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MicroRNA-9 induces defective trafficking of Nav1.1 and Nav1.2 by targeting Navβ2 protein coding region in rat with chronic brain hypoperfusion.

Sun LH, Yan ML, Hu XL, Peng LW, Che H, Bao YN, Guo F, Liu T, Chen X, Zhang R, Ban T, Wang N, Liu HL, Hou X, Ai J - Mol Neurodegener (2015)

Bottom Line: Intriguingly, miR-9 suppressed, while AMO-miR-9 enhanced, the trafficking of Nav1.1/Nav1.2 from cytoplasm to cell membrane.Further study showed that overexpression of miR-9 inhibited the Navβ2 expression by targeting on its coding sequence (CDS) domain by dual luciferase assay.However, binding-site mutation or miR-masks failed to influence Navβ2 expression as well as Nav1.1/Nav1.2 trafficking process, indicating that Navβ2 is a potential target for miR-9.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Harbin Medical University, No.157 Baojian Road, Nangang District,Harbin, Heilongjiang Province, 15008, China. sunlihua0219@163.com.

ABSTRACT

Background: Previous studies have demonstrated that the trafficking defects of Nav1.1/Nav1.2 are involved in the dementia pathophysiology. However, the detailed mechanisms are not fully understood. Moreover, whether the impaired miRNAs regulation linked to dementia is a key player in sodium channel trafficking disturbance remains unclear. The cognitive impairment induced by chronic cerebral ischemia through chronic brain hypoperfusion (CBH) is likely reason to precede dementia. Therefore, our goal in the present study was to examine the role of microRNA-9 (miR-9) in regulating Nav1.1/Nav1.2 trafficking under CBH generated by bilateral common carotid artery occlusion (2VO).

Results: The impairment of Nav1.1/Nav1.2 trafficking and decreased expression of Navβ2 were found in the hippocampi and cortices of rats following CBH generated by bilateral 2VO. MiR-9 was increased in both the hippocampi and cortices of rats following CBH by qRT-PCR. Intriguingly, miR-9 suppressed, while AMO-miR-9 enhanced, the trafficking of Nav1.1/Nav1.2 from cytoplasm to cell membrane. Further study showed that overexpression of miR-9 inhibited the Navβ2 expression by targeting on its coding sequence (CDS) domain by dual luciferase assay. However, binding-site mutation or miR-masks failed to influence Navβ2 expression as well as Nav1.1/Nav1.2 trafficking process, indicating that Navβ2 is a potential target for miR-9. Lentivirus-mediated miR-9 overexpression also inhibited Navβ2 expression and elicited translocation deficits to cell membrane of Nav1.1/Nav1.2 in rats, whereas injection of lentivirus-mediated miR-9 knockdown could reverse the impaired trafficking of Nav1.1/Nav1.2 triggered by 2VO.

Conclusions: We conclude that miR-9 may play a key role in regulating the process of Nav1.1/Nav1.2 trafficking via targeting on Navβ2 protein in 2VO rats at post-transcriptional level, and inhibition of miR-9 may be a potentially valuable approach to prevent Nav1.1/Nav1.2 trafficking disturbance induced by CBH.

No MeSH data available.


