Journal: Frontiers in Pharmacology

Article Title: Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer

doi: 10.3389/fphar.2022.890891

Figure Lengend Snippet: PFN1 is correlated with NSCLC metastasis and could promote NSCLC cell migration in vitro . (A) Representative IHC images of PFN1 expression on the NSCLC tissues. (B) The staining index of PFN1 on the tissue chip. ** p < 0.01. (C) Representative IHC images of PFN1 expression on the tissue chip. (D) The expression of PFN1 in TCGA LUAD data. ** p < 0.01. (E) The Kaplan–Meier survival analysis of PFN1 in NSCLC patients. (Data source: TCGA LUAD dataset) (F,G) Wound healing assays conducted to evaluate the migration ability of PFN1 -overexpressing (F) and PFN1 knockdown (KD) (G) H1299 cells. ** p < 0.01; scale bar, 500 μm. (H,I) Transwell migration assays conducted to evaluate the migration of PFN1 -overexpressing (H) and PFN1 KD (I) H1299 cells. ** p < 0.01; scale bar, 500 μm. EV, empty vector; OE, PFN1 overexpression; NC, negative control; si-1/ 2, PFN1 siRNA1 1/2.

Article Snippet: PFN1 siRNA and control scramble siRNA were synthesized by Guangzhou RiboBio Co. (Guangzhou, China), and the sequences are listed in .

Techniques: Migration, In Vitro, Expressing, Staining, Knockdown, Plasmid Preparation, Over Expression, Negative Control

Journal: Frontiers in Pharmacology

Article Title: Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer

doi: 10.3389/fphar.2022.890891

Figure Lengend Snippet: PFN1 could promote MVs secretion in NSCLC. (A) Heatmap of differentially expressed proteins between EV and PFN1 OE cells. (B) GO enrichment analysis of differentially expressed proteins. (C) COG/KOG analysis of differentially expressed proteins. (D) MVs extracted from EV-expressing and PFN1 -overexpressing cells, using continuous differential centrifugation, identified using transmission electron microscopy. Scale bar, 100 nm. (E,F) Flow cytometry (E) and western blotting (F) were used to quantify MVs in PFN1 -overexpressing and EV-expressing cells. ARF6 and actin were used as MV markers. (G) Expression of PFN1 and annexin A1 in lung tumor tissues detected using immunofluorescence. (H) The staining index of p-MLC on the tissue chip. ** p < 0.01. (I) Representative IHC images of p-MLC expression. (J) Spearman rank correlation analysis was used to assess the relationship between PFN1 and p-MLC expression on the tissue chip; p and r values are shown in the plot.

Article Snippet: PFN1 siRNA and control scramble siRNA were synthesized by Guangzhou RiboBio Co. (Guangzhou, China), and the sequences are listed in .

Techniques: Expressing, Centrifugation, Transmission Assay, Electron Microscopy, Flow Cytometry, Western Blot, Immunofluorescence, Staining

Journal: Frontiers in Pharmacology

Article Title: Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer

doi: 10.3389/fphar.2022.890891

Figure Lengend Snippet: MVs derived from PFN1 OE cells promote migration in NSCLC cells. (A) MVs collected from sera of patients with NSCLC quantified using flow cytometry. ** p < 0.01. (B) Protein expression of ARF6 and β-actin in MVs collected from sera of patients with NSCLC detected using western blotting. (C) Effect of PFN1 -overexpressing cell supernatants on cell migration evaluated through wound healing assays. ** p < 0.01; scale bar, 500 μm. (D) PKH67-labeled MVs taken up by H1299 cells. DAPI was used to stain the nuclei of H1299 cells. Scale bar, 500 μm. (E,F) Wound healing (E) and Transwell migration (F) assays conducted to evaluate the migration of H1299 cells after treatment with MVs derived from EV-expressing and PFN1 -overexpressing cells; ** p < 0.01; scale bar, 500 μm.

Article Snippet: PFN1 siRNA and control scramble siRNA were synthesized by Guangzhou RiboBio Co. (Guangzhou, China), and the sequences are listed in .

Techniques: Derivative Assay, Migration, Flow Cytometry, Expressing, Western Blot, Labeling, Staining

Journal: Frontiers in Pharmacology

Article Title: Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer

doi: 10.3389/fphar.2022.890891

Figure Lengend Snippet: PFN1 promotes in vivo NSCLC metastasis by elevating MV secretion. (A) Schematic illustration of the mouse model of metastatic tumor established to determine the role of PFN1 in tumor metastasis. (B) Body weight changes in mice after intracardiac injection of PFN1 -overexpressing and EV-expressing cell lines. (C,D) Representative images of lung (C) and liver (D) metastases of the mouse model. The number of metastases is displayed in the right-hand side graph. * p < 0.05, ** p < 0.01. (E) Representative images of HE-stained lung tissues of the mouse model. (F) Representative IHC images of PFN1 and p-MLC expression in lung tissues. The staining index is shown in the right-hand side graph. ** p < 0.01. (G) Representative images of HE-stained liver tissues of the mouse model. (H) Representative IHC images of PFN1 and p-MLC expression in liver tissues. The staining index is shown in the right-hand side graph. ** p < 0.01. (I) Body weight changes in mice after intracardiac injection of H1299 cells and MVs. (J) Representative images of lung metastases of the mouse model. The number of metastases is shown in the bottom graph. * p < 0.05. (K) Representative images of HE-stained lung tissues of the mouse model. (L) Representative IHC images of PFN1 and p-MLC expression in lung tissues. The staining index is shown in the right-hand side graph; * p < 0.05.

