Detection Of MiRNA-21 In Neuroblastoma By MRI Reporter Gene Imaging

Background and objectiveMicroRNA（miRNA）is a single-stranded non-coding RNA,containing about 20-25 nucleotides.It negatively modulates the target gene expression via induction of m RNA degradation or post-transcriptional gene silence to regulate many biological processes such as cell proliferation,differentiation and death.It has been confirmed that many kinds of miRNA are closely related to the occurrence and development of tumors.Among them,miR-21 is considered to be an important tumor promoting factor,which is ubiquitous in neuroblastoma,glioblastoma,breast cancer and other tumors,and can be used as a potential target for early diagnosis and treatment of tumors.In order to further understand the biological function of miRNA,it is necessary to use non-invasive imaging technology to monitor miRNA in vivo.In recent years,optical imaging based on reporter gene has been used in the visualization of miRNA,but optical imaging has some disadvantages,such as poor tissue penetration and easy quenching of light-emitting materials,which greatly reduces the potential of its clinical application.Magnetic resonance imaging（MRI）is not limited by tissue thickness,and has excellent soft tissue resolution,so it is easier to transform clinically than other imaging methods.With the continuous development of reporter gene imaging technology,MRI is widely used in the field of molecular imaging.At present,the main MRI reporter genes are tyrosinase,β-galactosidase,transferrin receptor and ferritin,among which FTH1 is the most commonly used.In previous studies,we have confirmed that FTH1-based MRI reporter gene imaging can be used to trace stem cells and tumor cells in vivo.In this experiment,MRI reporter gene and miR-21 were combined to construct a miR-21 responsive FTH1 gene reporter system.The gene reporter system was transferred into human neuroblastoma cells（SK-N-SH）,and intracellular miR-21 was interfered by miR-21 antisense oligonucleotides antagomiR-21.At the same time,MR imaging was performed to observe the signal changes of cells.To explore the feasibility of using reporter gene MRI to display the dynamic changes of miR-21 in SK-NSH cells in vivo and in vitro.Method1 Construction and identification of miR-21 responsive FTH1reporter geneThe FTH1 gene was amplified by PCR and inserted into the multi-clone site of the lentiviral vector p LVX-m CMV-Zs Green1-Puro to obtainthe lentiviral vector p LVX-m CMV-Zs Green1-Puro-FTH1（p LVX-FTH1）with overexpression of FTH1.The construction of the lentiviral vector p LVX-m CMV-Zs Green1-Puro-FTH1（p LVX-FTH1）was verified by DNA sequencing and restriction enzyme digestion.Then,the three target sequences（3×C＿miR-21）which are fully complementary to the miR-21gene are connected to the end of the FTH1 overexpression lentivirus vector by genetic engineering technology to obtain the FTH1-3×C＿miR-21overexpression lentivirus vector p LVX-m CMV-Zs Green1-Puro-FTH1-3×C＿miR-21（p LVX-FTH1-3×C＿miR-21）,which is identified by sequencing.The lentiviral vector plasmids of the two reporter genes were packaged and purified respectively and concentrated by ultrafiltration,and the lentivirus titer was determined by dilution titer.2 Expression and regulation of FTH1-3×C＿miR-21 gene reporter system in SK-N-SH cellsTwo lentiviruses（LVX-FTH1 and LVX-FTH1-3×C＿miR-21）were used to infect SK-N-SH cells.After three generations of puromycin screening,stable cell lines SK-N-SH/FTH1 and SK-NSH/FTH1-3×C＿miR-21 were obtained.SK-N-SH/WT without lentivirus transfection as the blank control group,SK-N-SH/FTH1 as the control group,and SK-N-SH/FTH1-3×C＿miR-21 was used as the experimental group.The expression of FTH1 in SK-N-SH,SK-N-SH/FTH1 and SK-N-SH/FTH1-3×C＿miR-21 cells was preliminarily identified by qPCR and Western blotting,and the cell viability was detected by CCK-8reagent.Then