LncRNA RASSF8-AS1 knockdown displayed antiproliferative and proapoptotic effects through miR-188-3p/ATG7 pathway in ox-LDL-treated vascular smooth muscle cells

Figure 1. Patients with AS and ox-LDL-treated HA-VSMCs exhibited increased level of RASSF8-AS1. (A) RASSF8-AS1 was detected with high expression in patients with AS compared with healthy participants. Dataset GSE146882 was downloaded from GEO database. The Y-axis represents the signal intensity of gene expression (log2-transformed). P value =0.01; N=10. *P<0.05. (B) ROC curve analysis of RASSF8-AS1 value in diagnosis of AS. AUC =0.77. (C) Healthy participants (normal; N=20) and patients with AS (N=20) were enrolled for assaying RASSF8-AS1 expression in peripheral blood. ***P<0.001 (t-test). (D) Relationship between RASSF8-AS1 and LDL-C. R=0.85; P<0.001. (E) ROC curve analysis of RASSF8-AS1 value in predicting LDL-C-treated HA-VSMCs. RASSF8-AS1 AUC =0.95; ox-LDL AUC =0.72. (F) HA-VSMCs (N=3) were assayed for RASSF8-AS1 expression after administration with ox-LDL (0, 25, 50, 75, and 100 µg/mL). NSP>0.05; *P<0.05; ***P<0.001 (t-test). HA-VSMC, human aortic vascular smooth muscle cell; AS, atherosclerosis; ox-LDL, oxidized low-density lipoprotein; LDL-C, low-density lipoprotein cholesterol; GEO, Gene Expression Omnibus; ROC, the receiver operation characteristic; AUC, area under the receiver…

Figure 2. ox-LDL increased cell viability and inhibited apoptosis in HA-VSMCs expressing RASSF8-AS1. (A) RASSF8-AS1 expression in HA-VSMCs. shRNA targeting RASSF8-AS1 (sh-lncRNA#1, #2, and #3) was introduced into HA-VSMCs. *P<0.05; **P<0.01; NSP>0.05 (t-test). N=3. (B) RASSF8-AS1 expression in HA-VSMCs transfected with sh-lncRNA#1 and treated by 75 µg/mL of ox-LDL. ***P<0.001 (t-test). N=3. (C) Cell viability of sh-lncRNA#1-treated HA-VSMCs. HA-VSMCs were treated by ox-LDL (75 µg/mL). ***P<0.001 (t-test). N=3. (D,E) HA-VSMCs were assayed for PCNA and Ki-67 expression after transfection with sh-lncRNA#1 and administration with ox-LDL (75 µg/mL). *P<0.05; **P<0.01 (t-test). N=3. (F,G) HA-VSMCs were detected for apoptosis rate after transfection with RASSF8-AS1 shRNA. *P<0.05; **P<0.01 (t-test). N=3. HA-VSMC, human aortic vascular smooth muscle cell; ox-LDL, oxidized low-density lipoprotein; sh-lncRNA, short hairpin RASSF8-AS1; sh-NC, short hairpin negative control; PCNA, proliferating cell nuclear antigen; FITC, fluorescein isothiocyanate; PI, prodium iodide.

Figure 3. RASSF8-AS1-induced targeted inhibition of miR-188-3p expression by RASSF8-AS1 in HA-VSMCs treated by ox-LDL. (A) Nuclear and cytoplasmic RASSF8-AS1 in HA-VSMCs (N=3). (B) HA-VSMCs were assayed for miR-188-3p expression after the treatment of ox-LDL (0, 25, 50, 75, and 100 µg/mL). NSP>0.05; **P<0.01; ***P<0.001. N=3. (C) The linear relationship based on miR-188-3p and RASSF8-AS1 expression in patients with AS. N=20; R =−0.75; P<0.001. (D) miR-188-3p-interacted RASSF8-AS1 (wild type and mutant type). (E) Confirmation of the interaction between RASSF8-AS1 and miR-188-3p by relative luciferase reporter assay. NSP>0.05; **P<0.01 (t-test). N=3. (F) miR-188-3p level in HA-VSMCs high or low expressing RASSF8-AS1. **P<0.01; ***P<0.001 (t-test). N=3. (G) miR-188-3p expression in RASSF8-AS1-deficient HA-VSMCs after ox-LDL treatment. **P<0.01; ***P<0.001 (t-test). N=3. HA-VSMC, human aortic vascular smooth muscle cell; ox-LDL, oxidized low-density lipoprotein; AS, atherosclerosis; sh-lncRNA, short hairpin RASSF8-AS1; sh-NC; short hairpin negative control; NC, negative control; WT, wild type; MUT, mutant type.

