Hypoxia Induces H19 Expression Through Direct And Indirect Hif-la Activity,Promoting Oncogenic Effects In Glioblastoma

Research background:Malignant glioblastoma,one of the most lethal type of human intracranial tumors,is characterized with high recurrence and incurable disease.Hypoxic condition is the key to the tumor pathological features,mainly due to the fast growth of tumor cells but with insufficient blood vessels to support oxygen,causing low local tissue blood perfusion and local low oxygen in tumor.GBM is often characterized as necrosis area in the center of tumor,due to the fast tumor growth usually with problem of providing blood oxygen in central area.In other word hypoxia state has been a common phenomenon in glioblastoma.Recently it has been extensively studied that hypoxia played a significant role in malignant functional phenotype of tumors,which promotes tumor progression,such as invasion,metastasis and angiogenesis,and tumor adaptability under hypoxic stress environment.As we know that hypoxia-inducible factor-1 complex(HIF-1)is the key mediator of hypoxic response,which is a heterodimeric transcription factor composed of Hif-1αand HIF-1α subunits.Under normoxic conditions,Hif-1α is hydroxylated by proline hydroxylase domain(PHD)proteins.Upon hydroxylation,Hif-1α is recognized by the von Hippel-Lindau(VHL)protein,and then be ubiquitinated and undergoes rapid degradation through the ubiquitin-proteasome system.Under hypoxic conditions,however,PHD activity is inhibited,and then Hif-1 aaccumulates and dimerizeswithHIF-1α,forming the active HIF-1 complex and translocating to the nucleus.In the nucleus,HIF-1 complex bind to the hypoxia response elements(HREs,5’-RCGTG-3’)in the promoters or enhancers of target genes,resulting in transcriptional activation of downstream target genes.Meanwhile in recent years,a growing number of evidences indicated IncRNAs were closely related to the tumorigenesis and progression in human GBM.Several types of 1ncRNAs were involved in the process of tumor alteration under hypoxia,which played a potential role in facilitating tumor progression and adaption under low oxygen stress.H19,as a classic oncogene among these IncRNAs,is highly expressed in a number of tumors,and tightly associated with the cycle,tumor,apoptosis and proliferation activities.According to the previous studies,H19 was upregulated tumor cell lines under hypoxia,but the specific mechanism remains dismal.The article aimed to explore the concrete mechanism of hypoxia-elevated H19,which was through direct and indirect effects on the H19 promoter region to activate DNA transcription,stimulating H19 expression under hypoxia to promote malignant effects in glioblastomas.Our study suggest H19 might be a promising diagnostic and therapeutic target,This novel molecular mechanism demonstrated in our study suggested H19 might be a promising diagnostic and therapeutic target and provides new insights for malignant brain tumors.Methods:1.MRNA expression data for gliomas and normal controls and survival data of patients with glioma were downloaded from The Cancer Genome Atlas(TCGA)data portal.2.Fluorescence quantitative PCR reactions were performed using Taqman reverse transcription reagents and S YBR Green PCR Master Mix according to manufacturer’s protocols.Various glioma cell lines under different period of hypoxia via physical anoxia way were cultivated and analyzed for H19 level at different point of hypoxic time through fluorescence quantitative PCR.3.The U87 and U251 cells were transfected with.Hif-1α plasmid or siRNA respectively.H19 relative level were analyzed within the treated cells by fluorescence quantitative PCR.Besides,nude mouse orthotopic tumor model was built to investigate the regulation of Hif-1α-stimulated level of H19.4.The sensitivity of hypoxia-upregulated H19 depending on PTEN status in various glioma cell lines was analyzed by fluorescence quantitative PCR and Western blot assays.Moreover the regulation of H19 under hypoxia was contributed to Hif-1α together with PTEN status in glioma cells.5.The expression of Hif-1α,PTEN and H19 levels were analyzed in 22 human glioblastoma tissues(GBM)and 15 adjacent normal brain tissues(NBT)removed from patients with fluorescence quantitative PCR and Western