CircKDM4B Suppresses Breast Cancer Progression Via The MiR-675/NEDD4L Axis

Purpose:The high incidence and mortality of breast cancer（BC）poses a serious threat to women’s health.Patients with metastatic BC have a poor prognosis,but the specific mechanism remains unclear.To identify the molecular mechanism of BC metastasis is helpful to find the therapeutic targets and improve patient outcomes.Circular RNA（circRNA）has a closed loop structure without 5’ caps and 3’ polyA tails.CircRNA can serve as microRNA（miRNA）sponges,interact with proteins or translate to peptides and thereby regulate tumor progression.At present,circRNA involved in the development and progression of breast cancer has been reported.However,the dysregulated circRNA involved in BC metastasis is unclear,and the underlying mechanism remains unknown.In this study,we identified a novel circRNA（hsa_circ₀002926,termed circKDM4B）by transcriptome high-throughput sequencing.The expression of circKDM4B was detected in BC cells and clinical samples.The effect of circKDM4B on BC growth and metastasis were explored both in vitro and in vivo.The mechanism of circKDM4B regulating BC metastasis was explained through the competing endogenous RNA（ceRNA）regulatory network.Methods:Transcriptome high-throughput sequencing was used to screen out circKDM4B.The expression of circKDM4B in cells and clinical samples was detected with Real-time quantitative polymerase chain reaction（RT-qPCR）.Cells were treated with actinomycin D and Ribonuclease R（RNase R）to detect the RNA stability of circKDM4B.The intracellular localization of circKDM4B was determined by RNA nucleoplasmic separation and fluorescent in situ hybridization（FISH）.Transwell assays were performed to value the effect of circKDM4B on the migration and invasive abilities of BC cells.CCK-8 and EdU assays were used to observe the effects of circKDM4B on the growth and proliferation of BC cells.The effects of circKDM4B on the growth and metastasis of BC in vivo were observed in nude mice.The miRNA that may bind to circKDM4B and the target genes of miRNA were predicted.RNA immunoprecipitation assay（RIP）and dual-luciferase reporter assay were used to confirmed that circKDM4B served as miRNA sponges.The mechanism of ceRNA regulatory network and its regulation on BC metastasis were further confirmed by western blot,immunoprecipitation assay（IP）,immunohistochemistry（IHC）,enzyme-linked immunosorbent assay（ELISA）,and tube formation assay.Results:1.CircKDM4B is significantly down-regulated in BC tissuesTranscriptome high-throughput sequencing was performed with three BC tissues with or without lymph node metastasis in each group.We identifed a novel circRNA hsa_circ₀002926（herein named circKDM4B）,which was down-regulated in the metastasis group.RT-qPCR was used to verify the expression of circKDM4B in clinical samples,and the results showed that the expression of circKDM4B was significantly down-regulated in BC tissues compared with non-tumor tissues.In addition,circKDM4B was highly expressed in immortalized mammary epithelial cells MCF-10A and lowly invasive BC cells（MCF-7 and T47D）,but expressed at a low level in highly invasive BC cells（MDA-MB-231 and MDA-MB-468）.To detect the stability of circKDM4B,we exposed cells to actinomycin D and found that the half-life of circKDM4B was more than its linear transcript KDM4B mRNA.Besides,circKDM4B was more resistant to RNase R digestion than its linear transcript.RNA nucleoplasmic separation and FISH assay both confirmed that circKDM4B was mainly localized in the cytoplasm.2.CircKDM4B inhibits cell migration and invasion abilities in vitroThe circKDM4B overexpression vector was constructed,and the interfering RNA specifically targeting circKDM4B back splicing site was synthesized.In MDA-MB-231 and MDA-MB-468 cells,we used the circKDM4B overexpression vector.In MCF 10A,MCF-7 and T47D cells,we knocked down circKDM4B with small interfering RNA.The overexpression efficiency and the interfering efficiency were confirmed and the linear transcript KDM4B mRNA had no significant change.The Transwell assay showed that overexpression of circKDM4B inhibited the migration and invasion ability of BC cells,while knockdown of circKDM4B promoted cell migration and invasion ability.CCK-8 assay manifested that circKDM4B had no significant effect on cell viability.EdU assay illustrated that circKDM4B had no obvious effect on cell proliferation.All these findings suggest that circKDM4B inhibits migration and invasion of BC cells.3.CircKDM4B serves as miR-675 sponge in BC cellsRIP assays showed that circKDM4B was significantly enriched in the anti-AG02 group compared with the IgG group.Using RegRNA 2.0 to analyze miRNA which circKDM4B may sponge,we screened out seven oncogenic miRNA including miR-18a,miR-20a,miR-27a,miR-187,miR-196a,miR-346,and miR-675.The miRNA mimics and pmirGLO-circKDM4B were co-transfected into BC cells.The dual-luciferase reporter assays showed that in MDA-MB-231 cells,miR-27a and miR-675 decreased the luciferase activity of circKDM4B by 30%and 25%,respectively.In MDA-MB-468 cells,miR-196a and miR-675 reduced the luciferase activity of circKDM4B by 33%and 