人舌癌耐药细胞株OSC-19/CDDP的多药耐药性检测及差异性表达microRNAs的筛选

doi:10.3969/j.issn.1005-4979.2020.02.004

《口腔颌面外科杂志》 ›› 2020, Vol. 30 ›› Issue (2): 80-84. doi: 10.3969/j.issn.1005-4979.2020.02.004

人舌癌耐药细胞株OSC-19/CDDP的多药耐药性检测及差异性表达microRNAs的筛选

杨晓红(), 胡小华(), 钟雯怡, 杨琨, 易杰, 姚礼, 张立刚

遵义医科大学附属口腔医学院，贵州遵义 563000

收稿日期:2018-08-09 修回日期:2019-12-15 出版日期:2020-04-28 发布日期:2020-04-24
通讯作者: 胡小华, 主任医师. E-mail: hxh2132000@163.com
作者简介:
杨晓红（1976—），女，贵州遵义人，博士，副教授. E-mail: 854936065@qq.com
基金资助:
贵州省科教青年英才培养工程项目(黔卫函（2012）239号); 贵州省科技厅资助项目(黔科合LH 字（2015）7510号); 遵义医学院学术新苗培养及创新探索专项后补助课题(黔科合平台人才（2017）5733-007); 遵义市科学技术局、遵义医学院附属口腔医院联合科技研发资金(遵市科合社字（2018）241号); 遵义市红花岗区科学技术基金项目(遵红科合社字（2015）07号)

Verification of the Multidrug Resistance of Human Tongue Squamous Carcinoma Cell Line OSC-19/CDDP and Its Differently Expressed MicroRNAs

YANG Xiaohong(), HU Xiaohua(), ZHONG Wenyi, YANG Kun, YI Jie, YAO Li, ZHANG Ligang

School and Hospital of Stomatology, Zunyi Medical University, Zunyi 563000, Guizhou Province, China

Received:2018-08-09 Revised:2019-12-15 Online:2020-04-28 Published:2020-04-24

摘要/Abstract

摘要：

目的：检测人舌鳞癌耐药细胞株OSC-19/CDDP是否具有多药耐药的生物特性，并筛选出与亲本细胞（OSC-19）显著差异表达的microRNAs（miRNAs），探讨其在舌癌耐药中的作用。方法：对前期实验建立的耐顺铂（cisplatin, CDDP）细胞株OSC-19/CDDP，采用CCK-8法检测其对不同化疗药物的耐药性; RT-PCR检测亲代与耐药株的多药耐药基因1（multidrug resistance 1, MDR1）表达。通过微阵列芯片筛查在OSC-19/CDDP与亲本细胞OSC-19中差异性表达的miRNAs，并通过RT-PCR 进一步验证基因芯片的结果。结果：人舌癌多药耐药细胞株OSC-19/CDDP对CDDP的耐药指数为16.7，交叉耐药的紫杉醇（PTX）、5-氟尿嘧啶（5-Fu）、平阳霉素（PYM）耐药指数分别为7.93、5.31和3.01；RT-PCR结果显示OSC-19/CDDP细胞中MDR1的表达是OSC-19的15.23倍。微阵列芯片结果显示：OSC-19/CDDP与OSC-19比较，分别有17个miRNAs的表达增高程度和12个miRNAs的表达降低程度超过10倍。结论：人舌鳞癌耐药细胞株OSC-19/CDDP具有多药耐药的生物学特性，可作为舌鳞癌的顺铂耐药机制及多药耐药机制研究的理想细胞模型。显著差异表达的miRNAs可能在舌鳞癌耐药的发生、发展中发挥重要作用。

关键词: 舌癌, 多药耐药, microRNA

Abstract:

Objective: To verify the multidrug resistance of OSC-19/CDDP cell line. Screening differently expressed microRNAs（miRNAs）between OSC-19/CDDP and its parental cell (OSC-19) and discussing their effect in drug resistance. Method: The drug-resistance index of cell line OSC-19/CDDP was detected to different chemotherapeutics with CCK-8,and the MDR1(multidrug resistance 1) expression in both OSC-19/CDDP and parental cell was detected with RT-PCR. Screening differently expressed miRNAs between them was detected with micro-array and confirmed RT-PCR. Results: The resistance index of OSC-19/CDDP to CDDP was 16.7 and its cross resistance index to PTX, 5-FU, and PYM were 7.93, 5.31 and 3.01 respectively. MDR1 mRNA expression level was 15.23 fold higher in OSC-19/CDDP than in parental cell. Micro-array revealed 17 miRNAs increased and 12 declined over 10-fold between OSC-19/CDDP and OSC-19. Conclusion: The multidrug resistance of OSC-19/CDDP was verified, making it an ideal model for mechanism research on human tongue squamous cell carcinoma cisplatin or multidrug resistance. Those differently expressed miRNAs may play vital roles in this resistance progress.

Key words: tongue squamous cell carcinoma, multidrug resistance, microRNA

中图分类号:

杨晓红, 胡小华, 钟雯怡, 杨琨, 易杰, 姚礼, 张立刚. 人舌癌耐药细胞株OSC-19/CDDP的多药耐药性检测及差异性表达microRNAs的筛选[J]. 《口腔颌面外科杂志》, 2020, 30(2): 80-84.

YANG Xiaohong, HU Xiaohua, ZHONG Wenyi, YANG Kun, YI Jie, YAO Li, ZHANG Ligang. Verification of the Multidrug Resistance of Human Tongue Squamous Carcinoma Cell Line OSC-19/CDDP and Its Differently Expressed MicroRNAs[J]. 《Journal of Oral and Maxillofacial Surgery》, 2020, 30(2): 80-84.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2020/V30/I2/80

图/表 6

表1 基因引物序列

Table 1 Primer sequences used for RT-PCR

基因	引物序列（5'-3'）
GAPDH	正向 AGGTCGGAGTCAACGGATTTGGTCG
	反向 TGGCCAGGGGTGCTAAGCAGT
MDR1	正向 GCAATGGAGGAGCAAAGAAG
	反向 AATGTAAGCAGCAACCAGCAC

图1 半数抑制浓度CDDP、PTX、5-FU、PYM作用下的OSC-19及OSC-19/CDDP细胞形态（×40）注：A.4.55 μg/mLCDDP作用下的OCS-19细胞；B.4.55 μg/mLCDDP作用下的OCS-19/CDDP细胞； C.3.56 μg/mLPTX作用下的OCS-19细胞；D.3.56 μg/mLPTX作用下的OCS-19/CDDP细胞；E.127.30 μg/mL5-Fu作用下的OCS-19细胞；F.127.30 μg/mL5-Fu作用下的OCS-19/CDDP细胞；G.1 650 μg/mLPYM作用下的OCS-19细胞；H.1 650 μg/mLPYM作用下的OCS-19/CDDP细胞。

Figure 1 Morphology of the OSC-19 and OSC-19/CDDP cells under the LD50 concentration of CDDP, PTX, 5-FU, PYM respectively（×40）

表2 抗肿瘤药物对于OSC-19及OSC-19/CDDP细胞的半数抑制浓度及耐药指数

药物	半数抑制浓度(μg/mL）		耐药指数
药物	OSC-19	OSC-19/CDDP	耐药指数
CDDP	4.55	79.06	16.70
PTX	3.56	28.23	7.93
5-Fu	127.30	676.10	5.31
PYM	1 650.00	4 963.10	3.01

图2 OSC-19与OSC-19/CDDP细胞MDR1的mRNA表达情况

Figure 2 The MDR1 gene expression level of OSC-19 and OSC-19/CDDP

表3 微阵列芯片筛查差异表达的miRNAs

Table 3 The miRNAs differentially expressed in micro-arraychip

miRNAs	表达量比值	表达量
miRNAs	表达量比值	OSC-19细胞	OSC-19/CDDP细胞
miR-302b-5p	10.927	8.446 11	92.292 83
miR-3912-3p	17.897	0.044 91	0.803 74
miR-126-3p	12.139	0.053 89	0.654 21
miR-571	0.051	0.607 78	0.031 15
miR-429	0.023	0.952 10	0.021 81

图3 OSC-19/CDDP及OSC-19细胞中miRNAs的表达水平

Figure 3 The results of miRNAs expression level of cell of OSC-19/CDDP and OSC-19