the different concentrations of anagomiR-21 were introduced into SK-N-SH/FTH1-3×C＿miR-21 cells,WB and MR imaging were used to detect the dose-effect relationship between antagomiR21concentration and FTH1 expression.Finally,three groups of cells were transfected with the optimal concentration of antagomiR-21,and the cells were examined by MR imaging,prussian blue staining and electron microscope.3 In vivo detection of dynamic changes of miR-21 in SK-N-SH cell xenografts by MRI based on miR-21 response reporter geneThree groups of cells,SK-N-SH/WT,SK-N-SH/FTH1 and SK-N-SH/FTH1-3×C＿miR-21,were implanted subcutaneously into the root of the right hindlimb of nude mice to establish the model of neuroblastoma.According to the dose of 30nm L/g injected into the tail vein of antagomiR-21,7.0T magnetic resonance T₂WI was performed before and 24 hours after administration,and the value of R₂ was determined.After MR scanning,the tumor tissue was stained with prussian blue.Results1 Construction and identification of miR-21 responsive FTH1 gene reporter systemRestriction endonuclease digestion and DNA sequencing of PCR products showed that the plasmids p LVX-FTH1 and p LVX-FTH1-3×C＿miR-21 were successfully constructed,and the virus titers obtained respectively were 1.5×10⁸TU/m L and 1.2×10⁸TU/m L,met the experimental requirements.2 Expression and regulation of FTH1-3×C＿miR-21 gene reporter system in SK-N-SH cellsThe results of qPCR showed that obvious expression of FTH1 could be detected in SK-N-SH/FTH1 and SK-NSH/FTH1-3×C＿miR-21 group in three groups before antagomiR-21 transfection,while there was no obvious expression in SK-N-SH/WT group.The results showed that before transfection of antagomiR-21,obvious FTH1 expression could be detected in only SK-NSH/FTH1 group,but there was no obvious expression in the other two groups.The results of CCK-8 assay revealed no significant difference of cell viability in three groups,indicating that gene transduction would not affect the normal growth of cells.After SK-N-SH/FTH1-3×C＿miR-21 cells were transfected with different concentrations of antagonmiR-21,the results of WB and MRI showed that the expression of FTH1 and the corresponding changes of MRI signal were dose-dependent,and the expression of FTH1 was the highest when the concentration of antagonmiR-21 was 40nmol/L.After the three groups of cells were transfected with the best antagomiR-21 concentration,the MRI signal of SK-N-SH/FTH1-3×C＿miR-21 group was significantly lower than that before transfection,and there was no obvious signal change in the other two groups before and after transfection.The results of intracellular iron detection were consistent with the results of MRI.3 In vivo detection of dynamic changes of miR-21 in SK-N-SH cell xenografts by MRI based on miR-21 response reporter geneThe nude mouse model of SK-N-SH cell transplantation tumor was successfully established.Before the use of antagomiR-21 intervention,compared with the SK-N-SH/WT group,the signal decreased in the SK-N-SH/FTH1 group,but there was no obvious signal change in the SK-N-SH/FTH1-3×C＿miR-21 group;after antagomiR-21 administration,the MRI signal decreased significantly in the SK-N-SH/FTH1-3×C＿miR-21 group than before antagomiR-21 administration.In the other two groups,no significant changes of MRI signal were foundbefore and after intervention.The results of Prussian blue staining of tumor tissue were consistent with the results of MRI.ConclusionThe miR-21-responsive FTH1 gene reporting system was successfully constructed.The expression of the system is"off"in SK-N-SH cells because of its binding to miR-21.AntogomiR-21 can"turn on"the reporter gene expression by inhibiting the activity of miR-21,thus causing the change of MRI signal.This provides a potential non-invasive imaging method for early stage 5 diagnosis and gene therapy evaluation of tumors targeted by miR-21.