与MUC1共同调控肿瘤化疗耐药的MUCIN家族成员的筛选

doi:10.3969/j.issn.1674-8115.2022.09.015

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (9): 1288-1295.doi: 10.3969/j.issn.1674-8115.2022.09.015

• 论著 · 基础研究 • 上一篇

与MUC1共同调控肿瘤化疗耐药的MUCIN家族成员的筛选

汤开然(), 吴琼, 黄思佳, 邱旭东, 李文彦, 邓华云(), 黄雷()

上海交通大学基础医学院组织胚胎学与遗传发育学系，上海 200025

收稿日期:2021-12-13 接受日期:2022-03-09 出版日期:2022-06-07 发布日期:2022-06-07
通讯作者: 邓华云,黄雷 E-mail:sjtu-tkr-01005@sjtu.edu.cn;denghuayun0796@shsmu.edu.cn;leihuang@shsmu.edu.cn
作者简介:汤开然（1999—），女，本科生；电子信箱：sjtu-tkr-01005@sjtu.edu.cn。
基金资助:
国家自然科学基金(81874197);上海交通大学医学院第十三期大学生创新训练计划(1319014);上海市教育委员会实验技术队伍建设计划(BJ1-3000-19-0135)

Screening of MUCIN family members synergistic with MUC1 in tumor chemoresistance

TANG Kairan(), WU Qiong, HUANG Sijia, QIU Xudong, LI Wenyan, DENG Huayun(), HUANG Lei()

Department of Histoembryology, Genetics and Developmental Biology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China

Received:2021-12-13 Accepted:2022-03-09 Online:2022-06-07 Published:2022-06-07
Contact: DENG Huayun,HUANG Lei E-mail:sjtu-tkr-01005@sjtu.edu.cn;denghuayun0796@shsmu.edu.cn;leihuang@shsmu.edu.cn
Supported by:
National Natural Science Foundation of China(81874197);The 13th Innovation Training Program for Students of Shanghai Jiao Tong University School of Medicine(1319014);Experimental Technical Team Construction Project of Shanghai Municipal Education Commission(BJ1-3000-19-0135)

摘要/Abstract

摘要：

目的·在黏蛋白（MUCIN）家族成员中，筛选与黏蛋白1（mucin 1，MUC1）协同调控肿瘤细胞化学治疗（化疗）耐药的蛋白。方法·利用2对细胞系（紫杉醇耐药细胞HeLa229/TR-CasCTL和MUC1敲除细胞HeLa229/TR-CasMUC1，母本细胞HeLa229/P和紫杉醇耐药细胞HeLa229/TR），应用实时荧光定量PCR（quantitative real-time PCR，qPCR）技术检测MUCIN家族成员的mRNA表达水平，分析其与MUC1表达的相关性；通过蛋白质印迹法（Western blotting）检测筛选出的MUCIN家族成员的蛋白表达水平，以筛选出在mRNA和蛋白水平均与MUC1表达存在相关性的靶蛋白；通过短发卡RNA（short hairpin RNA，shRNA）方法沉默靶基因，利用药物半抑制浓度（half-maximum inhibitory concentration，IC₅₀）实验检测细胞耐药性的变化；进一步利用GEPIA数据库分析多种肿瘤组织中MUC1和MUC13的平均表达水平；采用R2数据库分析在宫颈癌、结肠癌和胰腺癌组织中MUC1与MUC13表达的线性相关性；通过Kaplan-Meier Plotter数据库分析MUC1和MUC13的表达与胃癌患者生存率的关系。结果·qPCR检测结果显示，在MUCIN家族成员中，与MUC1在mRNA水平存在相关性的有MUC13和MUC18；Western blotting检测结果显示，在蛋白水平，只有MUC13随着MUC1的上调而上调，随着MUC1的敲除而下调；IC₅₀实验结果显示，随着MUC13的沉默，肿瘤细胞对紫杉醇的IC₅₀明显下降。进一步利用GEPIA数据库分析发现，在多种肿瘤组织中MUC1与MUC13存在同时高表达或低表达的特点；通过R2数据库分析发现，MUC1和MUC13的表达量在宫颈癌、结肠癌和胰腺癌肿瘤组织中呈正相关；通过Kaplan-Meier Plotter数据库分析发现，MUC1和MUC13的表达量与胃癌患者的生存率呈负相关。结论·MUC1与MUC13在肿瘤细胞化疗耐药中存在协同作用，同时与肿瘤患者的生存预后密切相关；临床上联合抑制MUC1和MUC13在肿瘤细胞化疗耐药治疗上具有可行性。

关键词: 黏蛋白家族, 黏蛋白1, 协同作用, 化学治疗耐药

Abstract:

