[1]张廷华a,胡友元b,袁 博.miR-100-5p 对甲状腺癌细胞增殖与凋亡调控作用的实验研究[J].现代检验医学杂志,2024,39(04):56-62.[doi:10.3969/j.issn.1671-7414.2024.04.011]
　ZHANG Tinghuaa,HU Youyuanb,YUAN Bo.Experimental Study on the Regulatory Effects of miR-100-5p on Proliferation and Apoptosis of Thyroid Cancer Cells[J].Journal of Modern Laboratory Medicine,2024,39(04):56-62.[doi:10.3969/j.issn.1671-7414.2024.04.011]
点击复制

miR-100-5p 对甲状腺癌细胞增殖与凋亡调控作用的实验研究()
分享到：

《现代检验医学杂志》[ISSN:/CN:]

卷:
第39卷
期数:
2024年04期
页码:
56-62
栏目:
论著
出版日期:
2024-07-15

文章信息/Info

Title:
Experimental Study on the Regulatory Effects of miR-100-5p on Proliferation and Apoptosis of Thyroid Cancer Cells
文章编号:
1671-7414（2024）04-056-07
作者:
张廷华1a; 胡友元1b; 袁 博2
（1. 怀化市第二人民医院a. 检验科；b. 病理科，湖南怀化 418000；2. 南方科技大学医院检验科，广东深圳 518055）
Author(s):
ZHANG Tinghua1a;  HU Youyuan1b;  YUAN Bo2
（1a. Department of Clinical Laboratory;1b. Department of Pathology Laboratory, the Second People’s Hospital of Huaihua City, Hunan Huaihua 418000, China; 2. Department of Clinical Laboratory, Southern University of Science and Technology Hospital, Guangdong Shenzhen 518055, China）
关键词:
微小核糖核酸-100-5p; 甲状腺癌; 细胞增殖; 细胞凋亡
分类号:
R736.1；R730.43
DOI:
10.3969/j.issn.1671-7414.2024.04.011
文献标志码:
A
摘要:
目的 通过实验探讨微小核糖核酸（microRNA，miR）-100-5p 在甲状腺癌细胞中的表达情况及其对细胞增殖与凋亡的调控作用。方法 使用荧光定量PCR 检测miR-100-5p 在甲状腺癌细胞系（TPC-1，KTC-1）与甲状腺正常细胞系（Nthy-ori3-1）中的相对表达情况。TPC-1 细胞分别转染miR-100-5p 模拟物（miR-100-5p mimic）、抑制物（miR-100-5pinhibitor）及相应阴性对照（miR-mimic NC，miR-inhibitor NC）后，用CCK-8 检测TPC-1 细胞增殖情况，流式细胞仪检测TPC-1 细胞凋亡情况。通过miRTarBase 和TargetScan7.2 数据库对miR-100-5p 的靶基因进行预测和功能富集分析，用蛋白印迹实验与双荧光素酶报告基因实验验证miR-100-5p 对成纤维细胞生长因子受体3（fibroblast growth factor receptor 3，FGFR3）的靶向调控作用。结果 与Nthy-ori3-1 细胞相比，miR-100-5p 在TPC-1 细胞中表达水平（1.87±0.03 vs 1.00±0.03）与KTC-1 细胞中表达水平（6.33±0.47 vs 1.00±0.03）均上调，差异具有统计学意义（t=-34.220，-19.588，均P<0.05）。转染miR-100-5p mimic 组在24，48，72h 细胞450nm 吸光度(A450nm) 均高于miR-mimic NC 组，差异具有统计学意义（t=-7.516，-17.828，-8.445，均P<0.05）；转染miR-100-5p inhibitor 组在24，48，72h A450nm 均低于miR-inhibitor NC 组，差异具有统计学意义（t=6.720，6.782，6.073，均P<0.05）。与miR-mimic NC组相比，转染miR-100-5p mimic后凋亡率（7.43%±0.49%vs 10.55%±0.80%）下降（t=5.767，P=0.004），与miR-inhibitor NC 组相比，转染miR-100-5p inhibitor 后凋亡率（3.19%±0.22%vs 2.64%±0.15%）上升（t=-3.606，P=0.023），差异均有统计学意义。蛋白印迹实验显示，与miR-mimic NC 组相比，FGFR3 在miR-100-5p mimic 组蛋白表达水平（0.78±0.12 vs 1.00±0.00）下调（t=3.071，P=0.037），与miR-inhibitor NC组相比，FGFR3 在miR-100-5p inhibitor 组蛋白表达水平（1.17±0.07 vs 1.00±0.00）上升（t=-4.509，P=0.046），差异均有统计学意义。与miR-mimic NC 相比，miR-100-5p mimic 没有降低FGFR33’UTR 野生型组荧光素酶活性（1.01±0.17 vs1.00±0.00）与突变型组荧光素酶活性（0.99±0.11 vs 1.00±0.00），差异无统计学意义（t=-0.057，0.181，P=0.96，0.873）。结论 miR-100-5p 在甲状腺癌细胞中表达上调，可促进甲状腺癌细胞增殖、抑制细胞凋亡，其可能成为甲状腺癌诊疗中新的生物标志物与调控靶点。
Abstract:
Objective To explore the expression of microRNA (miR) -100-5p in thyroid cancer cells and its regulatory effects on cell proliferation and apoptosis through experiments. Methods The relative expressions of miR-100-5p in thyroid cancer cell lines (TPC-1 and KTC-1) and normal thyroid cell lines (Nthy ori3-1) were detected using fluorescence quantitative PCR. After transfection of miR-100-5p mimic, miR-100-5p inhibitor, and corresponding negative controls (miR-mimic NC, miR-inhibitor NC) into TPC-1 cells, the proliferation condition of TPC-1 cells was detected using CCK-8, and the apoptosis condition of TPC-1 cells was detected using flow cytometry. Prediction and functional enrichment analysis of target genes of miR-100-5p were performed using the miRTarBase and TargetScan7.2 databases. The targeted regulatory effect of miR-100-5p on fibroblast growth factor receptor 3 (FGFR3) was validated using Western blot and dual luciferase reporter gene experiments. Results Compared with Nthy-ori3-1 cells, the expression levels of miR-100-5p in TPC-1 cells (1.87±0.03 vs 1.00±0.03) and KTC-1 cells (6.33±0.47 vs 1.00±0.03) were both up-regulated, with significant differences (t=-34.220, -19.588, all P<0.05). The 450nm absorbance (A450nm) of cells transfected with miR-100-5p mimic at 24, 48 and 72 h were higher than the miR-mimic NC group, with significant differences (t=-7.516，-17.828，-8.445，all P<0.05). Conversely, the A450nm values of cells transfected with miR-100-5p inhibitor at 24, 48 and 72 h were lower than the miR-inhibitor NC group, with significant differences (t=6.720, 6.782, 6.073, all P<0.05). The apoptosis rate after transfection with miR-100-5p mimic was decreased compared to miR-mimic NC group (7.43%±0.49% vs 10.55%±0.80%), with significant differences (t=5.767, P=0.004). Compared to miR-inhibitor NC group, the apoptosis rate after transfection with miR-100-5p inhibitor was increased (3.19%±0.22% vs 2.64%±0.15%), with significant differences (t=-3.606, P=0.023). Western blot experiments showed that FGFR3 protein expression levels in the miR- 100-5p mimic group were down-regulated compared to the miR-mimic NC group (0.78±0.12 vs 1.00±0.00), with significant differences (t=3.071, P=0.037). Compared to the miR-inhibitor NC group, FGFR3 protein expression levels in the miR-100-5p inhibitor group were up-regulated (1.17±0.07 vs 1.00±0.00), with significant differences (t=-4.509, P=0.046). There was no significant difference in the luciferase activity of the FGFR3 wild-type (1.01±0.17 vs 1.00±0.00) and mutant groups (0.99±0.11 vs 1.00±0.00) between miR-100-5p mimic and miR-mimic NC, and the differences were statistically significant (t=-0.057, 0.181,P=0.96, 0.873). Conclusion MiR-100-5p in thyroid cancer cells was up-regulated, which may promote cell proliferation and inhibit apoptosis. It may become a new biomarker and regulatory target in the diagnosis and treatment of thyroid cancer.

