Although large numbers of long noncoding RNAs (lncRNAs) expressed in the mammalian nervous system have been detected, their functions and mechanisms of regulation remain to be fully clarified. It has been reported that the lncRNA antisense transcript for β-secretase-1 (BACE1-AS) is elevated in Alzheimer’s disease (AD) and drives the rapid feed-forward regulation of β-secretase, suggesting that it is critical in AD development. In the present study, the senile plaque (SP) AD SH-SY5Y cell model was established using the synthetic amyloid β-protein (Aβ) 1–42
Long noncoding RNAs (lncRNAs), which are a type of noncoding RNA (ncRNA) varying in size from 200 bp to >100 kb, are transcribed by RNA polymerase II, and are often spliced and polyadenylated (
The AD SP cell model was generated as previously described (
Each group of SH-SY5Y cells was seeded at 2×103 cells per well in a 96-well plate until 85% confluent. MTT (Sigma-Aldrich) reagent (5 mg/ml) was added to the maintenance cell medium at different time-points and incubated at 37°C for an additional 4 h. The reaction was terminated with 150 μl dimethylsulfoxide (Sigma-Aldrich) per well, the cells were lysed for 15 min, and the plates were gently agitated for 5 min. The absorbance values were determined using an ELISA reader (Model 680; Bio-Rad, Hercules, CA, USA) at 490 nm.
Total RNA from each group was isolated with TRIzol reagent (Invitrogen Life Technologies), according to the manufacturer’s instructions. The RNA samples were treated with DNase I (Sigma-Aldrich), quantified, and reverse-transcribed into cDNA with the ReverTra Ace-α First Strand cDNA Synthesis kit [Toyobo (Shanghai) Biotech Co., Ltd., Shanghai, China]. qPCR was conducted using a RealPlex4 real-time PCR detection system from Eppendorf AG (Barkhausenweg, Hamburg, Germany), with SYBR-Green Real-time PCR Master mix [Toyobo (Shanghai) Biotech Co., Ltd.] as the detection dye. qPCR amplification was performed for >40 cycles with denaturation at 95°C for 15 sec and annealing at 57°C for 45 sec. Target cDNA was quantified with the Eppendorf BioSpectrometer (Eppendorf AG). A comparative threshold cycle (Ct) was used to determine gene expression relative to a control (calibrator), and steady-state mRNA levels are reported as an n-fold difference relative to the calibrator. For each sample, the marker gene Ct values were normalized using the following formula: ΔCt = Ct_genes − Ct_18S RNA. To determine relative expression levels, the following formula was used: ΔΔCt = ΔCt_samplegroups − ΔCt_controlgroup. The values used to plot the relative expression of the markers were calculated using the 2−ΔΔCt method. The mRNA levels were calibrated on the basis of levels of 18S rRNA. The cDNA of each gene was amplified with primers as previously described (
The cells were lysed using a 2X loading lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). The total amount of proteins from the cultured cells was subjected to 12% SDS-PAGE and transferred onto a hybrid polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). Following inhibition with 5% (w/v) non-fat dried milk in Tris-buffered saline with Tween-20 (TBST; Beyotime Institute of Biotechnology), the PVDF membranes were washed four times (15 min each) with TBST at room temperature and incubated with primary antibodies, including rabbit anti-human Ki67 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-human BACE1, Aβ1–40, Aβ1–42 and GAPDH antibodies (Cell Signaling Technology, Inc., Beverly, MA, USA). Following extensive washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin (Ig) G secondary antibody (1:1,000; Santa Cruz Biotechnology, Inc.) for 1 h. Following washing four times (15 min each) with TBST at room temperature, the immunoreactivity was visualized using an enhanced chemiluminescence kit from Perkin Elmer, Inc. (Norwalk, CT, USA).
