MiR-370 Transcriptionally Activated By Stat3 Promotes Cell Proliferation Through Down-regulation WTX In Wilms Tumor

Wilms tumor is the most common malignant solid tumor in children, which plays an important role in pediatric surgery. But so far, the molecular mechanisms in tumorigenesis remain challenging to determine. Therefore, it is of great significance to further explore the pathogenesis to provide new methods of treatment and improve survival rate. Stat3 is a transcription factor that can promote oncogenesis. Stat3 activation is com-monly viewed as crucial for multiple tumor proliferation and metastasis. Currently, however, there is no literature on the role of Stat3 in Wilms tumor. Based on the tumor tissue and tumor cell, this study explores the effect of Stat3 in the tumour cell proliferation, clarifies the mechanism of the regulatory effect of Stat3 in Wilms tumor, in an effort to provide a new target for the biotherapy of Wilms tumor.Part I Expression of Stat3 in tumor tissues of Wlims tumor and its effects on the viablity and proliferation of tumor cellsObjective: To explore the expression of Stat3 in Wilms tumor resection specimens and to reveal the effects of Stat3 on cell viability and cell proliferation of Wilms tumor cell strain G401. This study will reveal the role of Stat3 signaling pathway in the development of human Wlims tumor.Methods: 22 cases of Wlims tumor resection specimens were seleeted. Quantitative PCR(Q-PCR) and Western blot were applied to observe the stat3 expression in Wilms tumor issues. In G401 cells, Stat3 was over-expressed by IL-6 or CA-Stat3 eukaryotic expression plasmid, and MTT assay and Brd U assay were used to observe the viability and proliferation of G401 cells. On the contrary, the Stat3 pathway was blocked by si RNAs against Stat3, and the activity and proliferation of G401 cells were detected byMTT assay and Brd U assay.Results: Stat3 was expressed in every Wilms tuomer issue. And the level of Stat3 expression was significantly higher in the tumor area than in the peritumoral region(P <0.05). In the G401 cells, which were treated with IL-6 or transfected with CA-Stat3, Stat3 was significantly over-expressed(P <0.05). And the cell viability and proliferation were enhanced( P <0.05). Conversely, the cell viability and proliferation were suppressed with blocked Stat3 by si RNA(P <0.05).Conclusions: Stat3 is widely expressed in Wilms tumor issused and involves in the viability and proliferation of tumor cells. So Stat3 is important in the pathogenesis of Wilms tumor.Part II Stat3 transcriptionally modulates expression of mi R-370 in Wilms tumorObjective: To study the molecular mechanisms of Stat3 transcriptionally activating mi R-370 expression.Methods: Stat3 was activated by IL-6 in G401 cells, and then the differentially expressed mi RNAs were screened by the mi RNAs Microarray analysis. Then Bioinformatics was used to predict the mi RNA which contained binding sites in promoter region. Stat3 was over-expressed by IL-6 and CA-Stat3 eukaryotic expression plasmid or blocked by si RNA against Stat3, and then Q-PCR was applied to observe the stat3 expression. Q-PCR was used to detect the mi R-370 expression in Wilms tumor issues, and analyze the correlation between Stat3 and mi R-370 expression. The molecular mechanism of stat3 moduting mi R-370 expression in Wlims tumor was verified by using Luciferase reporter assay and chromosome coprecipitation technique.Results: More than triple times and P <0.05 differentially expressed mi RNA was under cluster analysis. Compared to the G401 cells without treated by IL-6, the number of upregulated expressed mi RNA was 5 while downregulation expressed mi RNA 4 in the G401 with activative Stat3 induced by IL-6. It is predicted the promoter region of mi R-370 encoding gene contained Stat3 binding sites via Bioinformatics. In the G401 cells, which were treated with IL-6 or transfected with CA-Stat3, mi R-370 was significantly overexpressed(P <0.05). Conversely, the mi R-370 expression was suppressed with blocked Stat3 by si RNA(P <0.05). The level of mi R-370 expression was significantly higher in the tumor area than in the peritumoral