Journal: Biology Direct

Article Title: LncRNA NORFA promotes the synthesis of estradiol and inhibits the apoptosis of sow ovarian granulosa cells through SF-1/CYP11A1 axis

doi: 10.1186/s13062-024-00563-1

Figure Lengend Snippet: NORFA is a novel inducer of E2 in sow GCs. ( A ) GSEA analysis based on RNA-seq data of NORFA knockdown showing a significant enrichment of the ovarian steroidgenesis pathway. ( B ) A significant positive correlation between the expression level of NORFA in the whole follicle and E2 levels in follicular fluid was identified by Pearson correlation analysis ( n = 16). ( C ) Images of the healthy follicles (HF) and paired adjacent atretic follicles (AF) (paired n = 8). ( D ) Alteration patterns of NORFA expression (left) and E2 levels (right) in the HFs and AFs were detected using RT-qPCR and ELISA (paired n = 8). ( E ) NORFA expression (left) and E2 levels (right) in GCs transfected with pcDNA3.1-NORFA (0, 0.4, 0.8, 1.6 and 3.2 µg) for 48 h were detected using RT-qPCR and ELISA ( n = 3). ( F ) After transfection with 1.6 µg pcDNA3.1-NORFA into GCs for different times (0, 12, 24, 48 and 72 h), NORFA expression (left) and E2 levels (right) were measured using RT-qPCR and ELISA ( n = 3). ( G ) NORFA expression (left) and E2 levels (right) in GCs transfected with NORFA-siRNA (0, 10, 20, 40 and 80 µM) for 48 h were detected using RT-qPCR and ELISA ( n = 3). ( H ) GCs were transfected with 20 µM NORFA-siRNA for the indicated times (0, 12, 24, 48 and 72 h), RT-qPCR and ELISA were performed to measure NORFA expression (left) and E2 levels (right) ( n = 3). Data in ( E - H ) were shown as mean ± SEM. Significance was analyzed by two-tailed Student’s t -test and ANOVA. ** P < 0.01

Article Snippet: Pearson correlation analyses were performed by GraphPad Prism v8.0.

Techniques: RNA Sequencing, Knockdown, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transfection, Two Tailed Test

Journal: Biology Direct

Article Title: LncRNA NORFA promotes the synthesis of estradiol and inhibits the apoptosis of sow ovarian granulosa cells through SF-1/CYP11A1 axis

doi: 10.1186/s13062-024-00563-1

Figure Lengend Snippet: NORFA induces the synthesis of PREG in sow GCs. ( A ) The levels of PREG in GCs transfected with indicated amount of pcDNA3.1-NORFA (0, 0.4, 0.8, 1.6 and 3.2 µg) for 48 h were detected by ELISA ( n = 3). ( B ) 1.6 µg pcDNA3.1-NORFA was transfected into GCs for different times (0, 12, 24, 48 and 72 h), and PREG levels were measured by ELISA ( n = 3). ( C ) PREG concentrations in GCs transfected with indicated amount of NORFA-siRNA (0, 10, 20, 40 and 80 µM) for 48 h were detected by ELISA ( n = 3). ( D ) 20 µM NORFA-siRNA was transfected into GCs for the indicated times (0, 12, 24, 48 and 72 h), ELISA was performed to measure PREG levels ( n = 3). ( E ) A significant positive correlation between NORFA expression in GCs and PREG levels in follicular fluid was identified by Pearson correlation analysis ( n = 16). ( F ) Alteration patterns of PREG levels in the HFs and AFs were detected by ELISA (paired n = 8). ( G - H) The effects of NORFA overexpression (NORFA OE ) co-treated with 10 µM ONO-2952 ( G ) or 20 µM StAR-siRNA ( H ) on the PREG levels in GCs were detected by ELISA ( n = 3). Data were shown as mean ± SEM with at least three independent replicates. Significance was analyzed by two-tailed Student’s t -test and ANOVA. ** P < 0.01

Article Snippet: Pearson correlation analyses were performed by GraphPad Prism v8.0.

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Expressing, Over Expression, Two Tailed Test

Journal: Biology Direct

Article Title: LncRNA NORFA promotes the synthesis of estradiol and inhibits the apoptosis of sow ovarian granulosa cells through SF-1/CYP11A1 axis

doi: 10.1186/s13062-024-00563-1

Figure Lengend Snippet: CYP11A1 is positively regulated by NORFA and mediates its function in sow GCs. ( A ) The expression patterns of nine steroid hormone synthesis-related genes in sow GCs after NORFA knockdown (NORFA KD ) were obtained from RNA-seq. ( B ) The FPKM values of CYP11A1 mRNA in control and NORFA-inhibited sow GCs were obtained from RNA-seq. ( C ) The expression correlation between NORFA and CYP11A1 mRNA in follicles was identified by Pearson correlation analysis ( n = 16). ( D ) Alteration patterns of CYP11A1 mRNA levels in the HFs and AFs were detected by RT-qPCR (paired n = 8). ( E-F ) The effects of NORFA overexpression (NORFA OE ) or knockdown (NORFA KD ) on the mRNA ( E ) and protein ( F ) levels of CYP11A1 in sow GCs were analyzed by RT-qPCR and western blotting ( n = 3). ( G - K ) pcDNA3.1-NORFA (NORFA OE ) was co-transfected with CYP11A1-siRNA (CYP11A1 KD ) into sow GCs for 48 h, the mRNA ( G ) and protein ( H ) levels of CYP11A1 were detected by RT-qPCR and western blotting ( n = 3), the concentration of PREG and E2 were measured using ELISA ( I , n = 3), and GC proliferation was analyzed by CCK-8 ( J ) and EdU staining ( K ). Data were shown as mean ± SEM with at least three independent replicates. Significance was analyzed by two-tailed Student’s t -test and ANOVA. ** P < 0.01

Article Snippet: Pearson correlation analyses were performed by GraphPad Prism v8.0.

