Additional file 1 of lncRNA Helf promotes hepatic inflammation and fibrosis by interacting with PTBP1 to facilitate PIK3R5 mRNA stabilization
Creators: name: Han, Xiaohui
name: Guo, Beichen
name: Zhao, Sicong
name: Li, Yehua
name: Zhu, Jing
name: He, Yifan
name: Wang, Jiajun
name: Yao, Qingbin
name: Shao, Shuai
name: Zheng, Lina
name: Shi, Zhemin
name: Han, Tao
name: Hong, Wei
name: Zhang, Kun
Publisher: name: figshare
Year: 2023
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Science Policy
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Hematology
subject: Virology

description: Additional file 1: Fig. S1, related to Fig. S1. (A) The fold change of lnc-Helf was shown according to the microarray data. (B, C) The nucleotide sequence of mouse lnc-Helf and human lnc-HELF. (D) The comparison between human and mouse lnc-Helf sequences. (E) The nuclei or cytoplasm of primary HSCs was isolated, and qRT–PCR analysis detected the expression of lnc-Helf, Neat1, and Gapdh. (F) The intracellular localization of lnc-Helf in primary HSCs was measured by RNA-FISH assays; scale bar is 10 μm. (G) qRT–PCR analysis of lnc-Helf in AML12 cells, LX-2 cells, RAW264.7 cells, BMMs, and HUVECs (H) qRT–PCR was used to assess the expression of lnc-Helf and Acta2 in primary HSCs cultured at day 2 treated with 10 ng/ml TGF-β for 24 h. (I–L) The correlations of lnc-HELF, ACTA2, COL1α1, ALT, and AST were assessed by Pearson correlation analysis, n = 34. *p &lt; 0.05, unpaired Student’s t test (H). Fig. S2, related to Fig. S2. Mice were divided into six groups: AAV8-NC, NC + CCl4, lnc-Helf-sh1#, lnc-Helf-sh1# + CCl4, lnc-Helf-sh2#, and lnc-Helf-sh2# + CCl4. Mice were injected with AAV8-lnc-Helf-shRNAs or AAV8-NC virus 2 weeks after the first injection of CCl4 via tail vein. After CCl4 treatment for 8 weeks, (A–D) the volcano map and GO analysis of differentially expressed mRNAs in lnc-Helf-sh1# + CCl4 mice and lnc-Helf-sh2# + CCl4 mice are shown, compared with NC + CCl4 mice. Fig. S3, related to Fig. S2. Mice were divided into six groups: AAV8-NC, NC + CCl4, lnc-Helf-sh1#, lnc-Helf-sh1# + CCl4, lnc-Helf-sh2# and lnc-Helf-sh2# + CCl4. Mice were injected with AAV8-lnc-Helf-shRNAs or AAV8-NC virus 2 weeks after the first injection of CCl4 via tail vein. After CCl4 treatment for 8 weeks (A), IHC for COL1α1 and TGFβ are shown; scale bar is 100 μm for 40× and 400 μm for 10× magnifications . (B) qRT–PCR was used to assess the expression of Acta2, Col1α1, Mmp2, Timp1, and Tgfβ1. (C, D) Serum ALT and AST was examined. Data are presented as mean ± SEM.*/#p &lt; 0.05. *p &lt; 0.05 for AAV8-NC. #p &lt; 0.05 for NC + CCl4, one-way ANOVA (B-D). Fig. S4, related to Fig. S2. Mice were divided into six groups: AAV8-NC, NC + CCl4, lnc-Helf-sh1#, lnc-Helf-sh1# + CCl4, lnc-Helf-sh2#, and lnc-Helf-sh2# + CCl4. Mice were injected with AAV8-lnc-Helf-shRNAs or AAV8-NC virus 2 weeks after the first injection of CCl4 via tail vein. After CCl4 treatment for 8 weeks, (A) western blot was used to determine the expression of CD11b, TNF-α, IL-1β, and MCP1. GAPDH was used as an internal control. (B, C) qRT–PCR was used to assess the RNA level of proinflammatory genes (Tnf-α, Mcp1, Il-1β, and Il-6) and proliferation-related genes (Pcna, Ki67, Cyclin D1, and Bcl-2) in livers of each group. (D) IHC for CD11b, F4/80, TNF-α, IL-1β, LY6C, and PCNA; scale bar is 100 μm for 40× and 400 μm for 10× magnifications. Data