Journal: Molecular Cancer Research

Article Title: RAIN Is a Novel Enhancer-Associated lncRNA That Controls RUNX2 Expression and Promotes Breast and Thyroid Cancer

doi: 10.1158/1541-7786.mcr-19-0564

Figure Lengend Snippet: Figure 7. Model of RAIN mechanism of action in controlling RUNX2 expression. A, Schematic representation of RAIN activ- ity in the organization of the RUNX2 genomiclocus. Aspreviouslyshown(21), theactivationoftheRUNX2P2promoter in thyroid and breast cancer cells requires the juxtaposition of the RUNX2 P2promotertooneproximal(ENH3)and two distal (ENH11 and ENH13) ENHs, which are brought together by the 3D looping of the chromatin. The transcrip- tional activation of each of the RUNX2 ENHs requires the binding of the TF AP1 and of the chromatin reader BRD4. RAIN is transcribed from two alternative TSS within ENH10 and ENH11, respectively. The 3D looping of the chromatin brings the 30end of l-RAIN in contact with the RUNX2 P2 promoter. In this region, l-RAINparticipatestoRUNX2expression regulation by at least two distinct mechanisms: (i) l-RAIN binds to WDR5 promoting its recruitment on the RUNX2 P2 promoter. WDR5 carrying MLL1 facil- itates the trimethylation of H3K4 trig- gering transcriptional activation and promoting RNA-PolII recruitment on the RUNX2 P2 promoter. (ii) l-RAIN seques- ters NELFe removing its binding on the RUNX2 promoter facilitating the transition of RNA-PolII into active transcription. Loss of RAIN expression disrupts this network resulting in a significant inhibition of RUNX2 expres- sion. B, Besides RUNX2, RAIN controls an independent panel of cancer- related TFs; we hypothesize with a similar molecular mechanism involving WDR5 and NELFe. Further experi- ments are needed to consolidate this second part of the model.

Article Snippet: Ten percent of total RNA was used as input sample, and 5 106 cells were used for each IP with 6 mg of NELFe (F-9, SC377052 Santa Cruz Biotechnology), 4 mg of WDR5 (A302-429A, Bethyl Laboratories), or BRD4 (A301-985A50, Bethyl Laboratories) antibodies, and the relative IgG control (mouse IgG SC2025 Santa Cruz Biotechnology and rabbit IgG 2729S; Cell Signaling Technology).

Techniques: Expressing, Activation Assay, Binding Assay, Inhibition

Journal: Cancer cell

Article Title: Oncogenic activation of the RNA binding protein NELFE and MYC signaling in hepatocellular carcinoma

doi: 10.1016/j.ccell.2017.06.002

Figure Lengend Snippet: Role of the RNA binding protein NELFE in hepatocellular carcinoma cells. (A) Kaplan-Meier survival analysis of LCI and TCGA-LIHC datasets based on segmentation values of NELFE (high: log2 >0.2, low: log2<0.2). (B–H) Cell proliferation rates measured by xCELLigence (B), colony formation (C), oncosphere formation measured by Algimatrix 3D assay (D), cell migration (E), cell invasion (F), cropped immunoblot (G), and proportions of cells in during G2/M phase measured by 7′AAD staining using flow cytometry (H) of Hep3B and Huh1 HCC cells after shNELFE or shCtrl via lentivirus. Statistical significance for the proliferation rate (B) is measured at time-point 72 hr, results shown in (E–H) were measured at 48 hr. For invasion assay (with matrigel), relative invasion index is calculated by normalizing to cells that have migrated (no matrigel). (I) Bioluminescence of NOD/SCID mice at eight weeks (middle panel). On the right panel, mean signal from shCtrl (n=4) or shNELFE (n=6). (J) Representative livers of three mice in each group. Arrows are pointing at tumor nodules. Scale bars, 1 cm. *p<0.05, **p<0.01, ***p<0.001. All data are mean ± SD. See also Figure S2.

Article Snippet: HHT4-SV40 cells were transfected with NELFE or vector lentivirus at MOI 5 (NELFE mGFP tagged ORF, Origene).

Techniques: RNA Binding Assay, Migration, Western Blot, Staining, Flow Cytometry, Invasion Assay

Journal: Cancer cell

Article Title: Oncogenic activation of the RNA binding protein NELFE and MYC signaling in hepatocellular carcinoma

doi: 10.1016/j.ccell.2017.06.002

Figure Lengend Snippet: NELFE enhances MYC tumorigenicity. (A) Bar graph of colony formation assay of HHT4 cells or HHT4 cells ectopically expressing the indicated proteins at day 10. (B) Representatie image and quantifiation of oncosphere formation assay at day 7 measured by Algimatrix 3D assay. Scale bar, 200 μM. (C) Proliferation rates of different cell lines up to 72 hr. (D) RT-PCR analysis of relative mRNA expression of MYC-related genes. (E) Hematoxylin and eosin and immunohistochemical staining of indicated tumors. Scale bars, 40 μM. (F) Number of tumor nodules four weeks aftr the injection of indicated cells. Short horizontal lines represent the mean. (G) RT-PCR analysis of relative mRNA expression of MYC-related genes in MYC or MYC+NELFE tumor tissues. Data were first normalized to β–actin to get dCt. Relative mRNA was then calculated using 2dCt. *p<0.05, **p<0.01. All data are mean ± SD. See also Figure S4.

Article Snippet: HHT4-SV40 cells were transfected with NELFE or vector lentivirus at MOI 5 (NELFE mGFP tagged ORF, Origene).

Techniques: Colony Assay, Expressing, Tube Formation Assay, Reverse Transcription Polymerase Chain Reaction, Immunohistochemical staining, Staining, Injection

Journal: Cancer cell

Article Title: Oncogenic activation of the RNA binding protein NELFE and MYC signaling in hepatocellular carcinoma

doi: 10.1016/j.ccell.2017.06.002

Figure Lengend Snippet: NELFE affects MYC-related genes by modulating MYC binding. (A) MYC transam assay on HCC cells treated with 48 hr of NELFE siRNA compared to scrm (*p<0.01). (B) ChIP-PCR of MYC-related genes of HCC cells after shNELFE compared shCtrl. Data is relative to 2% input. (C) ChIP-PCR of MYC-related genes of HHT4-SV40 cells overexpressed with Ctrl, NELFE, MYC or MYC+NELFE. Anti-MYC was used for IP. (D) ChIP-PCR of MYC-related genes in Hep3B cells after 48 hr of MYC siRNA or scrm. Anti-NELFE was used for IP. (E) Represented immunoblot of co-IP assay in HCC cells. MYC or Rabbit IgG was used for IP. All data are mean ± SD. See also Figure S6.

Article Snippet: HHT4-SV40 cells were transfected with NELFE or vector lentivirus at MOI 5 (NELFE mGFP tagged ORF, Origene).

Techniques: Binding Assay, Western Blot, Co-Immunoprecipitation Assay