MiR-183 Functions As An Oncogene By Targeting PDCD4 Expression In Esophageal Squamous Cell Carcinoma

Background:MicroRNAs(miRNAs),a class of 21-24 nucleotides small non-coding RNA,control specific gene expression by targeting mRNAs for translational repression or diminish mRNA stability,which involved in cell differentiation,proliferation,apoptosis,metabolism and other physiological process.More and more researches demonstrated miRN As regulated tumor progression and may act as tumor suppressors or oncogenes.As the research of esophageal squamous cell carcinoma(ESCC)miRNAs profile is still rare all over the world,we identified differential miRNAs expression in 3 pairs of ESCC tissues and paired normal controls,which found that miR-183 expression in ESCC was 4.24 times than normal esophageal tissues.MiR-183 is high expressed in various cancer types such as colon cancer,liver carcinoma,synovial sarcoma and rhabdomyosarcoma.However,the expression of miR-183 in retinoblastoma is down-regulated,which played a tumor suppressor role.There are reports indicated the relative expressions of miR-183 in ESCC tissues were significantly associated with increased risk for esophageal cancer,but its specific mechanism of action and expression in ESCC has not been clearly elucidated.Objetives:To investigate the expression of miR-183 in ESCC tissues,intraepithelial neoplasia tissues and normal controls,explore the effect of miR-183 on ESCC cells and the mechanisms in ESCC development.Methods:1.Using microarray analysis to screen aberrantly expressed miRNAs in 3 pairs of ESCC tissues and normal controls,miR-183 was selected as the objective of this study.2.The expression of miR-183 in 32 pairs of ESCC tissues and 30 pairs of esophageal intraepithelial neoplasia tissues were examined by Taqman qRT-PCR while pdcd4(mRN A and protein)was detected by SYBR Green qRT-PCR,Western blot and IHC assay.3.The target genes of miR-183 were searched with bioinformatics tools such as TargetScan/miRBase and Pictar,potential target genes expression were assessed by qRT-PCR,Western blot and Luciferase reporter assay.4.MiR-183 mimics,miR-183 inhibitors,si-pdcd4 and their corresponding negative controls were transfected into ESCC cells,CCK-8 assay for cell proliferation,colony formation assay for clonogenic growth,flow cytometry assay for cell apoptosis,transwell assay for cell migration and invasion.5.Differential concentrations of PI3K/AKT inhibitor LY294002(0μM、10μM、20pM、40μM)were added into ESCC cells and cultured for hours,miR-183 level was measured by qRT-PCR pdcd4,AKT,p-AKT expressions were detected by Western blot assay.Results:1.51 up-regulated and 17 down-regulated miRNAs in our microarray,with miR-183 level in ESCC was 4.24 times than normal controls.The expression of miR-183 in 32 paired of ESCC tissues was 3.98 times than normal controls(P<0.0001),while 2.93 times in intraepithelial neoplasia tissues(P<0.005).The expression of pdcd4 mRNA and protein in ESCC tissues were 0.46 and 0.27 times than normal tissues(P<0.05).According to the IHC assay,the staining ofpdcd4 protein in ESCC tissues was(0.178±0.021),which was significantly lower than that of normal tissues(0.267±0.031)(P<0.05).2.Pdcd4 was one target gene of miR-183 according to bioinformatics analysis.The expression of pdcd4 protein was reduced(increased)when transfected with miR-183 mimics(inhibitors)(P<0.05)while pdcd4 mRNA didn’t change(P>0.05).Luciferase activity was decreased when transfected pdcd4 wt-3’-UTR vector in miR-183 mimics compared to control(P<0.05),when the sequences mutated,luciferase activity showed no obvious change(P>0.05).3.The effects of miR-183 mimics on ESCC cells were described using CCK-8,colony formation,flow cytometry and transwell assay.The OD value of Eca109 cells of 24h,48h,72h,96h were 0.389±0.006,0.854±0.289,1.431±0.036 and 1.631±0.036 respectively;214±17 cells in colony formation assay,4.93±0.76 cell apoptosis rate,449±42 migration cells and 402±11 invasive cells.With negative controls,Eca109 cells showed 0.339±0.113,0.679±0.192,1.273±0.035 and 1.483±0.082 in CCK-8 assay,116±9 cells in colony formation assay,8.6±0.72 in flow cytometry,201±12 migration cells and 271±13 invasive cells(P<0.05).Treated with si-pdcd4,Eca109 cells showed nearly the same effect with miR-183 mimics(P>0.05).Accordingly,the results of TE13 cells were similar to Eca 109 cells.4.Differential concentrations of LY294002 could reduce miR-183 level of ESCC cells.Compared to blank control,miR-183 expression was 0.14,0.16,0.04 times and p-AKT expression was 0.67,0.81,0.85 times with 10μM,20 μM and 40μM(P<0.05).On the contrary,pdcd4 expression was increased with 1.24,1.13,1.21 times than blank control(P<0.05).Conclusions:1.MiR-183 was regulated in ESCC tissues and esophageal intraepithelial neoplasia tissues compared to normal controls,but the pdcd4 expression was decreased in ESCC tissues.2.According to the Western blot and Luciferase reporter assay,pdcd4 was a target gene of miR-183 in ESCC cells,which verified the biological predictions.3.MiR-183 may affect the development of ESCC by negatively regulating pdcd4 expression.4.Inhibition of PI3K/AKT signaling pathway may reduce the ESCC cells miR-183 and p-AKT expression,increase pdcd4 level,which suggested AKT may be an upstream regulator of miR-183.