Long noncoding RNA PiHL regulates p53 protein stability through GRWD1/RPL11/MDM2 axis in colorectal cancer

We identified a novel long noncoding RNA (lncRNA) upregulated in colorectal cancer (CRC). We elucidated its role and clinical significance in CRC carcinogenesis. Methods: LncRNA candidates were identified using TCGA database. LncRNA expression profiles were studied by qRT-PCR and microarray in paired tumor and normal tissues. The independence of the signature in survival prediction was evaluated by multivariable Cox regression analysis. The mechanisms of lncRNA function and regulation in CRC were examined using molecular biological methods. Results: We identified a novel long noncoding gene (PiHL, P53 inHibiting LncRNA) from 8q24.21 as a p53 negative regulator. PiHL is drastically upregulated in CRC and is an independent predictor of CRC poor prognosis. Further in vitro and in vivo models demonstrated that PiHL was crucial in maintaining cell proliferation and inducing 5-FU chemoresistance through a p53-dependent manner. Mechanistically, PiHL acts to promote p53 ubiquitination by sequestering RPL11 from MDM2, through enhancing GRWD1 and RPL11 complex formation. We further show that p53 can directly bind to PiHL promoter and regulating its expression. Conclusion: Our study illustrates how cancer cells hijack the PiHL-p53 axis to promote CRC progression and chemoresistance. PiHL plays an oncogenic role in CRC carcinogenesis and is an independent prognostic factor as well as a potential therapeutic target for CRC patients.


Western Blot
Western blot analysis was performed as previously described(2). Cells were suspended in RIPA lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor cocktail (Sigma-Aldrich). Cell lysates or retrieved proteins were analyzed by immunoblot with primary antibodies and HRP-conjugated secondary antibodies.

Colony formation assay
HCT116 and RKO cells were seeded into 6-well plates at a density of 1×10 3 and 500 cells per well, respectively. After two weeks, cells were fixed by 100% methanol and stained with 0.1% crystal violet.
Colonies were counted using Image J software (NIH, Bethesda, MD, USA).

Flow cytometry analysis
For the cell cycle analysis, 48 h after transfection, 1 × 10 6 cells in the log phase of growth were harvested by trypsinization, washed twice with cold PBS, fixed in ice-cold 70% ethanol, and incubated overnight at −20°C. Propidium iodide (PI,50 ug/ml,Sigma) and RNaseA (0.1 mg/ml, Sigma) were added to the cells and stained for 15 min. Cell cycle profiles were captured using a FACS Calibur flow cytometer (BD, Biosciences, CA, USA), and the data were analyzed using ModFit LT software (Verity Software House, Inc., Topsham, ME, USA).
For the cell apoptosis assay, an Annexin V-FITC Apoptosis Detection Kit II (BD, Biosciences, CA, USA) was used by measuring the membrane redistribution of phosphatidylserine. Cells were treated according to the manufacturer's instructions. The pre-labeled cells were detected and apoptosis was quantified using a FACS Calibur flow cytometer with Cell-Quest software (BD Biosciences). Annexin V-FITC and PI double stain was used to evaluate the percentages of apoptosis. Annexin V− and PI− cells were used as controls. Annexin V+ and PI− cells were designated as apoptotic and Annexin V+ and PI+ cells were designated as necrotic. Each test was repeated in triplicate.

Terminal deoxynucleotidyl transferase-mediated nick-end labelling (TUNEL) assay
Apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining using an In Situ Apoptosis Detection Kit (Takara Bio Inc., Shiga, Japan) according to the manufacturer's instructions. The sections were counterstained with Mayer's hematoxylin and TUNEL-positive cells were quantified in 3 randomly selected fields of each section at ×400 magnification.

Luciferase assays
HEK-293T cells were seeded in 96-well plates at a density of 5,000 cells per well 24 h before transfection.
The cells were co-transfected with a mixture of 45 ng PGL3-basic-PiHL promoter, 5 ng pRL-TK and 150 ng pCDH-p53 or control according to recommended instructions using Lipofectamine 3000 (Invitrogen). Twenty-four h after transfection, Firefly and Renilla luciferase activity was measured by the Dual-Luciferase Reporter Assay System (Promega, San Luis Obispo, CA, USA). Relative firefly luciferase activities were detected by a BD Monolight 3010 luminometer (BD Biosciences), and Renilla luciferase activities served as an internal control. The sequences for the primers are listed in Supplementary Table 1.

Chromatin immunoprecipitation (ChIP)
Cells were exposed to Dox or DMSO for 24h. Subsequently, ChIP assays were performed on chromatin extracts from these cells according to the manufacturer's specifications (EZ-ChIP kit-Millipore, Boston, MA, USA) with the following antibodies: rabbit anti-p53 (Abcam, Cambridge, UK) and normal rabbit IgG (Millipore, Boston, MA, USA). The immunoprecipitated DNA was purified and bound regions were identified by PCR analysis with primers (Supplementary Table 1) specific for the protein binding regions (BRs) within the promoter of PiHL.

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The coding potential of the PiHL transcript was analyzed using the Coding Potential Assessment Tool  Error bars represent ±s.e.m, n = 3. *P < 0.05 by two-tailed t-test.