Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Aug 7;8(42):72182-72196.
doi: 10.18632/oncotarget.20053. eCollection 2017 Sep 22.

Discovery and validation of the tumor-suppressive function of long noncoding RNA PANDA in human diffuse large B-cell lymphoma through the inactivation of MAPK/ERK signaling pathway

Yingjun Wang  1 , Mingzhi Zhang  1 , Huanan Xu  2 , Yifei Wang  3 , Zhaoming Li  1 , Yu Chang  1 , Xinhuan Wang  1 , Xiaorui Fu  1 , Zhiyuan Zhou  1 , Siyuan Yang  1 , Bei Wang  1 , Yufeng Shang  1
Affiliations

Discovery and validation of the tumor-suppressive function of long noncoding RNA PANDA in human diffuse large B-cell lymphoma through the inactivation of MAPK/ERK signaling pathway

Yingjun Wang et al. Oncotarget. .

Abstract

Diffuse large B-cell lymphoma (DLBCL) is one of the leading causes of cancer-related mortality, and responds badly to existing treatment. Thus, it is of urgent need to identify novel prognostic markers and therapeutic targets of DLBCL. Recent studies have shown that long non-coding RNAs (lncRNAs) play an important role in the development of cancer. By using the next generation HiSeq sequencing assay, we determined lncRNAs exhibiting differential expression between DLBCL patients and healthy controls. Then, RT-qPCR was performed for identification in clinical samples and cell materials, and lncRNA PANDA was verified to be down-regulated in DLBCL patients and have considerable diagnostic potential. In addition, decreased serum PANDA level was correlated to poorer clinical outcome and lower overall survival in DLBCL patients. Subsequently, we determined the experimental role of lncRNA PANDA in DLBCL progression. Luciferase reporter assay and chromatin immunoprecipitation assay suggested that lncRNA PANDA was induced by p53 and p53 interacts with the promoter region of PANDA. Cell functional assay further indicated that PANDA functioned as a tumor suppressor gene through the suppression of cell growth by a G0/G1 cell cycle arrest in DLBCL. More importantly, Cignal Signal Transduction Reporter Array and western blot assay showed that lncRNA PANDA inactivated the MAPK/ERK signaling pathway. In conclusion, our integrated approach demonstrates that PANDA in DLBCL confers a tumor suppressive function through inhibiting cell proliferation and silencing MAPK/ERK signaling pathway. Thus, PANDA may be a promising therapeutic target for patients with DLBCL.

Keywords: MAPK/ERK; diffuse large B-cell lymphoma; lncRNA PANDA; lncRNAs; p53.

PubMed Disclaimer

Conflict of interest statement

CONFLICTS OF INTEREST We declare no potential conflicts of interest.

