ALKBH5-Mediated m6A Modification of LncRNA KCNQ1OT1 Triggers the Development of LSCC via Upregulation of HOXA9

doi:10.21203/rs.3.rs-637073/v1

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ALKBH5-Mediated m6A Modification of LncRNA KCNQ1OT1 Triggers the Development of LSCC via Upregulation of HOXA9

https://doi.org/10.21203/rs.3.rs-637073/v1

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published 01 Dec, 2021

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Background

RNA epigenetic modification is the chemical basis of RNA-regulated gene expression, of which N6-methyladenosine (m6A) is one of the most common post-transcriptional modifications in mRNA. More and more studies show that m6A modification is involved in the development of complex human diseases, especially in the development of cancer. However, it is unclear whether the m6A modification of lncRNA modification plays an important role in the development of laryngeal squamous cell carcinoma (LSCC).

Methods

Microarray analysis was used to quantitatively detect the m6A apparent transcriptional modification level of lncRNA in LSCC tissue. Methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) was used to examine the m6A modification of KCNQ1OT1 transcript. Quantitative real-time PCR (qRT-PCR) was used to detect the expression levels of KCNQ1OT1, ALKBH5, HOXA9. Western blot was performed to measure the expression ALKBH5 and HOXA9 protein. The expression of KCNQ1OT1 in LSCC and non-tumor tissues was detected by In situ hybridization (ISH). The biological effects of KCNQ1OT1 in vivo and in vitro were tested, meanwhile, the effects of HOXA9 on LSCC cells were tested in vitro. The expression levels of ALKBH5 and HOXA9 in LSCC and non-tumor tissues, and the expression levels of Ki-67, MMP2 and MMP9 in xenograft tumors were detected by Immunohistochemistry(IHC). RNA immunoprecipitation (RIP) and RNA pull-down assay confirmed the interaction relationship between ALKBH5/YTHDF2 and KCNQ1OT1. Luciferase Reporter assay and rescue experiment verified that HOXA9 is the target of KCNQ1OT1.

Results

KCNQ1OT1 expression in LSCC tissues was low m6A-methylation and high expression than that in non-tumor tissues, KCNQ1OT1 knockdown inhibits the proliferation, invasion and metastasis of LSCC. Further studies showed that the N6-methyladenosine (m6A) demethylase ALKBH5 mediators KCNQ1OT1 expression via an m6A-YTHDF2-dependent manner. It was found that KCNQ1OT1 could directly bind to HOXA9 to further regulate the proliferation, invasion and metastasis of LSCC cells.

Conclusions

Our research indicates that ALKBH5 mediated m6A modification of LncRNA KCNQ1OT1 triggers the development of LSCC via up-regulation of HOXA9.

Cancer Biology

Oncology

m6A methylation

Laryngeal squamous cell cancer

ALKBH5

LncRNA KCNQ1OT1

Laryngeal squamous cell carcinoma (LSCC) is the most common pathological subtype (> 95%) of laryngeal carcinoma [1–2]. Meanwhile, the prevalence of LSCC was ranked as 2nd in head and neck squamous cell carcinoma. It is reported that in 2018, there were 177422 new cases of LSCC and 94771 deaths of LSCC in the world [3]. Notably, the incidence of LSCC in China is about four times that in the United States, with an estimated death toll of more than 15000 per year [4]. The high incidence rate and mortality rate of LSCC are seriously threatening human health. Although the current diagnosis of LSCC can be treated by surgery, radiotherapy, chemotherapy and immunotherapy, the prognosis of LSCC is not optimistic. The 5-year overall survival (OS) rate of patients with advanced LSCC is still lower than 50% [5]. Therefore, we urgently need to have a deeper understanding of the mechanism that promote LSCC progression, which may help to develop more effective treatment strategies for LSCC patients.

N6 methyladenosine (m6A) was first discribed in 1974 as a major internal modification of RNA in higher eukaryotes, which has aroused great interest in recent years [6–8]. The researchers suggested that the m6A methylation plays an important role in the regulation of gene expression by influencing RNA stability, mRNA degradation and translation [9–10]. This modification mainly depends on the dynamic regulation of RNA methyltransferases (writers), demethylases (erasers) and m6A-binding proteins (readers), which may play a key role in the occurrence and development of carcinogenesis [11–12]. The core components of RNA methyltransferase complex include methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14) and Wilms tumor 1-associated protein (WTAP) [13]. In turn, fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5) can remove the m6A modification of RNA [14]. In addition, m6A-binding proteins, including YTHDF1, YTHDF2, YTHDF3 and YTHDC1, have been identified as m6A modified "readers" and regulate the processing, translation and degradation of mRNA [15]. As a demethylase, ALKBH5 is involved in the regulation of methylation. In recent years, the overexpression of ALKBH5 in a variety of cancers has been found, such as breast cancer, glioblastoma and gastric cancer [7, 16–17]. However, the role of ALKBH5 in the development of LSCC has not been studied. Therefore, how ALKBH5 regulates the level of RNA m6A methylation, and thus affects the pathogenesis of LSCC deserves further investigation.

