Journal: International Journal of Surgery (London, England)

Article Title: LINC00476 cooperates with ARIH2 and suppresses pancreatic cancer progression by inducing VIM ubiquitination

doi: 10.1097/JS9.0000000000003596

Figure Lengend Snippet: LINC00476 is downregulated in PDAC and associated with poor prognosis. (A) Flowchart showing the process used to identify DElncRNAs in patients with PDAC compared to that of healthy individuals from public datasets. (B) The expression of LINC00476 in multi-tumors from the TCGA+GTEx database. The non-parametric Mann–Whitney U -test. (C) The correlation between LINC00476 and pathological stage ( P = 0.00044). Chi-square test or Fisher’s exact tests. (D) The expression level of LINC00476 in GSE16515 , GSE15471 , and GSE28735 dataset. Two-tailed Student’s t -test. (E) Kaplan‒Meier survival curves for overall survival (OS) in the TCGA dataset in the high LINC00476 and low LINC00476 group ( P < 0.00001). The log-rank test. Representative micrographs (F) and quantification (G) of LINC00476 expression in in situ hybridization (ISH) analysis in normal pancreatic tissue ( n = 30) and PDAC tissue ( n = 30). Scale bar = 100 μm; RS: risk score; Two-tailed Student’s t -test. (H) Kaplan–Meier analysis of the probability of overall survival of PDAC patients in the hospital cohort. The log-rank test. (I) Representative images of FISH of LINC00476 (red) in PDAC and adjacent normal tissues. DAPI (blue) was used for nuclear counterstaining. P values are shown as * P < 0.05, *** P < 0.001, **** P < 0.0001. All data represent the means ± SD from three independent assays.

Article Snippet: Briefly, for FISH, after counter staining with DAPI (Beyotime, Shanghai, China), slides were visualized under a Leica SP8 X confocal microscope (Leica, Germany).

Techniques: Expressing, MANN-WHITNEY, Two Tailed Test, In Situ Hybridization

Journal: International Journal of Surgery (London, England)

Article Title: LINC00476 cooperates with ARIH2 and suppresses pancreatic cancer progression by inducing VIM ubiquitination

doi: 10.1097/JS9.0000000000003596

Figure Lengend Snippet: LINC00476 inhibits proliferation, invasion, and migration in PDAC. (A and B) The efficiency of LINC00476 overexpression in PANC-1 cells and LINC00476 knockout (KO3 and KO6) in MIA PaCa-2 cells were assessed using qRT-PCR. (C and D) CCK-8 assays were used to determine the cell proliferation rates in LINC00476 overexpression and LINC00476 knockout cells. (E and F) The effect of LINC00476 overexpression and knockout on colony formation was investigated in PDAC cell lines. (G and H) Representative images of wound healing assays after LINC00476 overexpression and knockout in PDAC cell lines. Scale bar = 100 µM. (I and J) Representative images of Migration and Invasion assays after LINC00476 overexpression and knockout in PDAC cell lines. Scale bar = 5 µM. (K) Representative images of subcutaneous tumors formed by the indicated cells. (L) Tumor weights and volume are expressed as the mean ± SD of eight mice. (M) Representative images of FISH of LINC00476 (red) in mice tissues. DAPI (blue) was used for nuclear counterstaining. (N) The representative images of H&E staining and ISH staining with LINC00476 probe in tumors. Scale bar = 50 µM. (O) Metastasis assays in vivo was performed by splenic injection to evaluate the efficiency of KO3 on tumor metastasis. Representative images of H&E staining in metastatic tumors formed by MIA PaCa-2 cells. Scale bar = 50 µM. P values are shown as * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. All P -values were assessed using two-tailed Student’s t -test and one-way ANOVA followed by Tukey’s multiple comparisons test. All data represent the means ± SD from three independent assays.

Article Snippet: Briefly, for FISH, after counter staining with DAPI (Beyotime, Shanghai, China), slides were visualized under a Leica SP8 X confocal microscope (Leica, Germany).

Techniques: Migration, Over Expression, Knock-Out, Quantitative RT-PCR, CCK-8 Assay, Staining, In Vivo, Injection, Two Tailed Test

Journal: International Journal of Surgery (London, England)

Article Title: LINC00476 cooperates with ARIH2 and suppresses pancreatic cancer progression by inducing VIM ubiquitination

