The Expression And Mechanism Of CircHIPK3 In The Prostate Cancer

Background Prostate cancer(PCa)is the most common cancer in men,and a leading cause of death in the western world.Prostate cancer epidemic in China is characterized by rapid rates of increase over the last decade.Exposure to westernized diet and life style and aging population in combination contribute substantially to the increasing epidemic in this region.Despite the initial success of surgery and radiation therapy for localized prostate cancer,the effects of recent therapies are limited,and once patients progress to the incurable stage prostate cancer with overall survival significantly decreasing.Hence a new molecular target for early diagnosis and treatment of prostate cancer is still necessary.Circular RNAs(circ RNAs)are a class of endogenous RNAs that are extensively found in mammalian cells and exert critical functions in the regulation of gene expression.It has been demonstrated that circ HIPK3 is preferentially localized in the cytoplasm,stably and abundantly expressed in different human tumors,include prostate cancer.Circ HIPK3 has been proposed to be involved in both tumorigenic potential and carcinogenesis.However,its mechanism in prostate cancer has not been clearly reported.Objective This study aims to explore the specific mechanism of circ HIPK3 in the tumorigenesis and development of prostate cancer.Contents 1 The differently expressed circ RNAs in prostate cancer were screened according to the database,quantitative PCR(q PCR)was used to verify the expression of circ RNAs in prostate cancer and adjacent tissues.The definite subject was chose based on the above results.2 Identification and location of Circ HIPK3 in prostate cancer cells: c DNA and g DNA of prostate cancer cell lines were used as templates to determine whether the sequence was circ HIPK3 by PCR using divergent primer and convergent primer,respectively.At the same time,PCR products were sequenced by Sanger to confirm the splice junction.RNase R digestion experiments proved that circ HIPK3 has a certain stability in tolerance to digestion.Fluorescence in situ hybridization(FISH)and nuclear plasma separation experiments were used to determine the location of circ HIPK3 in prostate cancer cells.3 To explore the role of Circ HIPK3 in prostate cancer in vitro: CCK-8 and clone formation experiments were used to verify the effect of circ HIPK3 on proliferation capacity of prostate cancer cell lines;the effect of circ HIPK3 on the cell cycle and apoptosis were analyzed by flow cytometry in prostate cancer cell lines;Transwell assay and wound healing assay were used to analysis of the effects of circ HIPK3 on the migration and invasion of prostate cancer cell lines.4 To explore the role of circ HIPK3 in prostate cancer in vivo: To construct the lentiviral vector of circ HIPK3 and LNCa P cell line which stably transfected with circ HIPK3.Nude mice were injected with stable cells.Tumors were observed after injection.5 To investigate whether circ HIPK3 could act as a mi RNA sponge in prostate cancer: Firstly,the circular RNA Interactome database was used to screen mi RNAs that circ HIPK3 may sponge.RNA pull down assay,was used toselect which mi RNA could be sponge via circ HIPK3.Ago2-RIP experiment,double luciferase reporter gene assay and FISH assay were used to verify the interaction between the screened mi RNA and circ HIPK3.6 To screen target genes regulated by mi RNAs: Target genes that may be regulated by mi RNAs were screened through the Star Base database.The differentially expressed genes in prostate cancer from the TCGA database.The two results were verified by q PCR in prostate cancer tissues.The target gene expression after over-expression or sliencing with circ HIPK3 in prostate cancer cell lines were measured by q PCR and western blotting(WB).7 To explore the mechanism of circ HIPK3 to promote the development in prostate cancer: overexpression or sliencing of circ HIPK3 in prostate cell lines was added to mi RNA inhibitors or mimics,respectively.The changes in cell cycle and apoptosis were analyzed by flow cytometry.q PCR and WB were used to detect changes in the target genes.Results 1 Circ HIPK3 is increased in both prostate cancer tissues and whole blood.The high expressed circ HIPK3 is correlated with poor prognosis of prostate cancer.As well PCa patients with high expression of circ HIPK3 had higher Gleason scores than patients with low expression.2 Circ HIPK3 was derived from the second exon of HIPK3.The full length of circ HIPK3 was successfully amplified by the c DNA of prostate cancer cell lines.The sequence was turned out to be correct by Sanger.It was localized in the cytoplasm of prostate cancer cells by Nuclear separation assay and FISH.3 Circ HIPK3 promotes the proliferation,migration and invasion of prostate cancer cells,and inhibits the apoptosis of prostate cancer cells.4 Overexpression of circ HIPK3 could promote tumor growth in vivo.5 Circ HIPK3 acts as mi R-338-3p sponge in prostate cancer cells.6 Circ HIPK3 regulates Cdc25 B in prostate cancer by sponging mi R-338-3p.7 Circ HIPK3 facilitates the from G2/M transition in prostate cancer cell lines via the circ HIPK3 / mi R-338-3p / Cdc25 B axisConclusions Circ HIPK3 is highly expressed in whole blood and tissues of patients with prostate cancer.The high expression of circ HIPK3 in whole blood is not only positively related to the poor prognosis of prostate cancer,but also related to the level of total PSA in serum and the Gleason score.The higher the circ HIPK3 expression in the PCa tissues,the higher the Gleason score.In vitro and in vivo experiments confirm that circ HIPK3 is associated with prostate cancer proliferation,migration,invasion,and apoptosis.It promotes the transition from G2 to M phase of prostate cancer cells as well as inhibits apoptosis by sponging mi R-338-3p.It promotes the development of prostate cancer through the circ HIPK3 / mi R-338-3p / Cdc25 B axis.