Micro RNA-27a Promotes Proliferation And Matastasis Of Gastric Cancer Cells By Targeting PHLPP2

Background:Gastric cancer(GC)is a common digestive system tumor.Although recent advancement in gastric cancer early detection,therapy,and prevention partly enhanced the survival rate of early stage gastric cancer,advanced gastric cancer is still incurable and the mortality rate remains high.The unlimited proliferation,the strong metastasis capability of the tumor cells and the high recurrence rate are the main causes of death in patients with gastric cancer.The occurrence of gastric cancer is a multi factor,multi step biochemical process.The process involves multiple molecular mechanisms.Therefore,it is of great significance to further elucidate the molecular mechanisms of GC.Convincing evidencehasconfirmed that mi RNAs regulatea majority of cellular processes related tothe biological behavior of tumors,including cell proliferation,apoptosis,differentiation and metastasis.In general,mi RNAs perform their functions viasuppressionof specific target genes.The role of mi R-27 a in tumorigenesis differs in various cells and tissues.It is regarded as an oncogene in several types of tumors,such asosteosarcoma and laryngeal carcinoma.However,mi R-27 a is suggested to be a cancer suppressive mi RNA in esophageal squamous cell carcinoma and colorectal cancer.Accumulating evidence indicatesthat micro RNA-27a(mi R-27a)is involved in carcinogenesis and tumor progression.However,the exact function and molecular mechanism of mi R-27 a in gastric cancer remain unclear.Its functions and molecular mechanisms in GC need to be further investigated.PH Domain Leucine-rich Repeat Protein Phosphatase2(PHLPP2)is a protein phosphatase that acts as a tumor suppressor.It has been reported to induce cell cycle arrest and apoptosis and suppress tumor metastasis of various cancer types.It can directly dephosphorylate and inactivate Akt at Ser473 and subsequently inhibitthe PI3K/Akt signaling pathway.Here,we explored whether PHLPP2 was a novel target of mi R-27 a.Our study suggested that mi R-27 a exerts its functions of promoting proliferation and metastasis in GC cells by activating the Akt signaling pathway via targeting PHLPP2.Methods:Quantitative real-time PCR(q RT-PCR)was used to quantify the expression of mi R-27 aand its target gene.The function of mi R-27 a in gastric cancer was investigated through in vitro and in vivo assays(MTT assay,colony formation assay,flow cytometry assay,wound healing assay,transwell migration and invasion assay,immunohistochemistry(IHC),immunofluorescence(IF)and Western blot).A luciferase reporterassay was conducted to confirm the target gene of mi R-27 a.Results:PartⅠ Mi R-27 a promots the proliferation and metastasis of gastric cancer cellsWe found the mean expression level of mi R-27 a was higher in tumor tissues compared with matched non-tumor tissues.The higher mi R-27 a level was associated with advanced clinical stages,advanced T stages,advanced N stages and advanced M stages.We also found the expression level of mi R-27 a in gastric cancer cell lines was also generally increased.We chose SGC-7901 and AGS cell lines as model throughout the entire experiment.We further carried out the relevant functional experiments,to study the effects of mi R-27 a on the malignant biological behavior of gastric cancer cells.In SGC-7901 cells,downregulation of mi R-27 a can inhibit the proliferation,clone formation,scratch healing,transwell migration and invasion of gastric cancer cells and inhibit the EMT of gastric cancer cells.Meanwhile,downregulation of mi R-27 a in SGC-7901 cells could also induce cell cycle arrest and apoptosis,and influence the expression of Akt pathway related proteins.Contrary results were founded in AGS cells transfected when mi R-27 a was upregulated.Furthermore,our xenograft mouse model also unveiled the suppressive effects of mi R-27 a knockdown on tumor growthand metastasisin vivo.PartⅡ Mi R-27 a targets PHLPP2 in gastric cancerTo investigate the hidden molecular mechanism by which mi R-27 a modulates GC cells,we then investigated target genes of mi R-27 a using public bioinformatics tools.Among the predicted targets,PHLPP2 was chosen for further analysis because of its vital role in inhibiting tumor development and modulating cell proliferation and metastasis.Dual luciferase assays showed a decline of luciferase activity in AGS cells transfected with mi R-27 a agomir compared to the negative control.We found the m RNA and the protein level of PHLPP2 was decreased in AGS cells transfected with mi R-27 a agomirs.While in SGC-7901 cells transfected with mi R-27 aantagomir,we obtained the opposite results.In addition,a remarkable relationship between mi R-27 a levels and PHLPP2 levels was observed in these tumor samples.The lower expression of PHLPP2 was associated with advanced clinical stages,advanced T stages,advanced N stages and advanced M stages.Part Ⅲ Mi R-27 a activates the Akt/GSK3β pathway through PHLPP2 to exert its biologic effects on GC cellsWe next examined the role of mi R-27a-mediated inhibition of PHLPP2 in the development and maintenance of malignant phenotypes of GC cells.To examine whether mi R-27 a exerts its function via PHLPP2,SGC-7901 cellswere co-transfected with mi R-27 a antagomir and PHLPP2 si RNA.We found that silencing PHLPP2 can partiallyattenuatethe effects produced by mi R-27 a inhibition on cells proliferation,apoptosis,migration and invasion.Our results revealed that p-Akt,Cyclin D1,Vimentin and snailexpression was down-regulated,p21,p27 and E-cadherin was upregulated in SGC-7901 cells where mi R-27 a was suppressed,while PHLPP2 silencing could abolish these changes.These findings indicate that the PHLPP2/Akt axis contributes to the mi R-27a-mediated progression of GC Conclusion:In summary,our current study demonstrated that mi R-27 a possessed tumor inducing effectsongastric cancer through the novel target PHLPP2.Downregulation of mi R-27 a suppressed multiple malignant biological behaviors,including inhibition of tumor cell growth,and reduction of tumor cell migration and invasion.In addition,silencing of PHLLP2 could abolish malignant biological behaviors changes induced by inhibition of mi R-27 a.That is to say,mi R-27 a can promote proliferation and metastasis of gastric cancer cells by suppressing PHLPP2 and activatingthe Akt/GSK3β pathway.Conclusively,our findings indicated that mi R-27 a acted as an oncogene in GC and clarified its functional mechanism.Therefore,mi R-27 a may be a potential novel target for GC prevention and therapy.