The Study Of The Tumor Growth And Invasion Mechanism Regulated By MiRNA-140-5p Through TDF-? And IGF-1 Signaling Pathway In Nephroblastoma

Nephroblastoma,also called Wilm's tumor originated from the embryonic tissue,is one of the most common pediatric abdominal malignant tumors occurred in children under the age of 5,the annual incidence rate is about 3.3/1000000,that occupys 8-10%of children with malignant tumor and 95%of children with malignant kidney tumor.The peak incidence occurs in pre-school children,between 1 to 5 years of age.In recent years,due to the progress of comprehensive treatment,the survival rate of the disease has been greatly improved,but at the end of the disease there is still the possibility of malignant metastasis,there are still 5%of the mortality rate.Therefore,it is of great significance to find out the molecular mechanism of the occurrence and development of Wilms tumor,and to find a way to interfere with the occurrence and development of tumor,and to improve the treatment of tumor.miRNAs are endogenously produced,small non-coding single-stranded RNAs(17?25-nucleotides long)that play an essential role in a wide variety of biological processes including cell proliferation,apoptosis,the immune response and tumorigenesis,primarily by inhibiting gene expression.We will explore the regulation mechanism of miR-140-5p by detecting the changes of downstream protein phosphorylation level in TGF-? and IGF-1 pathway,siRNA and rescue experiments were also included.TGFBR1 and IGF1R as the direct target of miR-140-5p will further be confirmed.We confirmed that miR-140-5p may contribute to the occurrence and development of Wilms tumor through the interaction of TGF-? and IGF-1 signaling pathway.This study may provide the basis data for future study of pathogenesis of Nephroblastoma.Objective:To detect whether the expression of miRNA-140-5p is aberrantly in nephroblastoma.To investigate the regulation mechanism of miRNA-140-5p via the TGF-? and IGF-1 signal pathway.Method:1.miRNA array assay was conducted to explore the miRNA expression profile of nephroblastoma.2.quantitative Real-time PCR were conducted to verify the expression of miRNA-140-5p.3.Overexpression of miR-140-5p in nephroblastoma cell lines G401 and WT-CLS1 by lentivirus infection.MTS assay,flow cytometry and migration assay were performed for cell proliferation and invasion assay.4.The bioinformatics was used to predict the target gene for miRNA-140-5p,dual-luciferase reporting system was used for verifying TGFBR1 and IGF1R as a target gene of miRNA-140-5p.5.ELISA assay was used for FGF9 detection,Western-blot assay was performed to evaluate the expression of TGFBRI and downstream protein Smad2/3,ERK and AKT,IGF1R and downstream protein AKT.6.Rescue Experiment was used to confirme the inhibition effect of miR-140-5p on TGFBR1 and IGF1R.8.Statistical analysis was performed using SPSS 13.0.The Student's t-test was used for unpaired comparison.Statistical significance level was set at P<0.05.Result:1.The expression of miRNAs in nephroblastoma tissues and adjacent normal tissues was detected by miRNA microarray,and there were significant differences in the expression of miRNAs between two groups.miR-140-5p was selected as the key miRNAs by bioinformatics analysis.Q-PCR method was used to verify the low expression of miR-140-5p in nephroblastoma tumor tissue.2.Overexpression of miR-140-5p in nephroblastoma cell lines G401 and WT-CLS1 by lentivirus infection.Compared with the control group(miRNA-NC),the proliferation rate of miR-140-5p over expression group was significantly lower.Cell cycle arrest in G0/G1 phase.Migration assay showed that the cell migration rate of miR-140-5p group was significantly decreased.3.Double luciferase reporter gene experiments showed that miR-140-5p targets the 3'UTR of TGFBR1 and IGF1R.Western blot experiments further confirmed that miR-140-5p could inhibit the expression of TGFBR1 and IGF1R.4.Compared with the control group,the expression of TGFBR1 and IGF1R decreased in the over expression miR-140-5p cell group.At the same time,the expression of TGFBR1 downstream proteins Samd2/3,P-ERK and P-AKT also decreased,and the expression of IGF1R and its downstream protein P-AKT decreased.5.The expression of TGFBR1 and IGF1R were rescued in miR-140-5p over expressed cells.It was found that the expression of TGFBR1 and its downstream protein Samd2/3,P-ERK and P-AKT were rescued,and IGF1R and its downstream protein P-AKT were rescued.Conclusion:1.The miRNA-140-5p expression level is downregulated in nephroblastoma.2.miR-140-5p suppresses tumor growth in nephroblastoma at least partially by regulating TGF-beta and IGF-1 signal pathway.