Mechanisms Of Intergenic Type Hsa＿circ＿0007379 Underlying Inhibiting The Growth And Metastasis Of Colorectal Cancer Through RUNX1

Objectives: To screen and identify downregulated circular RNAs(circRNAs)in colorectal cancer(CRC).To investigate the expression features and biological functions of hsa＿circ＿0007379 in CRC and reveal the molecular mechanisms underlying its biogenesis and functions.To assess the potential value of hsa＿circ＿0007379 as a therapeutic molecule for CRC.Methods: The Arraystar Human circRNA Array V2 high-throughput chip was used to screen aberrantly expressed circRNAs in three paired CRC tissues and adjacent normal tissues.Quantitative real-time polymerase chain reaction(q RT-PCR)was used to detect the hsa＿circ＿0007379 expression levels in 55 matched CRC tissues and adjacent normal tissues as well as in one normal colonic epithelium cell line and six CRC cell lines.QRT-PCR,Agarose gel eletrophoresis(AGE),Sanger sequencing,RNase R and actinomycin D assays were utilized to identify the circular structure and high stability of hsa＿circ＿0007379.Fluorescence in situ hybridization(FISH),nuclear/cytoplasmic RNA isolation and q RT-PCR were used to determine the cellular distribution of hsa＿circ＿0007379 in CRC.Bioinformatics analysis,cyclization test and RNA binding protein immunoprecipitation(RIP)were used to explore the molecular mechanism underlying the biogenesis and downregulation of hsa＿circ＿0007379 in CRC.Lentivirus containing overexpressed plasmids or short hairpin RNAs(sh RNA)was used to transfect CRC cell lines and obtain cell lines steadily overexpressed hsa＿circ＿0007379or silenced hsa＿circ＿0007379,separately.A series of in vitro and in vivo functional assays,including cell counting kit-8(CCK-8),plate colony formation,5-Ethynyl-2’-deoxyuridine(Ed U)assays,Transwell assays and animal experiments,were performed to investigate the effects of hsa＿circ＿0007379 on the proliferation,migration,and invasion of CRC cells.High-throughput RNA-seq,Western blot(WB),RIP,RNA pull-down,co-immunoprecipitation(Co-IP),and dual luciferase reporter assay were used to investigate the molecular mechanisms of hsa＿circ＿0007379 underlying suppressing CRC growth and metastasis.CRC patient derived organoids(PDOs)and patient derived xenografts(PDXs)models were successfully constructed and used to evaluate the potential value of hsa＿circ＿0007379 as a therapeutic molecule for CRC.Results:(1)Three hundred and twenty six differentially expressed circRNAs(DECs)were screened out from CRC tissues(FC>1.5,P<0.05);among which,122 were upregulated and 204 were downregulated.The expression levels of hsa＿circ＿0007379 in CRC tissues were significantly lower than that in adjacent normal tissues(P <0.0001),and the expression levels of hsa＿circ＿0007379 in CRC cells were also significantly lower than that in human normal colon epithelial cells(P < 0.05).Hsa＿circ＿0007379 showed a progressive decreasing expression pattern in paracancer normal tissues,stage I+II CRC tissues,and stage III+IV CRC tissues(P < 0.05).The expression levels of hsa＿circ＿0007379 in CRC tissues were negatively correlated with tumor size(P = 0.037),invasion depth(P = 0.004)and TNM stage(P = 0.044).(2)Hsa＿circ＿0007379 was a unique intergenic type circRNA with high stability and long half-life.Hsa＿circ＿0007379 distributed in both the nucleus and the cytoplasm in CRC cells,and most of the hsa＿circ＿0007379 was located in the nucleus.There were reverse complementary matches(RCMs)in the upstream and downstream of the parent sequence of hsa＿circ＿0007379,which mediated the generation of hsa＿circ＿0007379 through base complementary pairing.ATP-dependent RNA helicase A(also known as DHX9),which was highly expressed in CRC,suppressed the biogenesis of hsa＿circ＿0007379 and down-regulated the expression of the hsa＿circ＿0007379 through blocking the binding of the RCMs.(3)Hsa＿circ＿0007379 could inhibit the proliferation,migration and invasion of CRC cells in vitro,and suppress the growth of subcutaneous transplanted tumors and the formation of lung metastases in nude mice in vivo.(4)Hsa＿circ＿0007379 specifically bound to the KH4 domain of RNA binding protein KSRP through the “GUCC” motif(site2000～2003).Hsa＿circ＿0007379 also specifically bound to pri-miR-320 a and pre-miR-320 a through base complementary pairing,respectively.In the nucleus,hsa＿circ＿0007379 formed a trimer complex with KSRP and pri-miR-320 a,and recruied Drosha enzyme complex through KSRP to process pri-miR-320 a and to promote the generation of pre-miR-320 a.Interestingly,hsa＿circ＿0007379 also formed a trimer complex with KSRP and pre-miR-320 a in the cytoplasm,and recruited Dicer enzyme complex through KSRP to process pre-miR-320 a and to promote the maturation of miR-320 a.Mi R-320 a blocked the activation of Wnt/β-catenin signaling pathway by inhibiting the expression of transcription factor RUNX1.(5)The introduction of exogenous hsa＿circ＿0007379 could significantly inhibit the growth of PDOs and PDXs derived from CRC patients.Conclusions:(1)Hsa＿circ＿0007379 is downregulated in CRC;its biogenesis is mediated by RCMs and negatively regulated by DHX9.(2)Hsa＿circ＿0007379 inhibits the growth and metastasis of CRC by blocking the activation of Wnt/β-catenin signaling pathway via KSRP/ miR-320 a /RUNX1 axis.(3)Hsa＿circ＿0007379 may be a potential therapeutic molecule for CRC.