参考文献/References:

[1] 仲亚东, 孔德桐, 马伯敏. 甲状腺癌患者组织及手术前后血清中NR3C2 和ZEB1 表达水平及其与预后价值研究[J]. 现代检验医学杂志, 2023, 38(4): 94-99. ZHONG Yadong, KONG Detong, MA Baimin. Study on the expression level of NR3C2 and ZEB1 in thyroid cancer tissue and serum of patients before and after surgery and their prognostic value[J]. Journal of Modern Laboratory Medicine, 2023, 38(4): 94-99.
[2] 刘星, 霍占江. 甲状腺乳头状癌组织中galecti-3 与miR-375 水平表达意义及相关性研究[J]. 现代检验医学杂志, 2023, 38(1): 49-52, 185. LIU Xing, HUO Zhanjiang. Expression significance and correlation of galectin-3 and miR-375 in papillary thyroid carcinoma tissue[J]. Journal of Modern Laboratory Medicine, 2023, 38(1): 49-52, 185.
[3] 张廷华, 胡友元. miRNA 在甲状腺癌中的研究进展[J]. 生命的化学, 2022, 42(12): 2231-2236. ZHANG Tinghua, HU Youyuan. Research progress of miRNA in thyroid cancer[J]. Chemistry of Life, 2022, 42(12): 2231-2236.
[4] PAPAIOANNOU M,CHORTI A G, CHATZIKYRIAKIDOU A, et al. MicroRNAs in papillary thyroid cancer: what is new in diagnosis and treatment[J]. Frontiers in Oncology, 2021, 11: 755097.
[5] ENIAFE J, JIANG Shuai. MicroRNA-99 family in cancer and immunity[J]. Wiley Interdisciplinary Reviews RNA, 2021, 12(3): e1635.
[6] 张廷华, 胡友元. miR-100 在人类癌症中的研究新进展[J]. 分子诊断与治疗杂志, 2024, 16(1): 195-198. ZHANG Tinghua, HU Youyuan. New progress in the research of miR-100 in human cancer[J]. Journal of Molecular Diagnosis and Therapy, 2024, 16(1): 195-198.
[7] GE Yiman, SHU Jia, SHI Gang, et al. MiR-100 suppresses the proliferation, invasion, and migration of hepatocellular carcinoma cells via targeting CXCR7[J]. Journal of Immunology Research, 2021, 2021:9920786.
[8] 邹艳花, 许可葵, 李灿, 等. 2019 年湖南省肿瘤登记地区恶性肿瘤发病与死亡及2015-2019 年变化趋势分析[J]. 中国肿瘤, 2023, 32(7): 483-491. ZOU Yanhua, XU Kekui, LI Can, et al. Cancer incidence and mortality in hunan cancer registration areas in 2019 and its trend from 2015 to 2019[J]. China Cancer, 2023, 32(7): 483-491.
[9] 赵芳芳, 郭红, 陈嘉. LncRNA TUG1 在甲状腺癌组织中的表达及其对细胞增殖和迁移的影响[J]. 现代检验医学杂志, 2020, 35(6): 42-47. ZHAO Fangfang, GUO Hong, CHEN Jia. Expression of LncRNA TUG1 in thyroid carcinoma tissues and its effect on cell proliferation and migration[J]. Journal of Modern Laboratory Medicine, 2020, 35(6): 42-47.
[10] LI Chen, GAO Yanping, ZHANG Kai, et al. Multiple roles of microRNA-100 in human cancer and its therapeutic potential[J]. Cellular Physiology and Biochemistry, 2015, 37(6): 2143-2159.
[11] XIE Haihui, XIAO Ruobing, HE Yaolin, et al. MicroRNA-100 inhibits breast cancer cell proliferation, invasion and migration by targeting FOXA1[J]. Oncology Letters, 2021, 22(6): 816.
[12] ZHANG Hongliang, YANG Kang, REN Tingting, et al. MiR-100-5p inhibits malignant behavior of chordoma cells by targeting IGF1R[J]. Cancer Management and Research, 2020, 12: 4129-4137.
[13] YE Yun, LI Suliang, WANG Jianjun. MiR-100-5p downregulates mTOR to suppress the proliferation, migration, and invasion of prostate cancer cells[J]. Frontiers in Oncology, 2020, 10: 578948.