Article Snippet: PFN1 siRNA and control scramble siRNA were synthesized by Guangzhou RiboBio Co. (Guangzhou, China), and the sequences are listed in .

Techniques: In Vivo, Injection, Expressing, Staining

Journal: Frontiers in Pharmacology

Article Title: Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer

doi: 10.3389/fphar.2022.890891

Figure Lengend Snippet: Mechanisms underlying the promotion of MLC phosphorylation by PFN1. (A,B) Protein expression after PFN1 overexpression (A) and knockdown (B) measured using western blotting. (C) Protein expression in PFN1 mutants measured using western blotting. (D) PFN1 interactions with ROCK1/2 confirmed using co-IP. (E) Protein expression after treatment with Y27632 (10 µM) measured using western blotting. (F) Effect of PFN1 on ROCK1 activity. ** p < 0.01. (G) Effect of PFN1 on ROCK2 activity. (H) Flow cytometry measuring changes in the amount of MVs after treatment with Y27632; * p < 0.05.

Article Snippet: PFN1 siRNA and control scramble siRNA were synthesized by Guangzhou RiboBio Co. (Guangzhou, China), and the sequences are listed in .

Techniques: Phospho-proteomics, Expressing, Over Expression, Knockdown, Western Blot, Co-Immunoprecipitation Assay, Activity Assay, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer

doi: 10.3389/fphar.2022.890891

Figure Lengend Snippet: ROCK1 inhibitor Y27632 partially reversed the promotion of lung cancer metastasis by PFN1 in vitro and in vivo . (A,B) Wound healing assays conducted to evaluate the effect of Y27632 (A) and Y27632 combined with MVs (B) on cell migration. ** p < 0.01; scale bar, 500 μm. (C) Transwell migration assays conducted to evaluate the effect of Y27632 and Y27632 combined with MVs on cell migration. ** p < 0.01; scale bar, 500 μm. (D) Schematic diagram of the mouse model of metastatic tumor established to determine the effect of Y27632 on PFN1-induced lung cancer metastasis. (E) Body weight changes in mice after intracardiac injection of PFN1 -overexpressing H1299 cells and intraperitoneal injection of Y27632 (10 mg/kg). (F) Representative images of lung and liver metastatic tissue in mice. The number of metastatic nodules is shown in the right-hand side graph. * p < 0.05. (G,H) Representative images of HE-stained lung (G) and liver (H) metastases. (I) Representative IHC images of PFN1 and p-MLC expression in lung tissues. The staining index is shown in the right-hand side graph. ** p < 0.01. (J) Representative IHC images of PFN1 and p-MLC expression in liver tissues. The staining index is shown in the right-hand side graph; ** p < 0.01.

Article Snippet: PFN1 siRNA and control scramble siRNA were synthesized by Guangzhou RiboBio Co. (Guangzhou, China), and the sequences are listed in .

Techniques: In Vitro, In Vivo, Migration, Injection, Staining, Expressing

Journal: Frontiers in Pharmacology

Article Title: Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer

doi: 10.3389/fphar.2022.890891

Figure Lengend Snippet: Schematic diagram of the role of PFN1 in NSCLC metastasis. In the initiation stage of NSCLC, cells with upregulated PFN1 secret more MVs through PFN1 interactions with the ROCK/p-MLC pathway. These MVs contain numerous oncogenenic moleculars, which could enhance migration abilities of PFN1 normal expressed NSCLC cells, and untimately promote progression and metastasis of NSCLC.

Article Snippet: PFN1 siRNA and control scramble siRNA were synthesized by Guangzhou RiboBio Co. (Guangzhou, China), and the sequences are listed in .

Techniques: Migration

Journal: International Journal of Oncology

Article Title: SERP1 is a novel marker of poor prognosis in pancreatic ductal adenocarcinoma patients via anti-apoptosis and regulating SRPRB/NF-κB axis

doi: 10.3892/ijo.2017.4111

Figure Lengend Snippet: Flow chart of the derivation and function analysis of stress associated endoplasmic reticulum protein 1 (SERP1). (A) Flow chart. (B) Heat map of differentially expressed mRNAs in pancreatic ductal adenocarcinoma (PDAC) from GEO profiles database. The 10 mRNAs with the most significant difference are shown.