The cultured cells were washed three times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min. Following inhibition, the cells were initially incubated with primary antibody overnight at 4°C, and then with fluorescein isothiocyanate- or Cy3-conjugated goat anti-rabbit IgG antibody (1:200; Sigma-Aldrich) and 5 μg/ml DAPI (Sigma-Aldrich) at room temperature for 30 min. Then, the cells were thoroughly washed with TBST and viewed through a fluorescence microscope (DMI3000; Leica, Allendale, NJ, USA).
The Aβ1–42 ELISA kit (Hermes Criterion Biotechnology, Vancouver, BC, Canada) was used according to the manufacturer’s instructions. Briefly, all the cells and supernatants were harvested and dissociated in 0.1 M Tris (pH 7.4) containing 1% Triton X-100 (Sigma-Aldrich) and 5 mM MgCl2 by sonication. The concentration of Aβ1–42 was measured and the data were normalized against the protein concentration and expressed as a nanogram of Aβ1–42 per milligram of total protein. All the samples were added to anti-Aβ1–42 antibody-precoated microtest wells and incubated for 60 min. Following washing three times, the HRP-conjugated detection antibodies were then added followed by the addition of the substrate solution. The absorbance was determined at a wavelength of 450 nm.
Northern blotting was performed as previously described (
As previously described (
An siRNA targeted lncRNA BACE1-AS expression plasmid was constructed as previously described (
Each experiment was performed as least three times and the data are expressed as the mean ± standard error. The differences were evaluated using Student’s t-test. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using the SPSS 10.0 statistical software package (SPSS, Inc., Chicago, IL, USA).
Firstly, the MTT assay was used to evaluate whether exogenous Aβ1–42 was able to suppress SH-SY5Y cell proliferation. Large aggregates of synthetic Aβ1–42 suppressed the proliferation of SH-SY5Y cells in a time-dependent manner (
The expression of the enzyme BACE1 in SH-SY5Y cells, which is closely associated with Aβ1–42 processing, was investigated prior to (day zero) and following (day six) Aβ1–42 treatment using qPCR, IF staining and western blot analysis. The qPCR analysis demonstrated that the expression of BACE1 mRNA in the Aβ1–42-treated group was significantly elevated compared with that in the two control groups on day six (Fig. ). However, no significant differences in the mRNA expression levels of BACE1 in the three groups at day zero were identified. Western blotting confirmed that the BACE1 protein was expressed at significantly higher levels in the Aβ1–42 treated group than in the WT and DMSO-treated control groups on day six (
The potential of siRNA silencing of lncRNA BACE1-AS expression to reduce the stability of BACE1 mRNA and to attenuate the ability of BACE1 to cleave APP was then investigated in SH-SY5Y cells. Northern blot analysis indicated that the lncRNA BACE1-AS hybridization signal was weaker in the siRNA-BACE1-AS-transfected cell group than that in the siRNA mock-transfected group (
The nature and functions of ncRNAs appear to be numerous and varied. A range of small ncRNAs, including siRNAs, microRNAs and piRNAs, have been implicated in a host of roles, including transcriptional regulation, control of chromatin structure, heterochromatin formation and proteomic status (
This study was supported by a grant from the National Natural Science Foundation of China (no. 81202811), Shanghai Municipal Health Bureau Fund (no. 20124320) and Project funded by the China Postdoctoral Science Foundation (no. 2014M550250) to Te Liu. In addition, this study was supported by the Budget Program of Shanghai Municipal Education Commission (no. 2011JW64) to Zhihua Yu.