are presented as mean ± SEM. */#p &lt; 0.05. *p &lt; 0.05 for AAV8-NC. #p &lt; 0.05 for NC + CCl4, one-way ANOVA (B and C). Fig. S5, related to Fig. S3. Mice were divided into six groups: AAV8-NC, NC + BDL, lnc-Helf-sh1#, lnc-Helf-sh1# + BDL, lnc-Helf-sh2# and lnc-Helf-sh2# + BDL. Mice were injected with AAV8-lnc-Helf-shRNAs or AAV8-NC virus 2 days before sham operation or bile duct ligate operation via tail vein. After 21 days of operation, (A) qRT–PCR was used to examine the RNA level of lnc-Helf in livers of each group. (B, C) Serum AST and ALT was examined. (D) qRT–PCR was used to assess the RNA level of Pcna, Ki67, Cyclin D1, and Bcl-2 in livers of each group. Data are presented as mean ± SEM. */#p &lt; 0.05. *p &lt; 0.05 for AAV8-NC. #p &lt; 0.05 for NC + BDL, one-way ANOVA (A-D). Fig. S6, related to Fig. S4. (A) Quantitative analysis of western blot of Fig. S4B. (B) Primary HSCs at day 2 were infected with lenti-control and lnc-Helf-shRNA or lenti-lnc-Helf for 48 h, following the treatment with 10 ng/ml TGF-β1. The expression and location of COL1α1 was assessed by confocal microscopy. Scale bar is 20 μm. (C) Quantitative analysis of western blot of Fig. S4F. (D) Primary HSCs at day 2 were infected with lenti-control and lenti-lnc-Helf for 48 h, following the treatment with 10 ng/ml TGF-β1. The expression and location of COL1α1 was assessed by confocal microscopy. Scale bar is 20 μm. (E–G) LX-2 cells were infected with lenti-control or lenti-lnc-HELF for 48 h, following the treatment with TGF-β1 for 24 h. qRT–PCR was used to assess the expression of lnc-HELF, ACTA2, COL1α1, COL1α2, TIMP1, TGF-β1, PCNA, KI67, and CYCLIN D1 (E); western blot was used to determine the expression of the protein level of α-SMA, MMP2, CYCLIN D1, CYCLIN B1, and PCNA (F). GAPDH was used as an internal control. Cell proliferation was detected by CCK8 (G). (H) Primary HSCs at day 3 were infected with lenti-control and lenti-lnc-Helf for 96 h, cell proliferation was detected by CCK8. Data are presented as mean ± SEM. */#p &lt; 0.05. *p &lt; 0.05 for LV-control. #p &lt; 0.05 for LV-control + TGF-β1, one-way ANOVA (A, C, and E) and unpaired Student’s t test (G and H). Fig. S7, related to Fig. S5. (A) Mouse primary HMs transfected with siRNA for 24 h, qRT–PCR analysis was used to detect the RNA level of Mrc1, Arg1, Cd163, and Il10. (B) HMs were transfected with pcDNA3.1-lnc-Helf or pcDNA3.1 for 48 h, qRT–PCR was used to assess the expression of Mrc1, Arg1, Cd163, and Il10. (C) RAW264.7 cells were transfected with pcDNA3.1 or pcDNA3.1-lnc-Helf for 48 h, qRT–PCR was used to assess the expression of lnc-Helf, Tnf-α, Mcp-1, Il-1β, Il-6, Pcna, Ki67, Cyclin D1, Cyclin E1, Mrc1, Arg1, and Il10. (D) RAW264.7 cells were transfected with pcDNA3.1 or pcDNA3.1-lnc-Helf for 48 h following treatment with 20 ng/ml IFN-γ for 24 h. Mature supernatant IL-1β level was detected by ELISA. Data are presented as mean ± SEM. */#p &lt; 0.05. *p &lt; 0.05 for pcDNA3.1. #p &lt; 0.05 for pcDNA3.1 + IFN-γ, unpaired Student’s t test (A–C) and one-way ANOVA (D). Fig. S8, related to Fig. S6. (A) Mice were divided into six groups: AAV8-NC, NC + BDL, lnc-Helf-sh1#, lnc-Helf-sh1# + BDL, lnc-Helf-sh2#, and lnc-Helf-sh2# + BDL. Western blot was used to determine the protein level of phos-AKT (Thr308), phos-c-Raf1 (Ser259), phos-GSK-3β (Ser9), and AKT in liver tissues of each group. (B) LX-2 cells