Article Snippet: The small interfering RNA targeting SERP1 and its negative control (NC) sequences were designed and synthesized by GenePharma Biotech, Co., Ltd. (Shanghai, China).

Techniques:

Journal: International Journal of Oncology

Article Title: SERP1 is a novel marker of poor prognosis in pancreatic ductal adenocarcinoma patients via anti-apoptosis and regulating SRPRB/NF-κB axis

doi: 10.3892/ijo.2017.4111

Figure Lengend Snippet: Expression level of the stress associated endoplasmic reticulum protein 1 (SERP1) in pancreatic ductal adenocarcinoma (PDAC) tissues is upregulated. (A) SERP1 was upregulated in six pairs of PDAC tissues compared with adjacent normal tissues. (B) The expression of SERP1 in eight pairs of PDAC tissues was upregulated compared with adjacent normal tissues from GEO database. (C and D) The Human Protein Atlas database results revealed that the expression level of SERP1 were upregulated in PDAC tissues compared with normal tissues. (E) Amplification of SERP1 was found in various tumors, including PDAC.

Article Snippet: The small interfering RNA targeting SERP1 and its negative control (NC) sequences were designed and synthesized by GenePharma Biotech, Co., Ltd. (Shanghai, China).

Techniques: Expressing, Amplification

Journal: International Journal of Oncology

Article Title: SERP1 is a novel marker of poor prognosis in pancreatic ductal adenocarcinoma patients via anti-apoptosis and regulating SRPRB/NF-κB axis

doi: 10.3892/ijo.2017.4111

Figure Lengend Snippet: Correlation between expression level of the stress associated endoplasmic reticulum protein 1 (SERP1) and progression of pancreatic ductal adenocarcinoma (PDAC) patients. (A and B) The expression level of SERP1 in T3/4 stage PDAC specimen was significantly higher than in T1/2 stage, while there was no significant difference between SERP1 and N stage. (C) Correlation analysis was performed between expression level of SERP1 and clinical stages. SERP1 was correspondingly increased with the increase of clinical AJCC stage of PDAC. (D) Constituent ratio with high level of SERP1 was drastically different between normal samples and I/II or III/IV stage of PDAC patients.

Article Snippet: The small interfering RNA targeting SERP1 and its negative control (NC) sequences were designed and synthesized by GenePharma Biotech, Co., Ltd. (Shanghai, China).

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: SERP1 is a novel marker of poor prognosis in pancreatic ductal adenocarcinoma patients via anti-apoptosis and regulating SRPRB/NF-κB axis

doi: 10.3892/ijo.2017.4111

Figure Lengend Snippet: The correlation between expression level of the stress associated endoplasmic reticulum protein 1 (SERP1) and prognosis of pancreatic ductal adenocarcinoma (PDAC) patients. (A) K-M curve for OS of patients with high (n=89) and low (n=87) SERP1 expression level. Results revealed that high SERP1 expression level was correlated with shorter OS for PDAC patients. (B) SERP1 high expression group (n=61) had shorter DFS than SERP1 low expression group (n=76). (C) The expression of SERP1 in OS-good and poor patients. (D) The expression of SERP1 in DFS-good and poor patients. (E and F) Multivariate Cox regression analyses showed SERP1 expression was an independent factor of patients' overall survival and disease-free survival.

Article Snippet: The small interfering RNA targeting SERP1 and its negative control (NC) sequences were designed and synthesized by GenePharma Biotech, Co., Ltd. (Shanghai, China).

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: SERP1 is a novel marker of poor prognosis in pancreatic ductal adenocarcinoma patients via anti-apoptosis and regulating SRPRB/NF-κB axis

doi: 10.3892/ijo.2017.4111

Figure Lengend Snippet: Stress associated endoplasmic reticulum protein 1 (SERP1) is mainly involved in regulating cell apoptosis. (A) Apoptosis gene sets were enriched in tumor group. The curve is the enrichment score of these genes by GSEA. (B) PANC-1 cells were transfected with three SERP1 siRNA. Forty-eight hours after transfection, the expression level of SERP1 was detected by western blotting, and results revealed that the expression level of SERP1 was obviously downregulated in PANC-1 cells transfected with SERP1-siRNA compared to negative control cells. (C and D) Compared to control group, PANC-1 cells transfected with SERP1-siRNA had a higher rate of apoptosis.

Article Snippet: The small interfering RNA targeting SERP1 and its negative control (NC) sequences were designed and synthesized by GenePharma Biotech, Co., Ltd. (Shanghai, China).

Techniques: Transfection, Expressing, Western Blot, Negative Control, Control

Journal: International Journal of Oncology

Article Title: SERP1 is a novel marker of poor prognosis in pancreatic ductal adenocarcinoma patients via anti-apoptosis and regulating SRPRB/NF-κB axis

doi: 10.3892/ijo.2017.4111

Figure Lengend Snippet: Protein-protein interaction (PPI) and GO analyses for interaction genes with the stress associated endoplasmic reticulum protein 1 (SERP1). (A and B) PPI analysis of interaction genes with SERP1. SRP receptor β subunit (SRPRB) was the main node protein for the interaction network. (C) GO analysis of the interaction gene with SERP1, and these genes were mainly involved in regulating cell death and apoptosis.

