miR-3940-5p reduces amyloid β production via selectively targeting PSEN1

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid beta (Aβ) in brain. Mounting evidence has revealed critical roles of microRNAs (miRNAs) in AD pathogenesis; however, the miRNAs directly targeting presenilin1 (PSEN1), which encodes the catalytic core subunit of γ-secretase that limits the production of Aβ from amyloid precursor protein (APP), are extremely understudied. The present study aimed to identify miRNAs targeting PSEN1 and its effect on Aβ production. This study first predicted 5 candidate miRNAs that may target PSEN1,through websites such as TargetScan, miRDB, and miRwalk. Subsequently, the targeting specificity of the candidate miRNAs towards PS1 was validated using dual-luciferase reporter assays. To investigate the regulatory effect of miR-3940-5p on gene expression based on its targeting of PS1, miR-3940-5p mimics or inhibitors were transiently transfected into SH-SY5Y cells. Changes in PSEN1 transcription and translation in the tested cells were detected using RT-qPCR and Western Blot, respectively. Finally, to explore whether miR-3940-5p affects Aβ production, SH-SY5Y APPswe cells overexpressing the Swedish mutant type of APP were transiently transfected with miR-3940-5p mimics, and the expression level of Aβ was detected using ELISA. The results are as follows: The dual-luciferase reporter assays validated the targeting specificity of miR-3940-5p for PSEN1. Overexpression of miR-3940-5p significantly reduced the mRNA and protein levels of PSEN1 in SH-SY5Y cells. Conversely, inhibition of miR-3940-5p led to an increase in PSEN1 mRNA levels. Transfection of miR-3940-5p mimics into SH-SY5Y-APPswe cells resulted in a significant reduction in Aβ42 and Aβ40. Lentiviral-mediated overexpression of miR-3940-5p significantly decreased the expression of PSEN1 and did not significantly affect the expression of other predicted target genes. Furthermore, stable overexpression of miR-3940-5p in SH-SY5Y-APPswe cells mediated by lentivirus significantly reduced the expression of PSEN1 and the production of Aβ42 and Aβ40. Therefore, our study demonstrates for the first time the functional importance of miR-3940-5p in antagonizing Aβ production through specific and direct targeting of PSEN1.


Introduction
Alzheimer disease (AD) is a progressive neurodegenerative disorder manifested as cognitive, mood, language, and memory impairments.As the primary cause of dementia in the elderly, AD has multiple contributing factors, with Aβ being widely acknowledged as one of the foremost factors influencing the onset and progression of AD (Hardy and Selkoe, 2002).Qi et al. 10.3389/fnagi.2024.1346978Frontiers in Aging Neuroscience 02 frontiersin.org Aβ is a series of peptide fragments formed after the cleavage of amyloid precursor protein (APP) by βand γ-secretase enzymes.The excessive aggregation of Aβ into amyloid plaques disrupt the structural integrity of dendritic spines and synapses, resulting in a cascade of deleterious effects that ultimately lead to neuronal death (Ingelsson et al., 2004).Additionally, Aβ oligomers have the potential to inflict direct damage upon the synapses and neurites of brain neurons, along with the activation of microglia and astrocytes (Selkoe and Hardy, 2016).Clinical trials utilizing monoclonal antibody (Donanemab) to selectively remove Aβ have demonstrated significant reductions in cognitive and functional decline in patients of early-stage AD (Mintun et al., 2021).Crucially, recent clinical trials involving three distinct Aβ antibodies-solanezumab, crenezumab, and aducanumab-indicate a potential deceleration in cognitive decline, as observed in post hoc analyses of individuals with mild AD (Selkoe and Hardy, 2016), confirming the critical role of Aβ in AD pathogenesis.Therefore, identifying molecular targets that specifically inhibit or modulate Aβ production has become a research focus in AD.
Since γ-secretase is a rate-limiting enzyme in Aβ biogenesis, studying how PSEN1 is regulated by miRNAs may provide a promising therapeutic approach for AD.To this end, this study aimed to investigate the regulatory role of miRNAs on the expression of PSEN1.
We utilized bioinformatics tools such as Target Scan and experimental techniques including dual-luciferase reporter analysis, transient and stable transfection, RT-qPCR, western blot, and ELISA to identify candidate miRNAs that potentially target PSEN1.We confirmed that miR-3940-5p directly targets the 3'UTR of PSEN1 and reduces its translation level in SH-SY5Y cells.Upon miR-3940-5p overexpression, the generation of Aβ 42 and Aβ 40 in SH-SY5Y-APP swe cells was significantly decreased.Our study highlights the regulatory role of miRNAs in AD pathogenesis and identifies miR-3940-5p as a potential therapeutic target for AD.

