The Tumor-suppressive Function Of CircHIPK3 In Renal Clear Cell Carcinoma

Background:Renal cell carcinoma?RCC?is one of the most common malignant tumors of the urinary system,representing approximately 2% to 3% of all adult malignancies.Clear cell renal cell carcinoma?CCRCC?is the most common type of RCC.Circular RNA?circ RNA?is a novel type of endogenous non-coding RNA?nc RNA?.Previously it was considered a transcriptional error or a mere byproduct,a variety of circ RNAs have been discovered with the development of high-throughput sequencing and bioinformatics,but their roles in CCRCC are still unknown.Methods 1.Firstly,we performed circ RNA-seq and data analysis from three human CCRCC tissues and matched adjacent normal renal tissues.2.Among all of the circ RNAs that RNA-seq detected,circ HIPK3?circ Base ID: hsa_circ₀000284?was the most abundant circ RNA in our circ RNA-seq data,then we selected circ HIPK3 for the next study.Furthermore,via q RT-PCR,we investigated the expression of circ HIPK3 in CCRCC cell lines.We established stable transfection with high expression levels of circ HIPK3.3.After establishing stable transfections in CCRCC cell lines,we assayed the invasion and metastasis of CCRCC cells via wound-healing assay and Transwell assay.Next,we further examined the proliferation of CCRCC cell lines by CCK-8 and Edu assay.Caki1 cells following stable transfection with circ HIPK3 or vector were injected into nude mice.To explore the circ RNA-mi RNA-m RNA axis,we predicted the relationship between all of the mi RNAs and circ HIPK3 in the mi Base database by mi Randa software.Results 1.In total,1184 differentially expressed circ RNAs were found in all of the tissues,of which 490 circ RNAs were upregulated and 694 circ RNAs were downregulated with fold-changes ?2 and p-value <0.05.2.circ HIPK3 exhibited low expression in most CCRCC tissues compared with that in pair-matched adjacent normal tissues.Furthermore,via q RT-PCR,we found that circ HIPK3 levels were downregulated in CCRCC tissues and cell lines compared with those in matched normal tissues and normal kidney epithelial cells.Next,we transfected these plasmids into Caki-1 and ACHN cell lines via lentiviruses.We established stable transfection with high expression levels of circ HIPK3.As expected,the expression level of circ HIPK3 in the over-expression group was increasing markedly compared to that in the empty-vector control group.3.Wound-healing assay suggested that overexpression of circ HIPK3 suppressed cellular migration in Caki1 and ACHN cells.Similarly,over-expression of circ HIPK3 also inhibited migration and invasion of CCRCC cells in transwell-migration and Matrigel-invasion assays.CCK-8 and Ed U assay indicated that the overexpression of circ HIPK3 significantly inhibited CCRCC cellular growth compared with that of the vector group.A nude mouse tumorigenicity assay showed that the growth and tumor weight of xenograft tumors in the circ HIPK3-overexpression group were decreased compared with those of the vector group.In total,we found at least 95 mi RNAs that had a total score >140,which may have one or two binding sites in circ HIPK3.Conclusions 1.A variety of differentially exoresssed circ RNAs was discovered in CCRCC and kidney tissues,which most originated from exons.2.Circ HIPK3 was lowly expressed in CCRCC tissues and cell lines,which was consistent with our circ RNA-seq data.3.Circ HIPK3 overexpression inhibited migration,invasion,and proliferation of CCRCC cells in vitro and in vivo.Via bioinformatics that circ HIPK3 may act as a mi RNA sponge.