The Effect Of LncRNA MIAT On Neuroblastoma Neuro2a Cells Under The Action Of Inflammatory Factors And Its Mechanism

Objective: Neuroblastoma derived from undifferentiated sympathetic ganglion cells,is one of the most common malignant tumors in children and is well-known for its wide spread,rapid progression and poor prognosis.It has always been a hot topic in clinical studies.Long non-coding RNA(lncRNA)is a kind of non-coding nucleotide sequences discovered in recent years.As a kind of new molecule,it does not code for proteins,but can act as a regulatory role by participating in a variety of life activities through different ways.LncRNA is widely discovered in tumors and can be targeted,which plays an important role in the early detection,early diagnosis,precise treatment and improving prognosis.LncRNA MIAT,widely expressed in the nervous system and in vitro cultured neurons,is related to the differentiation of neurons,but its regulatory role in neuro-related diseases is not fully understood.Neurite growth is also closely connected to neurological diseases.To explore the influence of MIAT on neuroblastoma Neuro2 a cells is helpful for further understanding of the disease.LncRNA can also play a regulatory role by regulating the expression of adjacent genes.Cryba4,Crybb1 and Sez6 L are all adjacent genes of MIAT.Detecting the influence of MIAT on the expression of adjacent genes is helpful to study the mechanism of neuroblastoma.In addition,the growth and progression of tumors is closely related to the microenvironment of tumor tissues and metastatic organs.To explore the regulatory effects of inflammatory factors on MIAT is of great significance for guiding the treatment of neuroblastoma.Methods: The logarithmic phase Neuro2 a cells were chosen as the research object.Through transfection of blank plasmids or MIAT knockdown plasmids,these cells are divided into negative control group and experimental group differently.By transwell,wound healing,MTT and flow cytometry tests,the cells ability of invasion,migration,proliferation and apoptosis is detected.By analyzing the statistics from living cell imaging analysis system,the total neurite length,longest neurite length,number of neurites and cell differentiation rate were analyzed.To explore whether they were regulated by MIAT,RT-PCR was used to analyze the expression of adjacent genes,Crybb1,Cryba4 and Sez6 L,after MIAT knocking down.To find suitable condition,the genes expression of Neuro2 a cells treated with different concentration of il-6 was detected by RT-PCR.Supernatant from microglia stimulated by right concentration of LPS was treated on Neuro2 a cells and the expression of related genes were detected by RT-PCR.Results: ShRNA was transfected into Neuro2 a cells with the help of plasmid.Observing neurite out-growth,we found that compared with the control group,the differentiation rate was significantly reduced in the MIAT knockdown group(P<0.05).At the same time,the total neurite length,the longest neurite length and the number of neurites decreased significantly(P<0.05)and the differences in each group over time were statistically significant(P<0.05).Cell function-related experiments showed that compared with the control group,apoptosis rate was significantly increased and proliferation,migration and invasion ability were significantly decreased(P<0.05)in the MIAT knockdown group.It was also found that the expression of Cryba4 and Crybb1 in the MIAT knockdown group had no significant change(P<0.05),but the expression of Sez6 L gene decreased significantly(P<0.05)compared with the control group.Furthermore,treated Neuro2 a cells with different concentrations of IL-6,we found that when the concentration of IL-6 was over 200ng/ml,the expressions of MIAT and Sez6 L were significantly increased(P<0.05).Similarly,the expression of MIAT and Sez6 L was significantly increased when the Neuro2 a cells were treated with supernatant from LPS stimulated microglia(P<0.05).Conclusion: 1.MIAT knockdown can deduce neuronal differentiation and neurite growth in Neuro2 a cell line.2.MIAT knockdown induced Neuro2 a cells apoptosis and inhibited its proliferation,migration and invasion.3.MIAT has no obvious effect on the expression of Cryba4 and Crybb1 genes,but can regulate the expression of Sez6 L gene.4.High concentration of il-6(> 200ng/ml)and supernatant of LPS stimulated microglia can regulate the gene expression of MIAT/Sez6 L in Neuro2 a cells.