Related in: MedlinePlus

MiR-9 downregulates the expression of Navβ2 proteins. a, Verification of uptake of miR-9 by NRNs after transfection, *P < 0.05 vs NC, #P < 0.05,  mean ± s.e.m, n = 3 independent RNA samples for each group. b, Effects of miR-9 on protein levels of endogenous Navβ2 in primary cultured neonatal rat neurons (NRNs), using western blot analysis. Cells were transfected with miR-9, AMO-9, miR-9 + AMO-9, or NC. mean ± s.e.m from 3 batches of cells for each group. *P < 0.05, **P < 0.01 vs NC; ##P < 0.01 vs miR-9. c, Effects of miR-9 on SCN2B in primary cultured neonatal rat neurons (NRNs) using qRT-PCR analysis. Cells were transfected with miR-9, AMO-9, miR-9 + AMO-9, or NC. mean ± s.e.m from 3 batches of cells for each group. d, Representative confocal microscope images showing primary culture hippocampus neuron stained for Tubulin (green, upper), Navβ2 (red, middle). A merged image depicting double positivity (yellow) is shown on the bottom after transfection with miR-9 mimics or/and AMO-miR-9, negative control, n = 3. e, Repression of Navβ2 by miR-9 using the miRNA-masking antisense oligodeoxynucleotides (ODNs) techniques in NRNs determined by Western blot analysis, n = 3 batches of cells for each group, mean ± s.e.m, **P < 0.01 vs NC; ##P < 0.01 vs miR-9, ODN1 (oligodeoxynucleotides, which masks the binding sites of miR-9, located in the position 336–358 of SCN2B CDS region); ODN-2 (oligodeoxynucleotides, which masks the binding sites of miR-9, located in the position of 575–597 of SCN2B CDS region)
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Fig4: MiR-9 downregulates the expression of Navβ2 proteins. a, Verification of uptake of miR-9 by NRNs after transfection, *P < 0.05 vs NC, #P < 0.05,  mean ± s.e.m, n = 3 independent RNA samples for each group. b, Effects of miR-9 on protein levels of endogenous Navβ2 in primary cultured neonatal rat neurons (NRNs), using western blot analysis. Cells were transfected with miR-9, AMO-9, miR-9 + AMO-9, or NC. mean ± s.e.m from 3 batches of cells for each group. *P < 0.05, **P < 0.01 vs NC; ##P < 0.01 vs miR-9. c, Effects of miR-9 on SCN2B in primary cultured neonatal rat neurons (NRNs) using qRT-PCR analysis. Cells were transfected with miR-9, AMO-9, miR-9 + AMO-9, or NC. mean ± s.e.m from 3 batches of cells for each group. d, Representative confocal microscope images showing primary culture hippocampus neuron stained for Tubulin (green, upper), Navβ2 (red, middle). A merged image depicting double positivity (yellow) is shown on the bottom after transfection with miR-9 mimics or/and AMO-miR-9, negative control, n = 3. e, Repression of Navβ2 by miR-9 using the miRNA-masking antisense oligodeoxynucleotides (ODNs) techniques in NRNs determined by Western blot analysis, n = 3 batches of cells for each group, mean ± s.e.m, **P < 0.01 vs NC; ##P < 0.01 vs miR-9, ODN1 (oligodeoxynucleotides, which masks the binding sites of miR-9, located in the position 336–358 of SCN2B CDS region); ODN-2 (oligodeoxynucleotides, which masks the binding sites of miR-9, located in the position of 575–597 of SCN2B CDS region)

Mentions: To observe the influence of miR-9 on the protein translation of SCN2B, we analyzed the protein levels of Navβ2 in primary cultured neonatal rat neurons (NRNs) co-transfected with miR-9 mimics. The successful transfection of miR-9 was identified (Fig. 4a) by qRT-PCR and Navβ2 protein levels were significantly decreased in the presence of miR-9 (Fig. 4b). AMO-9 rescued the downregulation of Navβ2 elicited by overexpression of miR-9 and scrambled negative control of microRNA failed to affect the protein levels, suggesting that miR-9 predominantly suppresses SCN2B translation (Fig. 4b,d). However, the mRNA level of Navβ2 was not changed in the presence of miR-9 (Fig. 4c), which may ascribed to transcriptional regulation. To verify the transcriptional mechanism of miR-9 on Navβ2, the target protector technique was applied and the results showed that the SCN2B target protector (oligodeoxynucleotides (ODNs)-miR) of miR-9 (the position of 336–358 of SCN2B CDS) attenuated the reduction in Navβ2 levels induced by miR-9 (Fig. 4e, P < 0.01), implying that the SCN2B shows a great potential as the target for miR-9.Fig. 4


MicroRNA-9 induces defective trafficking of Nav1.1 and Nav1.2 by targeting Navβ2 protein coding region in rat with chronic brain hypoperfusion.

