脂筏特征蛋白2对肝内胆管细胞癌生物学行为的调控作用

Regulation of biological behavior of intrahepatic cholangiocarcinoma by flotillin 2

目的观察脂筏特征蛋白2(FLOT2)在肝内胆管细胞癌中的表达,探讨FLOT2对肝内胆管细胞癌增殖,迁移的作用。方法收集2021年9月至2022年12月郑州大学人民医院健康体检人员20例、肝内胆管结石(HL)+肝炎(HA)患者20例和肝内胆管细胞癌患者10例共50例检测血液样本,行蛋白组学检测,结合GEPIA2数据库分析FLOT2在肝内胆管细胞癌中的表达。构建小干扰RNA(siRNA)沉默肝内胆管癌细胞HuCCT1中的目的基因,以NC-FLOT2为对照组,si-FLOT2为实验组,实时荧光定量聚合酶链式反应(qRT-PCR)及蛋白质印迹法(Western blot)检测肝内胆管癌细胞中FLOT2的表达水平。细胞计数试剂盒(CCK-8)检测细胞增殖能力及细胞毒性,划痕愈合实验检测细胞迁移能力,流式细胞术检测细胞凋亡程度,组间比较采用t检验。结果蛋白组学表明在肝内胆管细胞癌患者中,FLOT2表达较正常人群升高。qPCR实验中,HuCCT1细胞NC组表达量为(0.77±0.10),实验组表达明显下降,分别为0.29±0.09、0.15±0.01、0.11±0.02(t=11.634、11.725、13.432,P均<0.01)。Western blot实验表明,对照组FLOT2条带灰度值为(0.91±0.03),实验组表达降低(0.41±0.14,t=5.160,P<0.05)。划痕实验中下调FLOT2可降低肝内胆管癌细胞迁移能力,48 h后划痕愈合率对照组为(0.89±0.09)%,实验组分别为(0.49±0.08)%、(0.50±0.06)%、(0.60±0.07)%(t=11.170、5.626、3.838,P均<0.01)。CCK-8增殖实验对照组为5.49±0.53,实验组(4.19±0.22,t=7.164,P<0.05;3.33±0.25、1.83±0.28,t=10.065、10.593,P均<0.01)。凋亡实验中,实验组凋亡率[(6.92±0.89)%],敲低FLOT2后实验组较对照组凋亡明显增多[(10.03±0.31)%,t=-6.828,P<0.05;(12.43±0.83)%、(13.87±0.38)%,t=-11.709、-12.101,P均<0.01]。CCK-8毒性实验表明,RBE和HuCCT1对吉西他滨的IC50分别为(8.90±1.28)、(34.31±7.40)μg/ml,RBE对吉西他滨的IC50明显低于HuCCT1(t=-7.077,P<0.05)。qPCR结果表明,HuCCT1中FLOT2表达量明显高于RBE[(0.004±0.001)、(0.019±0.005),t=-5.187,P<0.05]。敲低FLOT2后,HuCCT1细胞株对吉西他滨的IC50值明显降低,实验组IC50[(7.30±0.60)、(6.52±0.61)、(7.03±1.08)μg/ml,t=-17.173、-15.079、-18.737,P均<0.01]。结论FLOT2在肝内胆管细胞癌中表达升高,并影响肝内胆管癌细胞HuCCT1的增殖、迁移、凋亡和耐药能力。

Objective To observe the expression of lipid raft characteristic protein 2(flotillin 2,FLOT2)in intrahepatic cholangiocarcinoma,and to explore the effect of FLOT2 on the proliferation and migration of intrahepatic cholangiocarcinoma.Methods Blood samples from 20 healthy physical examination personnel,20 hepatolithiasis(HL)+hepatitis(HA)patients and 10 patients with intrahepatic cholangiocarcinoma were collected from People’s Hospital of Zhengzhou University from September 2021 to December 2022 for proteomic detection.The expression of FLOT2 in intrahepatic cholangiocarcinoma was analyzed by GEPIA2 database.Small interfering RNA(siRNA)was constructed to silence the target gene of HuCCT1 in intrahepatic bile duct cancer cells,with NC-FLOT2 as the control group and si-FLOT2 as the experimental group.Real-time quantitative polymerase chain reaction(qRT-PCR)and Western blotting were used to detect FLOT2 expression in intrahepatic biliary duct cancer cells.Cell proliferation ability and cytotoxicity were detected by cell counting kit-8(CCK-8),cell migration ability was detected by scratch healing test,and apoptosis degree was detected by flow cytometry.The t test was used for comparison between groups,and P<0.05 was considered statistically significant.Results Proteomics showed that FLOT2 expression was higher in patients with intrahepatic cholangiocarcinoma than in the normal population.The qPCR results showed that the expression level of FLOT2 in HuCCT1 cells in NC group was(0.77±0.10),and that in the experimental group was significantly decreased[(0.29±0.09),(0.15±0.01),(0.11±0.02),t=11.634,11.725,13.432,P<0.01].Western blotting results showed that the gray value of FLOT2 bands in the control group was(0.91±0.03),and the expression of FLOT2 bands in the experimental group was decreased[(0.41±0.14),t=5.16,P<0.05].The scratch test showed that down-regulation of FLOT2 could reduce the migration ability of intrahepatic bile duct cancer cells,and the scratch healing rate after 48 h was(0.89±0.09)%in the control group and 0.60±0.07 in the experimental group(0.49±0.08)%,(0.50±0.06)%,(0.60±0.07)%(t=11.170,t=5.626,3.838;P<0.01).CCK-8 proliferation experiment showed was(5.49±0.53)in the control group and(4.19±0.22)(t=7.164,P<0.05),(3.33±0.25)and(1.83±0.28)in the experimental group(t=10.065,10.593,P<0.01),suggesting that the decrease of FLOT2 expression can significantly inhibit the proliferation of intrahepatic cholangiocarcinoma.The apoptosis experiment showed that the apoptosis rate in the experimental group was(6.92±0.89)%,which was significantly increased as compared with the control group after FLOT2 was knocked down[(10.03±0.31)%,t=-6.828,P<0.05;(12.43±0.83)%,(13.87±0.38)%,t=-11.709,-12.101,P<0.01].The toxicity test of CCK-8 showed that the IC50 of RBE and HuCCT1 for gemcitabine was(8.90±1.28)and(34.31±7.40)μg/ml,respectively,and the IC50 of RBE for gemcitabine was significantly lower than that of HuCCT1(t=-7.077,P<0.05).The qPCR results showed that the expression level of FLOT2 in HuCCT1 was significantly higher than that in RBE[(0.004±0.001),(0.019±0.005),t=-5.187,P<0.05].After FLOT2 was knocked down,the IC50 value of gemcitabine in HuCCT1 cell line was significantly decreased,and that in the experimental group was(7.30±0.60),(6.52±0.61),(7.03±1.08)μg/ml(t=-17.173,t=-15.079,-18.737,all P<0.01).Conclusion The expression of FLOT2 is increased in intrahepatic cholangiocarcinoma,which affects the proliferation,migration,apoptosis and drug resistance of HuCCT1.