Article Snippet: The small interfering RNA targeting SERP1 and its negative control (NC) sequences were designed and synthesized by GenePharma Biotech, Co., Ltd. (Shanghai, China).

Techniques:

Journal: International Journal of Oncology

Article Title: SERP1 is a novel marker of poor prognosis in pancreatic ductal adenocarcinoma patients via anti-apoptosis and regulating SRPRB/NF-κB axis

doi: 10.3892/ijo.2017.4111

Figure Lengend Snippet: Co-expression genes with SERP1 from cBioPortal database.

Article Snippet: The small interfering RNA targeting SERP1 and its negative control (NC) sequences were designed and synthesized by GenePharma Biotech, Co., Ltd. (Shanghai, China).

Techniques:

Journal: International Journal of Oncology

Article Title: SERP1 is a novel marker of poor prognosis in pancreatic ductal adenocarcinoma patients via anti-apoptosis and regulating SRPRB/NF-κB axis

doi: 10.3892/ijo.2017.4111

Figure Lengend Snippet: Downregulated stress associated endoplasmic reticulum protein 1 (SERP1) promotes apoptosis via increasing SRP receptor β subunit (SRPRB) expression. (A and B) The Human Protein Atlas database results revealed that the expression level of SRPRB were upregulated in stromal fibroblast compared with tumor tissues. (C) Western blot analysis confirmed that downregulated SERP1 can increase the expression of SRPRB in PANC-1 cells. (D) PANC-1 cells were transfected with SRPRB overexpression plasmid, and western blot analysis found that the expression level of SRPRB was significantly increased in PANC-1 cells. (E and F) Compared to control group, SRPRB overexpression significantly promoted apoptosis of PANC-1 cells.

Article Snippet: The small interfering RNA targeting SERP1 and its negative control (NC) sequences were designed and synthesized by GenePharma Biotech, Co., Ltd. (Shanghai, China).

Techniques: Expressing, Western Blot, Transfection, Over Expression, Plasmid Preparation, Control

Journal: International Journal of Oncology

Article Title: SERP1 is a novel marker of poor prognosis in pancreatic ductal adenocarcinoma patients via anti-apoptosis and regulating SRPRB/NF-κB axis

doi: 10.3892/ijo.2017.4111

Figure Lengend Snippet: Downregulated stress associated endoplasmic reticulum protein 1 (SERP1) promotes cell apoptosis via regulating SRP receptor β subunit (SRPRB) associated NF-κB activation. (A) NF-κB signaling pathway gene sets were enriched in the tumor group. (B) Correlation analysis revealed that the expression level of SERP1 positively correlated with NF-κB expression in 178 pancreatic ductal adenocarcinoma (PDAC) patients from TCGA database. (C) The expression level of SRPRB was negatively correlated with NF-κB expression in 178 PDAC patients from TCGA database. (D) Western blotting confirmed that downregulated SERP1 could suppress the expression of NF-κB and the phosphorylation NF-κB in PANC-1 cells.

Article Snippet: The small interfering RNA targeting SERP1 and its negative control (NC) sequences were designed and synthesized by GenePharma Biotech, Co., Ltd. (Shanghai, China).

Techniques: Activation Assay, Expressing, Western Blot, Phospho-proteomics

Journal: Cell Death and Differentiation

Article Title: A novel LncRNA HITT forms a regulatory loop with HIF-1α to modulate angiogenesis and tumor growth

doi: 10.1038/s41418-019-0449-8

Figure Lengend Snippet: HITT prevents tumor growth in vivo by inhibiting angiogenesis in a HIF-1α dependent manner. The expression levels of HITT in human colorectal tumors (T) of different stages (B) relative to their paired adjacent normal controls (N) (n = 46, a, b) were determined by qRT-PCR. Tumor volumes at the indicated dates (c), as well as images (left) and tumor weights (right) at 4 weeks (d) of HCT116/vector (Vect.), HIC116/HITT, HCT116/(Vect. + si-HIF-1α), and HCT116/(HITT + si-HIF-1α) xenografts. The average values are present as bar graphs (means ± SD) (n = 6 for each group). Representative immunohistochemistry staining for Ki-67 (top) and CD31 (bottom) in the indicated xenografts (e), and the quantification of bar graphs (f). g HIF-1α and VEGF protein (upper panel) and VEGF mRNA (lower panel) levels were measured by WB and qRT-PCR, respectively, in HCT116 xenografts. Representative images are presented. h, i VEGF protein levels were measured by ELISA in clone stably transfected with HITT and vector controls after HIF-1α knockdown (KD) or ectopic HIF-1α expression under hypoxia. The KD efficiency of HIF-1α was confirmed by WB. j Representative images of HUVECs are presented (left) and quantification of HUVEC tube formation assays is presented in the bar graph (right). Data derived from three independent experiments are presented as mean ± SEM in the bar graph. Values in controls were normalized to 1 (a, f, g). *P < 0.05; **P < 0.01; ##P < 0.01, compared with hypoxia-treated controls (h–j). Vect. vector control, Ctl. control. Scale bar 50 μm. See also Fig. S1–S3

Article Snippet: siRNA specifically targeting HITT, YB-1, HIF-1α, HIF-2α, or Ago2 , and nonspecific si-scramble control were synthesized by GenePharma (Shanghai, China).

