| Renal cell carcinoma(RCC)is the most prevalent malignancy of the adult kidney,accounting for approximately 90% of kidney tumors and 3% of cancers in adults.The morbidity and mortality of RCC have increased steadily in recent years.Despite advances in cancer therapies,RCC is an intractable disease,and most affected patients undergo metastasis development and relapse after treatment.The molecular mechanism of RCC pathogenesis is still elusive,hampering the development of effective therapeutics.Therefore,specifically targeted therapies to improve the survival rate of RCC based on molecular mechanisms are urgently need.Micro RNAs(miRNAs)are a class of conserved non-coding RNAs of ~22 nucleotides in length that play an important role in regulating gene expression.miRNAs can directly target the 3?-untranslated region(UTR)of the m RNA to induce m RNA degradation and translation inhibition.A growing body of evidence has indicated that miRNAs are critical regulators of cancer development and progression that regulate cell proliferation,apoptosis,differentiation,migration,and invasion by functioning as either oncogenes or tumor suppressors.Increasing evidence has reported that various miRNAs are dysregulated in RCC,contributing to the pathogenesis of RCC and can serve as potential diagnostic and prognostic biomarkers and therapeutic targets of RCC.However,the precise role of miRNAs in RCC remains largely unknown.An increasing number of studies have suggested that astrocyte elevated gene-1(AEG-1)is a novel oncogene in many types of cancers.AEG-1 can be induced by human immunodeficiency virus-1(HIV)and tumor necrosis factor-?.AEG-1 plays an important role in cancer progression,obesity,and neurodegenerative diseases.AEG-1 is highly expressed in many cancers,including gastric cancer,colorectal cancer,hepatocellular carcinoma,glioma,and prostate cancer.The next study found that AEG-1 could activated many oncogene pathway.AEG-1 activated the MAPK pathway,special ERK pathway,activated ERK pathway can cross effect the Wnt pathway which regulate the tumor metastasis.LEF-1 combine with ?-catenin to form the dimer which was activated in the Wnt/?-catenin pathway,Wnt combine with phosohorylated FZD inactivate GSK3?/?,increasing the level of ?-catenin,causing the activated gene cause transcription in the nucleus.AEG-1 can downregulate the inhibitor Wnt of APC and CTBP2,we also found that LEF-1 was inhibited,which effect Wnt to regulate AEG-1,decreased the invasion the of cancer cell.Frizzled7(FZD7)is an important co-receptor in the Wnt signaling pathway.Aberrant expression of FZD7 occurs frequently in numerous cancers and is associated with abnormal activation of the Wnt signaling pathway.FZD7 regulates cancer cell proliferation,invasion,and metastasis by promoting the Wnt signaling pathway.Wnt signaling is extensively involved in the progression of RCC.High expression of FZD7 has been found in RCC tissues and cell lines and contributes to Wnt-mediated RCC progression.Therefore,we believe that targeting Wnt signaling pathway is a possible treatment for renal cell carcinoma.A recent study has indicated that miRNAs is a tumor-associated miRNA in various cancer types.However,the role of miRNAs in RCC remains unknown.The purpose of this study was to investigate the mechanism of miRNAs in renal cell carcinoma(RCC)cell lines,to determine whether micro RNAs(miR-384 and miR-613)can affect the prognosis of RCC cells through targeted Wnt signaling pathway,to further determine whether they can be used as a potential new targeted therapy for RCC.Methods:Part 1: Study on targeting AEG-1 by miRNA-384 in RCC.1.The level of miR-384 m RNA was detected by RT-PCR assays in the RCC tissue and cell line.The ACHN and Caki-1 cells were transiently transfected with miR-384 mimics or miR-384 inhibitor,the level of miR-384 m RNA was detected by RTq PCR,Cell proliferation,differentiation and invasion were detected.2.Bioinformatics analysis to analysis whether miR-384 target 3-UTR of AEG-1,the dual-luciferase reporter assay to verify whether miR-384 binds to the AEG-1 3-UTR.The level of miR-384 m RNA and AEG1 m RNA was detected by RT-PCR assays in the RCC tissue.RCC cells were transiently transfected with miR-384 mimics or miR-384 inhibitor to observe the AEG-1 effect the pathway of Wnt.3.RCC were transfected with the pc DNA3.0/AEG-1 vector,to observer whether the inhibitory effect of Mi R-384 was reversed on RCC Cell growth and invasion.Part 2: Study on Targeting FZD7 by miRNA-613 in RCC.1.The level of miR-613 m RNA was detected by RT-PCR assays in the RCC cell line.We employed an expresses miR-613 vector,to obseve the level of miR-613 in ACHN and 786-O by RT-q PCR.The express vector of miR-613 was transfered into the RCC,the proliferation rates of RCC was detected by MTT in the cell of ACHN and 786-O.Colony-forming capacity was detected by colony formation assay.Cell invasion was detected by Transwell invasion assay.2.Bioinformatics analysis to analysis whether miR-613 target 3-UTR of FZD7,The relative luciferase activity was detected using the dual-Glo luciferase assay system in the ACHN and 786-O cells transfected with miR-613 mimics and FZD7 3'-UTR constructs.The expression of FZD7 was detected by RT-q PCR and Western Blot in the ACHN and 786-O cells.3.Restoration of FZD7 expression,the