Identification Of TRAIL Sensivity Associated MicroRNA And Establishment Of Targeted Collaborative Treatment Strategy In Glioblastoma Cells

Part?Identification of TRAIL sensivity associated miRNAs and underlying mechanism in glioblastoma cells [Background]Glioblastoma(GBM)is the most aggressive form of brain tumors,with a median survival time no more than 16 months.Despite considerable advances in GBM therapy,it remains one of the most challenging diseases.TRAIL(TNF-related apoptosis-inducing ligand)is a promising anticancer agent that can be potentially used as an alternative or complementary therapy because of its therapeutic potential as a tumor-specific apoptosis inducer without affecting normal cells.However,the subsequent studies showed that many tumors were resistant to TRAIL,and the clinical application of recombinant TRAIL,have been hampered by its short half-life and unstable property in vivo.However,recent studies showed that genetically modified MSCs with high levels of TRAIL expression have been used to specifically delivery TRAIL to tumor tissues.It has partially counteracted the shortcoming of TRAIL treatment.MicroRNAs(miRNAs),evolutionarily well-conserved,small non-coding RNA,play an important role in the post-transcriptional regulation of target mRNA via mRNA degradation or translational repression through binding with 3'-untranslated regions(UTRs)of target genes.Accumulating evidences demonstrated that miRNAs play a critical role in the regulation of malignant behaviors of cancer cells,and cell-secreted miRNAs are predominantly carried by exosomes and exert in post-transcriptional regulation of gene expression in recipient cells through mRNA silencing.Therefore,it provides a promising targeted therapy strategy using MSCs-microRNA-TRAIL combination.[Aims]To find microRNA(s)which could enhance TRAIL-induced cell apoptosis and then use MSCs as a vehicle loading both of them to perform targeted therapy.[Methods]To define the critical microRNAs that regulate susceptibility to TRAIL in GBM cells,we performed a genome-wide expression profiling of microRNAs in GBM cell lines with the distinct sensitivity to TRAIL-induced apoptosis.Then cell apoptosis ratio were detected 48 h after microRNA transfection and TRAIL treatment using FACS assays.Then bioinformatics prediction,dual-luciferase reporter assay,qRT-PCR and Western blot were used to confirm the direct target gene of microRNA.Also,gain and loss of functional experiments were employed to further confirm the important roles of target gene in TRAIL-induced apoptosis.Subsequently,MSCs were modified with TRAIL and microRNA.Then modified MSCs were used to co-cultivate with U87 in order to verify its pro-apoptotic effect on tumor cells in vitro.At the same time,we use exosome inhibitors to confirm if this effect was mediated via exosome way.Transwell assays in vitro and immunohistochemistry experiments in vivo were used to confirm if MSCs loaded with TRAIL and microRNA have good tumor tropism.Finally,we established the subcutaneous tumor model in nude mice.MSCs loading with TRAIL and microRNA were used to treat tumor via tail vein injection in order to confirm the therapeutic effect.[Results]We found that the expression pattern of miR-7 is closely correlated with sensitivity of GBM cells to TRAIL.Furthermore,our gain and loss of function experiments showed that miR-7 is a potential sensitizer for TRAIL-induced apoptosis in GBM cells.In the mechanistic study,we identified XIAP is a direct downstream gene of miR-7.Additionally,this regulatory axis could also exert in other types of tumor cells like hepatocellular carcinoma cells.Also,MSCs loading with TRAIL and miR-7 have good tropism to tumor cells and tissues,they could enhanced receipt cell apoptosis through downregulation of XIAP in an exosome dependent manner.More importantly,in the xenograft model,enforced expression of miR-7 in TRAIL-overexpressed mesenchymal stem cells increased tumor cell apoptosis and suppressed tumor growth.[Conclusions]MicroRNA-7 can significantly enhance TRAIL-induced apoptosis through down-regulating XIAP in tumor cells.MSCs loaded with TRAIL and miR-7 could be specifically recruited to the tumor localization,and suppress tumor growth.Part? The new tumor suppressive molecular mechanism of microRNA-7 in Hepatocellular carcinoma(HCC)cells[Background]Micro RNAs(miRNAs)are a class of small non-coding RNAs encoded by the genomes of a wide range of multicellular organisms.Mature mi RNAs are incorporated into the RNA-induced silencing complex(RISC)and then target the 3'untranslated region(3'UTR)of a specific m RNA by base pairing,leading to translational repression or m RNA degradation.Most importantly,the ability of individual mi RNAs to regulate hundreds of transcripts allows them to coordinate complex programs of gene regulation and consequently induces global changes in cellular physiology.Indeed,a large number of evidence has demonstrated that mi RNAs provide functions essential for normal development and cellular homeostasis and,accordingly,dysfunction of these molecules has been linked to several human diseases,including cancer.In recent years,a large number of studies demonstrated that down-regulation of tumor suppressor mi RNAs plays a critical role in development and progression of hepatocellular carcinoma(HCC).Among the micro RNAs that are implicated in HCC,mi R-7 has recently been found to be down-regulated in HCC tissues and inhibit proliferation and metastasis in HCC cells in vitro and in vivo.And some studies showed that mi R-7 suppresses tumor growth and metastasis by targeting PI3K/AKT pathway in HCC.However,the role of mi R-7 inhuman hepatocellular carcinoma(HCC)and its underlying mechanism remain elusive.[Aims]To investigate the new tumor suppressive molecular mechanism of microRNA-7 in HCC cells.[Methods]Firstly,HCC cells were transiently transfected miR-7 mimics,and then were analyzed by cell cycle analysis using FACS.Then we predicted the potential target genes of mi R-7 by bioinformatics prediction tool.Subsequently,double luciferase reporter assay was used to test the relationship between mi R-7 and target gene.Then m RNA and protein expression level were tested by q RT-PCR and Western blot assays 48 h after mi R-7 transfection.Also,gain and loss of functional experiments were employed to further confirm the important roles of target gene in cell cycle regulation.Finally,the correlation between mi R-7 and target gene expression was analyzed in cancer cell lines and clinical samples using RT-PCR.[Results]Firstly,we found that exogenous expression of miR-7 arrested cell cycle in G1 phase in HCC cells.By combinational use of bioinformatic prediction,reporter assay,quantitative real-time PCR(q RT-PCR)and Western blot,we confirmed that CCNE1,an important mediator in G1/S transition is one of new direct target genes of mi R-7.Further studies revealed that silencing of CCNE1 recapitulated the effects of mi R-7 overexpression,whereas enforced expression of CCNE1 reversed the suppressive effects of mi R-7 in cell cycle regulation.Finally,analysis of q RT-PCR showed a reciprocal relationship between mi R-7 and CCNE1 in clinical cancer tissues and multiple types of tumor cell lines.[Conclusions]MiR-7 can directly downregulate the expression of CCNE1,and lead to cell cycle arrest in G1 phase,and consequently inhibit the proliferation of hepatocellular carcinoma cells.