Regulation of biological behavior of intrahepatic cholangiocarcinoma by flotillin 2
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 mi-gration of intrahepatic cholangiocarcinoma.Methods Blood samples from 20 healthy physical examination personnel,20 hepatolithiasis(HL)+hepatitis(HA)patients and 10 patients with intrahepatic cholangio-carcinoma were collected from People's Hospital of Zhengzhou University from September 2021 to Decem-ber 2022 for proteomic detection.The expression of FLOT2 in intrahepatic cholangiocarcinoma was ana-lyzed 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 be-tween 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 popula-tion.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,respec-tively,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.
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