Techniques: In Vivo, Expressing, Quantitative RT-PCR, Plasmid Preparation, Immunohistochemistry, Staining, Enzyme-linked Immunosorbent Assay, Stable Transfection, Transfection, Knockdown, Derivative Assay, Control

Journal: Cell Death and Differentiation

Article Title: A novel LncRNA HITT forms a regulatory loop with HIF-1α to modulate angiogenesis and tumor growth

doi: 10.1038/s41418-019-0449-8

Figure Lengend Snippet: HITT inhibits HIF-1α expression and activity. HIF-1α protein was detected by WB of lysates from control and HITT stably transfected lines (a). Quantification of WB bands was conducted by ImageJ (b). HIF-1α mRNA relative to 18 s was detected by qRT-PCR (lower panel, b). The KD efficiency of HITT was confirmed by qRT-PCR (c). HIF-1α protein levels were measured by WB after HITT KD under normoxia in the presence or absence of proteasome inhibitor MG132 (d). Quantification of WB bands was conducted by ImageJ (e). HIF-1α mRNA relative to 18 s was detected by qRT-PCR (lower panel, e). Hypoxic response element (HRE) luciferase activity (f), and expression levels of VEGF and Angiogenin-1 (g), were measured by luciferase reporter assay or qRT-PCR, respectively, under the indicated conditions. Data derived from three independent experiments are presented as mean ± SEM in the bar graph. Values in controls were normalized to 1 (b, c, e–g). *P < 0.05; **P < 0.01; ##P < 0.01, compared with hypoxia-treated controls (b, f–g). Vect. vector control. See also Fig. S4

Article Snippet: siRNA specifically targeting HITT, YB-1, HIF-1α, HIF-2α, or Ago2 , and nonspecific si-scramble control were synthesized by GenePharma (Shanghai, China).

Techniques: Expressing, Activity Assay, Control, Stable Transfection, Transfection, Quantitative RT-PCR, Luciferase, Reporter Assay, Derivative Assay, Plasmid Preparation

Journal: Cell Death and Differentiation

Article Title: A novel LncRNA HITT forms a regulatory loop with HIF-1α to modulate angiogenesis and tumor growth

doi: 10.1038/s41418-019-0449-8

Figure Lengend Snippet: HITT inhibits HIF-1α translation. a Representative images of HIF-1α protein levels detected by WB (left) and quantification of HIF-1α band intensities by ImageJ (right) for vector control and HITT ectopic expression HCT116 cells, in the presence of CHX, for the indicate time periods under hypoxia. HIF-1α levels in the CHX untreated group were normalized to 1. b HIF-1α protein levels were determined by WB after ectopic HITT expression in the presence or absence of MG132 in HCT116 and HeLa cells under hypoxia. c, d Polysomes in cytoplasmic extracts were fractionated through sucrose gradients, and mRNA levels of HIF-1α and GAPDH in HCT116 cells were measured by qRT-PCR in gradient fractions after HITT overexpression or KD. Representative data, as a percentage of total RNA of interest in the gradient from three independent experiments, are presented. e, f Affinity purification of biotinylated l-azidohomoalanine (AHA)-labeled acutely synthesized proteins from cell lysates. Purified HIF-1α and α-tubulin proteins were detected by WB

Article Snippet: siRNA specifically targeting HITT, YB-1, HIF-1α, HIF-2α, or Ago2 , and nonspecific si-scramble control were synthesized by GenePharma (Shanghai, China).

Techniques: Plasmid Preparation, Control, Expressing, Quantitative RT-PCR, Over Expression, Affinity Purification, Labeling, Synthesized, Purification

Journal: Cell Death and Differentiation

Article Title: A novel LncRNA HITT forms a regulatory loop with HIF-1α to modulate angiogenesis and tumor growth