Objective ·To screen partners of mucin 1 (MUC1) in MUCIN family members that involve in chemoresistance. Methods ·Two pairs of cell lines were employed, including HeLa229/TR-CasCTL and HeLa229/TR-CasMUC1, and HeLa229/P and paclitaxel-resistant cell HeLa229/TR. Firstly, the cells were employed to detect the mRNA levels of MUCINs by quantitative real-time PCR (qPCR). The correlation of mRNA levels between MUC1 and MUCINs was analyzed. Then, Western blotting was performed to detect the protein levels of the selected MUCINs to get the proteins correlated with MUC1 at both mRNA and protein levels. IC₅₀ assay was further employed to detect the role of target protein in paclitaxel resistance by knockdown of the gene through shRNA in HeLa229/TR cells. The mean expression levels of MUC1 and MUC13 in a variety of cancer tissues were further explored in the GEPIA database. The R2 database was further employed to detect the linear correlation of the expression between MUC1 and MUC13 in cervical, colon and pancreatic cancer tissues. Finally, the expression of MUC1 and MUC13 was analyzed by the Kaplan-Meier Plotter database in relation to the survival rate of patients with gastric cancer. Results ·The detection of qPCR showed that MUC13 and MUC18 exhibited correlation with MUC1 at the mRNA level in MUCIN family members. Western blotting showed that only MUC13 protein was up-regulated with the up-regulation of MUC1, while down-regulated with the silence of MUC1. IC₅₀ assay displayed that silencing of MUC13 in HeLa229/TR decreased IC₅₀ to paclitaxel. By searching the GEPIA database, the expression levels of MUC1 and MUC13 were simultaneous high or low in a variety of cancer tissues. From the R2 database, MUC1 and MUC13 showed a positive linear correlation in cervical, colon and pancreatic cancer tissues. In addition, the negative relationship of the expression of MUC1 and MUC13 and survival rate of patients with gastric cancer from the Kaplan-Meier Plotter database was observed. Conclusion ·MUC13 is a partner for MUC1 in promoting tumor chemoresistance. The expression levels of MUC1 and MUC13 are negatively related with the survival rate of cancer patients. It's feasible for joint inhibiting MUC1 and MUC13 to overcome chemoresistance.

Key words: MUCIN family, mucin 1 (MUC1), synergistic effect, chemoresistance

中图分类号:

汤开然, 吴琼, 黄思佳, 邱旭东, 李文彦, 邓华云, 黄雷. 与MUC1共同调控肿瘤化疗耐药的MUCIN家族成员的筛选[J]. 上海交通大学学报（医学版）, 2022, 42(9): 1288-1295.

TANG Kairan, WU Qiong, HUANG Sijia, QIU Xudong, LI Wenyan, DENG Huayun, HUANG Lei. Screening of MUCIN family members synergistic with MUC1 in tumor chemoresistance[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(9): 1288-1295.

图/表 7

图1 qPCR检测MUCIN家族成员的mRNA表达水平Note： ①P=0.000, ②P=0.006, compared with HeLa229/TR-CasMUC1.

Fig 1 Detection of mRNA expressions of MUCIN family members by qPCR

图2 qPCR检测筛选出的MUCIN家族成员的mRNA表达水平Note： ①P=0.000, compared with HeLa229/TR.

Fig 2 Detection of mRNA expressions of selected MUCIN family members by qPCR

图3 MUC13与MUC1的蛋白表达Note： A. Detection of protein expressions of MUC1, MUC13 and MUC16 by Western blotting in HeLa229/P and HeLa229/TR cell lines. B. Detection of protein expressions of MUC1, MUC13 and MUC16 by Western blotting in HeLa229/TR-CasCTL and HeLa229/TR-CasMUC1 cell lines.

Fig 3 Protein expressions of MUC13 and MUC1

图4 下调 MUC13 增加耐药肿瘤细胞对紫杉醇的敏感性Note： A. Detection of the silence of MUC13 by Western blotting in HeLa229/TR cell line. B. Detection the paclitaxel sensitivity of HeLa229/TR MUC13 sh1 and MUC13 sh2 compared with HeLa229/TR by IC50.

Fig 4 Downregulation of MUC13 increasing the paclitaxel sensitivity of chemoresistance tumor cells

图5 利用GEPIA数据库分析 MUC1 和 MUC13 在多种肿瘤组织中的表达情况Note： COAD—colon adenocarcinoma; PAAD—pancreatic adenocarcinoma; READ—rectum adenocarcinoma; STAD—stomach adenocarcinoma; CHOL—cholangiocarcinoma; ESCA—esophageal carcinoma; PRAD—prostate adenocarcinoma; SKCM—skin cutaneous melanoma; TGCT—testicular germ cell tumors; THYM—thymoma; UVM—uveal melanoma; ACC—adrenocortical carcinoma; PCPG—pheochromocytoma and paraganglioma.

Fig 5 Correlations between the expression levels of MUC1 and MUC13 in multiple cancer tissues by GEPIA

图6 宫颈癌、结肠癌、胰腺癌中 MUC1 与 MUC13 表达的相关性

Fig 6 Correlation of MUC1 and MUC13 at the level of gene expression in cervical cancer, colon cancer and pancreatic cancer tissues

图7 Kaplan-Meier plotter数据库分析 MUC1 和 MUC13 的表达与胃癌患者生存率的关系

Fig 7 Relationships between expressions of MUC1 as well as MUC13 and survival rates of gastric cancer patients shown in Kaplan-Meier plotter database

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与MUC1共同调控肿瘤化疗耐药的MUCIN家族成员的筛选

Screening of MUCIN family members synergistic with MUC1 in tumor chemoresistance

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