参考文献 13

[1]	孙小雅, 严汉兴, 贺莎, 等. 舌癌多种治疗方式的临床疗效比较[J]. 中南医学科学杂志, 2018, 46(1):16-19.
[2]	蒋磊, 王代友, 陆新萍. siRNA抑制人Tca8113/CDDP细胞多药耐药相关蛋白表达的研究[J]. 口腔颌面外科杂志, 2011, 21(2):88-91.
[3]	杨晓红, 胡小华, 钟雯怡, 等. 人舌鳞癌耐顺铂细胞株的建立及鉴定[J]. 中国社区医师, 2019, 35(30):9-10.
[4]	Yu ZW, Zhong LP, Ji T, et al. MicroRNAs contribute to the chemoresistance of cisplatin in tongue squamous cell carcinoma lines[J]. Oral Oncol, 2010, 46(4):317-322. doi: 10.1016/j.oraloncology.2010.02.002 URL
[5]	Ren W, Wang X, Gao L, et al. MiR-21 modulates chemosensitivity of tongue squamous cell carcinoma cells to cisplatin by targeting PDCD4[J]. Mol Cell Biochem, 2014, 390(1-2):253-262. doi: 10.1007/s11010-014-1976-8 pmid: 24609942
[6]	Lin XJ, He CL, Sun T, et al. Hsa-miR-485-5p reverses epithelial to mesenchymal transition and promotes cisplatin-induced cell death by targeting PAK1 in oral tongue squamous cell carcinoma[J]. Int J Mol Med, 2017, 40(1):83-89. doi: 10.3892/ijmm.2017.2992 URL
[7]	Chen CJ, Chin JE, Ueda K, et al. Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrug-resistant human cells[J]. Cell, 1986, 47(3):381-389. doi: 10.1016/0092-8674(86)90595-7 pmid: 2876781
[8]	Chen J, Tian W, Cai H, et al. Down-regulation of microRNA-200c is associated with drug resistance in human breast cancer[J]. Med Oncol, 2012, 29(4):2527-2534. doi: 10.1007/s12032-011-0117-4 pmid: 22101791
[9]	Bitarte N, Bandres E, Boni V, et al. MicroRNA-451 is involved in the self-renewal, tumorigenicity, and chemoresistance of colorectal cancer stem cells[J]. Stem Cells, 2011, 29(11):1661-1671. doi: 10.1002/stem.741 pmid: 21948564
[10]	Sun KX, Jiao JW, Chen S, et al. MicroRNA-186 induces sensitivity of ovarian cancer cells to paclitaxel and cisplatin by targeting ABCB1[J]. J Ovarian Res, 2015, 8: 80. doi: 10.1186/s13048-015-0207-6 URL
[11]	Zhou S, Huang Q, Zheng S, et al. MiR-27a regulates the sensitivity of breast cancer cells to cisplatin treatment via BAK-SMAC/DIABLO-XIAP Axis[J]. Tumour Biol, 2016, 37(5):6837-6845. doi: 10.1007/s13277-015-4500-1 URL
[12]	Li Z, Hu S, Wang J, et al. MiR-27a modulates MDR1/P-glycoprotein expression by targeting HIPK2 in human ovarian cancer cells[J]. Gynecol Oncol, 2010, 119(1):125-130. doi: 10.1016/j.ygyno.2010.06.004 pmid: 20624637
[13]	Chen Z, Ma T, Huang C, et al. MiR-27a modulates the MDR1/P-glycoprotein expression by inhibiting FZD7/β-catenin pathway in hepatocellular carcinoma cells[J]. Cell Signal, 2013, 25(12):2693-2701. doi: 10.1016/j.cellsig.2013.08.032 pmid: 24018051

选择文件类型/文献管理软件名称

选择包含的内容

人舌癌耐药细胞株OSC-19/CDDP的多药耐药性检测及差异性表达microRNAs的筛选

Verification of the Multidrug Resistance of Human Tongue Squamous Carcinoma Cell Line OSC-19/CDDP and Its Differently Expressed MicroRNAs

RichHTML

PDF

可视化