Figures

Figure 1
Figure 1. The heat map shows expression of the 120 lncRNAs most up- or down-regulated in DLBCL patients compared with healthy controls
The top 60 lncRNAs up- and down-regulated in non-responding group are shown in the top and bottom halves, respectively. The heat map was generated with an R package using normalization across rows (serum samples).
Figure 2
Figure 2. Analysis and validation of selected lncRNA expression by RT-qPCR
(A–F) Concentrations of six identified serum lncRNAs in DLBCL patients (n = 68) and healthy controls (n = 68) using RT-qPCR assay in validation cohort. (G–H) The expression levels of lncRNA PANDA and TUG1 were further validated in five DLBCL cell lines and one normal cell line.
Figure 3
Figure 3. Decreased serum PANDA expression was associated with poor clinical outcome in DLBCL patients
(A) ROC curves for differentiating the DLBCL patients from healthy controls using lncRNA PANDA expression in validation cohort. (B–C) Kaplan-Meier curves for overall survival (B) and recurrence-free survival (C) according to serum levels of PANDA in DLBCL patients in validation cohort.
Figure 4
Figure 4. LncRNA PANDA is induced by p53 and p53 interacts with the p53 response element in the promoter region of PANDA
(A) The potential binding site of p53 at the promoter region of lncRNA PANDA, and the description of wild p53 and mutant p53. (B) RT-qPCR showed that p53 mRNA was significantly decreased in serum of DLBCL patients compared with healthy individuals. (C) Western blot experiment indicated that p53 protein was dramatically silenced in most DLBCL cell lines compared with normal cells. (D) Spearman correlation assay suggested a positive correlation between p53 mRNA and MALAT1expression. (E) p53 was significantly elevated by the transfection of p53 overexpression vector. (F) lncRNA PANDA was significantly up-regulated by p53. (G) Potential p53 binding region in the promoter region of PANDA used for construction of luciferase vector containing the binding region. (H) Luciferase activity was significantly increased in p53-transfected cells compared with control vector in OCI-Ly8 cells. (I) The p53 binding at the promoter regions of PANDA was assessed by ChIP analysis. Shown are representative images of three independent experiments.
Figure 5
Figure 5. LncRNA PANDA suppresses proliferation and induces cell-cycle arrest in DLBCL cells
(A–B) PANDA expression was significantly silenced by PANDA inhibitor (A) or promoted by PANDA vector (B). (C–D) si-PANDA significantly promoted cell proliferation rate (C) while PANDA overexpression suppressed cell growth (D). (E) Immunofluorescence analysis PANDA overexpression dramatically suppressed the cell proliferation marker Ki-67 protein. (F) Cell-cycle analysis indicated that overexpression of PANDA promoted cell cycle arrest in G0/G1 phase in OCI-Ly8 cells. (G) LncRNA PANDA suppressed the colony formation capacity of U2932 and OCI-Ly8 cells.
Figure 6
Figure 6. LncRNA PANDA regulates cell proliferation through inactivation of MAPK/ERK signaling pathway
(A) Histogram shows the fold changes for the activities of different signaling pathways, as indicated by reporter activity. (B) Western blot analysis of the expression levels of p38-MAPK and p-ERK protein. (C) CCK8 assay showed that treatment with MAPK/ERK agonist Anisomycin potently abolished PANDA-induced suppression of cell growth. (D) Cell proliferation marker Ki-67 was suppressed by PANDA, however, this effect was partially reversed by MAPK/ERK agonist Anisomycin.
See this image and copyright information in PMC

References

    1. Tilly H, Gomes da Silva M, Vitolo U, Jack A, Meignan M, Lopez-Guillermo A, Walewski J, Andre M, Johnson PW, Pfreundschuh M, Ladetto M. Diffuse large B-cell lymphoma (DLBCL): ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2015;26:v116–125. - PubMed
    1. Smith A, Howell D, Patmore R, Jack A, Roman E. Incidence of haematological malignancy by sub-type: a report from the Haematological Malignancy Research Network. Br J Cancer. 2011;105:1684–1692. - PMC - PubMed
    1. Feugier P, Van Hoof A, Sebban C, Solal-Celigny P, Bouabdallah R, Ferme C, Christian B, Lepage E, Tilly H, Morschhauser F, Gaulard P, Salles G, Bosly A, et al. Long-term results of the R-CHOP study in the treatment of elderly patients with diffuse large B-cell lymphoma: a study by the Groupe d'Etude des Lymphomes de l'Adulte. J Clin Oncol. 2005;23:4117–4126. - PubMed
    1. Lee HJ, Shin DH, Kim KB, Shin N, Park WY, Lee JH, Choi KU, Kim JY, Lee CH, Sol MY. Polycomb protein EZH2 expression in diffuse large B-cell lymphoma is associated with better prognosis in patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone. Leuk Lymphoma. 2014;55:2056–2063. - PubMed
    1. Vaidya R, Witzig TE. Prognostic factors for diffuse large B-cell lymphoma in the R(X)CHOP era. Ann Oncol. 2014;25:2124–2133. - PMC - PubMed