Techniques: Expressing, Knockdown, RNA Sequencing, Control, Quantitative RT-PCR, Over Expression, Western Blot, Transfection, Concentration Assay, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Staining, Two Tailed Test

Journal: Biology Direct

Article Title: LncRNA NORFA promotes the synthesis of estradiol and inhibits the apoptosis of sow ovarian granulosa cells through SF-1/CYP11A1 axis

doi: 10.1186/s13062-024-00563-1

Figure Lengend Snippet: SF-1, a transcriptional activator, induces CYP11A1 transcription, PREG and E2 synthesis in sow GCs. ( A ) Effects of NORFA overexpression (NORFA OE ) or knockdown (NORFA KD ) on CYP11A1 promoter activity were analyzed by luciferase activity assays ( n = 3). ( B ) TFs that potentially target CYP11A1 promoter were screened by JASPAR prediction and siNORFA-mediated DETFs obtained from previous RNA-seq data. ( C-D ) The mRNA and protein levels of CYP11A1 in NR5A1 over-expressed sow GCs were detected using RT-qPCR and western blotting ( n = 3). ( E ) The correlation between NR5A1 and CYP11A1 mRNA levels was analyzed by Pearson correlation analysis ( n = 16). ( F ) Diagram showing the reporter vector containing CYP11A1 promoter with wild-type (WT) or mutant (MUT) SF-1 binding element (SBE). ( G ) Effects of NR5A1 overexpression (NR5A1 OE ) on the activities of reporter vectors established in ( F ) were detected by luciferase activity assays ( n = 3). ( H ) Enrichment of SF-1 on the promoter of CYP11A1 in sow GCs transfected with or without siNORFA was analyzed by ChIP. F1/R1 and F2/R2 are the primer pairs for SBE and SBE-X (without SBE), respectively. M indicates DNA marker, B indicates blank, I indicates IgG, S indicates SF-1, In indicates input. ( I-K ) The expression of CYP11A1 and the concentrations of PREG and E2 in sow GCs after co-transfection with pcDNA3.1-NR5A1 (NR5A1 OE ) and CYP11A1-siRNA (CYP11A1 KD ) were detected by RT-qPCR, western blotting, and ELISA ( n = 3). Data were presented as mean ± SEM with at least three independent replicates. Significance was analyzed by two-tailed Student’s t -test and ANOVA. ** P < 0.01

Article Snippet: Pearson correlation analyses were performed by GraphPad Prism v8.0.

Techniques: Over Expression, Knockdown, Activity Assay, Luciferase, RNA Sequencing, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Mutagenesis, Binding Assay, Transfection, Marker, Expressing, Cotransfection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Biology Direct

Article Title: LncRNA NORFA promotes the synthesis of estradiol and inhibits the apoptosis of sow ovarian granulosa cells through SF-1/CYP11A1 axis

doi: 10.1186/s13062-024-00563-1

Figure Lengend Snippet: SF-1, induced by NORFA at the post-transcriptional level, mediates the regulation of NORFA to CYP11A1 . ( A ) The abundance of NR5A1 transcript in sow GCs after NORFA knockdown (NORFA KD ) was detected by RNA-seq. ( B ) Correlation between NORFA and NR5A1 expression levels was analyzed using Pearson correlation analysis ( n = 16). ( C-D ) The effects of NORFA overexpression (NORFA OE ) or knockdown (NORFA KD ) on the mRNA ( C ) and protein ( D ) levels of NR5A1 in sow GCs were detected using RT-qPCR and western blotting ( n = 3). ( E ) Effects of NORFA overexpression (NORFA OE ) or knockdown (NORFA KD ) on the stability of NR5A1 mRNA were analyzed using ActD chase assays ( n = 3). ( F ) The colocalization of NORFA and NR5A1 mRNA in sow GCs was detected using RNA-FISH. Scale bar = 10 μm. ( G ) Schematic view of truncated fragment of NORFA (left panel), and the physical interaction between NORFA and NR5A1 mRNA, as well as the interaction regions of NORFA were identified using RNA pull-down (right panel). ( H ) Effects of NORFA with IR1 deletion on the stability of NR5A1 mRNA were detected using ActD chase assays ( n = 3). ( I-K ) The mRNA ( I ) and protein ( J ) levels of CYP11A1, PREG and E 2 concentration ( K ) in sow GCs after co-transfection with NORFA-siRNA (NORFA KD ) and pcDNA3.1-NR5A1 (NR5A1 OE ) were detected by RT-qPCR, western blotting, and ELISA assays ( n = 3). Data were presented as mean ± SEM with three independent replicates. Significance was analyzed by two-tailed Student’s t -test and ANOVA. * P < 0.05, ** P < 0.01

Article Snippet: Pearson correlation analyses were performed by GraphPad Prism v8.0.

Techniques: Knockdown, RNA Sequencing, Expressing, Over Expression, Quantitative RT-PCR, Western Blot, Concentration Assay, Cotransfection, Enzyme-linked Immunosorbent Assay, Two Tailed Test