47%,respectively.However,miR-27a had no significant effect on the luciferase activity of circKDM4B in MDA-MB-468 cells and miR-196a had no significant effect on the luciferase activity of circKDM4B in MDA-MB-231 cells.Thus,miR-675 was selected for further study because of its effect both in MDA-MB-231 and in MDA-MB-468 cells.After the binding sites between miR-675 and circKDM4B were mutated,miR-675 had no effect on the luciferase activity of mutant circKDM4B.Moreover,circKDM4B did not influence the expression of miR-675,and miR-675 had no effect on circKDM4B expression.Biotin-labeled RNA pull-down assay showed that circKDM4B was bound to miR-675.Transwell assays showed that miR-675 significantly enhanced the migration and invasion abilities of BC cells.Further,miR-675 could reverse the restrained effect of circKDM4B on cell migration and invasion.The mutant circKDM4B had minor inhibitory effect on migration and invasion capabilities in BC cells.4.NEDD4L is a direct target of miR-675 and is positively regulated by circKDM4BWe used Targetscan to predict common targets of miR-27a,miR-196a and miR-675.Based on previous reports,we found that NEDD4L was a tumor suppressor and was the target of miR-675.Then the luciferase reporter vector of pmirGLO-NEDD4L-3’UTR was constructed.Dual-luciferase reporter assays showed that the luciferase activity of NEDD4L was reduced by miR-675 and increased by circKDM4B.Then,the biding sites between miR-675 and NEDD4L were mutated,and neither miR-675 nor circKDM4B had effect on the mutant NEDD4L.Additionally,NEDD4L expression was negatively regulated by miR-675 and positively regulated by circKDM4B in a dose-independent manner.RT-qPCR showed that NEDD4L was significantly down-regulated in BC tissues than non-tumor tissues in clinical samples.Then NEDD4L expression was detected with IHC in clinical BC tissues,and the expression of NEDD4L was positively correlated with circKDM4B.All of above,these results suggests that circKDM4B positively regulates NEDD4L expression by sponging miR-675.5.CircKDM4B negatively regulates PI3KCA by ubiquitination via miR-675/NEDD4L axisIP assays showed that knockdown of NEDD4L inhibited PI3KCA ubiquitination in BC cells.Overexpression of circKDM4B increased NEDD4L expression,promoted PI3KCA ubiquitination,while knockdown of circKDM4B decreased NEDD4L expression,inhibited PI3KCA ubiquitination.Besides,knockdown of circKDM4B decreased NEDD4L but enhanced PI3KCA,p-AKT（Ser473）and VEGFA.Overexpression of circKDM4B increased NEDD4L but decreased PI3KCA,p-AKT（Ser473）and VEGFA,and miR-675 reversed this effect.Then we collected the conditioned medium for ELISA assays after BC cells were transfected for 48 h.The results showed that overexpression of wild type circKDM4B not mutant type circKDM4B restrained VEGFA secretion,while knockdown of circKDM4B enhanced VEGFA secretion.We also collected the conditioned medium to culture Human Umbilical Vein Endothelial Cells（HUVECs）and performed Transwell assays and tube formation assays.Transwell assays showed that circKDM4B inhibited the migration ability of HUVECs,while knockdown of circKDM4B promoted the migration ability of HUVECs.Tube formation assays showed that circKDM4B inhibited the tube-formation ability of HUVECs,while knockdown of circKDM4B promoted the tube-formation ability of HUVECs.Together,these results suggest that circKDM4B negatively regulates PI3KCA by ubiquitination via the miR-675/NEDD4L axis,inhibits PI3K/AKT and VEGFA secretion,and thereby inhibits HUVECs migration and tube-formation in vitro.6.CircKDM4B may be a potential therapeutic target in BCTo observe the effect of circKDM4B in vivo,LV5-circKDM4B or LV5-NC lentivirus was transfected into MDA-MB-231 cells to construct circKDM4B stably overexpressed cell lines.Then cells were injected into nude mice.In subcutaneous xenograft model,compare with LV5-NC group,the growth and weight of xenograft tumors in the LV5-circKDM4B group were delayed.Besides,enhanced circKDM4B inhibited capsular invasion and pulmonary metastasis.In the caudal vein metastatic model,enhanced circKDM4B inhibited pulmonary metastasis.IHC assay in xenograft tumors showed that NEDD4L expression in the LV5-circKDM4B group was higher than that in the LV5-NC group.The correlation analysis showed that circKDM4B was positively correlated with NEDD4L protein expression.IHC assay with anti-CD34 antibody showed that compared with LV5-NC group,the microvessel number in LV5-circKDM4B group was less,suggesting that circKDM4B inhibits angiogenesis in vivo,which is consistent with our findings in vitro.Western blot showed that expression of NEDD4L was higher in the LV5-circKDM4B group than those in the LV5-NC group.Besides,compared with LV5-NC group,the expression of PI3KCA,p-AKT（Ser473）and VEGFA was lower.All above findings suggest that circKDM4B plays an important role in suppressing angiogenesis,tumor growth,and tumor metastasis in vivo,indicating that circKDM4B can be a promising target for BC therapy.Conclusion:Our study illustrates that the newly identified circKDM4B negatively regulates PI3KCA by ubiquitination through miR-675/NEDD4L axis,suppresses BC growth and metastasis.