Long non-coding RNA PVT1/microRNA miR-3127-5p/NCK-associated protein 1-like axis participates in the pathogenesis of abdominal aortic aneurysm by regulating vascular smooth muscle cells

Figures & data

Table 1. The sequences of the primers in this study

Primer	Sequences
PVT1	Forward: 5′-GCCCCTTCTATGGGAATCACTA-3′
	Reverse: 5′-GGGGCAGAGATGAAATCGTAAT-3′
NCKAP1L	Forward: 5′-GTGACGGAGGCTGTTCTCTC-3′
	Reverse: 5′-TCTGAGAGTTTGCGTTGG-3′
CSF1R	Forward: 5′-GAAGGGCAGACAGAGTGTCC-3′
	Reverse: 5′-GGATTCCCTGACCATGCCAA-3′
miR-3127-5p	Forward: 5ʹ-CGGGCTTGTGGAATGGTAAGC-3’
	Reverse: 5ʹ-CTGTCAGCTTCCCATTCC-3’
GAPDH	Forward: 5ʹ-CGCTCTCTGCTCCTCCTGTTC-3’
	Reverse: 5ʹ-ATCCGTTGCTCCGACCTTCAC-3’
U6	Forward: 5ʹ-GCTTCGGCAGCACATATACTAAAAT-3’
	Reverse: 5ʹ-CGCTTCACGAATTTGCGTGTCAT-3’

Figure 1. lncRNA PVT1/miR-3127-5p/NCKAP1L axis might be associated with AAA. (a) 335 differentially expressed genes (DEGs) from GSE7084 were screened out with adj.P value<0.05. (b) STRING was used to enrich the key biological processes for upregulated DEGs. (c-d) The expression of NCKAP1L and CSF1R in our collected AAA samples. (e) The expression of lncRNA PVT1 in our collected AAA samples. (f) Pearson correlation coefficient between lncRNA PVT1expression and NCKAP1L expression. (g). Pearson correlation coefficient between lncRNA PVT1expression and CSF1R expression. (h) miR-3127-5p predicted by starBase could bind to NCKAP1L and PVT1

Figure 2. lncRNA PVT1 is upregulated in AAA tissues and targets to miR-3127-5p. (a) RT-qPCR analysis examined the miR-3127-5p expression in in AAA tissues. (b) Pearson correlation analysis of lncRNA PVT1 expression with miR-3127-5p expression. (c) A binding site of lncRNA PVT1 to miR-3127-5p was presented by using starBase. (d) Binding of lncRNA PVT1 to miR-3127-5p confirmed by dual-luciferase reporter assay, vs. control, *P < 0.05,**P < 0.001

Figure 3. Establishment of VSMCs injury model for AAA using H2O2. (a and b) CCK-8 and EdU assays were used to detect the cell proliferation in VSMCs treated with H2O2 or not. (c and d) Caspase-3 activity, TUNEL and flow cytometry assays were performed to measure cell apoptosis in VSMCs treated with H2O2 or not, (e) Western blot analysis of IL-1, caspase-1, pro-caspase-1, Bax and Bcl-2 in VSMCs treated with H2O2 or not, vs. control, *P < 0.05,**P < 0.001

Figure 4. miR-3127-5p inhibitor abrogates the effect of lncRNA PVT1 silence on proliferation and apoptosis of H2O2-treating VSMCs

(a and b) The lncRNA PVT1 and miR-3127-5p expression in H2O2-treating VSMCs by RT-qPCR. (c) H2O2-treating VSMCs was transfected with si-PVT1, si-NC, miR-3127-5p inhibitor, miR-3127-5p inhibitor-NC, and si-PVT1+ miR-3127-5p inhibitor. After 48 h, RT-qPCR were perfomred to detect the transfection efficiency. (c) The transfected H2O2-treating VSMCs underwent CCK8 assays to test the cell proliferation. (d) Brudu assays were conducted to examine the proliferation of transfected H2O2-treating VSMCs. (e) Evaluation of caspase-3 activity in transfected H2O2-treating VSMCs. (f) TUNEL and flow cytometry assays were performed to measure cell apoptosis of VSMCs. (g) Western blot analysis of IL-1, caspase-1, pro-caspase-1, Bax and Bcl-2 in VSMCs. P < 0.05,** P < 0.001, vs.Si-NC; # P < 0.05, ## P < 0.001, vs.inhibitor-NC; $ P < 0.05, $$ P < 0.001, vs.si-PVT1+ inhibitor.

Figure 5. Identification of NCKAP1L as an effector of lncRNA PVT1/miR-3127-5p ceRNA activity

(a) Pearson correlation coefficient between miR-3127-5p expression and NCKAP1L expression. (b) Binding sequence of miR-3127-5p in NCKAP1L 3′ UTR predicted by starBase. (c) Normalized luciferase activity of H2O2-treating VSMCs nucleofected with the indicated miRNA mimics and 3′UT NCKAP1Lluciferase reporter vectors. (d) H2O2-treating VSMCs were transfected si-PVT1, si-NC, miR-3127-5p inhibitor, miR-3127-5p inhibitor NC, si-PVT1+ miR-3127-5p inhibitor. After 48 h, RT-qPCR were performed to examine NCKAP1LmRNA level. (e) Western blots were conducted to detect the NCKAP1Lprotein expression in cells (d) P < 0.05,** P < 0.001, vs.Si-NC; # P < 0.05, ## P < 0.001, vs.inhibitor-NC; $ P < 0.05, $$ P < 0.001, vs.si-PVT1+ inhibitor.

Figure 6. NCKAP1L silence recuses the effect of miR-3127-5p inhibitor on proliferation and apoptosis of H2O2-treating VSMCs

(a) RT-qPCR analysis of NCKAP1L expression in H2O2-treating VSMCs. (b) si-NC, si-NCKAP1L, miR-3127-5p inhibitor, miR-3127-5p inhibitor NC, miR-3127-5p inhibitor+si-NCKAP1L were introduced into H2O2-treating VSMCs for 48 h. The RT-qPCR was conducted to analyze the miR-3127-5p and NCKAP1L expression. (c and d). CCK-8 and EdU assays were used to detect the cell proliferation ability of VSMCs in different groups. (e and f). Caspase-3 activity, TUNEL and flow cytometry assays were performed to measure cell apoptosis of VSMCs in in different groups. (g) Western blot analysis of IL-1, caspase-1, pro-caspase-1, Bax and Bcl-2 in VSMCs. * P < 0.05, ** P < 0.001, vs.Si-NC; # P < 0.05, ## P < 0.001, vs.inhibitor-NC; $P < 0.05, $$P < 0.001, vs.si-NCKAP1L+inhibitor.

Data availability statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request ([email protected]).