Interaction Between DHCR24 and hsa_circ_0015335 Facilitates Cognitive Impairment in Cerebral Small Vessel Disease Patients

Abstract

Aims: The study attempted to determine the underlying role and regulation mechanism of 3β-hydroxysterol-Δ24 reductase (DHCR24) in the pathophysiology of cerebral small vessel disease-associated cognitive impairment (CSVD-CI). An RNA high-throughput sequencing and independent verification were conducted to identify potential circRNAs becoming the upstream regulator.

Methods: RNA sequencing was performed in whole-blood samples in cohort 1 (10 CSVD-CI and 8 CSVD with cognitively normal [CSVD-CN] patients). The DHCR24 and candidate circRNAs were verified in an independent cohort 2 (45 CSVD-CI participants and 37 CSVD-CN ones). The study also analyzed comprehensive cognitive assessments, plasma molecular index, and brain structure imaging.

Results: The expression of DHCR24 and has_circ_0015335 in whole-blood samples of CSVD-CI patients was significantly reduced compared to CSVD-CN patients in RNA sequencing and independent verification. Furthermore, the levels of DHCR24 and has_circ_0015335 were significantly related to global cognitive impairment in CSVD-CI patients. Meanwhile, DHCR24 could regulate the correlation between has_circ_0015335 expression and alterations in brain cortex in surface area, thickness, and volume in CSVD-CI patients. Additionally, hsa_circ_0015335 interacted with DHCR24 for plasma 24(S)-hydroxycholesterol levels among CSVD-CI patients.

Conclusion: Interaction between DHCR24 and hsa_circ_0015335 cognitively impaired CSVD by affecting brain cholesterol metabolism and brain structural changes.

Keywords: brain magnetic resonance imaging; cerebral small vessel disease; cholesterol metabolism; circRNA; cognitive impairment; mRNA.

FIGURE 1

Discovery and validation of differentially expressed DHCR24 and candidate circRNAs in cohort 1. (A) Cluster heat map shows in cohort 1 (8 CSVD‐CN patients and 10 CSVD‐CI patients), 681 differentially expressed mRNAs with a |log₂ (fold change)| > 0.58 and p < 0.05 and an effective test value in any one sample. The bold mRNA is DHCR24. (B) Volcano plot of 681 differentially expressed mRNAs. Horizontal coordinates represent mRNAs with log₂ (fold change) (CSVD‐CN/CSVD‐CI) > 0.58 or < −0.58. The vertical axis displays −log₁₀ (p‐value) > 1.30 (mean p < 0.05) of differentially expressed mRNAs. Each point represents an individual mRNA. DHCR24 is regulated mRNA in cohort 1. (C) RT‐qPCR was performed to verify the expression of DHCR24 in cohort 1 (8 CSVD‐CN patients and 10 CSVD‐CI patients). Each sample was tested in triplicate. Independent‐sample t‐test was used for data analysis and all data represents means ± standard deviation. (D) CircRNA‐associated DHCR24 mRNA networks. (E) Volcano plot of 897 differentially expressed circRNAs. Horizontal coordinates represent circRNAs with |log₂ (fold change)| > 0.58 (CSVD‐CN/CSVD‐CI). The vertical axis displays −log₁₀ (p‐value) > 1.30 (mean p < 0.05) of differentially expressed circRNAs. Each point represents an individual circRNA. Nine candidate circRNAs were showed in this figure. (F) RT‐qPCR was performed to verify the expression of nine candidate circRNAs in cohort 1 (8 CSVD‐CN patients and 10 CSVD‐CI patients). Each sample was tested in triplicate. Independent‐sample t‐test was used for data analysis and all data represents means ± standard deviation. CSVD‐CI, cerebral small vessel disease‐cognitive impairment; CSVD‐CN, cerebral small vessel disease‐cognitively normal; RT‐qPCR, real‐time quantitative polymerase chain reaction.

FIGURE 2

Replication and the clinical application of DHCR24 and has_circ_0015335 in the independent replication cohort 2. (A, B) Expression levels of DHCR24 and has_circ_0015335 between CSVD‐CN and CSVD‐CI groups were determined by RT‐qPCR. (C) Correlations between DHCR24 and has_circ_0015335 expression levels in two groups. (D) ROC curves of DHCR24, has_circ_0015335, and the combination of these two indices. CSVD‐CI, cerebral small vessel disease‐cognitive impairment; CSVD‐CN, cerebral small vessel disease‐cognitively normal; RT‐qPCR, real‐time quantitative polymerase chain reaction; ROC, receiver operating characteristic.

FIGURE 3

Association analyses of DHCR24 and has_circ_0015335 levels with cognitive assessments and 24(S)‐OHC levels in CSVD‐CI patients. (A) Heatmap of correlation analyses of cognitive assessments with DHCR24 and has_circ_0015335 levels. *0.01 ≤ p < 0.05; **0.001 ≤ p < 0.01; ***p < 0.001. (B) Correlation analysis of 24(S)‐OHC levels. (C) Interaction analysis of DHCR24 and has_circ_0015335 for the 24(S)‐OHC levels. 24(S)‐OHC, 24(S)‐hydroxycholesterol; AVLT‐20 min DR, auditory verbal learning test‐20‐min delayed recall; AVLT‐IR, auditory verbal learning test‐immediate recall; CDT, Clock Drawing Test; CSVD‐CI, cerebral small vessel disease‐cognitive impairment; CSVD‐CN, cerebral small vessel disease‐cognitively normal; DST, Digit Span Test; MMSE, mini‐mental state examination; MoCA, montreal cognitive assessment; Stroop‐A, Stroop Color and Word Test A; Stroop‐B, Stroop Color and Word Test B; Stroop‐C, Stroop Color and Word Test C; SVD, small‐vessel disease; TMT‐A, Trail Making Test A; TMT‐B, Trail Making Test B.

FIGURE 4

Association analyses of DHCR24 and has_circ_0015335 levels with brain MRI features in CSVD‐CI patients. (A–C) Significant difference of cortical surface area (mm²), cortical thickness (mm), and gray matter volume (mm³) in brain between CSVD‐CN and CSVD‐CI patients (p < 0.05, correction based on Monte Carlo Simulation). (D) Heatmap of correlation analyses of brain MRI features with DHCR24 and has_circ_0015335 levels. *0.01 ≤ p < 0.05; **0.001 ≤ p < 0.01; ***p < 0.001. (E) The mediation effects of DHCR24 levels on has_circ_0015335 and brain structure changes. “a,” “b,” and “c′” represent the direct effect between the two variables, and “c” represents the total effect between the two variables. CSVD‐CI, cerebral small vessel disease‐cognitive impairment; MRI, magnetic resonance imaging.