[14] JAKOB M, MATTES L M, K?FFER S, et al. MicroRNA expression patterns in oral squamous cell carcinoma: hsa-mir-99b-3p and hsa-mir-100-5p as novel prognostic markers for oral cancer[J]. Head & Neck, 2019, 41(10): 3499-3515.
[15] OTTAVIANI S, CASTELLANO L. MicroRNAs: novel regulators of the TGF-β pathway in pancreatic ductal adenocarcinoma[J]. Molecular Cellular Oncology, 2018, 5(6): e1499066.
[16] FUSO P, DI SALVATORE M, SANTONOCITO C, et al. Let-7a-5p, miR-100-5p, miR-101-3p, and miR-199a-3p hyperexpression as potential predictive biomarkers in early breast cancer patients[J]. Journal of Personalized Medicine, 2021, 11(8): 816.
[17] XUE Yao, YANG Xiaoyun, HU Shaoyan, et al. A genetic variant in miR-100 is a protective factor of childhood acute lymphoblastic leukemia[J]. Cancer Medicine, 2019, 8(5): 2553-2560.
[18] FIGUEROA-GONZ?LEZ G, CARRILLO-HERN?NDEZ J F, PEREZ-RODRIGUEZ I, et al. Negative regulation of serine threonine kinase 11 (STK11) through miR-100 in head and neck cancer[J]. Genes, 2020, 11(9): 1058.
[19] BI Yunlong, JING Yu, CAO Yang. Overexpression of miR-100 inhibits growth of osteosarcoma through FGFR3[J]. Tumour Biology, 2015, 36(11): 8405-8411.
[20] LUO Jie, CHEN Bin, JI Xianxiu, et al. Overexpression of miR-100 inhibits cancer growth, migration, and chemosensitivity in human NSCLC cells through fibroblast growth factor receptor 3[J]. Tumour Biology, 2016, 37(12): 15517-15524.
[21] LUAN Yongxin, ZHANG Shuyan, ZUO Ling, et al. Overexpression of miR-100 inhibits cell proliferation, migration, and chemosensitivity in human glioblastoma through FGFR3[J]. Onco Targets and Therapy, 2015, 8: 3391-3400.
[22] LI Zhipeng, LI Xu, YU Chao, et al. MicroRNA-100 regulates pancreatic cancer cells growth and sensitivity to chemotherapy through targeting FGFR3[J]. Tumour Biology, 2014, 35(12): 11751-11759.
[23] BAE J S, JUNG S H, HIROKAWA M, et al. High prevalence of DICER1 mutations and low frequency of gene fusions in pediatric follicular-patterned tumors of the thyroid[J]. Endocrine Pathology, 2021, 32(3): 336-346.
[24] JONKER P K C, VAN DAM G M, OOSTING S F, et al. Identification of novel therapeutic targets in anaplastic thyroid carcinoma using functional genomic mRNA-profiling: paving the way for new avenues?[J]. Surgery, 2017, 161(1): 202-211.
[25] TIEDJE V, TING S, WALTER R F, et al. Prognostic markers and response to vandetanib therapy in sporadic medullary thyroid cancer patients[J]. European Journal of Endocrinology, 2016, 175(3): 173-180.
[26] ST BERNARD R, ZHENG Lei, LIU Wei, et al. Fibroblast growth factor receptors as molecular targets in thyroid carcinoma[J]. Endocrinology, 2005, 146(3): 1145-1153.
[27] ONOSE H, EMOTO N, SUGIHARA H, et al. Overexpression of fibroblast growth factor receptor 3 in a human thyroid carcinoma cell line results in overgrowth of the confluent cultures[J]. European Journal of Endocrinology, 1999, 140(2): 169-173.
[28] PARKER B C, ENGELS M, ANNALA M, et al. Emergence of FGFR family gene fusions as therapeutic targets in a wide spectrum of solid tumours[J]. The Journal of Pathology, 2014, 232(1): 4-15.

