Long Non-coding RNA NORAD Affects Cisplatin Resistance In Esophageal Squamous Cell And The Related Mechanisms

Part one Establishing of cisplatin resistant cells and detecting the expression of long non-coding RNA NORAD in esophageal squamous cell carcinoma cellsObjective: To establish cis-dichloro diammine platinum(CDDP)resistant cell line of esophageal squamous cell cancer(ESCC)by intermittent administration of CDDP.The expression of long non-coding RNA(lnc RNA)DNA damage-activated RNA(NORAD)and the effects on cell biological behavior were detected.Methods:By means of intermittent induction in vitro and increasing drug concentration,a CDDP-resistant cell line YES-2/CDDP-R was constructed which was capable of maintaining growth in 0.5 ?g/ml CDDP culture medium.Morphological changes of YES-2 and YES-2/CDDP-R cells was observed by an inverted microscope.In YES-2 and YES-2 / CDDP-R cells,MTT detected the half-inhibitory concentration of CDDP in two kinds of cells.Quantitative real-time polymerase chain reaction(q RT-PCR)and western blotting detected the multidrug resistance-associated protein 1(MRP1)in two types of cells.Cell scratch healing experiments were performed to test the migration ability of the two cells.q RT-PCR was used to detect NORAD expression in two kinds of cells.Results:After 9 months of CDDP concentration gradient treatment,esophageal cancer CDDP resistant cell line YES-2/CDDP-R was established.Under an inverted microscope,the boundary of YES-2/CDDP-R cell membrane was unclear,the refractive index of the nucleus decreased,the number of intracellular vacuoles and black particles increased significantly,and giant cells appeared.The IC50 of CDDP of YES-2/CDDP-R cells was significantly higher than that of YES-2 cells by MTT assay(P<0.05).After CDDP resistance in YES-2 cells,the expression level of MRP1 increased(P<0.05),the cell migration ability increased(P<0.05),and the expression level of NORAD increased(P<0.05).Conclusions:1.The CDDP-resistant cell line YES-2/CDDP-R of esophageal cancer YES-2 cells was successfully established.After drug resistance,the sensitivity to CDDP decreased,MRP1 expression was increased,and migration ability was enhanced.2.The expression of NORAD in YES-2/CDDP-R cells was higher than that in parental cells.Part two Long non-coding RNA NORAD regulates cisplatin resistance in esophageal squamous cell carcinoma by targeting miR-101-3pObjective: Database search was used to predict the possible downstream signal molecules of NORAD,and to explore the effect of NORAD on cisplatin resistance in esophageal cancer YES-2 cells through micro RNA(miRNA).Methods:The small interfering RNA(si RNA)of lnc RNA NORAD was used to knock down NORAD expression in esophageal cancer CDDP-resistant cell line YES-2/CDDP-R.Set the group to(Con group,si-NORAD-1 group,si-NORAD-2 group and si-NORAD-3 group),q RT-PCR detected NORAD knockout efficiency of each group.After NORAD knockdown of YES-2/CDDP-R cells,MTT detected changes in cell proliferation ability,cell scratch healing experiments detected changes in cell migration ability,and transwell experiments detected changes in cell invasion ability.The DIANA-Lnc Base v.2 database queries NORAD downstream targets.q RT-PCR was used to detect miR-101-3p expression in YES-2/CDDP-R cells before and after NORAD knockdown.The Target Scan database retrieves miR-101-3p downstream targets.After knockdown of NORAD and miR-101-3p,western blotting was used to detect the expression of MRP1 protein in cells,and MTT was used to detect the tolerance of cells to CDDP.Results:The si-NORAD group cells and the Con group cell were successfully set up(P<0.05 or P<0.01,respectively).The cell proliferation ability in si-NORAD groups were decreased than that in the Con group(P<0.05).Also,the migration ability and invasion ability in si-NORAD groups were decreased than that in the Con group(P<0.05).By DIANA-Lnc Base v.2 database,seven predicted binding sites for miR-101-3p to NORAD were found out,which indicating that miR-101-3p may be a downstream target gene of NORAD.The miR-101-3p expression in YES-2/CDDP-R cells was down-regulated than that in the YES-2 cells(P<0.05);but in si-NORAD groups was up-regulated than that in the Con group(P<0.05).Target Scan database search found binding site between miR-101-3p and 3'UTR of MRP1,indicating that miR-101-3p may play a role in MRP1 post-transcriptional regulation.The expression of MRP1 protein in the si-NORAD groups was significantly lower than that in Con group(P<0.05),but in the si-NORAD+miR-101-3p inhibitor group was higher than that in si-NORAD group(P<0.05).The IC50 value of the si-NORAD group was lower than that of the Con group(P<0.05),but in the si-NORAD+miR-101-3p inhibitor group was higher than that in si-NORAD group(P<0.05).Conclusions:1.NORAD affects the cell proliferation,migration,and invasion behavior of esophageal cancer CDDP-resistant cell line YES-2/CDDP-R.2.NORAD regulates CDDP resistance in YES-2 cells through miR-101-3p / MRP1.