| Objective:1.To explore the effects of mi R-29 a, mi R-29b1, BACE1 and Aβ1-42 after rat cortical neurons exposed to Al(mal)3.2.To investigate the regulation mechanism of mi R-29 a and mi R-29b1 on the expression of BACE1 during the deposition of amyloid-β induced by Al(mal)3 through establishing a high expression of mi R-29 a or mi R-29b1 cell model.3.To investigate the effect of NF-κB on the abnormal expression mi R-29 a and mi R-29b1 induced by aluminum in vitro.Methods:1.The Cerebral cortex of newborn SD rats( ≤ 24h-old) was used for primary neuronal cultures, and 10μmol / L of cytarabine(Ara-C) was added on the third day. The specific protein Neuron-specific class Ⅲβ-Tublin was used to detect neuron purity by Immunohistochemical methods. The primaryneuronal cells were exposed in different doses of aluminum-maltolate( Al(mal)3).The experimental groups were divided into the control groups(0μmol/L), the low dose groups(20μmol/L), the middle dose groups(40μmol/L), the high dose groups(80μmol/L). After 24 h, collecting cells and the cellculture medium, mi R-29 a, mi R-29b1 and BACE1m RNA were detemined by RT-PCR,and the expression of BACE1 and Aβ1-42 were detected by ELISA.2.The exogenous mi R-29 a or mi R-29b1 was transferred into cortical neurons to establish a high expression cell model through the method of slow virus infection.The experimental group were divided into control group, 20μmol/L Al(mal)3, 40μmol/LAl(mal)3, 80μmol/L Al(mal)3, negative transfection group and the target gene transfection group+phosphate buffered saline(PBS), 20μmol/L Al(mal)3, 40μmol/L Al(mal)3,80μmol/L Al(mal)3 group. After24 h, collecting cells andthe cellculture medium, mi R-29 a,mi R-29b1 and BACE1m RNA were detemined by RT-PCR,and the expression of BACE1 and Aβ1-42 were detected by ELISA.3.HEK293 cell were cultured in vitro. The exogenous mi R-29 a or mi R-29b1 was transferred into HEK293 cell to establish a high expression cell model using liposome transfection technology. The experimental group were divided into control group,100μmol/L Al(mal)3, 200μmol/L Al(mal)3, 400μmol/L Al(mal)3, negative transfection group and the target gene transferred group+PBS, 100μmol/L Al(mal)3, 200μmol/L Al(mal)3, 400μmol/L Al(mal)3 group.After 24 h, collecting cells andthe cellculture medium,mi R-29 a, mi R-29b1 and BACE1m RNA were detemined by RT-PCR,and the expression of BACE1 and Aβ1-42 were detected by ELISA.4.The rat cortical neurons were cultured in vitro.Intervention experiments were performed on cells by selecting NF-κB agonist(TPA) and NF-κB inhibitor(PDTC). The experimental group were divided into control group, intervention group with 1μmol/L TPA, intervention group with 50μmol/L PDTC, exposed group with 40μmol/L Al(mal)3and intervention group with 40μmol/L Al(mal)3 and 50μmol/L PDTC.After 24 h,collecting cells andthe cellculture medium, mi R-29 a, mi R-29b1 and BACE1m RNA were detemined by RT-PCR,and the expression of BACE1 and Aβ1-42 were detected by ELISA.Results:1.The immunohistochemical method results shows: the purity of neurons was 95%.After the neuronal cells were exposed in different doses of Al(mal)3, compared with the control groups, the expression of BACE1 m RNAand BACE1 protein increased with increasing doses(P<0.05), but there were not statistical difference in the low dose group.The levels of Aβ1-42 increased, there were statistical difference in the middle dose andVII high dose group(P<0.05). However, theexpression of mi R-29 a and mi R-29b1 decreased insignificantly, compared with the control groups, there were statistical difference in the middle dose and high dose group(P<0.05).2.After the exogenous mi R-29 a or mi R-29b1 was transferred into cortical neurons,compared with the control groups, the expression of BACE1 m RNA and BACE1 protein were not significantly changed in the negative transfection group(P>0.05). The