doi: 10.1038/s41418-019-0449-8

Figure Lengend Snippet: HITT inhibits HIF-1α translation by competing with YB-1 for binding with HIF-1α 5′-UTR. a The expression levels of HIF-1α were measured by WB in HITT stable lines, with or without YB-1 KD, under hypoxia in HCT116 and HeLa cells. The KD efficiency of YB-1 was confirmed by WB. b Schematic of the positions of high-stringency YB-1 binding sites in HITT predicted by a bioinformatics online tool (http://rbpmap.technion.ac.il/). c Biotin-HITT pull-down using HeLa cell lysates showed that YB-1 bands were present in Biotin-HITT precipitates. YB-1-associated HITT (d) and HIF-1α 5′-UTR (e, f), as measured by qRT-PCR following CLIP YB-1 after HITT overexpression or KD, under normoxia or hypoxia, in HCT116 cells, as indicated in the figures. GAPDH mRNA and CLIP IgG were used as negative controls. g Schematic of the positions of high-stringency YB-1 binding sites in HITT (left). CLIP assay was used to detect the binding of YB-1 with full-length or fragmented HITT in 4T1 cells, a cell line with no endogenous HITT, after ectopic expression of the indicated HITT. CLIP IgG and 18 s were used as negative controls (lower panel). Similar expression levels of HITT were confirmed by qRT-PCR (upper panel). h Schematic of full-length and fragmented HITT, and the predicted YB-1 binding sites (upper panel). HIF-1α protein levels were measured by WB after transfection with full-length (1–2050) HITT and MT (1–1029) HITT (lower right panel). Similar expression levels of HITT were confirmed by qRT-PCR (lower left panel). i Schematic of positions of all potential YB-1 binding sites in HIF-1α 5′-UTR predicted by a bioinformatics online tool (http://rbpmap.technion.ac.il/)(Left). Luciferase reporter containing HIF-1α 5′-UTR (1–256) or two truncated mutants (1–138: M1 and 139–256: M2) was used to assess translational efficiency after HITT overexpression with or without YB-1 KD (right). Data derived from three independent experiments are presented as mean ± SEM in the bar graph (d–i). Values in controls were normalized to 1. **P < 0.01 (d–i); #P < 0.05, ##P < 0.01, compared with hypoxia-treated controls (d, e, and i). N.S. not significant (d–g). See also Fig. S5

Article Snippet: siRNA specifically targeting HITT, YB-1, HIF-1α, HIF-2α, or Ago2 , and nonspecific si-scramble control were synthesized by GenePharma (Shanghai, China).

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Over Expression, Transfection, Luciferase, Derivative Assay

Journal: Cell Death and Differentiation

Article Title: A novel LncRNA HITT forms a regulatory loop with HIF-1α to modulate angiogenesis and tumor growth

doi: 10.1038/s41418-019-0449-8

Figure Lengend Snippet: HIF-1α lowers HITT’s stability via negative feedback. a Hypoxia-responsive lncRNAs reported previously and HITT were measured by qRT-PCR under hypoxia relative to normoxia. HIF-1α expression was determined by WB, and HITT was measured by qRT-PCR in samples derived from cells subjected to dose- (b) and time-dependent (c) hypoxia treatment. The expression levels of HITHITT and HITT promoter luciferase reporter activity were detected by qRT-PCR and luciferase reporter assay, respectively, in HCT116 cells after HIF-1α KD (d) or overexpression (e) under hypoxia. The efficiency of HIF-1α overexpression and KD was confirmed by WB. f The half-lives of HITT and GAPDH mRNA were measured by qRT-PCR in the presences of ActD in HCT116 cells. Data derived from three independent experiments are represented as mean ± SEM in the bar graph. *P < 0.05, **P < 0.01, N.S. not significant. See also Fig. S6

Article Snippet: siRNA specifically targeting HITT, YB-1, HIF-1α, HIF-2α, or Ago2 , and nonspecific si-scramble control were synthesized by GenePharma (Shanghai, China).

Techniques: Quantitative RT-PCR, Expressing, Derivative Assay, Luciferase, Activity Assay, Reporter Assay, Over Expression

Journal: Cell Death and Differentiation

Article Title: A novel LncRNA HITT forms a regulatory loop with HIF-1α to modulate angiogenesis and tumor growth