Cell culture
In this study, we used four cell lines, namely HEK 293, HEK 293 T, SH-SY5Y, and SH-SY5Y-APP swe .SH-SY5Y-APP swe was genetically modified to overexpress APP swe in SH-SY5Y.These cells were obtained from Kunming Institute of Zoology of the Chinese Academy of Sciences (HEK 293, or Kunming Medical University (SH-SY5Y-APP swe ), were cultured in DMEM supplemented with 10% fetal bovine serum (Gbico, Waltham, MA, USA).Prior to experimentation, the cells were harvested using trypsinization and counted using the trypan blue exclusion method when they reached approximately 70% confluency.For the experiments, 150,000 cells were seeded into each well of a 24-well plate (Corning, New York, USA), while 50,000 cells were seeded into each well of a 96-well plate (Corning, New York, USA).

Target prediction
To predict miRNAs targeting the PS1 3'UTR, this study employed three prediction websites: TargetScan (Agarwal et al., 2015), miRDB (Wong and Wang, 2015), and miRwalk (Dweep et al., 2011).TargetScan, a miRNA target gene prediction database, yielded results for various mammalian miRNA target genes.It relies on the principle of sequence complementarity to identify conservative 8-mer, 7-mer, or 6-mer sites (seed match sequences) within the target gene's 3'UTR.Further refinement, based on thermodynamic stability, was applied to select miRNAs targeting the gene of interest, with a preference for those with a context score percentile exceeding 90 for subsequent validation.miRDB, a database for predicting mammalian miRNA target genes, assigned higher credibility to results with scores above 80, considered relatively reliable.miRWalk, a comprehensive miRNA target gene database, encompassed information for multiple species, including Human, Mouse, Rat, Dog, Cow, etc.It employed four different algorithms (RNA22, miRanda, miRWalk, and TargetScan) to predict gene-targeting miRNAs and integrated this information with predictions from 12 existing miRNA target prediction programs.To reduce false positives, predictions from each of the three databases regarding miRNAs targeting the PS1 3'UTR were consolidated.The Whitehead BaRC public tools website was then used to obtain their intersection.Ultimately, five miRNAs with high scores and no prior literature reports were selected for further validation: miR-9-5p, miR-302a-3p, miR-520c-3p, miR-4507-5p, and miR-3940-5p. 10.3389/fnagi.2024.1346978Frontiers in Aging Neuroscience 03 frontiersin.org

Dual luciferase report analysis
HEK 293 cells were seeded onto a 96-well plate at a density of approximately 50,000 cells per well, 1 day prior to transfection.For the dual-luciferase reporter assay, 800 ng of the psiCHECK2 plasmid containing either wild-type or mutated PSEN1 3′UTR was independently co-transfected with 100 nM miR-3940-5p mimics (Ribobio, Guangzhou, China) using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA).After 48 h of incubation, the cells were lysed and subjected to the reporter assay (Promega, Wisconsin, USA).

Transient transfection
For SH-SY5Y cells transfections, ~150,000 cells/well were seeded onto a 24-well plate.The following day, Lipofectamine 2000 (5 μL) were made with 100 nM miRNA mimics, inhibitor, negative control or 100 nM siRNA (PSEN1 siRNA, general biol, Anhui, China) (Guo et al., 2023) in Opti-MEM media (Gbico, Waltham, MA, USA) complexes were allowed to form for 20 min before being added to cells.Then 500 μL of media (no antibiotics) was added to each well.Cells were incubated 48 h (RT-qPCR) or 72 h (western blot) until lysis.