were infected with lenti-control or lenti-lnc-HELF for 48 h following treatment with 10 ng/ml TGF-β1 for 24 h. Western blot was used to determine the protein level of phos-AKT (Thr308) and AKT. GAPDH was used as an internal control. (C) RAW264.7 cells treated with or without the AKT inhibitor MK2206 were transfected with pcDNA3.1 or pcDNA3.1-lnc-Helf for 72 h. qRT–PCR was used to detect the expression of lnc-Helf, Tnf-α, Mcp-1, Il-1β, Il-6, Pcna, Cyclin D1, and Cyclin E1. Data are presented as mean ± SEM. */#p &lt; 0.05. *p &lt; 0.05 for pcDNA3.1 + DMSO. #p &lt; 0.05 for pcDNA3.1-lnc-Helf + DMSO, one-way ANOVA (C). Fig. S9, related to Fig. S7. The expression and location of α-SMA in lnc-Helf-increased HSCs simultaneously transfected with siPTBP1 was assessed by confocal microscopy. Scale bar is 20 μm. Fig. S10, related to Fig. S8. (A, B) Prediction of the interaction probabilities between lnc-Helf and PTBP1 using the RPIseq and prediction of lncRNA–protein interactions database. (C) qRT–PCR was used to detect the RNA level of PTBP1 in livers of healthy people (n = 6) and fibrotic patients (n = 28). (D, E) The correlation of PTBP1, lnc-HELF, and PIK3R5 was assessed by Pearson correlation analysis, n = 34. (F–I) Mice were divided into six groups: AAV8-NC, NC + CCl4/BDL, lnc-Helf-sh1#, lnc-Helf-sh1# + CCl4/BDL, lnc-Helf-sh2#, and lnc-Helf-sh2# + CCl4/BDL. qRT–PCR was used to detect the RNA level of Pik3r5 and Ptbp1 in livers of each group. Data are presented as mean ± SEM. */#p &lt; 0.05. *p &lt; 0.05 for AAV8-NC. #p &lt; 0.05 for NC + CCl4/BDL, one-way ANOVA (F–I). Fig. S11, related to Fig. S8. (A, B) Primary HSCs at day 2 were infected with lenti-control or lnc-Helf-shRNA or lenti-lnc-Helf for 48 h, following the treatment with TGF-β1 for 24 h. qRT–PCR was used to detect the RNA level of Pik3r5. (C) LX-2 cells were infected with lenti-control or lenti-lnc-HELF for 48 h, following the treatment of TGF-β1 for 24 h. qRT–PCR was used to assess the expression of PIK3R5. (D) Mouse primary HMs were transfected with lnc-Helf siRNA for 24 h following treatment of 20 ng/ml IFN-γ for 24 h. qRT–PCR was used to assess the expression of Pik3r5. (E, F) Mouse primary HMs and RAW264.7 cells were transfected with pcDNA3.1 or pcDNA3.1-lnc-Helf for 48 h, qRT–PCR was used to assess the expression of Pik3r5. (G) qRT–PCR was used to assess the expression of Pik3r5 in lnc-Helf-overexpressed RAW264.7 cells simultaneously transfected with siPTBP1. Data are presented as mean ± SEM. */#p &lt; 0.05. *p &lt; 0.05 for LV-control or si-NC or pcDNA3.1or pcDNA3.1 + si-NC. #p &lt; 0.05 for LV-control + TGF-β1/IFN-γ or pcDNA3.1-lnc-Helf + si-NC, one-way ANOVA (A–D, and G) and unpaired Student’s t test (E and F). Fig. S12. Schematic diagram shows the function and mechanism of lnc-Helf in the progression of hepatic inflammation and fibrosis. Upon liver injury, increased lnc-Helf binds with PTBP1 to promote its interaction with PIK3R5 mRNA, resulting in increased stability and activating the AKT pathway, thus promotes HSCs and HMs activation and proliferation, which augments hepatic inflammation and fibrosis. Table S1. Clinical characteristics of patients. Table S2. The results of mass spectrometry from proteins pulled down by the sense and antisense of lnc-Helf. Table S3. Cloning primers for lnc-Helf. Table S4 siRNA sequences. Table S5 qRT–PCR primers. Table S6 RACE primers for lnc-Helf.