Sun LH, Yan ML, Hu XL, Peng LW, Che H, Bao YN, Guo F, Liu T, Chen X, Zhang R, Ban T, Wang N, Liu HL, Hou X, Ai J - Mol Neurodegener (2015)

MiR-9 downregulates the expression of Navβ2 proteins. a, Verification of uptake of miR-9 by NRNs after transfection, *P < 0.05 vs NC, #P < 0.05,  mean ± s.e.m, n = 3 independent RNA samples for each group. b, Effects of miR-9 on protein levels of endogenous Navβ2 in primary cultured neonatal rat neurons (NRNs), using western blot analysis. Cells were transfected with miR-9, AMO-9, miR-9 + AMO-9, or NC. mean ± s.e.m from 3 batches of cells for each group. *P < 0.05, **P < 0.01 vs NC; ##P < 0.01 vs miR-9. c, Effects of miR-9 on SCN2B in primary cultured neonatal rat neurons (NRNs) using qRT-PCR analysis. Cells were transfected with miR-9, AMO-9, miR-9 + AMO-9, or NC. mean ± s.e.m from 3 batches of cells for each group. d, Representative confocal microscope images showing primary culture hippocampus neuron stained for Tubulin (green, upper), Navβ2 (red, middle). A merged image depicting double positivity (yellow) is shown on the bottom after transfection with miR-9 mimics or/and AMO-miR-9, negative control, n = 3. e, Repression of Navβ2 by miR-9 using the miRNA-masking antisense oligodeoxynucleotides (ODNs) techniques in NRNs determined by Western blot analysis, n = 3 batches of cells for each group, mean ± s.e.m, **P < 0.01 vs NC; ##P < 0.01 vs miR-9, ODN1 (oligodeoxynucleotides, which masks the binding sites of miR-9, located in the position 336–358 of SCN2B CDS region); ODN-2 (oligodeoxynucleotides, which masks the binding sites of miR-9, located in the position of 575–597 of SCN2B CDS region)
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Fig4: MiR-9 downregulates the expression of Navβ2 proteins. a, Verification of uptake of miR-9 by NRNs after transfection, *P < 0.05 vs NC, #P < 0.05,  mean ± s.e.m, n = 3 independent RNA samples for each group. b, Effects of miR-9 on protein levels of endogenous Navβ2 in primary cultured neonatal rat neurons (NRNs), using western blot analysis. Cells were transfected with miR-9, AMO-9, miR-9 + AMO-9, or NC. mean ± s.e.m from 3 batches of cells for each group. *P < 0.05, **P < 0.01 vs NC; ##P < 0.01 vs miR-9. c, Effects of miR-9 on SCN2B in primary cultured neonatal rat neurons (NRNs) using qRT-PCR analysis. Cells were transfected with miR-9, AMO-9, miR-9 + AMO-9, or NC. mean ± s.e.m from 3 batches of cells for each group. d, Representative confocal microscope images showing primary culture hippocampus neuron stained for Tubulin (green, upper), Navβ2 (red, middle). A merged image depicting double positivity (yellow) is shown on the bottom after transfection with miR-9 mimics or/and AMO-miR-9, negative control, n = 3. e, Repression of Navβ2 by miR-9 using the miRNA-masking antisense oligodeoxynucleotides (ODNs) techniques in NRNs determined by Western blot analysis, n = 3 batches of cells for each group, mean ± s.e.m, **P < 0.01 vs NC; ##P < 0.01 vs miR-9, ODN1 (oligodeoxynucleotides, which masks the binding sites of miR-9, located in the position 336–358 of SCN2B CDS region); ODN-2 (oligodeoxynucleotides, which masks the binding sites of miR-9, located in the position of 575–597 of SCN2B CDS region)
Mentions: To observe the influence of miR-9 on the protein translation of SCN2B, we analyzed the protein levels of Navβ2 in primary cultured neonatal rat neurons (NRNs) co-transfected with miR-9 mimics. The successful transfection of miR-9 was identified (Fig. 4a) by qRT-PCR and Navβ2 protein levels were significantly decreased in the presence of miR-9 (Fig. 4b). AMO-9 rescued the downregulation of Navβ2 elicited by overexpression of miR-9 and scrambled negative control of microRNA failed to affect the protein levels, suggesting that miR-9 predominantly suppresses SCN2B translation (Fig. 4b,d). However, the mRNA level of Navβ2 was not changed in the presence of miR-9 (Fig. 4c), which may ascribed to transcriptional regulation. To verify the transcriptional mechanism of miR-9 on Navβ2, the target protector technique was applied and the results showed that the SCN2B target protector (oligodeoxynucleotides (ODNs)-miR) of miR-9 (the position of 336–358 of SCN2B CDS) attenuated the reduction in Navβ2 levels induced by miR-9 (Fig. 4e, P < 0.01), implying that the SCN2B shows a great potential as the target for miR-9.Fig. 4