doi: 10.1097/JS9.0000000000003596

Figure Lengend Snippet: LINC00476 binds to VIM. (A) KEGG pathway analysis of LINC00476 based on transcriptome sequencing. (B) FISH staining showed that LINC00476 (red) was mainly localized in the cytoplasm and cytoskeleton. DAPI (blue) was used for nuclear counterstaining. (C) RNA pull-down assay was performed to detect the binding protein with biotinylated LINC00476. LINC00476 antisense RNA was used as the negative control. (D) GO-CC (cellular component) analysis predicting the potential proteins binding to LINC00476. (E) Western blotting was performed to evaluate the specific association of VIM with biotinylated LINC00476. Lysates of AsPC-1 and PANC-1 cells were harvested for RNA pull-down assays. LINC00476 antisense RNA was used as the negative control. (F and G) RNA-binding protein immunoprecipitation (RIP) assay was performed using an antibody against VIM. QRT-PCR was used to detect LINC00476 enrichment. IgG was used as the isotype control. Two-tailed Student’s t -test. (H) FISH staining showed that LINC00476 (red) was colocalized with VIM (green). (I) Secondary structure of LINC00476 ( http://www.Lncipedia.Org/ ). (J) Western blotting of VIM in samples precipitated by biotinylated full-length LINC00476 (FL) or LINC00476 truncations (Δ1: 1–485 bp; Δ2: 486–1024 bp; Δ3: 1025–1270bp). FL LINC00476 was used as the positive control. (K) Flag-RIP assay for LINC00476 showing its fold enrichment in cells transiently transfected with plasmids containing Flag-tagged full-length and truncated VIM constructs (Del1: 1–101 aa; Del2: 101–410 aa; Del3: 101–466 aa). IgG-RIP was used as the internal control. P values are shown as *** P < 0.001; **** P < 0.0001. All data represent the means ± SD from three independent assays.

Article Snippet: Briefly, for FISH, after counter staining with DAPI (Beyotime, Shanghai, China), slides were visualized under a Leica SP8 X confocal microscope (Leica, Germany).

Techniques: Sequencing, Staining, Pull Down Assay, Binding Assay, Negative Control, Western Blot, RNA Binding Assay, Immunoprecipitation, Quantitative RT-PCR, Control, Two Tailed Test, Positive Control, Transfection, Construct

Journal: International Journal of Surgery (London, England)

Article Title: LINC00476 cooperates with ARIH2 and suppresses pancreatic cancer progression by inducing VIM ubiquitination

doi: 10.1097/JS9.0000000000003596

Figure Lengend Snippet: ARIH2 is the E3 ligases for VIM. (A) Venn diagram for identifying nine candidate E3 ligases overlapping with the potential binding proteins of LINC00476. (B) The protein level of ARIH2 in LINC00476 overexpression and knockout cells. (C and D) A RNA-binding protein immunoprecipitation (RIP) assay was performed using an antibody against VIM. QRT-PCR was used to detect LINC00476 and VIM enrichment. IgG was used as the isotype control. Two-tailed Student’s t -test. (E) Western blotting was performed to evaluate the specific association of ARIH2 with biotinylated LINC00476. Lysates of AsPC-1 and MIA PaCa-2 cells were harvested for RNA pull-down assays. LINC00476 antisense RNA was used as the negative control. ( F ) FISH staining showed that LINC00476 (red) was colocalized with ARIH2 (yellow) and VIM (green) in the LINC00476 overexpression and knockout cells. DAPI (blue) was used for nuclear counterstaining. P values are shown as **** P < 0.0001. All data represent the means ± SD from three independent assays.

Article Snippet: Briefly, for FISH, after counter staining with DAPI (Beyotime, Shanghai, China), slides were visualized under a Leica SP8 X confocal microscope (Leica, Germany).

Techniques: Binding Assay, Over Expression, Knock-Out, RNA Binding Assay, Immunoprecipitation, Quantitative RT-PCR, Control, Two Tailed Test, Western Blot, Negative Control, Staining

Journal: International Journal of Surgery (London, England)

Article Title: LINC00476 cooperates with ARIH2 and suppresses pancreatic cancer progression by inducing VIM ubiquitination

doi: 10.1097/JS9.0000000000003596

Figure Lengend Snippet: ARIH2 decreases VIM and suppresses tumor growth in a PDX model. (A) Graphical diagram of intratumoral injection of in vivo optimized ARIH2 overexpression and control lentivirus in PDX-bearing mice. (B) Representative images of xenograft tumors derived from PDX in each mouse group. Mice bearing xenograft tumors were treated with ARIH2 overexpression (ARIH2-OE) and control (Vector) lentivirus. (C and D) Xenograft tumor growth was monitored by measuring the tumor volume and weighing the tumors. Tumor volume and weight are expressed as the mean ± SD of five mice. Two-tailed Student’s t -tests. (E and F) Representative images of H&E staining, ISH staining of LINC00476 and IHC staining of CDH1, CDH2, Ki67, and VIM of xenograft tumors. Scale bar = 50 µM. Two-tailed t -tests. (G) Representative images of FISH and IF staining showed that the expression of LINC00476 (red) was lower compared to ARIH2 (green) overexpression in PDX models. DAPI (blue) was used for nuclear counterstaining. (H) Schematic diagram of the effect of the LINC006476/VIM axis on the tumorigenicity and progression and metastasis of PDAC. P values are shown as* P <0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. All data represent the means ± SD from three independent assays.

Article Snippet: Briefly, for FISH, after counter staining with DAPI (Beyotime, Shanghai, China), slides were visualized under a Leica SP8 X confocal microscope (Leica, Germany).

Techniques: Injection, In Vivo, Over Expression, Control, Derivative Assay, Plasmid Preparation, Two Tailed Test, Staining, Immunohistochemistry, Expressing