STAT3/miR-130b-3p/MBNL1 Feedback Loop Regulated By MTORC1 Signaling Promotes Angiogenesis And Tumor Growth

BackgroundThe mammalian target rapamycin(mammalian target of rapamycin,mTOR)signaling pathway is involved in multiple metabolic processes in mammals and plays an important role.Dysregulation of mTOR signaling presents in different pathological conditions such as diabetes,cancer,and Alzheimer’s disease.mTOR signaling is often dysregulated in various human cancers,such as breast cancer,prostate cancer,and kidney cancer.Upregulation of mTOR signal can promote tumor growth and progression by promoting growth factor receptor signal,angiogenesis,glucose metabolism and inhibition of autophagy.Meanwhile,it has been reported that PI3K/Akt/mTOR pathway is overactivated in more than 80% of head and neck squamous cell carcinoma(head and neck squamous cell carcinoma,HNSCC),thus mTOR provides a potential key molecular target for clinical treatment of HNSCC.MTOR has two complexes with different functions,mTOR complex 1(mammalian target of rapamycin complex 1,mTORC1)and mTOR complex 2(mammalian target of rapamycin complex 2,mTORC2).Abnormally activated mTORC1 plays an important role in tumor angiogenesis,but the exact mechanism still unclear.MicroRNAs(miRNAs)are a class of non-coding RNAs that are highly conserved in eukaryotes.Most of the miRNAs have hairpin structure,which is a small single-stranded RNA composed of about 18-24 nucleotides.miRNAs generally exist in three forms,the original Pri-miRNA,the Pre-miRNA formed by pri-miRNA after processing,and the mature miRNA that is cleaved by Dicer enzymes.Functionally,miRNAs silence the expression of target genes by complementary binding with target genes.However,the role of miRNAs in mTORC1-activated tumors is rarely reported,and it is still unclear whether non-coding RNA is involved in mTORC1-mediated angiogenesis.ObjectivesIn this study,we explored the expression changes of micro-RNA-130b-3p(miR-130b-3p)mediated by mTORC1 activation and its role and molecular mechanism in the development and progression of TSC tumors and head and neck tumors.MethodsTSC2 and TSC1 deficient mouse embryonic fibroblasts(MEFs)(Tsc2-/-and Tsc1-/-MEFs)and control cells(Tsc2+/+ and Tsc1+/+ MEFs)are widely used to study TSCrelated diseases and the mTOR signaling pathway.We first detected the expression of miR-130b-3p in mTORC1-activated cells and control cells by quantitative real-time PCR(q RT-PCR).Then we explored the effect of miR-130b-3p on angiogenesis using tube formation assay and chick embryo chorioallantoic membrane(CAM)assay.Next,we used bioinformatics analysis,Western blot,RNA immunoprecipitation,immunofluorescence,luciferase reporter gene,chromatin immunoprecipitation(CHIP),tube formation assay,CAM assay,HNSCC patient-derived xenotransplantation(patientderived xenograft,PDX)model and other experiments,furtherly explored the regulatory mechanism and function of signal transducer and activator of transcription 3(activator of transcription 3,STAT3),miR-130b-3p and Muscleblind-like protein 1(muscleblind-like protein 1,MBNL1)in MEFs and human tumors.ResultsWe first proved that mTORC1 activation up-regulated the expression of miR-130b-3p,and the increased miR-130b-3p enhanced the angiogenesis and tumorigenesis of mTORC1-activated cells both in vitro and in vivo.Then we found that mTORC1 upregulated the expression of miR-130b-3p by activating STAT3,while STAT3 could activate miR-130b-3p by directly binding to the promoter of miR-130 b gene.Subsequently we determined that MBNL1 is the direct target of miR-130b-3p,and functionally overexpression of MBNL1 will inhibit angiogenesis.Then,we demonstrated MBNL1 depletion rescued the compromised angiogenesis and tumor growth caused by miR-130b-3p inhibition.We also found that the expression of miR-130b-3p was significantly up-regulated in HNSCC and positively correlated with mTORC1 signal transduction.And the inhibition of miR-130b-3p will affect angiogenesis and tumor growth in the HNSCC PDX model.Finally,we found that MBNL1 feedback inhibited the activation of STAT3 in mTORC1-activated cells.ConclusionThe STAT3/miR-130b-3p/MBNL1 feedback loop plays a vital role in mTORC1-mediated angiogenesis and tumor progression.This pathway could be targeted for therapeutic intervention of mTORC1-related cancers.