| Renal cell carcinoma(RCC)is one of the most common urological malignancy,accounting for 4%of all new cancers and 2.4%of all cancer deaths worldwide.RCC is notorious for its resistance to routine chemotherapy and radiotherapy.Surgery is the most commonly used treatment of RCC,however,it has a poor effect on patients with recurrence or metastasis.Up to 30%of RCC patients present with metastatic disease at the time of diagnosis,with a very poor 5-year survival rate.Clear-cell renal cell carcinoma(ccRCC)is the most commonly observed RCC subtype,which accounts for approximately 75-85%of all cases of RCC with the highest rates of local invasive,metastasis,mortality,and refractory to current treatments.However,the mechanisms involved in the development and progression of RCC were largely unknown,and more effective therapeutic approaches are urgently required.Speckle-type POZ protein(SPOP)is a BTB/POZ domain protein that encodes an E3 ubiquitin ligase component.Recently study regard SPOP as a regulatory hub to promote RCC tumorigenesis and an attractive target specific to RCC that may yield novel drug discovery efforts.However,the regulation of SPOP expression and function in RCC remains incompletely understood.Further investigation of the SPOP regulation mechanisms is critical for better understanding of RCC pathogenesis.The discovery of microRN As(miRNAs)has added another layer of complexity to the control of gene expression.miRNAs are recently discovered small(19-22 nucleotides)noncoding RNAs that function as repressors of gene activity by binding to complementary sequences in the 3' untranslated region(3'-UTR)of target gene transcripts,leading to mRNA degradation and/or translational repression.Our previous study has shown that miRNAs are frequently aberrantly expressed,and play crucial roles in diverse regulation pathways in RCC.However,whether certain miRNAs are involved in the regulation of SPOP during RCC progression is not clear.Therefore,the purpose of this study is to explore the miRNAs that regulate SPOP,and to illuminate the role and molecular mechanism of miRNA-SPOP axis in the development and progression of RCC.In our present study,we firstly measured the expression of SPOP in ccRCC tissues and matched normal adjacent tissues(NATs)from a cohort of 120 ccRCC patients by immunohistochemistry(IHC)and Western Blot assay,and found that SPOP expression levels were markedly upregulated in ccRCC tissues(P<0.001)and correlated with poor prognosis in ccRCC(P=0.001).Furthermore,we knocked down SPOP expression in two human renal carcinoma cell lines A498 and ACHN with two siRNAs(si-SPOP-1 and si-SPOP-2),then performed CCK8 assay,EdU assay,transwell invasion assay,and wound healing assay in these RCC cells,and demonstrated that SPOP promoted proliferation,migration,and invasion of renal carcinoma cells,suggesting that SPOP play as a critical oncogene in ccRCC tumourigenesis.Subsequently,we performed computational predictions and predicted 10 candidate miRNAs targeting the SPOP 3'-UTR.Afterwards,we examined the expression of these miRNAs in ccRCC tissues using qRT-PCR,and found 5 miRNAs downregulated,among which,miR-520c-3p,miR-372-3p and miR-373-3p from miR-520/372/373 family were identified to be the most significantly downregulated and inversely correlated with SPOP protein levels in ccRCC tissues.In addition,luciferase reporter assay and Western Blot assay demonstrated that miR-520/372/373 family suppressed SPOP protein expression post-transcriptionally,and restored the expression of PTEN and DUSP7,two downstream genes demonstrated to be degraded by SPOP.These results suggest that miR-520/372/373 family is a mighty regulator of oncogene SPOP.To explore the biologic effects of the miR-520/372/373 family-SPOP axis on renal carcinoma cells,miR-520/372/373 family expression was manipulated in vitro,and cellular function analyses were conducted in both A498 and ACHN cell lines by transient transfection of miRNA mimics or stable transfection with lentiviral expression vectors of miR-520c-3p,miR-372-3p or miR-373-3p.Then we found that miR-520/372/373 family suppressed renal carcinoma cell proliferation,invasion and migration through suppressing SPOP expression.Furthermore,we established subcutaneous xenografted model,orthotopic xenograft model,and high metastatic orthotopic xenograft model,and found that miR-520/372/373 family significantly suppressed tumour growth,invasion and lung metastasis,and effectively attenuated the stimulative effect of SPOP on tumour progression in vivo.These results revealed that miR-520/372/373 family suppressed SPOP expression,leading to elevation of PTEN and DUSP7 levels,consequently decreased proliferation,invasion,and migration of RCC cells in vitro and in vivo,suggesting great potential of miR-520/372/373 family in the treatment of RCC.Moreover,to test the transcriptional regulatory mechanisms of the miR-520/372/373 family in RCC,common putative transcription factors binding to the promoter regions of miR-520c and miR-372/373 were predicated using bioinformatics.Subsequently,qRT-PCR,chromatin immunoprecipitation(ChIP),luciferase reporter assay,and western blot assay indicated that transcription factor E2F1 suppressed the expression of miR-520/372/373 family,thus indirectly regulated downstream SPOP signalling pathway.In clinical ccRCC tissues,E2F1 protein was significantly elevated,and inversely correlated with miR-520/372/373 family.These results showed that E2F1 specifically regulated miR-520/372/373 family expression transcriptionally,and that upregulated E2F1 expression might be the reason why miR-520/372/373 family decreased in RCC.In conclusion,through clinical sample studies,in vitro cellular function analyses,and multiple tumor-bearing mouse models,our present study demonstrates a critical tumour suppressor role of the miR-520/372/373 family in RCC by regulating the SPOP pathways,and reveals that E2F1-miR-520/372/373-SPOP axis functions as a key signalling pathway in RCC progression and metastasis,which may offer promising therapeutic opportunities. |
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