blot assays.The correlation of Hif-1α and H19 levels were also analyzed in GBM tissues,with effects of PTEN status on this correlation.Immunohistochemitry and FISH in situ hybridization assays were implemented to investigate visually the level of Hif-1α and H19 in two representative specimens including NBT and GBM tissues.6.Bioinformatics,Chromatin immune coprecipitation(ChIP)assays,Luciferase reporter assay,QRT-PCR and western blot assay were used to analysis whether Hif-1α can bind to H19 promoter region and sequential activation of SP1 by Hif-1α can regulate H19 transcription.7.Selected human glioma cell line U87 and U251 were analyzed during logarithmic phase for cell experiments.The effects of migration and invasion induced by hypoxia and H19 were evaluated by wound healing assay and-transwell assay.8.Bioinformatics,Luciferase reporter assay,QRT-PCR and western blot was performed to identify whether H19 can regulate α-catenin expression by binding mir-181d and inhibiting the miRNA repression of its target genes.Results:1.H19 expression was elevated gradually with hypoxic time compared to normaxia in U87 and U251 cells.Further,H19 expression was significant higher in glioma cells treated with Hif-1α plasmid,and vice versa.The phenomenon of Hif-1α-upregulated H19 was also observed in nude mouse orthotopic tumor model.2.In multiple GBM cell lines including two primary GBM cells(GP1 and GP2)collected from two patients,the results showed that the significant elevated H19 level under hypoxia was not observed in cells with normal level of PTEN(Ln229 and PG1),but in cells with very low level of PTEN(U87,U251,U118,U373 and PG2).Rescue assays showed that PTEN could impact the stability of Hif-1α thereby influencing H19 level under hypoxia.3.Among 22 human glioblastoma tissues(GBIM)and 15 adjacent normal brain tissues(NBT)removed from patients,upregulation of Hif-1α in human GBM was significantly obvious relative to NBT,which were analogous to the results of H19 between GBM and NBT.The Hif-1α levels was positively correlated with H19 levels in GBM specimens.And PTEN status is an important factor beneficial for the correlation of Hif-1α and H19.4.Bioinformatics,ChIP assays and Luciferase reporter assay demonstrated that Hif-1α not fully but at least partly promoted H19 level via directly binding sites in the H19 promoter.Even when all HRE sequences were mutated,in both U87 and U251 cells,Hif-1α expression resulted in increased luciferase activity in all groups compared to the negative control plasmid.5.It has also been shown that elevated SP1 protein levels could drive binding of Hif-1α to the HREs within the SP1 promoter.Bioinformatics,ChIP assays and Luciferase reporter assay demonstrated that SP1 binds strongly to the H19 promoter region through its specific binding sites(GC-boxes)and that this interaction is required for optimal H19 transcriptional activation.Thus,SP1 plays a pivotal role along with Hif-la in H19 regulation under hypoxia.6.It is indicated that H19 contributed to the migration and invasion induced by hypoxia in glioma cells by wound healing assays and transwell assays.Meanwhile H19 played an important role in mediating the EMT marker proteins under hypoxia.7.Bioinformatics and Luciferase reporter assay showed that H19 expression upregulated α-catenin expression via binding and inhibiting miR-181d,which negatively regulated α-catenin mRNA,thereby contributing to H19 oncogenic functions in glioblastomas.Conclusions:1.H19 is elevated under hypoxia in glioblastoma cells,mainly due to the effects of Hif-1α.And PTEN is an advantageous condition for hypoxia-driven H19 induction.2.Hif-1α can directly bind to H19 promoter to activate the transcription.Besides Hif-1α promote SP1 mRNA and protein expression under hypoxia,and that SP1 has a critical intermediary role in Hif-1α-driven H19 expression,binding GC-boxes in the H19 promoter.3.In turn hypoxia-stimulated H19 can contribute to the function of migration and invasion of glioblastoma cells induced under hypoxia,which is involved to EMT marker proteins.We identified that H19 sponge activity towards miR-181d affects α-catenin levels in U87 and U251 cells.This novel mechanism offers a new understanding of how H19 and miRNAs interact to control gene transcription.