LncRNA HULC Regulating The Expression Of ABCC1 Through MTOR Effected Of Drug Sensitivity In Hepatocellular Carcinoma

Objective: To explore the regulatory function of HULC on ABCC1 and m TOR,and their relations concerning drug sensitivity.Methods: Real Time Fluorescent Quantitative PCR(q RT-PCR)is applied to test the RNA level of HULC and ABCC1.Western blotting was utilized to verify the protein expression of ABCC1,m TOR,p-m TOR.MTT was used to test the sensitivity of cancer cells to 5-FU and DDP.Cell cycle and apoptosis are measured by Flow cytometry and cell growth was determined by EDU.Results: Compared with normal liver cell line L-O2 and liver cancer cell line SMMC-7721 Hep G2 expresses more HULC by q RT-PCR.Knocking down of HULC by si RNA(si HULC)significantly reduced ABCC1 m RNA in Hep G2 cells(1.08±0.07 vs 0.77±0.33).MTT result shows that si-HULC significantly decreased cell survival rate compared to si-NC(0.76 times less).Hep G2 cells transfected with si-HULC showed lower cell viability when treated with 5FU(0;50;500;5,000;50,000 μM,p<0.05)or DDP(0;12.5;25;50;100μM,p<0.05)compared with si-NC by MTT.DDP and5-FU significantly decreased viability at 50 and 5,000μM respectively,so these concentrations are adopted in later experiments.When treated with 5,000μM 5-FU or50μM DDP for 24/48/72 hours,si-HULC showed lower cell viability compared with si-NC and the difference became very clear at 48 h in Hep G2 cells.Especially,si-HULC showed significantly lower(0.62/0.75 times less respectly)cell viability when treated with 5,000μM 5-FU or 50μM DDP compared with si-NC in 48 hours.EDU assay showed the significant inhibition of cell growth with si HULC.Cells were treated with 5,000μM 5FU or 50μM DDP for 48 h and si-HULC showed lower cell growth rate compared with si-NC.(p<0.05).Western blotting showed more ABCC1 expressed in Hep G2 cells than L-O2.And si-HULC decreased ABCC1 protein level(0.8 fold of si-NC).Hep G2 cells express more m TOR and p-m TOR compared with L-O2,and 800 n M of Rapamycin significantly decreased ABCC1 protein expression by western blotting.In HULC knockdown cells,m TOR and p-m TOR are markedly decreased(0.85 and 0.43 fold vs si-NC respectively).Rapamycin also decreased m TOR and p-m TOR at 800 n M(0.48 and 0.69 fold vs control respectly).These results indicate that HULC increased m TOR and p-m TOR expression in Hep G2 cells,and 800 n M of Rapamycin showed similar effects.So knockdown of HULC by si-HULC inhibits m TOR signaling pathway.Flow cytometry showed si-HULC also play a role in cell cycle and apoptosis.Compared with si-NC,si HULC arrested cell cycle at G1/S phase,and increased cell apoptosis.si-HULC cells treated with 5,000μM 5-FU or 50μM DDP showed more cell cycle arrested at G1/S phase and apoptosis rate.Conclusion:1.Knockdown of lnc RNA HULC inhibited liver cell proliferation and decreased cell survival.2.Knockdown of lnc RNA HULC down regulated m TOR,thus decreasing ABCC1 expression.3.5-FU and DDP Drug sensitivity of Hep G2 cells is increased by lnc RNA HULC knockdown,which inhibited tumorigenesis,promoted cell apoptosis,and arrested cell cycle at G1/S phase.