Multiple MicroRNAs Modulate P21^Cip1/Waf1 Expression By Directly Targeting Its 3' Untranslated Region

AIM: To screen and identify miRNAs which can directly target p21 and regulate its expression experimentally through a high-throughput luciferase reporter system, then the biological function and significance of these miRNAs was explored. Finally the value on prediction of different target prediction tools was compared and evaluated.METHODS: Molecular cloning technique was used to construct and identify two lentivirus-expressing vectors—pWPXL-Luc and pWPXL-Luc-p21-3'UTR, virus particles were collected after the pWPXL-Luc or pWPXL-Luc-p21-3'UTR vectors were co-transfected with the psPAX2 packaging plasmid and the envelope plasmid pMD2.G into HEK-293T cells. Furthermore, two stable cell lines expressing luciferase singly or co-expressed luciferase and p21-3'UTR were established by transducing HEK-293 cells with recombinant lentivirus; the former was used as control in the following expriments. After si-Dicer1 transfection into HEK 293 cells line by liposome, real-time PCR and Western blot were applied to identify the downregulation of Dicer1 mRNA and the expression of p21 protein respectively. Using four different miRNA target-prediction tools, a library of 266 miRNAs were predicted to target p21-3'UTR, then these miRNAs were individually transfected into the two stable cells by liposome. Luciferase activities were assayed after transfection, comparing with the control, miRNAs which can directly target p21 were screened. The location of the target sites in the 3'UTR of p21 was analyzed, then a serial mutations of these potential target sites were performed and placed to the downstream of a luciferase construct immediately. HEK 293T cells were transiently cotransfected with NC or miRNA mimics, luciferase reporter and pRL-CMV Renilla luciferase reporter. The luciferase activity was measured 36 hr after transfection using the dual-luciferase reporter assay system. Next, these p21-regulating miRNA mimics were introduced into HEK 293 cells, then real-time PCR and Western blot were applied to identify the downregulation of mRNA and protein expression of p21 respectively. Considering our results and the current predictions, the value on prediction of different prediction tools was compared and evaluated. CCK-8 and cell cycle assay were applied to investigate the effects of eight p21-regulating miRNAs on cell proliferation and cell cycle.RESULTS: Enzyme cutting and sequencing results confirm the construction of pWPXL-Luc and pWPXL-Luc-p21-3'UTR successfully. The establishment of two stable cells was verified by virus titre measuration and luciferase activity assay. Compared to NC, mRNA expression of Dicer1 was significantly decreased and protein expression of p21 was substantially increased by si-Dicer1. A pool of 266 miRNAs that could potentially target p21 was selected, then these miRNAs were individually transfected into the two stable cells. Luciferase activities were assayed 36h after transfection. Of these 266 miRNAs, 28 significantly decreased the luciferase activity of the 293-luc-p21UTR reporter compared with the control. Of note, our results appropriately included the previously identified miRNAs (miR-93, miR-106a/b, miR-17 and miR-20a/b) that had been reported to target p21. The relative luciferase activities did not drop as sharply in cells transfected with the mutated p21 3'UTRs compared with cells with their wild-type counterparts. Analysis of the location of the target sites in the 3'UTR of p21 showed that eighteen target sequences distribute quite differently and some miRNAs share the same target sites. Although, in some cases, the level of p21 mRNA was not affected by miRNAs, even elevated slightly, the level of p21 protein was consistently and substantially downregulated. Of these miRNAs, miR-298, miR-515-3p, miR-363, miR-639 and miR-132 showed the most obviously inhibitory effects (more than 4-fold downregulation). Considering our experimental results and the theoretical predictions, TargetScan and PicTar performed the best on prediction. Also the nonconserved target sites should not be ignored and GU pairing was allowed in the development of target-prediction tools. CCK-8 and cell cycle assay demonstrated that eight p21-regulating miRNAs locating on chromosome 19 miRNA cluster could promote cell proliferation and G1-S transition by regulating p21.Conclusion: Twenty-eight miRNAs could posttranscriptionally modulate the expression of p21 by directly binding to its 3'UTR. Some of these miRNAs were invovled in the development of some disease and tumor, and might have some physiological and pathological significance. Of the target-prediction tools, TargetScan and PicTar performed the best on prediction. In the development of target-prediction tools, more attention should be pay to the nonconserved target sites and GU pairing.