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- Matherials and Methods
- Disclosure Statement
- Supporting Information
Identification of new nasopharyngeal carcinoma (NPC) biomarkers is of great clinical value for the diagnosis and treatment of NPC. HOTAIR, a cancer-related long non-coding RNA, was tested and its prognostic value for NPC was evaluated. As determined using in situ hybridization (ISH), 91 of 160 (56.87%) paraffin-embedded NPC biopsies showed high expression levels of HOTAIR (staining index score ≥ 6). HOTAIR was upregulated in tumors with a large size (P = 0.021), more advanced clinical stage (P = 0.012) and increased lymph node tumor burden (P = 0.005). Quantified using real-time PCR, HOTAIR expression levels in fresh tissue and paraffin-embedded samples were 5.2 ~ 48.4-fold higher compared with non-cancer tissue samples. Moreover, HOTAIR expression levels increased with clinical stage progression, which was consistent with ISH findings in the paraffin-embedded tissue. Most importantly, NPC patients with higher HOTAIR levels had a poor prognosis for overall survival using univariate and multivariate analysis. In addition, HOTAIR mediated the migration, invasion and proliferation of NPC cells in vitro. HOTAIR is a potential biomarker for the prognosis of NPC, and dysregulation of HOTAIR might play an important role in NPC progression.
Nasopharyngeal carcinoma (NPC) is a rare tumor that arises from the nasopharynx epithelium. Nasopharyngeal carcinoma occurs around the world; however, it is vastly more common in certain regions of Southeast Asia than elsewhere. The incidence of NPC in South China, especially in the Cantonese region around Guangzhou, is approximately 100-fold higher compared with Europe and North America, with viral, environmental and genetic factors implicated in its etiology.[2-5] Although treatment with radiotherapy temporarily controls the primary tumor, frequent tumor recurrence and distant metastases remain major obstacles for long-term patient survival.[6, 7] Therefore, identifying more accurate predictive biomarkers is of great clinical value to further understand NPC cell biology and develop novel therapeutic strategies.
The discovery of numerous non-coding RNA (ncRNA) transcripts in human cells has dramatically altered our understanding of the biology of normal and malignant cells. A large class of small ncRNA, microRNA, has been characterized as oncogenes or tumor suppressors through post-transcriptional regulation of protein expression. However, the findings that long non-coding RNA (lncRNA) more than 200 nt in length are differentially expressed in cancer cells and bind to various regulatory proteins have increased the complexity of ncRNA involvement in tumor biology regulation.[8-10] Dysregulated lncRNA expression levels characterize the entire spectrum of disease, and aberrant lncRNA function drives cancer through the disruption of cell processes, typically by facilitating transcriptional regulation. This process can occur through the targeting of either genomically local (cis-regulation) or genomically distant (trans-regulation) genes. Of the cis-regulatory lncRNA (imprinting lncRNA), H19 has been most extensively studied in cancer.[12-14] In model systems, silencing H19 expression impaired cell growth and clonogenicity in lung cancer cell lines in vitro and decreased xenograft tumor growth of Hep3B hepatocellular carcinoma cells in vivo. Other cis-regulatory lncRNA, such as HOTAIRMI and HOTTIP, play important roles in differentiation status of cancer cells in leukemias. Similar to most cis-acting lncRNA, trans-regulatory lncRNA typically facilitate the epigenetic regulation of gene expression. In cancer, trans-regulatory lncRNA gained widespread attention through the characterization of HOTAIR. HOTAIR is located in the HoxC cluster and was found to regulate the HoxD cluster genes through a trans-regulatory mechanism. Recently, upregulated expression of HOTAIR was observed in numerous solid tumors, including breast cancer, hepatocelluar carcinoma and colon cancer. In breast cancer, overexpression of HOTAIR facilitates aberrant polycomb repressive complex (PRC2) function by increasing PRC2 recruitment to the genomic positions of target genes and mediates the epigenetic repression of PRC2 target genes. Clinically, overexpression of HOTAIR is an independent predictor of overall survival and progression-free survival for several cancers.[17, 18] Thus, lncRNA represent a novel but poorly characterized aspect of cancer biology. While a biological understanding of HOTAIR in breast and hepatocellular carcinomas has progressed, increased understanding of the functional role of HOTAIR in other human cancers, such as NPC, is needed.
In the present study, we examined HOTAIR expression in normal and malignant human nasopharyngeal tissue and cell lines. Our results demonstrated that high HOTAIR expression is associated with NPC progression and predicts a poor patient prognosis. In addition, we studied how HOTAIR influences the invasiveness of NPC cells in vitro.