Exogenous Aβ1–42 affected SH-SY5Y cell proliferation and gene expression. (A) MTT assays demonstrated that large aggregates of synthetic Aβ1–42 inhibited SH-SY5Y cell proliferation in a time-dependent manner (**P<0.01 and #P>0.05 vs. WT group; n=3). (B) Results of quantitative polymerase chain reaction analysis demonstrated that the mRNA expression of BACE1 and APP in the Aβ1–42 treatment group was markedly elevated, while the Ki67 expression in this group was markedly decreased compared with that in the other two groups on day six. However, no significant differences in the mRNA expression levels (normalized against 18S rRNA levels) of BACE1, APP and Ki67 were identified between the Aβ1–42-, the WT- and the DMSO- treated groups on day 0 (**P<0.01 and #P>0.05 vs. WT group; n=3). (C) Western blot analysis confirmed that the expression of the BACE1, Aβ1–42 and Aβ1–40 proteins was significantly increased in the Aβ1–42 treatment group, compared with the WT- and DMSO-treated groups, while the expression of Ki67 in this group was markedly decreased on day six. GAPDH was used as a loading control (**P<0.01, *P<0.05 and #P>0.05 vs. WT group; n=3). (D) Immunofluorescent staining confirmed that the expression of the BACE1, Aβ1–42 and Aβ1–40 proteins was significantly increased in the Aβ1–42-treated group on day six, while the expression of these proteins was not detected on day zero (original magnification, ×200). Aβ, amyloid β-protein; WT, untreated group; APP, amyloid precursor protein; BACE1, β-secretase-1; DMSO, dimethylsulfoxide.
Exogenous Aβ1–42 induced the expression of the BACE1 enzyme and lncRNA BACE1-AS. (A) Northern blotting demonstrated that the BACE1 mRNA hybridization signals were higher in Aβ1–42-treated cell extracts than those in the other two groups. Strong lncRNA BACE1-AS hybridization signals were also detected only in Aβ1–42-treated cells. (B) RNA duplex formation. RNase protection assays were conducted on BACE1 mRNA to evaluate RNA duplex formation. Northern blot analysis revealed that non-overlapping probe hybridization signals were weaker than overlapping probe hybridization signals in SH-SY5Y cells following treatment with RNase, indicating that the overlapping part of BACE1 mRNA and lncRNA BACE1-AS transcripts was protected from degradation. These observations confirm that BACE1 and BACE1-AS form RNA duplexes. Aβ, amyloid β-protein; BACE1, β-secretase-1; lncRNA, long noncoding RNAs; BACE1-AS, antisense transcript for β-secretase-1; DMSO, dimethyl sulfoxide; WT, untreated group.
Attenuation of the ability of BACE1 to cleave APP by siRNA suppression of the expression of BACE1-AS. (A) Northern blotting demonstrated that BACE1-AS hybridization signals were weaker in siRNA-BACE1-AS-transfected cells than in siRNA-mock transfected cells. Strong BACE1 hybridization signals were detected in siRNA-mock transfected cells; however, not in siRNA-BACE1-AS transfected cells. (B) Quantitative polymerase chain reaction analysis demonstrated that the mRNA expression of APP and BACE1, but not that of Ki67, was significantly lower in siRNA-BACE1-AS-transfected cells than in siRNA mock-transfected cells on day six of Aβ1–42 treatment. However, no significant difference in the mRNA expression levels of APP, BACE1 and Ki67 (normalized against 18S rRNA expression) was identified in the two groups on day zero (**P<0.01 and #P>0.05 vs. siRNA-mock transfected cells; n=3). APP, amyloid precursor protein; BACE1, β-secretase-1; BACE1-AS, antisense transcript for β-secretase-1; siRNA, small interfering RNA.
Inhibition of protein expression by siRNA suppression of BACE1-AS. (A) Western blot analysis revealed that the protein expression levels of BACE1, Aβ1–42 and Aβ1–40 were significantly decreased in siRNA-BACE1-AS transfected cells compared with siRNA-mock transfected cells. (**P<0.01 and #P>0.05 vs. the siRNA-mock transfected cells group; n=3). GAPDH was used as a loading control. (B) Immunofluorescent staining confirmed that the expression of BACE1, Aβ1–42 and Aβ1–40 proteins was significantly decreased in siRNA-BACE1-AS-transfected cells compared with that in siRNA mock-transfected cells. BACE1, β-secretase-1; BACE1-AS, antisense transcript for β-secretase-1; Aβ, amyloid β-protein; siRNA, small interfering RNA.