Lentivirus packaging and stable cell line generation
To package the slow virus, the virus genome is cloned into a specialized vector containing all the essential elements for viral replication and packaging.Specifically, a chemical synthesis method is used to clone the fragment of the precursor structure of miR-3940-5p (miR-3940-5p-pre), which encodes it, into the slow virus vector (pCDH-CMV-MCS-EF1-copGFP-T2A-Puro vector).The precursor sequence that encodes miR-3940-5p (miR-3940-5p-pre): 5'TCTAGACGAATAGCTCCTTTTCTAGCTCCACCCAACCCCGC CCGAGACCCCTAAACCAGAGTGTCGGGCCTAGGGTCGGGTG AATGGAACCAATGAGAGGAAGGAAGATTTTTG GATCC3' .The vector is then transfected into a packaging cell line that contains a helper virus.The helper virus provides the proteins and enzymes required for the slow virus to replicate and package its genome.The slow virus genome is integrated into the host cell genome through a process known as transduction.Subsequently, the packaging cell line is infected with the helper virus, which initiates the production of slow virus particles.The slow virus particles are then released from the packaging cell line and can infect target cells.
To overexpress miR-3940-5p, SH-SY5Y cells were seeded in triplicate in a six-well plate in the following order: blank group, overexpressing miR-3940-5p group, and negative control group.When the cells reached about 70% confluency, a mixture of complete culture medium, corresponding virus solution, and polybrene (10 mg/ mL mother liquor, 10 μg/mL final concentration) (Beyotime, Shanghai, China) was added to the six-well plate, replacing the original culture medium.The volume of each component was 1 mL, 1 mL, and 2 μL, respectively.Normal culture medium was substituted 24 h later.After 48 h, the culture medium was replaced with medium containing puromycin (Beyotime, Shanghai, China), and the concentration was adjusted based on the screening outcomes (Final concentration of 1 μg/mL).Following 3-4 days of puromycin drug treatment, wild-type SH-SY5Y cells perished, and cells stably overexpressing miR-3940-5p were obtained and denoted as SH-SY5Y-miR-3940-5p-pre cells.We used the same procedures to obtain SH-SY5Y-APP swe cells stably overexpressing miR-3940-5p, which were denoted as SH-SY5Y-APP swe -miR-3940-5p-pre cells.

Western blotting
After transfection miR-3940-5p mimic for 5 h, the cells were exchanged with normal culture medium and incubated for an additional 72 h.In addition, during the logarithmic growth phase, total protein was extracted from SH-SY5Y cell lines overexpressing miR-3940-5p, western blot technique can be used to detect protein expression levels, which involves protein extraction, protein concentration measurement, protein electrophoresis, protein transfer, blocking, primary antibody incubation (Anti-PSEN1, 1: 6000, Abcam, Cambridge, UK, Anti-GAPDH, 1: 2000, Beyotime, Shanghai, China), washing, secondary antibody (Abcam, Cambridge, UK) incubation, detection, and data analysis using software such as Image J.

Enzyme labeled immunosorbent assay (ELISA)
The levels of both soluble peptides were measured in the conditioned media of transfected cells using ELISA kits (Jiangsu Enzyme Label Biotechnology, Jiangsu, China) for Aβ 40 and Aβ 42 .A standard curve was generated according to the protocol to obtain the concentration of each Aβ peptide (pg/μg).In this context, "pg" represents the amount of Aβ measured in each well, while "μg" represents the total protein content in each well.