Figure 4. Targeted sponging of miR-188-3p by RASSF8-AS1 regulated proliferative activity and apoptotic process of HA-VSMCs in response to ox-LDL. (A) RASSF8-AS1 and miR-188-3p expression in HA-VSMCs treated by ox-LDL after transfection with sh-lncRNA#1 or miR inhibitor. **P<0.01; ***P<0.001 (t-test). N=3. (B) HA-VSMCs were assayed for cell viability after transfection with sh-lncRNA#1 or miR inhibitor or treatment by ox-LDL (75 µg/mL). **P<0.01; ***P<0.001 (t-test). N=3. (C) PCNA expression in HA-VSMCs after transfection with sh-lncRNA#1 or miR inhibitor and treatment of ox-LDL (75 µg/mL). ***P<0.001 (t-test). N=3. (D) Apoptosis rate of HA-VSMCs after transfection with sh-lncRNA#1 or miR inhibitor and treatment with ox-LDL (75 µg/mL). ***P<0.001 (t-test). N=3. PCNA, proliferating cell nuclear antigen; shRNA, short hairpin RNA; HA-VSMC, human aortic vascular smooth muscle cell; ox-LDL, oxidized low-density lipoprotein; sh-lncRNA, short hairpin RASSF8-AS1; miR inhibitor, miR-188-3p inhibitor; NC, negative control; FITC, fluorescein isothiocyanate; PI, prodium iodide.

Figure 5. ATG7 was a downstream target of miR-188-3p in HA-VSMCs. (A) HA-VSMCs were assayed for ATG7 mRNA and protein after ox-LDL treatment (75 µg/mL). *P<0.05 (t-test). N=3. (B) Serum ATG7 mRNA in healthy individuals (normal) and patients with AS. **P<0.01 (t-test). N=20. (C) HA-VSMCs were assayed for AGT7 mRNA after transfection with miR mimic or inhibitor. **P<0.01; ***P<0.001 (t-test). N=3. (D) Targeted sites between the wild type or mutant type of miR-188-3p and ATG7. (E) The direct interaction between ATG7 mRNA and miR-188-3p was confirmed by relative luciferase reporter assay. NSP>0.05; **P<0.01 (t-test). N=3. (F) miR-188-3p mimic decreased ATG7 transcription in ox-LDL-treated HA-VSMCs. ***P<0.001 (t-test). N=3. ox-LDL, oxidized low-density lipoprotein; ATG7, autophagy-related gene 7; HA-VSMC, human aortic vascular smooth muscle cell; AS, atherosclerosis; sh-lncRNA, short hairpin RASSF8-AS1; miR inhibitor, miR-188-3p inhibitor; miR mimic, miR-188-3p mimic; NC, negative control; WT, wild type; MUT, mutant type.

Figure 6. ATG7 expression and autophagy induced by RASSF8-AS1-mediated sponging of miR-188-3p in HA-VSMCs after ox-LDL treatment. (A) Western blotting showing autophagy protein expression in HA-VSMCs transfected with the indicated vehicles and treated by ox-LDL. (B-F) Quantification of ATG7 (B), LC3 II (C), Beclin-1 (E), and P62 (F) protein, as well as the ratio of LC3 II and LC3 I (D). NSP>0.05; *P<0.05; **P<0.01; ***P<0.001 (t-test). N=3. ox-LDL, oxidized low-density lipoprotein; ATG7, autophagy-related gene 7; HA-VSMC, human aortic vascular smooth muscle cell; sh-lncRNA, short hairpin RASSF8-AS1; miR inhibitor, miR-188-3p inhibitor.

Figure 7. RASSF8-AS1 modulated cell proliferation and apoptosis by regulating the ATG7-mediated autophagy pathway. (A) Western blotting showing autophagy protein expression in HA-VSMCs after transfection with the indicated vehicles (sh-lncRNA and ATG7) and treated by ox-LDL. (B-E) Quantification of the protein expression of ATG7 (B), Beclin-1 (D), and P62 (E), and the ratio of LC3 II and LC3 I (C). *P<0.05; **P<0.01; ***P<0.001 (t-test). N=3. (F) Cell viability of HA-VSMCs after transfection with sh-lncRNA or ATG7 and treatment with ox-LDL (75 µg/mL). **P<0.01; ***P<0.001 (t-test). N=3. (G) PCNA and Ki-67 protein expression in HA-VSMCs after transfection with sh-lncRNA or ATG7 and treatment with ox-LDL (75 µg/mL). **P<0.01; ***P<0.001 (t-test). N=3. (H,I) Apoptosis of HA-VSMCs transfected with sh-lncRNA or ATG7 and treated by ox-LDL (75 µg/mL). **P<0.01; ***P<0.001 (t-test). N=3. PCNA, proliferating cell nuclear antigen; sh-lncRNA, short hairpin RASSF8-AS1; ox-LDL, oxidized low-density lipoprotein; ATG7, autophagy-related gene 7; HA-VSMC, human aortic vascular smooth muscle cell; NC, negative control; FITC, fluorescein isothiocyanate; PI, prodium iodide.