- Top of page
- Matherials and Methods
- Disclosure Statement
- Supporting Information
Nasopharyngeal carcinoma is one of the most common cancers in southern China and Southeast Asia. This remarkable racial and geographic distribution of NPC indicates that the development of this cancer might be related to genetic factors. Identification of new NPC biomarkers will be helpful for the diagnosis and treatment of NPC and might provide new insight into its pathogenesis. In the present study, we found that clinically, high expression levels of HOTAIR, a cancer-related lncRNA, correlate with NPC progression. HOTAIR is an indicator for predicting the prognosis of advanced-stage NPC patients. In addition, HOTAIR contributes to the malignant character of NPC cells through involvement in diverse cellular processes, including migration, invasion and proliferation.
Recently, the roles for lncRNA as drivers of tumor suppressive and oncogenic functions have appeared in prevalent cancer types. It has been demonstrated that HOTAIR, a transregulatory lncRNA, is upregulated in breast cancer, hepatocellular carcinoma and colorectal cancer.[17-19] The HOTAIR expression levels assessed were found to be higher in cancerous tissue than in the corresponding non-cancerous tissue in primary breast tumors and colorectal cancers, and upregulated HOTAIR has also been detected in hepatocellular carcinoma (HCC) compared with the adjacent non-tumorous tissue in HCC patients.[18, 21] Similarly, the present study showed that higher levels of HOTAIR staining were observed in NPC tissue than in non-cancerous tissue; furthermore, the expression levels of HOTAIR were upregulated in samples with a higher tumor burden, including larger tumor size, advanced clinical staging and increased tumor burden of lymph nodes, as well as the presence of distant metastases. Therefore, HOTAIR is crucial for the oncogenesis and development of NPC.
Dysregulated expression of lncRNA in cancer marks the spectrum of disease progression and might serve as an independent predictor for patient outcome. HOTAIR is one of the few biologically well-studied lncRNA. Previous studies have demonstrated that high HOTAIR expression strongly correlates with the presence of liver metastasis. Similarly, overexpression of HOTAIR is linked to earlier recurrence in HCC patients who underwent surgical resection. Tumor recurrence and distant metastases are also responsible for poor survival of NPC patients. Here, the present study demonstrated that high expression levels of HOTAIR correlate with a poor LRFS, DMFS, DFS and overall survival in NPC patients. Multivariate analyses revealed that HOTAIR expression levels were a powerful independent prognostic factor of clinical outcomes, suggesting that HOTAIR could be a candidate biomarker for predicting tumor recurrence in NPC patients.
To date, in addition to tumor size and status of lymph nodes, molecular markers are considered important factors in predicting treatment and prognosis. Heterogeneous ribonucleoprotein K and thymidine phosphorylase are therapeutic markers for NPC. Epstein-Barr virus (EBV) is widely used for screening and early diagnosis of NPC. Centromere protein H might function as a prognostic marker of NPC at earlier clinical stages. Interestingly, the prognostic value of HOTAIR expression was improved for advanced-stage NPC patients. According to our data, NPC patients with tumors exhibiting high HOTAIR expression had significantly shorter overall survival compared with patients with low expression of HOTAIR in the subgroup with large tumors and high tumor burden in the lymph nodes. The sensitivity and specificity of HOTAIR for NPC patients was slightly improved for the III–IV and N2–N3 subgroups. These results suggest that HOTAIR might serve as a better prognostic marker for predicting the prognosis of advanced-stage NPC patients, which differs from many biomarkers that are usually used for early stage NPC.
Emerging evidence suggests that lncRNA constitute an important component of tumor biology. Suppression of HOTAIR in liver cancer cells reduces cell viability and invasion, sensitizes TNF-α-induced apoptosis and increases chemotherapeutic sensitivity. In breast cancer, overexpression of HOTAIR increases lung metastases in mice. Upregulation of HOTAIR drives the malignant character of gastrointestinal stromal tumors and promotes cell invasiveness by altering the expression of reported HOTAIR-targeted genes. Similarly, our data demonstrate that HOTAIR is upregulated in NPC cell lines with high invasive potential and the capacity for migration, invasion and proliferation was suppressed after knocking down HOTAIR expression. However, further studies are required to investigate the potential mechanisms of HOTAIR involvement in the development of NPC. The turnover of HOTAIR is not well understood and one study reported that depletion of HOTAIR induced a significant change in the gene expression profile, suggesting that HOTAIR might regulate a spectrum of genes by mechanisms other than directly interacting with histone modification complexes.
The above findings not only suggest a useful candidate molecular marker for NPC and an indicator for advanced-stage NPC but also provide new insights into the role of lncRNA in cancer biology. In addition to the prognostic value for NPC, HOTAIR combined with other biomarkers might be useful to stratify patients for individual therapies.