The Mechanism Of MiR-181a-mediated Glutamate/GABA Receptors In Amphetamine Type Stimulant Use Disorder

Background Glutamate is the most important excitatory neurotransmitter of the central nervous system,and neurotransmitter in the brain,plays the most important central inhibitory effect throughout the GABA receptor.There are a lot of research suggests that glutamate and GABA receptor play an important role in addictive substance use disorders.However,there are lack the reporte on the GRIA2 and GABRA1 expression of amphetamines type stimulant use disorder patients.Objective The study explore the expression of GRIA2 and GABRA1 of amphetamines type stimulant patients.Then look for the possible regulatory mechanism of abnormal expression.Provide theoretical basis for amphetamines type stimulant use disorder patients.Method 1.One hundred twenty-four ATS use disorder patients and Fifty-seven gender and age matched healthy controls were recruited.The GRIA2 and GABRA1 serum levels were assessed using the ELISA kits.2.Chronic methamphetamine treatments were performed to SH-SY5 Y cells,and then total RNA or protein of cells will be isolated to detect the expression of GRIA2 and GABRA1 after methamphetamine exposure.3.The psiCheck-2-GRIA2 and psiCheck-2-GRIK2 vector was used in the Dual Luciferase reporter gene assays in HEK 293 T cells and western blot in SH-SY5 Y cells,to examine whether miR-181 a negative regulate glutamate receptor genes GRIA2 and GABRA1.4.Twelve previously studied common SNPs of GABRA1 were selected to be genotyped using Sequenom MassARRY genotype assays.To investigate the associations between common genetic polymorphisms of GABRA1 and ATS use disorder,and detectd the associations between polymorphisms and aberrant expression of GABRA1.Result 1.The GRIA2 gene encoding protein expression of ATS patients(32.58±10.46)higher than that of healthy controls(23.56±9.68).GABRA1 gene encoding protein expression was downregulated among ATS use disorder patients.2.Reverse transcriptase PCR analysis showed that 2mM methamphetamine exposure increased GRIA2 and GABRA1 mRNA expression,respectively.GRIA2 protein expression is also increased in SH-SY5 Y cells following treatment with amphetamine.3.Based on the Dual Luciferase reporter gene assays result,GRIA2 is a direct target of miR-181 a.The relative luciferase activity of the reporter containing GRIA2 3’-UTR was significantly suppressed when miR-181 a was cotransfected.Western blot was performed to examine the effect of overexpression or knockdown of miR-181 a on the protein levels of GRIA2 in SH-SY5 Y cells.Based on the Dual Luciferase reporter gene assays result,GABRA1 maybe not direct targets of miR-181 a.4.No association between the SNPs of GABRA1 and ATS use disorders risk.Conclution 1.The GRIA2 gene encoding protein expression of ATS use disorder patients higher than that of healthy controls.2.2mM and 4mM MA exposure for 48 h increased GRIA2 mRNA expression.3.GRIA2 is a direct target of miR-181 a.miR-181 a may act as a suppressor through downregulating the expression of GRIA2.GABRA1 maybe not direct targets of miR-181 a.4.No association between the SNPs of GABRA1 and ATS use disorders risk.