Long noncoding RNA (LncRNA) is an endogenous transcription RNA molecule with a length of more than 200 nucleotides [18]. LncRNA has no protein-coding function and has a great influence on biological processes. In the gene regulatory network of tumor development, abnormal regulation of LncRNA has become an indispensable part. More and more studies have proved that LncRNAs play a crucial role in the multi-steps of tumor development [19]. The LncRNAs are often dysregulated in a variety of cancer types including LSCC. For instance, LncRNA growth arrest-specific 5 (GAS5) suppresses LSCC progression through the negative regulation of miR-21 and its targets involved in cell proliferation and apoptosis [20]. Our previous research has demonstrated that LncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) regulates CDK6 expression mediated by miR-107 in LSCC, which may be a potential target for therapeutic intervention of LSCC [21]. It has been proved that m6A methylated level can affect the expression of lncRNA, and then regulate the development of tumor [22–24]. Whether the m6A methylation of LncRNA affects its expression in LSCC is worth further study.

Here, we found that KCNQ1OT1was a valuable prognostic predictor of LSCC patients and revealed a new regulatory mechanism of KCNQ1OT1 up-regulation. The increase of ALKBH5 can promote the development of LSCC in a m6A-YTHDF2-dependent manner by promoting KCNQ1OT1-HOXA9 signaling. ALKBH5-KCNQ1OT1-HOXA9 may be a promising target for the treatment of LSCC.

Patients and specimens

86 pairs of LSCC and adjacent non-tumour tissues were collected from patients who received partial or total laryngectomy in the otolaryngology department of the Second Affiliated Hospital of Harbin Medical University from December 2017 to June 2019. Within 5 minutes after the operation, the specimens were labeled and stored in liquid nitrogen. They are refrigerated and transported to the laboratory for long-term storage at − 80 ℃. And these Patients in the study did not receive any cancer treatment before admission. Differentially m6A-methylated LncRNAs in three pairs of LSCC and non-tumor tissues were detected by microarray assay and the level of m6A modification in the top four low m6A methylation modified lncRNAs was detected by MeRIP-qPCR. In addition, we also detected the expression of ALKBH5 and HOXA9 in 86 pairs of LSCC and non-tumor tissues by IHC and qRT-PCR. This study was approved by the ethics committee of Harbin Medical University and obtained informed consent.

Microarray analysis

The Arraystar Human m6A-mRNA&lncRNA Epitranscriptomic microarray analysis were from Arraystar Arraystar Company (Rockville, MD, USA). Total RNA from each sample was quantified using the NanoDrop ND-1000. The sample preparation and microarray hybridization were performed based on the Arraystar’s standard protocols. Briefly, the total RNAs were immunoprecipitated with anti-N6-methyadenosine (m6A) antibody. The modified RNAs were eluted from the immunoprecipitated magnetic beads as the “IP”. The unmodified RNAs were recovered from the supernatant as “Sup”. The “IP” and “Sup” RNAs were labeled with Cy5 and Cy3 respectively as cRNAs in separate reactions using Arraystar Super RNA Labeling Kit. The cRNAs were combined together and hybridized onto Arraystar Human mRNA&lncRNA Epitranscriptomic Microarray (8x60K, Arraystar). After washing the slides, the arrays were scanned in two-color channels by an Agilent Scanner G2505C.

Methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR)

The methylated RNA immunoprecipitation was performed to evaluate the m6A modification levels of KCNQ1OT1, SPATA6L, RP11-150O12.5 and MECP2. 1–3 µg of total RNA and m6A labeled control mixture were added into 1ip buffer containing 2 µg of anti-m6A rabbit polyclonal antibody (synaptic systems, 202003). 20 µL Dynabeads were incubated at 4 ℃ for 2 hours. The suspension of M-280 Sheep anti rabbit IgG (11203d) was sealed at 4 ℃ for 2 hours with newly prepared 0.5% BSA. Wash with 300 µL 1×IP buffer for three times, then resuspend in the prepared total RNA antibody mixture, and rotated the RNA bound to the antibody beads for 2 hours at 4 ℃. The beads were then washed three times with 500 µL 1×IP buffer and twice with 500 µL washing buffer. The enriched RNA was eluted with 200 µL elution buffer at 50 ℃ for 1 hour. RNA was extracted by acidic phenol chloroform and ethanol precipitation. qRT-PCR detection was performed according to the above mentioned method.

