The Role And Mechanism Of Long Non-coding RNA UCA1 In Oxaliplatin Resistance In Hepatocellular Carcinoma

ObjectivesTo investigate the role and mechanism of long non-coding RNA Urothelial carcinoma associated 1(UCA1)in the oxaliplatin resistance in Hepatocellular Carcinoma(HCC).And to explore the correlations between UCA1 and oxaliplatin resistance,UCA1 and clinicopathological features in HCC patients.Methods1.Oxaliplatin-resistant HCC cells,HepG2/OXA and SMMC-7721/OXA,were established from HCC parental cells HepG2 and SMMC-7721 by stepwise exposure to gradually increasing concentrations of oxaliplatin.The oxaliplatin IC50 of the above cells were detected by MTT method,and the drug resistance index was calculated as equation of Drug resistance index=drug resistant cell IC50/sensitive cell IC50.UCA1 expression in HL-7702 cells and the above cells were detected by qRT-PCR.2.UCA1-overexpressed plasmids and UCA1-depleted plasmids were constructed.And then overexpressing UCA1 to HCC parental cells,HepG2 and SMMC-7721,were conducted by transfecting with lentiviral carrier.Then interfering UCA1 of the oxaliplatin-resistant HCC cells,HepG2/OXA and SMMC-7721/OXA,were conducted by transfected with lentiviral carrier.The oxaliplatin IC50 of the above cells were detected by MTT method.3.Dual-luciferase reporter assay was used to test whether miR-138-5p shared complementary bound at 3'-UTR of UCA1 by binding sites.Therefore pSI-check2-h-lncRNA-UCA1-WT and pSI-check2-h-lncRNA-UCA1-Mut were constructed and transfected into 293T cells together with miR-138-5p mimics or miR-138-5p negative control(miR-NC).Firefly luciferase value(internal reference value)and Renilla luciferase value(reporter gene luminescence value)were detected.4.Oxaliplatin-resistant HCC cells HepG2/OXA and SMMC-7721/OXA were transfected with miR-138-5p mimics,and oxaliplatin IC50 of the above cells were detected by MTT.5.Western Blot was used to detect the p-mTOR,mTOR,p-Akt and Akt expression of the above cells that overexpressed or interfered UCA1,and the cells that transfected with miR-138-5p mimics.6.HepG2/OXA-shUCA1 cells and HepG2/OXA-NC cells were subcutaneously inoculated in the right flank of nude mice.Each mice was inoculated with 2×10~6 cells(n=5).Oxaliplatin(10 mg/kg/w)or saline were injected intraperitoneally for 4 w eeks.Then the size and weight of tumors in each group were compared after sacrifice.The expression of UCA1,p-mTOR,mTOR,p-Akt and Akt in tumor tissues were detected by qRT-PCR and Western Blot.7.The expression of UCA1 in paraffin sections of tumor tissues from 75HCC patients were detected by in si tu hybridization.Then we compared the expression of UCA1 between oxaliplatin-resistant HCC patients and oxaliplatin-sensitive HCC patients.And the correlation between UCA1 and clinicopathological features of HCC patients was further explored.Results1.HepG2 and SMMC-7721(48h-oxaliplatin IC50 were 9.15±0.34uM and58.01±4.19uMrespectively)weresuccessfullytransforminto oxaliplatin-resistant HCC cells HepG2/OXA and SMMC-7721/OXA(48h-oxaliplatin IC50 were 19.37±0.87uM and 41.23±2.00uM respectively,with drug resistance index of 6.34 times and 2.13 times higher than their parental cells).The results of qRT-PCR showed that the expression of UCA1 in HepG2/OXA and SMMC-7721/OXA were significantly higher than HepG2 and SMMC-7721 respectively.2.The results of qRT-PCR and MTT showed that the expression of UCA1in HepG2-UCA1 and SMMC-7721-UCA1 cells were significantly higher than those in HepG2-NC and SMMC-7721-NC cells(P<0.01),and the oxaliplatin IC50 were significantly higher as well(P<0.05 or P<0.01).The expression of UCA1 in HepG2/OXA-shUCA1 and SMMC-7721/OXA-shUCA1 cells were significantly lower than those in HepG2/OXA-NC and SMMC-7721/OXA-NC cells(P<0.05 or P<0.01),and the oxaliplatin IC50 were significantly lower as well(P<0.01 or P<0.001).3.The results of dual-luciferase reporter gene assay showed that the fluorescent signal value of the miR-138-5p mimics group was significantly lower than the miR-NC group when the pSI-check2-h-lncRNA-UCA1-WT plasmid was co-transfected(P<0.001).The fluorescence signal values of miR-138-5p mimics group were not significantly different from those of miR-NC group when the pSI-check2-h-lncRNA-UCA1-Mut plasmid was co-transfected(P>0.05).4.The qRT-PCR and MTT results showed that the expression of miR-138-5p in HepG2/OXA and SMMC-7721/OXA which were transfected with miR-138-5p mimics were significantly higher than those transfected with miR-NC(P<0.01).And the oxaliplatin IC50 were significantly lower in t he miR-138-5p mimics group(P<0.01).5.Western Blot results showed that the expression of p-mTOR and p-Akt of HepG2 and SMMC-7721 cells were significantly increased after overexpressed UCA1,while the expression level of p-mTOR and p-Akt of HepG2/OXA and SMMC-7721/OXA cells were significantly decreased after interfered UCA1.And the expression level of p-mTOR and p-Akt of HepG2/OXA and SMMC-7721/OXA cells were significantly decreased after transfected with miR-138-5p mimics.6.As in animal model,when compared with the HepG2/OXA-NC group,the HepG2/OXA-shUCA1 group had significantly smaller tumors(P<0.001 or P<0.01),lighter tumor weight(P<0.001 or P<0.01),and lower UCA1(P<0.01)under the same conditions of oxaliplatin or normal saline.7.The results of in situ hybridization showed there were 32(82%)of the paraffin sections of 39 oxaliplatin-resistant HCC patients with high UCA1expression.And in the paraffin section of 36 oxaliplatin-sensitive HCC patients,it was only 9(25%)cases with high UCA1 expression.When HCC patients have high expression of UCA1,their serum AFP levels were relatively higher,tumors were larger,and distant metastasis was more likely to occur(P<0.05).ConclusionsLong non-coding RNA UCA1 can promote the resistance of oxaliplatin of HCC cells by regulating miR-138-5p and activating Akt/mTOR signaling pathway.And there is a positive correlation between the expression of UCA1and oxaliplatin resistance,serum AFP levels,tumor size,and distant metastasis of HCC patients.