Function Of MEG2 Regulation By MiR-181a-5p In Gastric Cancer

Gastric cancer(GC)is one of the leading causes of cancer-related death in the world.Recently,clinicians have systemically measured the activity of potential genetic targets in tumours for GC therapy.But there is no internationally accepted standard of care,and survival remains poor.Therefore,more effective therapeutic approaches to GC are much needed.It is important to understand the molecular mechanisms of GC to help develop new therapeutic strategies.Protein-tyrosine phosphatase MEG2(MEG2)is a member of the protein tyrosine phosphatase(PTP)family.MEG2 directs dephosphorylation of STAT3(signal transducer and activator of transcription 3)and the EGFR family,including EGFR(epidermal growth factor receptor)and HER-2,to inhibit receptor tyrosine kinase(RTK)activation.Since EGFR,HER-2 and STAT3 pathways play important roles in the development of GC,MEG2 may be a good tumour suppressor in GC by dephosphorylating EGFR,HER-2 and STAT3.In this study,we examined the expression of MEG2 protein by western blotting and that of MEG2 mRNA by qRT-PCR.We used bioinformatic analyses to search for microRNAs that potentially target MEG2.We performed a luciferase reporter assay to investigate the interaction between miR-181a-5p and MEG2.In addition,we assessed the effects of MEG2 and miR-181a-5p on gastric cancer cells in vitro and in vivo.Part IMEG2 functions as a tumour suppressor gene in gastric cancerObjective:the aim of this study was to determine the level of MEG2 expression in human gastric cancer tissues and investigate its function in gastric cancer.Methods:we measured the expression of MEG2 protein by western blotting and that of MEG2 mRNA by qRT-PCR in 20 pairs of gastric cancer tissues and 6 gastric cell lines.CCK-8 and Transwell assays were performed to analyse the effect of MEG2 on the proliferation and migration of gastric cancer cells.Results:the expression levels of MEG2 protein were strikingly lower both in gastric cancer tissues and in 5 gastric cancer lines but not mRNA,compared to adjacent noncancerous tissues and one normal gastric epithelial cell line(GES-1),respectively.MGC803 cells transfected with MEG2 siRNA showed an increase in cell proliferation and migration,while those transfected with the MEG2-overexpressing plasmid suppressed cell proliferation and migration.Conclusions:these results suggest that MEG2 may function as a tumour suppressor gene and suppress proliferation and migration in gastric cancer.The inconsistency between MEG2 mRNA and protein in gastric cancer tissues suggested that a post-transcriptional mechanism is involved in the regulation of MEG2 protein in gastric cancer.Part ⅡmiR-181a-5p reduces MEG2 expression directly at the post-transcriptional levelObjective:the purpose of this study was to further investigate the potential mechanism of downregulation of MEG2 in gastric cancer.Methods:we scanned three computational algorithms(TargetScan,miRanda and PicTar)to predict potential miRNAs that target MEG2.We chose miR-181a-5p for further experimentation and measured its levels in the same 20 pairs of gastric cancer tissues and 6 gastric cell lines.We used western blotting and qRT-PCR to evaluate MEG2 expression levels in human gastric cancer cells after miR-181a-5p overexpression or knockdown.Then,we used Luciferase reporter assay to determine whether miR-181a-5p regulated MEG2 expression by directly interacting with the binding site in the MEG2 3’-UTR.Results:miR-181a-5p was predicted as a regulator of MEG2 by all three software models.The minimum free energy value was-21.0 kcal/mol.The,miR-181a-5p levels were higher in the gastric cancer tissues and 5 gastric cancer cell lines.In vitro,MEG2 expression levels significantly decreased upon miR-181a-5p overexpression and increased upon knockdown of miR-181a-5p in MGC803 cells.To confirm the robustness,we used another gastric cell line(SGC7901)to repeat the above experiments and observed consistent results.In luciferase reporter assay,miR-181a-5p overexpression led to an approximately 80%decrease in luciferase reporter activity,whereas miR-181a-5p inhibition led to a nearly three-fold increase in reporter activity.As a negative control,the luciferase activity of the mutated reporter was not obviously affected by either miR-181a-5p overexpression or knockdown.Conclusions:these results suggest that miR-181a-5p directly recognizes and binds to the 3 ’-UTR of the MEG2 transcript and inhibits MEG2 translation.Part ⅢThe regulation of miR-181a on the tumour suppressor activity of MEG2 in gastric tumourigenesisObjective:the aim of this study was to assess the effects of MEG2 and miR-181a-5p on gastric cancer cells in vitro and in vivo.Methods:we investigated the effect of miR-181a-5p on cell proliferation and migration using CCK-8 and Transwell assays.We subsequently investigated the effect of miR-181a-5p/MEG2 on gastric cancer cell proliferation and migration by a gain-of-function approach.We co-transfected gastric cancer cells with the following:(1)pre-miR-control and control plasmid,(2)pre-miR-control and MEG2 overexpression plasmid with full-length ORF lacking the miR-181a-5/p-responsive 3’-UTR,(3)pre-miR-181a-5p and control plasmid,and(4)pre-miR-181a-5p and MEG2 overexpression plasmid.Conversely,we also performed a loss-of-function approach in vitro.To further confirm the above findings in vivo,we established gastric cancer xenografts in mice to observe the effects of miR-181a-5p and MEG2.MGC803 cells were infected with(1)control lentivirus,(2)miR-181a-5p overexpression lentivirus,(3)MEG2 overexpression plasmid,and(4)miR-181a-5p overexpression lentivirus together with MEG2 overexpression plasmid.Results:In vitro,MGC803 cells transfected with pre-miR-181a-5p had significantly greater capabilities to proliferate and migrate,while miR-181a-5p inhibition showed the opposite effect.Gastric cancer cells co-transfected with pre-miR-181a-5p and MEG2 overexpression plasmid showed significantly lower capabilities to proliferate and migrate compared to the ones transfected with pre-miR-181a-5p alone.Conversely,gastric cancer cells co-transfected with anti-miR-181a-5p and MEG2 siRNA showed significantly improved capabilities to proliferate and migrate compared to the ones transfected with anti-miR-181a-5p alone.In vivo,,the group with miR-181a-5p-overexpression showed a significant increase in both parameters compared to the control group,whereas the MEG2-overexpressing group exhibited a dramatic decrease.In addition,MEG2 overexpression attenuated the promotion of miR-181a-5p in vivo.Conclusions:the regulation of MEG2 by miR-181a-5p is able to promote gastric cancer cell proliferation and migration in vitro and enhance tumour growth in vivo.