The Expression Of MicroRNA-145 In Wilms' Tumor And Its Regulation Of Biological Behavior In Wilms' Tumor Cell

BackgroundWilms' tumor(WT)is the most common childhood renal cancer.WT accounts for about 6 percent of childhood tumors,and 0.1‰ in children and adolescents younger than 15 years.MicroRNAs(miRNAs)is about 18-25 nt noncoding RNA,those small RNA can involve almost the whole of life activity,its proliferation of cell differentiation,cycle regulation,cell apoptosis and the regulation function of autophagy get more and more attention.Recent findings of mutations in miRNA processing proteins suggest a pivotal role of miRNAs in WT genesis.MicroRNA-145(miR-145),one important component of the mircroRNA familys,has been proved a low expression in various human cancers,such as gastric cancer,colorectal cancer,esophageal cancer,breast cancer,prostate cancer.Research suggests that miR-145 as a tumor suppressor gene in intrahepatic bile duct cancer,bladder cancer and other tumors significant impact on various biological behavior of tumor cells,including cell growth,migration ability by phosphati-dylinositol 3-kinase/protein kinase B,(PI3K/Akt)signaling pathway;but its role is still not clear in the WT.Our study aims to investigate the expression of miR-145 in WT,the relationship with clinicopathological parameters,and the effect of PI3K/Akt in miR-145 regulatory mechanism with WT cell SK-NEP-1 about proliferation,apoptosis,autophagy and metastasis.Materials and MethodsWT tissues and adjacent non-tumor tissues from 32 WT patients were collected and qRT-PCR was applied to detect miR-145 expression in tumor tissue and adjacent normal tissue.The expression level of miR-145 in the cell line was detected by qRT-PCR,compared with 293T cells.The miR-145 lentivirus transfection or corresponding negative control were transfected into SK-NEP-1 cells.It was divided into blank control group,airborne virus transfection group,miR-145 overexpression group and miR-145+Akt-stimulate(Akt-s)group.GFP expression in cells was observed under a fluorescence microscope,and with the help of qRT-PCR detection of miR-145.Cell viability was measured by CCK-8 colorimetry.Apoptotic cell was assessed by acridine orange/ethidium bromide(AO/EB)staining and flow cytometry,Transwell assay were performed to examine the ability of cell migration.Western blotting was used to detect the expressions of proteins associated with PI3K/Akt signaling pathway(PI3K,Akt and p-Akt),apoptosis proteins(Bax,Bcl-2,caspase)and autophagy proteins(p62,LC3B and Beclinl)repectively.Results1.The study of miR-145 in the clinical specimens of Wilms' tumor.Compared with the corresponding normal tissue adjacent to carcinoma(relative expression was 22.75±3.78),the leval of miR-145 in wilms' tumor tissue(relative expression was 3.41 ± 1.56)is significantly lower,which express the difference was statistically significant(P<0.05);the level of miR-145 has obvious correlation with clinical stage,the level of miR-145 in the phase I children is significantly higher than the children with phase ?/?,the difference was statistically significant(P<0.05).2.Expression of miR-145 in Wilms' tumor cells and its regulation of Akt signaling pathway.Before transfection of miR-145 virus,determination of miR-145 in human kidney tumor cells SK-NEP-1 the relative expression(2.15 ±0.02),significantly lower than the human kidney embryonic epithelial cells(293T),the relative expression quantity(57.23 ±1.78),the difference was statistically significant(P<0.05).After transfection with miR-145,the expression of miR-145 in the blank control group was not statistically significant(P>0.05)compared with the viral idling group;Fluorescence detection and qRT-PCR confirmed the increased expression of miR-145 in miR-145 overexpression group(1.17±0.07)compared with the blank control group(12.6±0.24).The results of Western blotting showed that the level of PI3K and p-Akt protein decreased in the SK-NEP-1,and the PI3K/Akt signaling pathway was inhibited.3.Regulation of the biological behavior of miR-145 in Wilms' tumor cells.1.CCK-8 results showed that the proliferation rate of miR-145 overexpression group was slower than that in the blank control group and the difference was statistically significant(P<0.05).overexpression group was lower than that in the blank control group and the the virus idlThe results of Western blotting showed that the expression of Ki67 protein in miR-145ing group.2.The result of flow cytometry showed that the apoptosis rate of miR-145 overexpression group(1.87± 0.06)was significantly increased compared with the control group(0.17 ±0.04),and the difference was statistically significant(P<0.05).The results of AO/EB fluorescence staining showed that compared with the control group(4.69 ±4.79)%,the death rate of miR-145 overexpression group(19.36±3.14)%was increased,and the difference was statistically significant(P<0.05).Western blotting showed that in miR-145 overexpression group,the expression of apoptosis-related protein caspase-3 and ratio of Bax/Bcl-2 increased,autophagy-realted protein P62 decreased,LC3B and Beclinl are increased.3.Transwell migration experiment results show that migration ability of miR-145 overexpression group dropped significantly,compared with blank control group and the virus idling group,the difference was statistically significant(P<0.05).4.The role of Akt-s in regulation of the biological behavior of miR-145 in Wilms'tumor cells.In CCK-8,AO/EB fluorescence staining,Western blotting,Transwell migration experiment,Akt activator(2?g/ml)can partially restore the inhibitory effect of miR-145 in Wilms' tumor cells.ConclusionThe expression of miR-145 was down-regulated in the tumor tissues of Wilms'tumor,and was closely related to the clinical stages of the children.Up-regulation of miR-145 could inhibit the PI3K/Akt signaling pathway,thereby inhibiting the ability of proliferation and migration in Wilms' tumor cell SK-NEP-1,at the same time increase level of autophagy and apoptosis in the Wilms' tumor cell,which promote the tumor cell death,playing the role of tumor suppressor gene.