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

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,IC^(50))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;IC^(50)实验结果显示,随着MUC13的沉默,肿瘤细胞对紫杉醇的IC^(50)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。

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 paclitaxelresistant cell HeLa229/TR. Firstly, the cells were employed to detect the mRNA levels of MUCINs by quantitative real-time PCR(q PCR). 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. IC^(50)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 upregulated with the up-regulation of MUC1, while down-regulated with the silence of MUC1. IC^(50)assay displayed that silencing of MUC13 in HeLa229/TR decreased IC^(50)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.