoma and its different etiologies[J].Hepatoma Res,2016,2:151-162.
[12]Li D,Liu X,Lin L,et al.MicroRNA-99a inhibits hepatocellular carcinoma growth and correlates with prognosis of patients with hepatocellular carcinoma[J].J Biol Chem,2011,286(42):36677-36685.
[13]Turcatel G,Rubin N,El-Hashash A,et al.MIR-99a and miR-99b modulate TGF-βinduced epithelial to mesenchymal plasticity in normal murine mammary gland cells[J/OL].PLo S One,2012[2017-10-13].https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22299047.
[14]Mei LL,Qiu YT,Huang MB,et al.MiR-99a suppresses proliferation,migration and invasion of esophageal squamous cell carcinoma cells through inhibiting the IGF1R signaling pathway[J].Cancer Biomark,2017,20(4):1-11.
[15]Li D,Liu X,Lin L,et al.MicroRNA-99a inhibits hepatocellular carcinoma growth and correlates with prognosis of patients with hepatocellular carcinoma[J].J Biol Chem,2011,286(42):36677-36685.
[16]Zhang J,Jin H,Liu H,et al.MiRNA-99a directly regulates AGO2 through translational repression in hepatocellular carcinoma[J/OL].Oncogenesis,2014[2017-12-13].https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24732044.
[17]Xu Y,Huang J,Ma L,et al.MicroRNA-122 confers sorafenib resistance to hepatocellular carcinoma cells by targeting IGF-1R to regulate RAS/RAF/ERK signaling pathways[J].Cancer Lett,2016,371(2):171-181.
[18]Xia M,Li H,Wang JJ,et al.MiR-99a suppress proliferation,migration and invasion through regulating insulin-like growth factor 1 receptor in breast cancer[J].Eur Rev Med Pharmacol Sci,2016,20(9):1755-1763.