Target Regulation Of Hsa-miRNA-9-5p In Glioma Cells U87 On MACC1 Experimental Research

ObjectiveTo investigate the targeting effect of Hsa-miRNA-9-5p on MACC1 in human glioma U87 cell line,we studied the culture of glioma U87 cells and the rat model of experimental glioma.Methods1?To construct MACC1 luciferase vectors:according to the sequence published on GeneBank,MACC1 and 3'UTR respectively on the Hsa-miRNA-9-5p core region mutation primers were designed according to the sequence of primers both contain SpeI and MIU-1 restriction sites and protective base,connecting pMIR-REPORT plasmid the PCR and luciferase expression product can.2?Verify the dual luciferase system:inoculated with 1�10⁵ HEK-293T cell and96 cell culture plate,according to the DNA 0.2g plasmid and TransLipid 0.5?l were diluted to 25?l DMEM medium at room temperature,the static 5min will both gentle mixing,the formation of the DNA-TransLipid complex,and then added to the inoculation the cells,after gently shaking culture plate,the composite dispersion.At37?,5%CO₂ was cultured,and after 4-6h,the culture medium was replaced,and48h was continued.After 48h,the plate was taken out in the self culture box and the method of the double Luciferase Report system was used to detect the cell luminescence.3?The expression of MACC1 mRNA:the culture bottle was inoculated with U87cells for the night,and the infection of lentivirus was carried out when it was up to90%.After infection,fresh 10%FBS-DMEM medium was replaced every 24h.Until third days after infection,the cultured cells were harvested.PBS was washed for two times,and the total RNA was extracted.The first strand cDNA synthesis kit was reverse transcribed into cDNA template.Detection of DC-LAMP expression level by Real-time PCR.4?90 rats of Sprague-Dawley?SD?were randomly divided into 5 groups.Glioma group?group A:18?,saline group?sham operation group B:18?,normal control group?group C:18?,empty vector group?group D:18?,vector transfected virus?Hsa-miRNA-9-5p?group?group E:18?.Rat U87 glioma model was established by glioma group and virus transfection group.Rats in normal saline group were injected with 10?l normal saline intracranially.Normal control group was normal rats.The vector-transfected virus group was injected with Hsa-miRNA-9-5p transfection solution in situ 4 and 11 days after U87 glioma cells were inoculated.The empty vector group was injected with the untransfected vector 4 and 11 days after U87glioma cells were inoculated.All the rats in the five groups were examined by MRI at the corresponding time points on the 9th,15th and 21st day after inoculation of U87glioma cells.When necessary,the rats were executed painlessly.The glioma was removed completely under the microscope.The diameter of U87 glioma cells and the number,area and blood vessel of glioma tissue were measured.The expression of MACC1 was detected by RT-PCR and Western blot.Results1?Using cDNA as template,PCR was amplified to the core locus containing Hsa-miRNA-9-5p and MACC1.After 2%agarose gel electrophoresis,clear target bands were observed.The recombinant plasmid was verified by SpeI,MIU-1 double enzyme digestion,and the size of the recombinant plasmid was normal,suggesting that the construction of the vector was successful.2?The interference vector built and carrying luciferase vector was transfected into HEK-293T cells,and the establishment of interf ence vector transfected group,as well as the core role of mutation group as the control group,all PRL-TK fluorescence expression plasmid was used as a reference,results showed that MACC13,UTR mutation group?Mut?and green fluorescent protein the expression compared to the control group no significant change.Compared with the control group,the expression of MACC1 3'UTR?Wt?green fluorescent protein was significantly lower than that in the control group?P<0.05?.3?The lentivirus particles with Hsa-miRNA-9-5p were infected with U87 cells,72h harvested cells,and the total RNA was extracted to detect the expression of mRNA.Real-time PCR results showed that the expression of MACC1 mRNA in lentivirus infection was significantly lower than that in the normal control group?P<0.05?.4?There was no tumor growth in normal saline group and normal control group.On the 9th day after inoculation of U87 glioma cells,the growth of tumors in glioma group and Hsa-miRNA-9-5p group was slow?P>0.05?.From the 15th day after inoculation of U87 glioma cells to the end of the experiment,the tumor volume of glioma group increased rapidly,while the growth rate of tumor volume of Hsa-miRNA-9-5p group slowed down significantly from the 15th day after inoculation of U87 glioma cells,which was significantly different from the other three groups?P<0.01?.5?Vascular sparse distribution was observed in the transfection group?vascular area density 0.55�0.22?and the number of vascular increased significantly in the glioma group?vascular area density 2.76�0.11?.Compared with the transfection group,the normal saline group?vascular area density 1.43�0.21?,the empty carrier group?vascular area density 1.41�0.11?,the normal group?vascular area density1.39�0.18?had slightly more vascular distribution,but the difference was not statistically significant.Because of the growth characteristics of gliomas,the growth of microvessels was mainly initiated,the blood was detected.The number of tubes can also reflect the treatment of gliomas,inhibition of angiogenesis is also an important consideration direction,can also reflect the role of virus vector intervention,and the direction of treatment of blood vessels.6?Hsa-miRNA-9-5p recombinant adenovirus transfected into U87 glioma cells on the 9th,15th and 21st days,the tumor growth was slow in the transfection vector group,compared with the empty vector group and the control group,there was statistical significance,P=0.009,P<0.01,MACC1 expression was significantly reduced,the effect was obvious,with the treatment time migration,inhibition.The growth rate and the expression of MACC1 protein were more obvious in the tumor-producing group than in the control group.7?On the 9th,15th and 21st day after transfection,the expression of MACC1mRNA in U87 glioma cells was significantly decreased in the transfection vector group,compared with the empty vector group and the control group.The expression of MACC1 was significantly decreased in the transfection vector group?P=0.021,P<0.05?.With the migration of treatment time,the tumor growth was inhibited.The expression of mRNA was further decreased,and the trend was consistent with that of protein expression.Conclusion1?MACC1 gene is a metastasis related gene.It is a key regulator in the HGF/c-Met signal transduction pathway,and it can promote the chemotaxis,invasion and migration of tumor cells.2?The expression of MACC1 in human glioblastoma is positively correlated with the degree of malignancy.Inhibition of MACC1 expression may inhibit the malignant development of human glioblastoma.3?Bioinformatics analysis showed that Hsa-miRNA-9-5p could regulate the expression of MACC1,and it was preliminarily verified in human glioma cell line U87.4?The rat model of glioma was established by U87 glioma cells.The tumor grew rapidly,which was proved by imaging and fresh glial tissue.5?Hsa-miRNA-9-5p recombinant transfection vector was injected into U87glioma cells on the 9th,15th and 21st days.In the transfection vector group,the tumor grew slowly,which could inhibit the expression of MACC1 gene and the growth of tumor.6?Hsa-miRNA-9-5p can regulate and inhibit the expression of MACC1targeting,and it is one of the important factors of inhibiting the development of glioma.