Statistical approach
The data are presented as mean ± standard error.The comparison of means between two groups was performed using a t-test.Statistical analysis was conducted using SPSS 20.0 software, and graphical representations were created using GraphPad Prism 5.
To verify this, the seed sequence of miR-3940-5p in the PSEN1 3'UTR was mutated and constructed into the reporter vector.No changes in luciferase activity were seen when the constructs contained a mutated miR-4507-5p seed region (Figure 1C), indicating that the targeting effect of miR-3940-5p on the mutated PSEN1 was lost.Overall, these findings confirmed that miR-3940-5p directly targets PSEN1.

miR-3940-5p inhibits the PSEN1 transcription and translation in SH-SY5Y cells
To investigate the role of miR-3940-5p on PSEN1 further, we used the chemically synthesized miR-3940-5p mimics and inhibitors to overexpress and down express miR-3940-5p in SH-SY5Y cells.48 h after miR-3940-5p mimics transfection, the RT-qPCR analysis (Figure 2A) revealed that the expression of miR-3940-5p is increased by approximately 2000 times compared to the control group, indicating the successful overexpression of miR-3940-5p in the cells.Conversely, 48 h after miR-3940-5p inhibitor transfection, the expression of miR-3940-5p is decreased by 30% (Figure 2A).These data confirmed that miR-3940-5p mimics and inhibitors are functional in SH-SY5Y cells.
Next, miR-3940-5p mimics and inhibitors were transfected into SH-SY5Y cells and the expression levels of PSEN1 mRNA and protein were analyzed.RT-qPCR results (Figure 2B) revealed a significant decrease in PSEN1 mRNA levels in the miR-3940-5p group compared to the control group (p < 0.01).Western blot results (Figures 2C, D) showed that PSEN1 protein expression was significantly inhibited in the miR-3940-5p mimics group compared to the control group, which was consistent with the observed decrease in PSEN1 mRNA expression.When miR-3940-5p inhibitor was transfected into SH-SY5Y cells, the mRNA (p < 0.05; Figure 2B) and protein (p = 0.051; Figure 2C) levels of PSEN1 were significantly increase, although the increase in protein was marginally significant.Altogether, these results FIGURE 1 miR-3940-5p specifically targets the 3′UTR of PSEN1.(A) Schematic representation of the experimental design.The wild-type (WT) PSEN1 3′UTR was cloned downstream to the renilla luciferase gene in psiCheck 2 vector to generate the reporter constructs.The HEK293 cells were transfected with the luciferase reporter constructs and with candidate miRNA (miR-9-5p, miR-302a-3p, miR-520c-3p, miR-3940-5p, or miR-4507-5p) or negative controls (NC).After 48 h, dual luciferase assay (B) was performed.As shown in (A), the PSEN1 3′UTR with mutated miR-3940-5p target site (MUT) was constructed into psiCheck 2 vector.Then, HEK293 cells were transfected with constructs containing WT or MUT 3′UTR and miR-3940-5p or NC.After 48 h, dual luciferase assay (C) was performed (n = 3, t test, data represent mean ± SEM., **p < 0.01).

