The Functional Study Of Long Non-coding RNA-LLEST In Leukemia

[Objective]1.To study the mechanism of inhibiting BCL2 expression in leukemia cells by long non-coding RNA-LLEST.2.To study the effect of long non-coding RNA-LLEST on AMPK activity in leukemia cells.[Method]Our previous data declared that long non-coding RNA-LLEST was low expressed in malignant blood diseases compared with iron deficiency anemia,aplastic anemia and other benign blood diseases and it could inhibit the expression of BCL2 in leukemia cell lines,but the mechanism was still unclear.First we determined the subcellular localization of LLEST in leukemia cell lines K562 and SHI-1 by RNA FISH assay.RNA antisense purification(RAP)assay and quantitative PCR were done to determined whether LLEST directly interacts with BCL2 3'UTR.Target site were predicted by bioinformatics analiysis.The target sequence of LLEST on BCL23'UTR was verified by double luciferase reporter assay.The stable cell line K562 with high expression of LLEST and the stable cell line U937 with LLEST knockdown were constructed,and the expression of LLEST in the corresponding cell line was verified by qRT-PCR.Western Blot was used to detect the effect of LLEST on AMPK activity in leukemia cell lines K562 and U937 when glucose supply was adequate or insufficient.Western Blot was used to determine the effect of LLEST on AMPK activity in prostate cancer cell line PC-3 and non-neoplastic cell lines HEK293 and HUVEC when glucose supply was adequate or insufficient.[Results]1.RNA FISH results showed that in leukemia cell lines K562 and SHI-1,LLEST was mainly located in the cytoplasm.RAP and quantitative PCR results showed that LLEST directly interacts with BCL2 3'UTR.By analyzing miRBase database and the TargetScanHuman database,we found that GCCTGTAA nucleotide sequence on BCL23'UTR may be the target of LLEST.The results of dual-luciferase reporter gene assay confirmed that the target sequence of LLEST on BCL2 3'UTR was GCCTGTAA,indicating that LLEST inhibited the expression of BCL2 in a dose-dependent manner.2.In dextrose deficient energy stress,the AMPK activation(p-AMPK)level of K562 cell lines with high expression of LLEST was significantly lower than that of empty carrier cell lines.After glucose supply was restored,the p-AMPK level of both K562 cell lines with high expression of LLEST and empty carrier cell lines was lower than that of unsweetened cell lines,and the p-AMPK level of the former was still lower than that of the latter.In the U937 cell line with LLEST knockdown,p-AMPK level was significantly higher than that of the control cell line when glucose was deficient,and p-AMPK level of both the U937 cell line with LLEST knockdown and the control cell line' decreased after glucose supply was restored,and p-AMPK level of the former was higher than that of the latter,and the results were statistically different.3.In PC-3 cell lines of prostate cancer,the p-AMPK level of cell lines with high LLEST expression was lower than that of empty carrier cell lines when glucose was deficient.After glucose supply was restored,the p-AMPK level of both cell lines was lower than that of unsweetened cell lines,and the p-AMPK level of cell lines with high LLEST expression was still lower than that of empty carrier cell lines.In the non-neoplastic cell lines HEK293 and HUVEC,there was no significant difference in the p-AMPK level between the cell lines with high LLEST expression and the empty carrier cell lines when glucose was deficient.When glucose supply was restored,the p-AMPK level in the cell lines with high LLEST expression was lower than that of the empty carrier cell lines,and the results were statistically different.[Conclusion]1.Long non-coding RNA-LLEST directly inhibits BCL2 expression by acting on BCL23'UTR,and this inhibitory effect is dose-dependent.2.Long non-coding RNA-LLEST inhibits the activation of AMPK in leukemia cell lines,and this inhibitory effect is not affected by glucose level.3.The inhibition of phosphorylation of AMPK by long non-coding RNA-LLEST on non-tumor cell lines was influenced by glucose level.