Increasing evidence has revealed that specific lncRNAs might give a significant contribution in the progression, metastasis and chemoresistance of cancer[35]. These multiple functional tumor-associated lncRNAs have either oncogenic or tumor-suppressive properties in cancer [36]. In particular, accumulating research data have demonstrated that the lncRNAs alterations in HPV infected cells was also crucial for the progression of cervical cancer. For example, a microarray analysis revealed that thousands of host lncRNAs had differential expression in oncogenic HPV-positive cells compared to the HPV-negative cervical cancer cell line[37]. Lots of lncRNAs were found to be differentially expressed in the HPV18 positive HeLa cells respect to C33A cell line[38]. In addition, accumulating research data have demonstrated the high-risk HPV E6 and E7 oncoproteins could alter the expression of these lncRNAs and their downstream miRNAs or targets.
In general, HOTAIR has been shown to recruits chromatin-modifying proteins and to affect cancer epigenome modulation[39]. In cervical cancer, limited data suggested that HOTAIR could act as sponge for several miRNAs and cause deregulation of the respective target genes. However, the exact mechanism remains unclear. Furthermore, HOTAIR levels have shown to be strictly controlled by the HPV E7 protein in cervical cancer [22]. In this study, our results revealed that HOTAIR and miR-214-3p were both differentially expressed in HPV16 positive cervical cancer cells, HOTAIR showed significantly higher expression level, while miR-214-3p remains a lower expression level in cervical cancer cells. However, they showed exactly reverse expression tendency. Meanwhile, datasets mining also found the higher expression of HOTAIR in cancer tissues[ ]. These findings were consistent with previous studies[23, 24].
For better investigate the function of HOTAIR and miR-214-3p in HPV16 positive cervical cancer, we knocked down HOTAIR and overexpressed miR-214-3p in HPV16 positive cells, respectively. Our results indicated that no matter knocking down of HOTAIR or overexpressing miR-214-3p, cell proliferate ability was significantly inhibited accompanied by cellular apoptosis increased. That represents HOTAIR played a promoting role in HPV16 cervical carcinogenesis and the function of miR-214-3p was totally reverse. We supposed that HOTAIR was responsible for miR-214-3p down-regulation in HPV16 positive cervical cancer by serving as a competitive endogenous RNA through sponging mature miR-214-3p in cells. Based on bioinformatics analysis, we identified a potential binding site on HOTAIR transcript against miR-214-3p. Eventually A seven bp seed sequences (CAGCAGG) at 5’ end of miR-214-3p were fully complementary to the 1807–1813 bp on HOTAIR. Moreover, the function of this binding site as a microRNA response elements (MREs)on HOTAIR was also verified by dual luciferase assay. To further confirm the interaction between HOTAIR and miR-214-3p, we detected the change of miR-214-3p expression when HOTAIR expression was interfered in cancer cells. As shown in the results, miR-214-3p expression decreased accompany with the up-regulation of HOTAIR. Our results revealed a strong negative correlation between HOTAIR and miR-214-3p expression.
In above results, we had confirmed that HPV16E7 could upgrade HOTAIR expression. Next, when down or up-regulated expression of HPV16 E7 respectively, the expression of HOTAIR showed the same trend. In addition, miR-214-3p expression was significantly increased when HPV16 E7 expression was knocked down, but once HPV16 E7 gene was up-regulated expression again in cancer cells, HOTAIR expression went up also and the expression trend of miR-214-3p was obviously reversed. The results suggest that the infection status of HPV16 played an important role in regulating expression of HOTAIR or miR-214-3p in cervical cancer cells. Consequently, we speculated that the main cause of the above phenomenon was attributed to losing the sponge effect of HOTAIR against miR-214-3p.
Based on bioinformatics analysis, Wnt/β-catenin signaling pathway was one of the key pathways implicated in miR-214-3p regulation network in cervical cancer. Interestingly, Wnt/β-catenin signaling pathway contains some key genes, such as CTNNB1(β-catenin), PLCB4, PSEN1, PPP2CB, were all the well-recognized target gene for miR-214-3p by bioinformatics analysis. The studies found that miR-214-3p could degrade or inactivate the β-catenin gene by binding to the 3'UTR of β-catenin mRNA in various malignant tumors including breast cancer and esophageal cancer [40, 41]. In our study, β-catenin was significantly decreased at both mRNA and protein level when miR-214-3p was up expressed in HPV16 cervical cancer cells. And once HOTAIR was knocked down in HPV16 positive cervical cancer cells, miR-214-3p could escape from the absorption of HOTAIR and further lead to its target gene β-catenin been degraded. HOTAIR in HPV-negative C-33A cells remains an extremely lower level, so when we overexpressed the HOTAIR functional region in C-33A cells, the free miR-214-3p lost its inhibitory effect against β-catenin mRNA due to the adsorption of HOTAIR against miR-214-3p, eventually caused the expression of β-catenin was significantly up-regulated in cervical cancer cells.
Wnt/β-catenin dependent pathway also known as the canonical pathway. When Wnt/β-catenin dependent pathway was activated, β-catenin protein was accumulated in the cytoplasm, then it was transferred to the nucleu and interacted with T cell factor (TCF)/Lymphoid Enhancer Factor (LEF) transcription factors, and promotes the transcription of downstream targets, such as cyclin D1, c-Myc, and matrix metalloproteinase 1[42]. On this basis, Wnt/β-catenin signaling pathway played modulation roles in cancer cells apoptosis, proliferation, invasion, and migration in the progression of various cancers[43]. In this study, we found when HOTAIR was knocked down in HPV16 positive cervical cancer cells, the expression of β-catenin was significantly inhibited. We supposed that the lower expression of HOTAIR lost the sponge effect against miR-214-3p, further lead to degradation of β-catenin mRNA, then wnt/β-catenin signaling pathway was inhibited in cells. Oppositely, in HPV16-positive cervical cancer cells, HOTAIR was highly expressed due to the presence of HPV16E7, then miR-214-3p was adsorbed and loses its effect on target genes, and β-catenin expression was released, resulting in malignant phenotype of cervical cancer cells. In summary, HOTAIR and miR-214-3p might form a regulated axis and play important regulation roles in the malignant behaviour of HPV16-positive cervical cancer cells. Also, canonical wnt/β-catenin signaling pathway was a key part of this ceRNA regulatory network in cervical cancer.
At present, cervical cancer continues to represent an important health problem for in women worldwide. Although the epigenetic alterations in HPV infected cells indeed be crucial for the progression to cervical cancer, studies on the correlation between HPV and intracellular ncRNA were very limited so far. Meanwhile, the different investigations provided conflicting results with a significant proportion of ncRNAs being upregulated in one study but downregulated in another study. Therefore, characterization of the complex relationship between ncRNAs and target genes were needed to further carried out, help to improve the early diagnosis and treatment of cervical cancer.