Journal: Cellular & Molecular Biology Letters

Article Title: tsRNA-3040b accumulates R -loop to regulate Trim35 transcription, which leads to disordered glycolysis and promotes PAECs proliferation

doi: 10.1186/s11658-025-00816-5

Figure Lengend Snippet: tsRNA-3040b promotes R -loop formation in Trim35 gene to inhibit its transcription. A-B Cluster heatmap and volcano plot of the RNA sequencing results reveal that TRIM35 levels decrease under hypoxic conditions. C-D RT-qPCR and western blot analysis detect the Trim35 transcription levels ( C , n = 5) and protein levels ( D , n = 4). β -actin was used as an internal reference. E–F RT-qPCR and western blot analysis examines the Trim35 transcription levels ( E , n = 5) and protein levels ( F , n = 3) in mPAECs treated with tsRNA-3040b mimics or inhibitors. G-H RT-qPCR and western blot analysis show the Trim35 transcription levels ( G , n = 6) and protein levels ( H , n = 6) in lung tissue from mice treated with tsRNA-3040b agomir or antagomir. I Dot blot analysis shows reduced S9.6 expression after RNase H treatment. J RT-qPCR detect the Trim35 mRNA expression in mPAECs treated with tsRNA-3040b mimics and RNase H treatment. β -actin was used as internal reference. K Immunofluorescence detects S9.6 following the addition of tsRNA-3040b mimics or inhibitors. Nuclei stained with DAPI (blue). L The schematic diagram shows the binding sites of the Trim35 promoter and coding region (upper panel), and ChIP-PCR indicates R -loop at the Trim35 promoter 3′ region (left lower panel) and at coding region 1 and 2 sites (right upper panel). M ChIP-PCR indicates that tsRNA-3040b forms an R -loop, which increased under hypoxic conditions. N Diagram illustrating the mechanism of R -loop formation with tsRNA-3040b and with Trim35 . Data are presented as mean ± SEM. One-way ANOVA was employed for comparisons. CDS, coding sequence; Hyp, hypoxia; Inh, tsRNA-3040b inhibitor; mimics, tsRNA-3040b mimic; NC, negative controls; Nor, normoxia

Article Snippet: The TRIM35 overexpression plasmid was synthesized by Genechem (Shanghai, China), and the WNT3A overexpression plasmid was synthesized by Miaoling Biotech (Wuhan, China).

Techniques: RNA Sequencing, Quantitative RT-PCR, Western Blot, Dot Blot, Expressing, Immunofluorescence, Staining, Binding Assay, Sequencing

Journal: Cellular & Molecular Biology Letters

Article Title: tsRNA-3040b accumulates R -loop to regulate Trim35 transcription, which leads to disordered glycolysis and promotes PAECs proliferation

doi: 10.1186/s11658-025-00816-5

Figure Lengend Snippet: tsRNA -3040b/Trim 35 regulates the ubiquitination of Wnt3a and affects glycolysis. A RT-qPCR validation of knockdown efficiency by small interference RNA targeted to Trim35 ( siTrim35 ) ( n = 4). B , C Western blot analysis revealed reduced WNT3A protein expression in mPAECs ( n = 5) and in mouse lung tissues ( n = 6). D Western blot detects WNT3A protein expression upon Trim35 knockdown under normoxia, or Trim35 overexpression under hypoxia ( n = 6). E Schematic of molecular docking between Trim35 and Wnt3a. F Immunofluorescence demonstrated coexpression of Trim35 and Wnt3a. G Co-IP experiment confirmed the interaction of TRIM35 with WNT3A and reduced ubiquitination levels after treatment with si Trim35 . H Western blot analysis showed restored WNT3A protein levels after MG132 treatment. I RT-qPCR confirmed increased Hk2 transcription levels following siTrim35 ( n = 6). J Measurement of HK2 enzyme activity following siTrim35 ( n = 4). K–M The production of pyruvate, ATP, and lactate were measured by the corresponding assay kit upon Trim35 knockdown under normoxia, or Trim35 overexpression in mPAECs under hypoxia. β -actin was used as an internal reference. Data are presented as mean ± SEM. Unpaired two-tailed t -tests and one-way ANOVA were employed for comparisons. Agomir, tsRNA-3040b agomir; antagomir, tsRNA-3040b antagomir; HK, hexokinase; Hyp, hypoxia; mimics, tsRNA-3040b mimic; NC, negative controls; Nor, normoxia

Article Snippet: The TRIM35 overexpression plasmid was synthesized by Genechem (Shanghai, China), and the WNT3A overexpression plasmid was synthesized by Miaoling Biotech (Wuhan, China).

Techniques: Ubiquitin Proteomics, Quantitative RT-PCR, Biomarker Discovery, Knockdown, Western Blot, Expressing, Over Expression, Immunofluorescence, Co-Immunoprecipitation Assay, Activity Assay, Two Tailed Test

Journal: Cellular & Molecular Biology Letters

Article Title: tsRNA-3040b accumulates R -loop to regulate Trim35 transcription, which leads to disordered glycolysis and promotes PAECs proliferation

doi: 10.1186/s11658-025-00816-5

Figure Lengend Snippet: tsRNA-3040b influences the glycolytic pathway by regulating Wnt3a ubiquitination via Trim35, thereby affecting mPAECs proliferation. mPAECs were transfected with negative control plasmid, Trim35 plasmid, or Trim35 plasmid combined with Wnt3a plasmid, then exposed to hypoxia for 24 h. A RT-qPCR validation of glycolytic kinase Hk2 mRNA levels ( n = 5). B Assessment of HK2 enzyme activity ( n = 7). C Determination of pyruvate content ( n = 7). D Determination of ATP content ( n = 6). E Determination of lactate acid content ( n = 5). F Western blot analysis detects the PCNA protein expression in mPAECs with Wnt3a overexpression. G , H Representative images and summarized data of EdU staining and Ki67 staining in mPAECs. The nuclei are stained with Hoechst or DAPI (blue). Data are presented as mean ± SEM. One-way ANOVA were employed for comparisons. HK, hexokinase; Hyp, hypoxia; Nor, normoxia; plasmid, negative control plasmid

Article Snippet: The TRIM35 overexpression plasmid was synthesized by Genechem (Shanghai, China), and the WNT3A overexpression plasmid was synthesized by Miaoling Biotech (Wuhan, China).

Techniques: Ubiquitin Proteomics, Transfection, Negative Control, Plasmid Preparation, Quantitative RT-PCR, Biomarker Discovery, Activity Assay, Western Blot, Expressing, Over Expression, Staining

Journal: Cellular & Molecular Biology Letters

Article Title: tsRNA-3040b accumulates R -loop to regulate Trim35 transcription, which leads to disordered glycolysis and promotes PAECs proliferation

doi: 10.1186/s11658-025-00816-5

Figure Lengend Snippet: tsRNA-3040b promotes R -loops in Trim35 , affecting glycolysis during the hypoxia-induced pulmonary hypertension. Trim35 lead to the downregulation of Wnt3a through the ubiquitination under normoxic conditions, when exposure to hypoxia, the increasing generation of tsRNA-3040b suppresses Trim35 transcription by promoting or stabilizing R -loop structure in the Trim35 gene, leading to the increasing expression of Wnt3a, which activities the glycolysis enzymes and stimulated PAECs proliferation

Article Snippet: The TRIM35 overexpression plasmid was synthesized by Genechem (Shanghai, China), and the WNT3A overexpression plasmid was synthesized by Miaoling Biotech (Wuhan, China).

Techniques: Ubiquitin Proteomics, Expressing