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 (χ² = 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.

Keywords: CRISPR/Cas9; DNMT3A; laryngeal squamous cell cancer (LSCC); long non-coding RNA; microRNA.

FIGURE 1

The RP11‐159K7.2 expression level was up‐regulated in LSCC tissues and cell lines. In situ hybridization assay was used to determine the expression of RP11‐159K7.2. A, LSCC tissue; B, adjacent non‐tumorous tissue; C, positive control; D, negative control. E, RT‐qPCR was performed to validate RP11‐159K7.2 expression in 86 pairs of LSCC tissue and adjacent non‐tumorous tissue (***P < 0.001). F, RP11‐159K7.2 expression was higher in LSCC cells compared with a normal cell line (***P < 0.001). G‐J, Tumours with advanced clinical stages, with T3‐4 grade or with lymph node metastasis expressed higher levels of RP11‐159K7.2 ***P < 0.001. K, Kaplan‐Meier survival analysis indicated that high RP11‐159K7.2 expression levels in LSCC were significantly associated with worse OS (***P < 0.001)

FIGURE 2

RP11‐159K7.2 inhibition reduces LSCC cell proliferation and invasion. A‐B, The transfection and knockout efficiency were estimated 72 h after transfection with the lentivirus‐sgRNA‐EGFP. Enhanced green fluorescent protein (EGFP) expression in transfected cells was observed by light and fluorescence microscopy, respectively. C‐D, The proliferation of LSCC cells transfected with LentiCRISPR/Cas9 system was decreased at the different time point (24, 48 and 72 h, respectively) compared with the controls. CON, no transfection group; NC, negative control group; KO, RP11‐159K7.2 knockout group. E‐F, The transfection of lentivirus‐sgRNA‐EGFP inhibited the proliferation and invasion of LSCC cells ***P < 0.001. G, Efficiency of RP11‐159K7.2 expression in RP11‐159K7.2 down‐regulated TU‐212, and AMC‐HN‐8 cells were evaluated via RT‐qPCR

FIGURE 3

Knockout of RP11‐159K7.2 by CRISPR/Cas9 inhibits LSCC growth in vivo. A, Representative mouse injected with EGFP negative control lentivirus or RP11‐159K7.2 lentivirus‐sgRNA‐EGFP. B, Tumours were dissected after 6 weeks. Tumour volume (C) and tumour weight (D) in RP11‐159K7.2 lentivirus‐sgRNA‐EGFP group were significantly less than in the control group (***P < 0.001). n = 9 mice per group. RP11‐159K7.2 targets miR‐206 to interact with miR‐206 negatively. E, Distribution of differentially methylated regions (DMRs) among different chromosomes after knockout of RP11‐159K7.2. F, Candidate downstream microRNAs of RP11‐159K7.2 predicted by miRcode and the most down‐regulated microRNAs acquired from TCGA, and three microRNAs were screened out. G, microRNAs (with fold change <−0.5 and FDR < 0.1) plotted as a volcano plot. H, RT‐qPCR was performed to determine the relative expression of the three microRNAs in AMC‐HN‐8 cells transfected with RP11‐159K7.2 lentivirus‐sgRNA‐EGFP or EGFP negative control lentivirus. I, Kaplan‐Meier survival curves for miR‐206 associated with overall survival in HNSC. J, miR‐206 was observed to be significantly decreased in LSCC lines (TU‐212, AMC‐HN‐8) compared with the HEK‐293T cell line using RT‐qPCR, normalized to U6 as an endogenous control. K, A schematic diagram showing the binding sites between miR‐206 and RP11‐159K7.2. L, AMC‐HN‐8 cells were transfected with RP11‐159K7.2‐WT or RP11‐159K7.2‐Mut. RP11‐159K7.2‐WT significantly decreased luciferase activity of miR‐206, and there was no significant difference in the activity of miR‐NC *P < .05. M, Relative expression of miR‐206 in AMC‐HN‐8 cells transfected with LentiCRISPR/Cas9 system against RP11‐159K7.2 was decreased compared with the controls, measured using RT‐qPCR

FIGURE 4

RP11‐159K7.2 expression was suppressed in LSCC cells transfected LentiCRISPR/Cas9 system targeting RP11‐159K7.2, and this could be partly rescued by the cotransfection of miR‐206 inhibitors. A, LSCC cells were transfected with RP11‐159K7.2 NC + miR‐206, RP11‐159K7.2 KO + miR‐NC, RP11‐159K7.2 KO + miR‐206 inhibitor. Colony formation assay (B) and transwell assay (C) showed that miR‐206 inhibitor reversed the inhibition effect of LentiCRISPR/Cas9 system targeting RP11‐159K7.2 on the proliferation and invasion of LSCC cells

FIGURE 5

DNMT3A was identified as a direct target of miR‐206. A, Relative expression of the DNMTs in LSCC cells transfected with miR‐206 or miR‐NC measured by RT‐qPCR **P < .01. B, DNMT3A expression was negatively correlated with miR‐206 expression in HNSC. C, Starbase database predicted the target site between DNMT3A and miR‐206. D, AMC‐HN‐8 cells were transfected with DNMT3A‐WT or DNMT3A‐Mut. DNMT3A‐WT significantly decreased luciferase activity of miR‐206, and there was no significant difference in the activity of miR‐NC *P < .05. E, DNMT3A is up‐regulated in LSCC cell lines compared with normal cell line ***P < .001. F, Relative expression of DNMT3A in TU‐212 cell line, AMC‐HN‐8 cell line and HEK‐293T cells was detected by Western blots. G, Relative expression of DNMT3A in AMC‐HN‐8 cells transfected with LentiCRISPR/Cas9 system against RP11‐159K7.2 was decreased compared with the control groups (***P < .001). H, IHC detection of DNMT3A in paraffin‐embedded tissue sections of nude mice xenografts

FIGURE 6

miR‐206 inhibits proliferation and invasion by targeting DNMT3A in LSCC cells. (A) DNMT3A expression was suppressed in LSCC cells transfected siRNA against DNMT3A, and this could be partly rescued by the cotransfection of miR‐206 inhibitors. LSCC cells transfected with siRNA against DNMT3A reduced the ability of proliferation (B) and invasion (C), and the effect could be rescued by the cotransfection of miR‐206 inhibitors as our data suggested that miR‐206 binds to DNMT3A to negatively regulate cell proliferation and invasion

FIGURE 7

Negative feedback interaction between miR‐206 and DNMT3A. (A) miR‐206 expression levels in LSCC cells treated with 5‐Aza‐2’‐deoxycytidine. **P < .01. (B) Methylation profile in TU‐212 and AMC‐HN‐8 cell lines. The open and filled circles symbolized the unmethylated and methylated CpGs, respectively. (C) The methylation percentage of each cell line was shown. Error bars correspond to the mean ± SD (**P < .01). (D) Diagram summarizing the mechanism of action of RP11‐159K7.2/miR‐206/DNMT3A in LSCC cells. RP11‐159K7.2 competitively binds to miR‐206, which could directly combine with DNMT3A. DNMT3A also suppress miR‐206 through DNA methylation. The green arrows indicate promotive function, and red lines indicate suppressive function