Bottom Line: Intriguingly, miR-9 suppressed, while AMO-miR-9 enhanced, the trafficking of Nav1.1/Nav1.2 from cytoplasm to cell membrane.Further study showed that overexpression of miR-9 inhibited the Navβ2 expression by targeting on its coding sequence (CDS) domain by dual luciferase assay.However, binding-site mutation or miR-masks failed to influence Navβ2 expression as well as Nav1.1/Nav1.2 trafficking process, indicating that Navβ2 is a potential target for miR-9.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Harbin Medical University, No.157 Baojian Road, Nangang District,Harbin, Heilongjiang Province, 15008, China. sunlihua0219@163.com.

ABSTRACT

Background: Previous studies have demonstrated that the trafficking defects of Nav1.1/Nav1.2 are involved in the dementia pathophysiology. However, the detailed mechanisms are not fully understood. Moreover, whether the impaired miRNAs regulation linked to dementia is a key player in sodium channel trafficking disturbance remains unclear. The cognitive impairment induced by chronic cerebral ischemia through chronic brain hypoperfusion (CBH) is likely reason to precede dementia. Therefore, our goal in the present study was to examine the role of microRNA-9 (miR-9) in regulating Nav1.1/Nav1.2 trafficking under CBH generated by bilateral common carotid artery occlusion (2VO).

Results: The impairment of Nav1.1/Nav1.2 trafficking and decreased expression of Navβ2 were found in the hippocampi and cortices of rats following CBH generated by bilateral 2VO. MiR-9 was increased in both the hippocampi and cortices of rats following CBH by qRT-PCR. Intriguingly, miR-9 suppressed, while AMO-miR-9 enhanced, the trafficking of Nav1.1/Nav1.2 from cytoplasm to cell membrane. Further study showed that overexpression of miR-9 inhibited the Navβ2 expression by targeting on its coding sequence (CDS) domain by dual luciferase assay. However, binding-site mutation or miR-masks failed to influence Navβ2 expression as well as Nav1.1/Nav1.2 trafficking process, indicating that Navβ2 is a potential target for miR-9. Lentivirus-mediated miR-9 overexpression also inhibited Navβ2 expression and elicited translocation deficits to cell membrane of Nav1.1/Nav1.2 in rats, whereas injection of lentivirus-mediated miR-9 knockdown could reverse the impaired trafficking of Nav1.1/Nav1.2 triggered by 2VO.

Conclusions: We conclude that miR-9 may play a key role in regulating the process of Nav1.1/Nav1.2 trafficking via targeting on Navβ2 protein in 2VO rats at post-transcriptional level, and inhibition of miR-9 may be a potentially valuable approach to prevent Nav1.1/Nav1.2 trafficking disturbance induced by CBH.

No MeSH data available.


Related in: MedlinePlus