Regulation of laryngeal squamous cell cancer progression by the lncRNA RP11‐159K7.2/miR‐206/DNMT3A axis

Abstract Long non‐coding RNAs (lncRNAs), which are longer than 200 nt, have been proved to play a role in promoting or inhibiting cancer progression. The following study investigated the role and underlying mechanisms of lncRNA RP11‐159K7.2 in laryngeal squamous cell carcinoma (LSCC) progression. Briefly, in situ hybridization (ISH) and real‐time quantitative PCR (RT‐qPCR) showed higher expression of RP11‐159K7.2 in LSCC tissues and cell lines. Patients with low expression level of RP11‐159K7.2 lived longer compared to those with high expression of RP11‐159K7.2 (χ 2 = 39.111, ***P < 0.001). Multivariate Cox regression analysis suggested that lncRNA RP11‐159K7.2 was an independent prognostic factor for LSCC patients (HR = 2.961, ***P < 0.001). Furthermore, to investigate the potential involvement of RP11‐159K7.2 in the development of LSCC, we knocked out the expression of endogenous RP11‐159K7.2 in TU‐212 cells and AMC‐HN‐8 cells via CRISPR/Cas9 double vector lentiviral system. RP11‐159K7.2 knockout decreased LSCC cell growth and invasion both in vitro and in vivo. Mechanically, we found that RP11‐159K7.2 could positively regulate the expression of DNMT3A by sponging miR‐206. In addition, a feedback loop was also discovered between DNMT3A and miR‐206. To sum up, these findings suggest that lncRNA RP11‐159K7.2 could be used as a potential biomarker for prognosis and treatment of LSCC.

understood. 6 The lack of valuable biomarkers and the recurrence and metastasis are the significant problems in LSCC diagnosis and treatment. Therefore, identification of new, sensitive and specific LSCC biomarkers is urgent for the early diagnosis and prognostic evaluation LSCC patients. In addition, it is crucial to explore the molecular mechanisms leading to the pathogenesis of LSCC and discover effective therapeutic targets for suppression of the progression.
Long non-coding RNA (lncRNA) is non-coding, with more than 200 nucleotides in length that have an essential role in imprinting, epigenetic regulation, transcriptional and translational regulation. 7,8 LncRNAs were primitively thought to be the 'dark matter' or 'noise' of genomic transcription and to be dysfunctional. 9,10 With the rapid development of molecular biology, accumulating evidence has revealed that lncRNAs are involved in almost every aspect of cellular processes such as cell proliferation, apoptosis and migration. 11,12 In recent years, an increased understanding of lncRNAs has revealed that they function as potent oncogenes or tumour suppressors in various cancer types. 13,14 The aberrant expression of lncRNAs has been identified to play a key role in the tumorigenesis and development of laryngeal cancer. 15,16 Our previous studies also revealed that lncRNA HOTAIR, LET and NEAT1 participate in LSCC progression. [17][18][19] Nevertheless, the overall pathophysiological contribution of lncRNAs to LSCC needs to be further investigated. Over recent years, ceRNA effect has become a research hotspot. 20 LncRNAs can mutually regulate one another by competitively binding to microRNAs as long as they share common microRNA-binding sites. Previous studies have reported the effects of ceRNAs in the lncRNA-microRNA-mRNA regulatory network in LSCC. 21,22 Zhao et al have elucidated the complex regulated mechanism in LSCC via network construction approach. 23 With microarray analysis, our preliminary experiments showed that RP11-159K7.2 level was significantly increased in LSCC tissues (data not shown). In the present study, we explored the role and underlying mechanism of a novel lncRNA, RP11-159K7.2 in LSCC progression. Results showed that RP11-159K7.2 was overexpressed in LSCC and that RP11-159K7.2 levels were significantly correlated with cervical lymph node metastasis and prognosis of LSCC patients. Furthermore, we found that down-regulation of RP11-159K7.2 could suppress LSCC cell proliferation and invasion both in vitro and in vivo. Moreover, RP11-159K7.2 was evaluated as a molecular sponge for miR-206, an additional regulator of DNMT3A expression. Additionally, a feedback loop was also discovered between DNMT3A and miR-206. We first proposed the important molecular marker role of RP11-159K7.2 in laryngeal cancer and for the first time demonstrated that it is involved in the regulation of DNA methylation in laryngeal cancer. No RP11-159K7.2 research report has been seen so far.