region(P <0.05), and in Wilms tumor tissue Stat3 and mi R-370 expression were positively correlated. CA-Stat3 and reportor plasmid PGL4-370.WT were co-transfected into G401, and then Luciferase assay found that the fluorescences activity of PGL4-370.WT were ehanced(P<0.05) in the G401 cells with over-expressed Stat3, which suggested that Stat3 can target mi R-370 and regulate it in the level of transcription. Stat3 was activated by IL-6 or knock-downed via si RNAs in G401 cells, and then Ch IP assay revealed that Stat3 protein bound the mi R-370 promoter was significantly increased(P<0.05) in G401 cells treated with IL-6 or decreased(P<0.05) in G401 cells transfected with si RNAs, which implied that Stat3 can directly bind the mi R-370 promoter and transcriptionally activate mi R-370.Conclusions: The level of mi R-370 expression is significantly higher in the Wlims tumor tissues, and Stat3 and mi R-370 expression are positively correlated. Mi R-370 is a potential target gene for Stat3, and Stat3 transcriptionally activates mi R-370 expression.Part III The mechanism of mi R-370 in regulating tumor cell viability and proliferation in Wilms tumorObjective: To investigate the roles of mi R-370 in tumorigenicity and pathogenesis of Wilms tumors. To predict and confirm the target gene of mi R-370,and then to identify its role in cell viability and proliferation of Wilms tumor.Methods: Mi R-370 mimic or mi R-370 inhibitor were used to over-express or knockdown mi R-370 in G401 cell. Then MTT assay and Brd U assay were used to observe the cell viability and proliferation, and flow cytometry(FCM) was used to observe Cell cycle distribution. Meanwhile, Western blot was used to detect the expression of p21, p27 and Cyclin D1, which could relate cell cycle. Bioinformatics methods were also applied to predict the target genes of mi R-370 while DAVID database to do enrichment analysis on gene function and signal transduction pathway. Then, the mechanism of the interaction between mi R-370 and WTX, which was potential target gene for mi R-370, was analyzed by using Luciferase reporter gene system. After mi R-370 was over-expressed or knockdowned by mi R-370 mimic or inhibitor, Q-PCR and Western blot were applid to measure the WTX expression from m RNA transcription to protein translation. At last, MTT assay and Brd U assay were used to observe the viability and proliferation of the G401 cells, in which WTX expression was blocked via si RNAs.Results: The After G401 cells were transfected with mi R-370 mimic, MTT and Brd U assay showed that cell viability and proliferation were enhanced(P<0.05), FCM showed that cells in S phase increased(P<0.05), and Western blot showed that p21 and p27 protein expression decreased but Cyclin D1 increased. On the contrary, when mi R-370 was blocked by mi R-370 inhibitor, cell viability, cell proliferation and cells in S phase decreased(P<0.05), p21 and p27 protein expression increased(P<0.05), and Cyclin D1 protein expression decreased(P<0.05). Bioinformatics predicted that WTX was a potential target gene of mi R-370. And Luciferase assay was applied to confirm that mir-370 inhibited WTX transcription through 3’-UTR region of WTX(P < 0.05). Enrichment analysis on gene function and signal transduction pathway based on DAVID database showed that function of mi R-370 had a connection with cell cycle. After WTX was silenced by si RNAs,G401 cell viability and proliferation were enhanced(P<0.05), which showed that WTX knockdowned could produce the same biological effects as mi R-370 over-expressed. The results proved that mi R-370 could affect the viability and proliferation of G401 cells by inhibiting WTX.Conclusions: Mi R-370 can enhance cell viability, promote cell proliferation and regulate cell cycle by targeting WTX. Therefore, mi R-370 transcriptionally activated by Stat3 promotes cell proliferation through down-regulation WTX in Wilms tumor. Understand the precise roles played by Stat3/micro RNA-370/WTX regulatory axis will not only advance our knowledge of Wilms tumor biology, but also will help determine if Stat3 and mi R-370 has potential as a novel therapeutic target for the treatment of Wilms tumor.