PSEN1 is down-expressed in cells stably overexpressing miR-3940-5p
To further investigate the regulatory role of miR-3940-5p on PSEN1, we constructed a lentiviral vector by inserting the 119 bp sequence encoding the precursor structure of miR-3940-5p.This was achieved through xbaI/BamH I double enzyme digestion.The lentiviral plasmid carrying the correct miR-3940-5p sequence was then packaged into lentivirus and used to overexpress miR-3940-5p in SH-SY5Y cells.In addition to the miR-3940-5p sequence, the lentiviral vector also contained a green fluorescent protein gene (GFP).These features allowed us to select miR-3940-5p-overexpressing SH-SY5Y cells using puromycin and visualize them under a fluorescence microscope.
Following puromycin selection, SH-SY5Y cells with miR-3940-5p overexpression (termed as SH-SY5Y-miR-3940-5p-pre) displayed green fluorescence when observed under a fluorescence microscope (Figure 3A).We detected the expression level of miR-3940-5p in SH-SY5Y-miR-3940-5p-pre cells using RT-qPCR.Compared to the LV-miR-NC group, the expression of miR-3940-5p in the LV-miR-3940-5p-pre group was increased by 20%, confirming a successful overexpression of miR-3940-5p (Figure 3B).The level of miR-3940-5p in SH-SY5Y-miR-3940-5p-pre cells was not very high, probably due to the complexity of the transcriptional regulation of miR-3940-5p precursor that ultimately leads to the formation of both mature miR-3940-5p and miR-3940-3p.Subsequently, both RNA and protein were extracted from SH-SY5Y-miR-3940-5p-pre cells.The mRNA level of PSEN1 was measured using RT-qPCR, and the results indicated that the overexpression of miR-3940-5p considerably reduced the expression of PSEN1 (Figure 3C).In addition, the protein level of PSEN1 was analyzed through western blot, and the data showed that the overexpression of miR-3940-5p significantly reduced PSEN1 expression (Figures 3D, E).These results demonstrated that the mRNA and protein expression of PSEN1 were significantly declined in SH-SY5Y-miR-3940-5p-pre cells.
To validate the specificity of miR-3940-5p targeting PSEN1 in SH-SY5Y cells, we employed miRNA target prediction websites, including Target Scan, miRDB, and miRwalk, to screen for the potential miR-3940-5p targets.The top five genes targeted by miR-3940-5p with high prediction scores were TENM3, CPNE8, ATXN1, CUL7, and KCNA5.RT-qPCR was carried out to assess the expression levels of these five genes in SH-SY5Y-miR-3940-5p-pre cells.The results (Figure 3F) showed that, compared to the control group, the expression level of KCNA5 was significantly upregulated in SH-SY5Y-APP-miR-3940-5p cells, while the expression levels of other three genes remained unchanged (Figure 3F).Thus, miR-3940-5p does not target these 5 genes, to some extent, had specificity toward PSEN1.

miR-3940-5p inhibits Aβ production In SH-SY5Y-APP swe cells
PSEN1 is a crucial component of the γ-secretase complex, which plays a rate-limiting role in regulating Aβ production.Considering this, inhibiting PSEN1 expression may represent a potential therapeutic strategy for AD by reducing brain Aβ levels.To explore the impact of miR-3940-5p on Aβ production, SH-SY5Y-APP swe cells were utilized as a research model.This cell line is an AD cell model that can produce neurotoxic Aβ.RT-qPCR (Figure 4A) was employed to evaluate the expression of APP in this cell line and we found the expression of APP in SH-SY5Y-APP swe cells was 120 times higher than that in SH-SY5Y cells.Moreover, ELISA was used to quantify the expression level of Aβ in both SH-SY5Y and SH-SY5Y-APP swe cells.The data (Figure 4B) depicts the results of Aβ 40 and Aβ 42 content analysis in SH-SY5Y and SH-SY5Y-APP swe cells, which demonstrates that the Aβ 40 content in SH-SY5Y-APP swe cells is significantly higher  Frontiers in Aging 07 frontiersin.orgcompared to SH-SY5Y cells.Although the Aβ 42 content is also higher in SH-SY5Y-APP swe cells, the difference is not statistically significant.These results confirmed that SH-SY5Y-APP swe cells have a high expression of APP and Aβ.
To explore the downstream impact of miR-3940-5p on Aβ production, miR-3940-5p mimics were transiently transfected into SH-SY5Y-APP swe cells and ELISA was utilized to assess the expression levels of Aβ 40 and Aβ 42 thereafter.Meanwhile, a specific small interference RNA to PSEN1 (Guo et al., 2023) was used as a control.The results (Figure 4C) revealed that both the miR-3940-5p mimics and PSEN1-siRNA could significantly suppress the production of Aβ 40 and Aβ 42 in SH-SY5-APP swe cells.Since the increase in the relative ratio of Aβ 42 to Aβ 40 (Aβ 42 /Aβ 40 ) is a common result of familial AD-associated PSEN1 mutations (Sun et al., 2017) and plays an important role in AD pathogenesis (Kwak et al., 2020), we determined the effect of miR-3940-5p on the ratio of Aβ 42 /Aβ 40.The data showed that there is no significant difference in the ratio of Aβ 42 /Aβ 40 cross the miR-3940-5p mimics group, PSEN1-siRNA group, and the control group (Figure 4D).
Next, the lentiviruses system constructed above was used to stably overexpress miR-3940-5p in SH-SY5Y-APP swe cells.The success of miR-3940-5p overexpression was confirmed through fluorescence microscopy.The levels of Aβ 40 and Aβ 42 in SH-SY5Y-APP swe cells stably expressing miR-3940-5p-pre were analyzed using ELISA.The results (Figure 4E) showed a significant reduction in the levels of Aβ 40 and Aβ 42 in SH-SY5Y-APP swe -miR-3940-5p-pre cells compared to the control group.Regarding the ratio of Aβ 42 /Aβ 40 , there were no significant differences observed between the SH-SY5Y-APP swe -miR-3940-5p group and the control group (Figure 4F).These findings were consistent with the results obtained from transient miR-3940-5p mimics transfection.

