Abstract
BackgroundThe human genome encodes many long non-coding RNAs (lncRNAs). However, their biological functions, molecular mechanisms, and the prognostic value associated with pancreatic ductal adenocarcinoma (PDAC) remain to be elucidated. Here, we identify a fundamental role for the lncRNA HOXA transcript at the distal tip (HOTTIP) in the progression and chemoresistance of PDAC.MethodsHigh-throughput microarrays were performed to detect the expression profiles of lncRNAs and messenger RNAs in eight human PDAC tissues and four pancreatic tissues. Quantitative real-time PCR was used to determine the levels of HOTTIP and HOXA13 transcripts in PDAC cell lines and 90 PDAC samples from patients. HPDE6 cells (immortalized human pancreatic ductal epithelial cells) and corresponding adjacent non-neoplastic tissues were used as controls, respectively. The functions of HOTTIP and HOXA13 in cell proliferation, invasion, and epithelial-mesenchymal transition were evaluated by targeted knockdown in vitro. CCK-8 assays, colony formation assays, and xenografts in nude mice were used to investigate whether targeted silencing of HOTTIP could sensitize pancreatic cancer cells to gemcitabine. Immunohistochemistry was performed to investigate the relationship between HOXA13 expression and patient outcome.ResultsMicroarray analyses revealed that HOTTIP was one of the most significantly upregulated lncRNAs in PDAC tissues compared with pancreatic tissues. Quantitative PCR further verified that HOTTIP levels were increased in PDAC cell lines and patient samples compared with controls. Functionally, HOTTIP silencing resulted in proliferation arrest by altering cell-cycle progression, and impaired cell invasion by inhibiting epithelial-mesenchymal transition in pancreatic cancer. Additionally, inhibition of HOTTIP potentiated the antitumor effects of gemcitabine in vitro and in vivo. Furthermore, knockdown of HOXA13 by RNA interference (siHOXA13) revealed that HOTTIP promoted PDAC cell proliferation, invasion, and chemoresistance, at least partly through regulating HOXA13. Immunohistochemistry results revealed that higher HOXA13 expression was correlated with lymph node metastasis, poor histological differentiation, and decreased overall survival in PDAC patients.ConclusionsAs a crucial tumor promoter, HOTTIP promotes cell proliferation, invasion, and chemoresistance by modulating HOXA13. Therefore, the HOTTIP/HOXA13 axis is a potential therapeutic target and molecular biomarker for PDAC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0442-z) contains supplementary material, which is available to authorized users.
Highlights
The human genome encodes many long non-coding RNAs
Representative photos of invasive cells on the membrane are shown. (B) The effect of HOXA transcript at the distal tip (HOTTIP) knockdown on the invasion ability of MIA PaCa-2 cells and SW1990 cells was quantified by counting the number invasive cells. (C) (D) The effect of HOTTIP knockdown on expression of E-cadherin, Vimentin, and Snail 1 was evaluated by quantitative real-time Reverse Transcription-PCR (qRT-PCR). (E) (F) The effect of HOTTIP-knockdown on E-cadherin, Vimentin, and Snail 1 protein levels was confirmed by western blotting. (G) (H) (I) Immunofluorescence staining for the epithelial-mesenchymal transition (EMT) makers in SW1990 cells
The number of invading cells was analyzed. (G) (H) The expression levels of EMT-related genes (E-cadherin, Vimentin, and Snai1) was evaluated by qRT-PCR 48 h after transfection in both cell lines. (I) (J) The effect of HOXA13 silencing on E-cadherin, Vimentin, and Snail 1 protein levels was confirmed by western blotting. (K) Immunofluorescence staining for the EMT makers in SW1990 cells
Summary
The human genome encodes many long non-coding RNAs (lncRNAs). Their biological functions, molecular mechanisms, and the prognostic value associated with pancreatic ductal adenocarcinoma (PDAC) remain to be elucidated. We identify a fundamental role for the lncRNA HOXA transcript at the distal tip (HOTTIP) in the progression and chemoresistance of PDAC. Pancreatic ductal adenocarcinoma (PDAC), which accounts for more than 80% of pancreatic cancer cases, is one of the most malignant diseases world-wide. Chemotherapy plays a critical role in improving symptoms and prolonging overall survival of PDAC patients. Despite constant efforts to improve the diagnosis and treatment of PDAC, this devastating disease still has a dismal prognosis [4,5,6,7]. A better understanding of the genetic alterations and molecular mechanisms involved in PDAC development and chemoresistance will facilitate the identification of novel diagnostic and therapeutic targets [8]
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