expression of BACE1 m RNA decreased in positive transfection group, but the difference was not statistically significant(P>0.05). The expression of BACE1 protein decreased in positive transfection group, the difference was statistically significant(P<0.05). The expression of BACE1 m RNA and BACE1 protein decreased in the target gene transferred groups compared the same dose group which the target gene was not transferred, both the middle dose group and high dose group have statistical difference(P<0.05). The level of Aβ1-42 were not significantly changed in the negative transfection group compared with the control groups(P>0.05). The level of Aβ1-42 was decreased in positive transfection group, the difference was statistically significant(P<0.05). The level of Aβ1-42 decreased in the target gene transferred groups compared the same dose group which the target gene was not transferred, there were statistical difference in the middle dose and high dose group(P<0.05).3.The exogenous mi R-29 a or mi R-29b1 was transferred into HEK293 cell using liposome transfection technology. The relative expression of mi R-29 a and mi R-29b1 were 427 times and 480 times in 36 h.The cells were exposed in different doses of Al(mal)3 after the transfection. The results show:the expresions of BACE1 m RNA,BACE1 protein and Aβ1-42 were significantly increased compared with the control group(P<0.05). The expression of BACE1 m RNA, BACE1 protein and Aβ1-42 were not significantly changed in the negative transfection group(P>0.05). The expression of BACE1 m RNA, BACE1 protein and Aβ1-42 decreased in mi R-29 a transferred groups compared the control group, but BACE1 m RNA was not statistically significant(P>0.05),BACE1 protein and Aβ1-42 were statistically significant(P<0.05). The expression of BACE1 m RNA, BACE1 protein and Aβ1-42 decreased in mi R-29b1 transferred groupscompared the control group. The expression of BACE1 m RNA, BACE1 protein and Aβ1-42 decreased in the target gene transferred groups compared the same dose group which mi R-29 a or mi R-29b1 were not transferred(P<0.05).4.After the intervention experiments were performed on the rat cortical neurons,the expression of mi R-29 a and mi R-29b1 decreased significantly in exposed group with respectively 1μmol/L TPA and 40μmol/L Al(mal)3 compared with the control groups(P<0.05). Theexpression of mi R-29 a and mi R-29b1 increased in intervention group with 50μmol/L PDTC, but mi R-29 a was statistically significant(P<0.05),mi R-29b1 was not statistically significant(P>0.05). Theexpression of mi R-29 a and mi R-29b1 increased in intervention group with 40μmol/L Al(mal)3 and 50μmol/L PDTC compared with exposed group with 40μmol/L Al(mal)3(P<0.05). The expression of BACE1 m RNA, BACE1 protein and Aβ1-42 increased in exposed group with respectively1μmol/L TPA and 40μmol/L Al(mal)3 compared with the control groups(P<0.05). The expression of BACE1 m RNA, BACE1 protein and Aβ1-42 decreased in intervention group with 50μmol/L PDTC, but BACE1 protein and Aβ1-42 were statistically significant(P<0.05), BACE1 m RNA was not statistically significant(P>0.05). The expression of BACE1 m RNA, BACE1 protein and Aβ1-42 decreased in intervention group with 40μmol/L Al(mal)3 and 50μmol/L PDTC compared with exposed group with40μmol/L Al(mal)3(P<0.05).Conclusion:1.In rat cortical neurons, Al(mal)3 decreases the expression of mi R-29 a andmi R-29b1, increases the level of BACE1 which plays a key role in the generation of amyloid-β, and finally causes the deposition of amyloid-β-42.2.mi R-29 a and mi R-29b1 are involved in the regulation of BACE1 during the deposition of amyloid-β-42 induced by aluminum.3. The activation of NF-κB may participate in the process of abnormal expression of mi R-29 a and mi R-29b1 in rat cortical neurons induced by aluminum. |
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