Discussion
The pathogenesis of AD is multifaceted and not yet fully understood.Mounting evidence has highlighted the crucial role of miRNAs in regulating gene expression in the brain, where they constitute a significant regulatory system (Dehghani et al., 2018).Several studies have demonstrated that miRNAs regulate Aβ generation by targeting BACE1.For instance, miR-29c-3p (Cao et al., 2021), miR-195 (Zhu et al., 2012), miR-186 (Kim et al., 2016), miR-200a-3p (Wang et al., 2019).While miRNAs have been shown to play a crucial role in regulating β-secretase, miRNAs targeting γ-secretase are rarely reported.In current study, we provided sold evidence that miR-3940-5p is one miRNA specifically target PSEN1 and regulate the γ-secretase function of PSEN1.
We initially used bioinformatics screening to discover the potential miRNAs with high mRNA: miRNA binding affinity toward PSEN1.With five candidate miRNAs in hand, we used various experimental approaches to validate the computational predictions and demonstrate that the miR-3940-5p recognize specific binding sites in the 3'UTR of PSEN1 mRNA and suppress PSEN1 protein expression in SH-SY5Y cells.
Lentiviral-mediated overexpression of miRNAs is a principal method for studying miRNA functions as it has lower cell toxicity and allows for stable overexpression of the target miRNA in a short period of time (Herrera-Carrillo et al., 2009).Compared with the control cell line, the expression level of miR-3940-5p in the LV-miR-3940-5p-pre group was increased by 20%.The mild increase of miR-3940-5p in SH-SY5Y-miR-3940-5p cells is possibly due to the complexity of the miRNA transcription process when the exogenous encoding miR-3940-5p precursor sequence is inserted into the genome DNA, resulting in the formation of two mature miRNAs, miR-3940-5p and miR-3940-3p (O'Brien et al., 2021).Despite miR-3940-5p was mildly increased, the mRNA and protein levels of PSEN1 were significantly reduced, indicating the 20% increase of miR-3940-5p in SH-SY5Y-miR-3940-5p cells was functional.
In present study, transient and stable transfection methods were used to investigate the regulatory effects of miR-3940-5p on the production of Aβ Transfection of miR-3940-5p mimics and PSEN1-siRNA into SH-SY5Y-APP swe cells resulted in a decrease in both Aβ 40 and Aβ 42 levels.Notably, the stable overexpression of miR-3940-5p in SH-SY5Y-APP swe cells led to a decrease in both Aβ 40 and Aβ 42 levels, without altering the Aβ 42 /Aβ 40 ratio, which is consistent with the results of transient transfection.Notably, the stable overexpression of miR-3940-5p in SH-SY5Y-APP swe cells led to a decrease in both Aβ 40 and Aβ 42 levels, without altering the Aβ 42 /Aβ 40 ratio, which is consistent with the results of transient transfection.These findings demonstrate that, by suppressing the expression of PSEN1 and the consequent γ-secretase activity, miR-3940-5p is involved in reducing the production of Aβ.In future studies, it would be worthwhile to overexpress PSEN1 in SH-SY5Y cells and transfect them with miR-3940-5p to explore whether overexpression of PSEN1 can rescue the inhibitory effect of miR-3940-5p on Aβ levels.