相似文献/References:

[1]彭继英a,杨红杰b,石晓欣a,等.细针穿刺细胞学检查在甲状腺癌碘-131 治疗后复发诊断中的应用价值[J].现代检验医学杂志,2020,35(01):127.[doi:10.3969/j.issn.1671-7414.2020.01.033]
　PENG Ji-yinga,YANG Hong-jieb,SHI Xiao-xina,et al.Application Value of Fine Needle Aspiration Cytology in Recurrence Diagnosis of Thyroid Cancer after Iodine-131 Treatment[J].Journal of Modern Laboratory Medicine,2020,35(04):127.[doi:10.3969/j.issn.1671-7414.2020.01.033]
[2]赵芳芳,郭　红,陈　嘉.LncRNA TUG1 在甲状腺癌组织中的表达及其对细胞增殖和迁移的影响[J].现代检验医学杂志,2020,35(06):42.[doi:doi:10.3969/j.issn.1671-7414.2020.06.011]
　ZHAO Fang-fang,GUO Hong,CHEN Jia.Expression of LncRNA TUG1 in Thyroid Carcinoma Tissues and Its Effect onCell Proliferation and Migration[J].Journal of Modern Laboratory Medicine,2020,35(04):42.[doi:doi:10.3969/j.issn.1671-7414.2020.06.011]
[3]仲亚东,孔德桐,马伯敏.甲状腺癌患者组织及手术前后血清中NR3C2 和ZEB1 表达水平及其与预后价值研究[J].现代检验医学杂志,2023,38(04):94.[doi:10.3969/j.issn.1671-7414.2023.04.017]
　ZHONG Yadong,KONG Detong,MA Bomin.Study on the Expression Level of NR3C2 and ZEB1 in Thyroid Cancer Tissue and Serum of Patients before and after Surgery and Their Prognostic Value[J].Journal of Modern Laboratory Medicine,2023,38(04):94.[doi:10.3969/j.issn.1671-7414.2023.04.017]
[4]谈玖婷,傅聿明,刘 婧,等.甲状腺癌组织中DCBLD2 和MAP4K3 的表达及与临床病理特征及预后的关系[J].现代检验医学杂志,2024,39(02):34.[doi:10.3969/j.issn.1671-7414.2024.02.007]
　TAN Jiuting,FU Yuming,LIU Jing,et al.Expression of DCBLD2 and MAP4K3 in Thyroid Cancer Tissue and Their Relationship with Clinico-pathological Features and Prognosis[J].Journal of Modern Laboratory Medicine,2024,39(04):34.[doi:10.3969/j.issn.1671-7414.2024.02.007]

备注/Memo

备注/Memo:
基金项目：怀化市科技计划项目(2021R3113)。
作者简介：张廷华（1989-），男，硕士，副主任技师，研究方向：肿瘤分子诊断，E-mail:zth19890820@163.com。
通讯作者：袁博（1988-），男，硕士，主管技师，研究方向：肿瘤分子诊断，E-mail:503309099@qq.com。
更新日期/Last Update: 2024-07-15