| Colony formation assay
After transfection, TU-212 and AMC-HN-8 cells were seeded at an appropriate density into 6-well culture plates and maintained in DMEM containing 10% FBS. The colonies were fixed with methanol after 2 weeks and stained at room temperature with crystal violet for 30 minutes. The formed colonies were counted in every well.

| In vivo experiment
Eighteen BALB/c mice, age 5 to 6 weeks, were purchased from Vital

| Luciferase reporter assay
The downstream of the firefly luciferase (f-luc) in the pGL3 plasmid was

| Western blot analysis
As previously stated, LSCC cells were gathered and analysed using

| Immunohistochemistry
Xenografts removed from the mice were fixed in paraformalde-

| LncRNA RP11-159K7.2 is overexpressed in LSCC tissues and cell lines
In situ hybridization assay was performed to identify the localization  The same pathologist semi-quantitatively appreciated the level of inflammation on microscopic sections on a scale from 0 to 3, 0: none; 1: <10%; 2: 10%-50%; and 3: >50%. A score of 2 was used to distinguish between low (<2) and high (≥2) levels of RP11-159K7.2 gene expression. Data were analysed by chi-squared test. P-value with * indicates statistically significant.

| Correlations between the expression of RP11-159K7.2 and clinicopathological parameters
We analysed the correlation between RP11-159K7.2 expression and the clinicopathological parameters of patients with LSCC. As shown in Table 2

| RP11-159K7.2 knockout inhibited the proliferation and invasion of LSCC cells
To explore the potential involvement of RP11-159K7.  Figure 2F). In addition, the knockout efficiency was verified by using RT-qPCR ( Figure 2G).

| RP11-159K7.2 knockout inhibits the growth of LSCC in vivo
To further investigate the effects of RP11-159K7.2 on the regula-

| DNMT3A suppressed miR-206 in a DNA methylation-dependent manner
Given that we often observe abnormal hypermethylation in carcinogenesis, that is critical for down-regulating microRNAs, and that Compared with DMSO, treatment with 5-Aza-dC had a significant effect on the expression of miR-206, indicating that miR-206 expression in LSCC cells might be regulated by DNA methylation (**P < 0.01; Figure 7A). As shown in Figure 7B,C, we carried out BSP to evaluate the original methylation status of the identified CpG sites in LSCC cells.
The percentage of miR-206 promoter methylation in DNMT3A knockout cells in LSCC was 36.7% (TU-212) and 36.3% (AMC-HN-8), respectively, which was significantly lower than in control groups. The above data indicated that the high methylation status of CpG sites might inhibit the expression of miR-206 in LSCC. As summarized in Figure 7D, the obtained results confirmed our hypothesis, that is a negative feedback interaction between miR-206 and DNMT3A.

| D ISCUSS I ON
Laryngeal cancer is one of the most common malignant tumours in the head and neck region. In 2015, there were 26 300 new cases and 14 500 deaths in China. 28 According to the SEER Cancer Statistics Review, the 5-year overall survival rate is approximately 60% in the United States. 29,30 Consequently, it is of utmost importance to find a reliable molecular marker and elucidate its molecular mechanisms.
Protein-coding sequences account for only a tiny fraction (1%) of the genome, while the rest are associated with non-coding RNA genes. 31,32 Several studies have reported that the lncRNA is essential for many critical cell regulatory processes, such as genomic imprinting, chromatin modification, transcriptional regulation and post-transcriptional regulation. [33][34][35] LncRNA and proteins form a complex regulatory network, through which they regulate cell proliferation, differentiation and apoptosis, and participate in many life activities. 36,37 Recently, the role of lncRNA in human cancers has aroused much attention, and increasing evidence has emphasized the clinical significance of lncRNA in neoplasms. [38][39][40] Our previous studies suggested that the lncRNA HOTAIR, HOXA11-AS and  miR-206 plays a regulatory role in the process of DNA methylation of LSCC cells. Our previous study has shown that the expression of miR-206 is decreased in LSCC and that the loss of miR-206 is important for the proliferation, invasion and apoptosis of LSCC. 27 As a key DNA methylation catalytic enzyme, DNMT3A has pivotal roles in de novo DNA methylation. DNMT3A was predicted to be a downstream target gene of miR-206, which was further validated by experiments.
In addition, DNMT3A has been reported to induce aberrant CpG site methylation in human cancer cells. 43

ACK N OWLED G EM ENTS
This study was supported by the scientific funds of National Natural Science Foundation of China (Grant no. 81772874 and 81272965 to Yanan Sun).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The authors declare that the data in this article are available.