doi: 10.1038/s41418-019-0449-8

Figure Lengend Snippet: HIF-1α promotes HITT decay via MiR-205-mediated RNA degradation. a MiR-205 was predicted to be a potent microRNA that can bind HITT by two independent bioinformatics tools: http://www.mircode.org/index.php and http://annolnc.cbi.pku.edu.cn/index.jsp. b Expression levels of MiR-205 normalized to U6 were measured by qRT-PCR after HIF-1α KD under hypoxia. Relative expression levels of HITT were determined by qRT-PCR after transfection of MiR-205 inhibitors (Inh., c) or MiR-205 mimics (d) into HCT116 cells. MiR-205 levels normalized to U6 were measured by qRT-PCR. e The expression levels of HITT were determined by qRT-PCR in HCT116 cells after HIF-1α KD with or without MiR-205 inhibitor treatment. f–h Schematic of the hypothetical duplexes formed by interactions between the binding site in HITT (top), MiR-205 (middle), and mutated HITT (bottom) (f). The activities of wild type (WT) or MiR-205-binding defective mutant (MT) HITT luciferase reporters were detected in HCT116 cells after transfection of MiR-205 inhibitor (Inh., g) or mimics (h) under normoxia or hypoxia, as indicated in the figures. i Biotinylated precursor MiR-205 was transfected into HCT116 cells. MiR-205-associated endogenous HITT relative to total HITT was determined by qRT-PCR following CLIP biotin. CLIP GAPDH and nonbiotinylated MiR-205 were used as negative controls. j The expression levels of HITT were determined by qRT-PCR in HCT116 cells after Ago2 KD (right). KD efficiency was confirmed by WB (left). k Ago2-associated HITT levels relative to total HITT were determined by qRT-PCR following formaldehyde cross-linked IP of Ago2 in HCT116 cells under normoxia and hypoxia, with or without treatment with MiR-205 inhibitors (Inh.). GAPDH and IgG were used as negative controls. l Relative expression levels of HITT determined by qRT-PCR after treatment with MiR-205 mimics in control and Ago2 KD HCT116 cells. m HITT’s half-life was determined by qRT-PCR after treatment with MiR-205 inhibitor (Inh.) alone, Ago2 KD alone, or a combination of the two in the presences of RNA synthesis inhibitor ActD under hypoxia in HCT116 cells. Data derived from three independent experiments are presented as mean ± SEM in the bar graph. Values in controls were normalized to 1. *P < 0.05; **P < 0.01; #P < 0.05, compared with hypoxia treatment controls (c, e, g, k). N.S. not significant (e, g–i, k, l). See also Fig. S7

Article Snippet: siRNA specifically targeting HITT, YB-1, HIF-1α, HIF-2α, or Ago2 , and nonspecific si-scramble control were synthesized by GenePharma (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Binding Assay, Mutagenesis, Luciferase, Control, Derivative Assay

Journal: Cell Death and Differentiation

Article Title: A novel LncRNA HITT forms a regulatory loop with HIF-1α to modulate angiogenesis and tumor growth

doi: 10.1038/s41418-019-0449-8

Figure Lengend Snippet: The association of HITT with HIF-1α and VEGF levels in human colon cancer tissues. a Representative image of HIF-1α protein expression in human colon cancer tissue samples (T) and paired adjacent normal controls (N). The average fold-change of HIF-1α protein (b) and VEGF mRNA (c) levels normalized to 18S rRNA were detected by qRT-PCR and WB in human colorectal cancer tissue samples, (T) and paired adjacent normal controls (n = 46). Correlation analysis of HITT with HIF-1α protein (d) and VEGF mRNA (e). f Schematic diagram of the proposed HITT–HIF-1α feedback loop that controls hypoxia-induced angiogenesis and tumor growth. Under normoxia, HITT does not undergo HIF-1α/MiR-205-mediated destabilization. Thus, HITT acts as a molecular decoy for YB-1, such that it is associated with YB-1 via favorable binding sequences, titrating YB-1 away from HIF-1α 5′-UTR, resulting in the attenuation of HIF-1α protein translation. Under hypoxia, HITT is targeted by Ago2/MiR-205 for degradation in a HIF-1α-dependent manner, consequently diminishing the inhibitory effect of HITT on HIF-1α transcription, resulting in angiogenesis and tumor growth

Article Snippet: siRNA specifically targeting HITT, YB-1, HIF-1α, HIF-2α, or Ago2 , and nonspecific si-scramble control were synthesized by GenePharma (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Binding Assay

Journal: Virologica Sinica

Article Title: HSV-2-encoded miRNA-H4 Regulates Cell Cycle Progression and Act-D-induced Apoptosis in HeLa Cells by Targeting CDKL2 and CDKN2A

doi: 10.1007/s12250-019-00101-8

Figure Lengend Snippet: Production of HSV-2 LAT-encoded miRNA-H4. A Schematic of the HSV-2 genome expanded with details of the LAT locus. miRNA-H4 maps with antisense to the noncoding region of ICP34.5. L long, S short, IR internal repeat, TR terminal repeat, UL unique long region, US unique short region. B Secondary structure adopted by pri-miRNA precursor of miRNA-H4 which includes miRNA-H4-5p and miRNA-H4-3p according to the miRbase.

Article Snippet: miRNA-H4-5p mimics (MC-5p), miRNA-H4-5p inhibitor (MI-5p), miRNA-H4-3p inhibitor (MI-3p), miRNA inhibitor negative control (MI-NC), miRNA-H4 negative control (miRNA-NC), siRNAs against human CDKN2A (siRNA-p16), and CDKL2 (siRNA-p65) were all synthesized and purified by RiboBio (Guangzhou, China). siRNAs targeting nonspecific sequences were used as negative control (siRNA-NC). miRNA and siRNAs were all transfected using Lipofectamine 2000 (Invitrogen).

