肝细胞核因子1A对人胰腺癌PANC1细胞的吉西他滨联合白蛋白紫杉醇耐药性的影响

Effects of HNF1A inhibition on drug resistance of human pancreatic cancer PANC1 cells to gemcitabine plus abraxane

目的探讨肝细胞核因子1A (HNF1A)对人胰腺癌PANC1细胞株吉西他滨联合白蛋白紫杉醇耐药性的影响及其作用机制。方法选取2012年3月至2017年5月间中山大学孙逸仙纪念医院胆胰外科78例行手术切除后接受吉西他滨联合白蛋白紫杉醇化疗方案的局部晚期或远处转移的胰腺癌患者,采用qPCR法检测胰腺癌组织HNF1A表达水平,根据HNF1A表达水平的中位数将患者分为高表达组(39例)和低表达组(39例),分析HNF1A表达与肿瘤临床病理参数及患者生存期的相关性;采用qPCR法检测3株敏感胰腺癌细胞株(BxPC-3、CFPAC-1和L3.6pl)和4株耐药胰腺癌细胞株(PANC1、MIA PaCa-2、Hs766T和Mpanc96)HFN1A mRNA表达水平。以携带插入HNF1AcDNA质粒的慢病毒感染方法构建过表达HNF1A的PANC1 (HNF1A组),以携带空质粒的慢病毒感染的PANC1为对照组,采用qPCR和蛋白质印迹法分别检测两组PANC1细胞HNF1A和ATP结合盒转运蛋白家族中的ABCC1 mRNA及蛋白表达;采用MTT法检测两组细胞对吉西他滨和白蛋白紫杉醇的半抑制浓度(IC50);采用流式细胞术检测两组细胞凋亡率。结果HNF1A高表达组患者平均生存期显著长于低表达组(17.9个月比12.4个月),差异有统计学意义(P<0.001);胰腺癌组织HNF1A低表达与TNM分期晚、有神经浸润、患者生存期短相关。耐药的PANC1细胞HNF1A mRNA表达水平较敏感的BxPC-3细胞下调6.73倍,差异具有统计学意义(P<0.001)。HNF1A组的ABCC1 mRNA和蛋白表达水平较对照组显著下降(0.012±0.004比0.047±0.008、0.281±0.040比0.832±0.046,P=0.003、P<0.001)。HNF1A组PANC1细胞对吉西他滨和白蛋白紫杉醇的IC50较对照组显著下降[(26.31±2.91)μmol/L比(72.63±4.07)μmol/L],细胞凋亡率显著升高[(40.18±1.64)%比(21.31±1.98)%],差异均有统计学意义(P值均<0.01)。结论HNF1A可能通过下调ABCC1表达介导胰腺癌细胞产生吉西他滨和白蛋白紫杉醇耐药性。

Objective To explore the effects of hepatocyte nuclear factor 1 homeobox A(HNF1A) on drug resistance of PANC1 cells to gemcitabine plus abaraxane and explore the potential mechanism. Methods 78 pancreatic cancer patients with locally advanced or distant metastasis who received gemcitabine plus abaraxane chemotherapy after surgery in Biliary and Pancreatic Surgery Department of Sun Yat-sen Memorial Hospital from March 2012 to May 2017 were enrolled. qPCR was used to detect HNF1A mRNA levels in pancreatic cancer tissue. The patients were divided into high-expression group (n=39) and low-expression group (n=39) according to the median expression level of HNF1A, and the correlation of HNF1A expression with cancer clinicopathologic parameters and survival was analyzed. qPCR was used to detect HNF1A mRNA of 3 drug-sensitive cell lines (BxPC-3, CFPAC-1 and L3.6pl) and 4 drug-resistant pancreatic cancer cell lines (PANC1, MIA PaCa-2, Hs766T and Mpanc96). Lentivirus with plasmids carrying HNF1AcDNA infection was used to establish HNF1A overexpressing PANC1 cells ( HNF1A group), and lentivirus with empty plasmids were used to infect PANC1 cells to construct the control group. The mRNA and protein expression of HNF1A and ATP binding cassette transporter family ABCC1 in HNF1A group and control group were measured by qPCR and Western Blot, respectively. The half inhibition concentration (IC50) of gemcitabine plus abaraxane was detected by MTT, and cell apoptosis was examined by flow cytometry. Results Pancreatic cancer patients with high HNF1A expression had a better overall survival than those with low HNF1A expression (17.9 months vs 12.4 months), and the difference was statistically significant (P<0.001). HNF1A low expression in pancreatic cancer tissue was significantly associated with advanced TNM stage, perineural invasion (PNI) and short overall survival. The expression level of HNF1A was significantly down-regulated in drug-resistant PANC1 cells compared to drug-sensitive BxPC-3 cells by an average fold change of 6.73, and the difference was statistically significant (P<0.001). In HNF1A group, the mRNA and protein levels of ABCC1 were significantly decreased compared with those in control group (0.012±0.004vs0.047±0.008, 0.281±0.040vs0.832±0.046, P=0.003, P<0.001). IC50 of HNF1A group to gemcitabine plus abraxane was decreased compared with that of control group [(26.31±2.91)μmol/L vs (72.63±4.07)μmol/L], and the cell apoptosis rate of HNF1A group was increased compared with that of control group [(40.18±1.64)% vs(21.31±1.98)%], and the differences were statistically significant (P<0.01). Conclusions HNF1A may induce resistance of pancreatic cancer cell to gemcitabine plus abraxane by downregulating ABCC1.