RNA-targeted therapy, which regulates protein synthesis by designing antisense oligonucleotides (ASOs), is a promising approach for treating AD.ASOs based on miRNAs, including antagomiRs and miRNA mimics, have been shown to be potential targets.Several miRNAs based ASOs drugs have been tested in preclinical studies and demonstrated promising results (Grabowska-Pyrzewicz et al., 2021).By employing several in vitro functional experiments, we have shown that miR-3940-5p indirectly reduces the generation of Aβ by targeting PSEN1, providing a foundation for future in vivo experiments.Interestingly, one study has revealed that, by injecting a lentivirus-packaged miR-31 overexpression plasmid into the hippocampus of AD mice, the expression of App and Bace1 are declined, which finally reduces Aβ deposition and improves cognitive ability (Barros-Viegas et al., 2020).This study has inspired us to test whether overexpressing miR-3940-5p in AD mice would exert similar benefits.However, among the organisms present in the TargetScan database, including mouse, chimpanzee, and other nine species, no homologous miRNA for miR-3940-5p has been identified.This indicates that miR-3940-5p is evolutionarily non-conserved and is a human-specific miRNA.To substantiate the presumed human specificity of miR-3940-5p and its potential association with the onset and progression of Alzheimer's disease, a more rigorous inquiry is required.In subsequent investigations, we plan to meticulously compare and analyze the genomes of humans and other pertinent species (such as chimpanzees, mice, etc.) to conclusively establish whether this miRNA is uniquely present in humans.Following this, we will employ high-throughput sequencing technology to conduct an extensive miRNA expression profiling, comparing the expression patterns of miR-3940-5p between humans and other species.
Besides, in vivo validating the function of miR-3940-5p in currentavailable mouse model is challenging.The seed sequence of miR-3940-5p targeting to PSEN1 locates on the 3'UTR.In most transgenic AD mice based PSEN1, like APP/PSEN1 mice, only the coding sequence of mutated PSEN1 is transferred.Therefore, the human derived 3D brain organoids may be an ideal model to address this issue.Considering that the PSEN1 in brains of sporadic AD is upregulated (Borghi et al., 2010), it is worth to investigate whether miR-3940-5p in AD brains or serum is correspondingly downregulated.Upon confirmation of the human specificity of miR-3940-5p, we intend to delve into its origin and evolutionary trajectory in the human lineage.This endeavor is poised to enhance our comprehension of genetic disparities between humans and other species, shedding light on how these distinctions may impact biological functionalities and adaptability.
Previously, miR-3940-5p has found to target several genes.However, most of them were validated in cancer.For example, miR-3940-5p suppresses colorectal cancer metastasis by targeting integrin alpha 6 (Tao et al., 2021).miR-3940-5p suppresses the proliferation of non-small cell lung cancer cells by targeting cyclin D1 and ubiquitin specific peptidase-28 (Ren et al., 2017).In addition, miR-3940-5p is a hub miRNA upregulated in granulosa cells from patients with polycystic ovary syndrome and promotes granulosa cell proliferation by targeting KCNA5 (Gao et al., 2020).Coupled with our present study, these findings demonstrate that miR-3940-5p has a pleiotropic role in human health.However, the therapeutic use of miRNAs with a multitude of targets raises the concern of potential side effects.Given the intricate regulatory networks in which these miRNAs participate, targeting multiple genes may lead to unintended consequences, impacting cellular processes beyond the intended scope.Therefore, it is crucial to consider and discuss the possibility of side effects when exploring treatments based on miRNAs with diverse target profiles.
In conclusion, the present study shows, for the first time, that miR-3940-5p strongly suppresses the production of Aβ by specifically targeting PSEN1, the catalytic core subunit of γ-secretase.Our study is the first to link miR-3940-5p with AD and suggest miR-3940-5p could have potential to be a novel target in AD therapy.