Techniques:

Journal: Virologica Sinica

Article Title: HSV-2-encoded miRNA-H4 Regulates Cell Cycle Progression and Act-D-induced Apoptosis in HeLa Cells by Targeting CDKL2 and CDKN2A

doi: 10.1007/s12250-019-00101-8

Figure Lengend Snippet: miRNA expression and Act-D-induced apoptosis in HeLa-H4 cells. A Different cell lines were subjected to qRT-PCR to detect the relative expression of miRNA-H4-5p. B Different cell lines were subjected to qRT-PCR to detect the relative expression of miRNA-H4-3p. C The activity of caspase-3 in HeLa cells transfected with or without transfection, accompanied by Act-D treatment. D Apoptosis was assessed using FAM in HeLa cells with or without transfection, accompanied by Act-D treatment. E HeLa cells with or without transfection treated with Hoechst 33342 were imaged under a fluorescent microscope. Cells with bright blue nucleus were recognized as apoptotic.

Article Snippet: miRNA-H4-5p mimics (MC-5p), miRNA-H4-5p inhibitor (MI-5p), miRNA-H4-3p inhibitor (MI-3p), miRNA inhibitor negative control (MI-NC), miRNA-H4 negative control (miRNA-NC), siRNAs against human CDKN2A (siRNA-p16), and CDKL2 (siRNA-p65) were all synthesized and purified by RiboBio (Guangzhou, China). siRNAs targeting nonspecific sequences were used as negative control (siRNA-NC). miRNA and siRNAs were all transfected using Lipofectamine 2000 (Invitrogen).

Techniques: Expressing, Quantitative RT-PCR, Activity Assay, Transfection, Microscopy

Journal: Virologica Sinica

Article Title: HSV-2-encoded miRNA-H4 Regulates Cell Cycle Progression and Act-D-induced Apoptosis in HeLa Cells by Targeting CDKL2 and CDKN2A

doi: 10.1007/s12250-019-00101-8

Figure Lengend Snippet: Validation of miRNA-H4-5p targets. A Sequence alignment between miRNA-H4-5p and 3′-UTR of CDKN2A or CDKL2. Binding sites are indicated in dotted lines and the corresponding mutants are labeled in solid lines. All complementary pairing bases are indicated in the vertical curve. B, C Changes in luciferase enzyme viability. HeLa cells co-transfected with luciferase reporter containing either wild type (WT) or mutant type (mut) 3′-UTR of (B) CDKN2A or (C) CDKL2 along with MC-5p or miRNA-H4 negative control. Relative viability of luciferase enzyme = Renilla reniformis/firefly luciferase. D Effects of MC-5p or MI-5p on the expression of CDKN2A and CDKL2 in HeLa cells were examined using Western blot analysis with GAPDH as the internal control. Differences were judged to be statistically significant when *P < 0.05, **P < 0.01. E, F Expression of CDKN2A and CDKL2 mRNA was analyzed using qRT-PCR. G The effects of siRNAs on cell proliferation were determined by MTT assay 24 h and 36 h after transfection. H The relative number of EdU positive cells was estimated from siRNA-p16 and siRNA-p65 transfections, cells with positive EdU were found to have accumulated in the S phase.

Article Snippet: miRNA-H4-5p mimics (MC-5p), miRNA-H4-5p inhibitor (MI-5p), miRNA-H4-3p inhibitor (MI-3p), miRNA inhibitor negative control (MI-NC), miRNA-H4 negative control (miRNA-NC), siRNAs against human CDKN2A (siRNA-p16), and CDKL2 (siRNA-p65) were all synthesized and purified by RiboBio (Guangzhou, China). siRNAs targeting nonspecific sequences were used as negative control (siRNA-NC). miRNA and siRNAs were all transfected using Lipofectamine 2000 (Invitrogen).

Techniques: Biomarker Discovery, Sequencing, Binding Assay, Labeling, Luciferase, Transfection, Mutagenesis, Negative Control, Expressing, Western Blot, Control, Quantitative RT-PCR, MTT Assay

Journal: Virologica Sinica

Article Title: HSV-2-encoded miRNA-H4 Regulates Cell Cycle Progression and Act-D-induced Apoptosis in HeLa Cells by Targeting CDKL2 and CDKN2A

doi: 10.1007/s12250-019-00101-8

Figure Lengend Snippet: miRNA expression and its regulatory function on target genes in HSV-2-infected HeLa cells. A Expression of miRNA-H4-5p in different cell lines after HSV-2 infection were detected using qRT-PCR. B Expression of CDKN2A and CDKL2 in different cell lines after HSV-2 infection were examined using Western blot analysis with GAPDH as the internal control.

Article Snippet: miRNA-H4-5p mimics (MC-5p), miRNA-H4-5p inhibitor (MI-5p), miRNA-H4-3p inhibitor (MI-3p), miRNA inhibitor negative control (MI-NC), miRNA-H4 negative control (miRNA-NC), siRNAs against human CDKN2A (siRNA-p16), and CDKL2 (siRNA-p65) were all synthesized and purified by RiboBio (Guangzhou, China). siRNAs targeting nonspecific sequences were used as negative control (siRNA-NC). miRNA and siRNAs were all transfected using Lipofectamine 2000 (Invitrogen).

Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot, Control