expression of FZD7 was detected by RTq PCR and Western Blot in the ACHN and 786-O cells.The proliferation rates and cell invasion was detected.Part 3:Effects of combination of miR-384 and miR-613 on ACHN.The cell proliferation was detected by CCK8,cell invasion was detected by Colony Formation Assay,and the level of Wnt was detected by western bolt in ACHN cell treated by combination of miR-384 mimics and miR-613 mimics.Result: Part 1:Study on targeting AEG-1 by miRNA-384 in RCC.1.RT-q PCR results showed that miR-384 expression was significantly decreased in clinical RCC specimens compared with adjacent normal renal tissues(P < 0.01).miR-384 expression was markedly downregulated in RCC cell lines compared with normal kidney cells(P<0.01).The results showed that miR-384 expression was increased in cells transfected with miR-384mimics(P<0.01),whereas miR-384 expression was decreased in miR-384 inhibitor-transfected cells(P<0.05).The CCK-8 assay showed that compared with NC group,the proliferation was decreased in the group of miR-384mimics(P<0.05),and increased in the group of miR-384 inhibitor(P<0.05);The colony formation assay and cell invasion showed that compared with NC group,colony number and cell invasion of was decreased in the group of miR-384mimics(P<0.05),and increased in the group of miR-384 inhibitor(P<0.05);2.Bioinformatics analysis showed that the 3-UTR of AEG-1 contains putative targeting sites for miR-384.The results showed that overexpression of miR-384 significantly decreased the luciferase activity of the reporter vector containing the wildtype AEG-1 3-UTR,whereas miR-384 inhibition significantly increased.RT-q PCR and results showed that AEG-1 m RNA expression was significantly decreased in clinical RCC specimens compared with adjacent normal renal tissues(P < 0.01).RT-q PCR and Western Blot results showed that compared with NC group,AEG-1 expression was markedly downregulated in the group of miR-384mimics(P < 0.01),and increased in the group of miR-384 inhibitor(P<0.05);The dual-Glo luciferase assay system results showed that compared with NC group,the relative luciferase activity of Wnt was downregulated in the group of miR-384 mimics,and increased in the group of miR-384 inhibitor(P<0.05);3.Overexpression of AEG-1,The dual-Glo luciferase assay system results showed that compared with miR-384 mimics group,the relative luciferase activity of Wnt was increased in the group of miR-384mimics+ AEG-1 group(P<0.05);Compared with miR-384 mimics group,the proliferation and invasion was increased in the group of miR-384mimics+ AEG-1 group(P<0.05)by CCK8 and cell invasion assay.Part 2:Study on Targeting FZD7 by miRNA-613 in RCC.1.RT-q PCR results showed that miR-613 expression was downregulated in RCC cell lines compared with normal kidney cells(P<0.05);The results showed that miR-613 expression was increased in cells transfected with miR-613mimics(P<0.01).The MTT assay showed that compared with blank and NC group,the proliferation was decreased in the group of miR-613mimics(P<0.05);The colony formation assay and cell invasion showed that compared with NC group,colony number and cell invasion of was decreased in the group of miR-384mimics(P<0.05);2.Bioinformatics analysis showed that the 3-UTR of FZD7 contains putative targeting sites for miR-613.The results showed that overexpression of miR-613 significantly decreased the luciferase activity of the reporter vector containing the wildtype FZD7 3-UTR.RT-q PCR and Western Blot results showed that compared with blank and NC group,FZD7 expression was downregulated in the group of miR-613 mimics(P < 0.01);3.Overexpression of FZD7,Compared with miR-613 mimics group,the proliferation and invasion was increased in the group of miR-384mimics+ fzd7 group(P<0.05)by MTT and cell invasion assay.Part 3:Effects of combination of miR-384 and miR-613 on ACHN.We combine miR-384 mimics with miR-613mimics to deal with the cell line of ACHN,the CCK8 results show that compare with the group of miR-NC,the proliferation was decreased in the miR-384 mimics or miR-613mimics group.A??compare with miR-384 mimics or miR-613mimics group,the proliferation was significantly decreased in the miR-384+miR-613mimics group(P<0.01).The cell invasion results show that compare with the group of miR-NC,cell invasion was inhibited in the miR-384 mimics or miR-613mimics group;When we compared with the group of miR-384 mimics or miR-613mimics,found that miR-384+miR-613mimics can significantly inhibit the cell invasion(P<0.05).Western Blot show that compare with the group of miR-NC,the level of Wnt was decreased in the group of miR-384 mimics or miR-613mimics.When we compared with the group of miR-384 mimics or miR-613mimics,found that the level of Wnt was decreased in the group of miR-384+miR-613mimics(P<0.01).Conclusion:1.miR-384 and miR-613 have a low level expression in RCC,inhibit the proliferation,differentiation and invasion.2.miR-384 directly targets the 3'-UTR of AEG1 and inhibits AEG1 expression,and miR-613 directly targets the 3'-UTR of FZD7 and inhibits FZD expression in RCC cells.3.miR-384 can inbihit the pathway of Wnt by targeting AEG1.4.miR-384 combine with miR-613 can inhibit the proliferation rates and invasion in RCC cells,that may be relate to the pathway of Wnt. |
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