Quantitative real-time PCR (qRT-PCR)

According to the manufacturer’s protocol, total RNAs was isolated from LSCC samples and cell lines using Trizol Reagents (Invitrogen, Carlsbad, CA, USA). The purified RNA was performed by the Reverse Transcription Kit (Takara, China) to reverse-transcribed into cDNA. The expression level of RNAs were quantified using the SYBR Green Master Mix (Roche, Switzerland) and normalized to internal control GAPDH mRNA, finally, the 2^−ΔΔCt method was executed to detect the relative RNA expression level of RNAs. Three independent experiments were carried out by qRT-PCR for each sample to ensure the accuracy. All primers’ sequences used in the qRT-PCR were shown in Additional file 1: Table S1.

Subcellular Fractionation Location

According to the manufacturer's instructions, the PARIS Kit (Life Technologies, USA) is used to separate cytoplasmic and nuclear components.

In situ hybridization

ISH was performed on paraffin-embedded specimens of 86 pairs of LSCC and adjacent tissues to detect the expression of KCNQ1OT1. The RNA scope® 2.5 Assay and HybEZ™ Hybridization System employing in ISH,as well as KCNQ1OT1 target, positive and negative control probes were provided by Advanced Cell Diagnostics (ACD). The common housekeeping protein, peptidyl-prolyl isomerase B (PPIB) and the bacterial protein dihydrodipicolinate reductase (DapB) were used as positive and negative control probes, respectively. Slices were scored microscopically according to the manufacturer's instruction: 0: none; 1: each tumor cell has 1–3 brown signal points; 2: each tumor cell has 4–9 brown signal points; 3: each tumor cell has 10–15 brown signal points, and < 10 % of tumor cells have clusters of brown signal points; 4: each tumor cell has more than 15 brown signal points, and > 10% of tumor cells have clusters with many brown signal points. The low expression of KCNQ1OT1 is represented by 0 and 1 scores, while 2, 3 and 4 scores indicated the high expression of KCNQ1OT1.

Cell culture and Cell transfection

Human oral keratinocytes (HOK) and human laryngeal cancer cells (TU212 and AMC-HN8) were provided by Tongpai (Shanghai) Biotechnology Co., Ltd. (Shanghai, China), all of which were cultivated conventionally in DMEM medium (HyClone) containing 10% FBS (Biological Industries) and penicillin-streptomycin (Solaibao) in a humidified incubator at 37°C with an atmosphere with 5% CO2 in air (Thermo Fisher). Lentiviruses knocking down and over-express KCNQ1OT1, ALKBH5, HOXA9 and YTHDF2 as well as respective negative control vectors designed by Genechem Co., Ltd. (Shanghai, China) were transfected into human laryngeal cancer cells (TU212 and AMC‐HN8) in logarithmic growth period. The stable cell lines which selected by high glucose medium with 2.0 µg/mL puromycin for 2 weeks were used in subsequent experiments after verification by qRT-PCR. The transfection protocol was carried out according to the transfection reagent manufacturer's instructions.

Cell Proliferation assay

CCK-8 assay and EDU assay were implemented to measure cell proliferation. LSCC cells as the control group and lentivirus-transfected cells as the experimental group were respectively seeded into 96-well plates at a density of 5000 cells per well. After 24h in a humidified incubator at 37°C with 5% CO2, according to the cell counting kit 8 (CCK-8, Sigma-Aldrich, MO, US) manufacturer's instructions, 10 µL CCK8 solution was added into each well and incubated with cells. At the appointed time, the absorbance at 450nm was measured. In addition, a 5-ethynyl-20-deoxyuridine assay (EdU) kit (BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 555, shanghai, china) was used for detection of cell proliferation. Every step was carried out in strict accordance with the instructions of the kit.

Colony Formation assay

In order to further determine the long-term proliferation ability, cells in logarithmic growth phase were taken to prepare cell suspension and seeded in cell culture dishes for 2–3 weeks, and the fresh medium was replaced timely. At the appropriate time,4% paraformaldehyde was used to fix cell colony for 15 min, then 0.1% crystal violet stained for 20 ~ 30min. Images of colony formation were saved and analyzed.

Migration and invasion assay

Cell invasion ability was determined by Transwell assay. LSCC cells of different experimental groups in logarithmic growth phase were cultured in serum-free medium for 24h. After that, cells were resuspended in serum-free medium at a density of 1×10⁵ cells/ml. 200 µL of cell suspension was added into each Transwell chamber coated with diluted Matrigel(BD Biosciences), as well as, 600 µL of high glucose medium containing 20% FBS was added into each well of 24-well culture plate. Transwell plates were incubated at 37 ° C for 24h. Finally, the invaded cells on the lower surface of Transwell chambers were fixed with 4% paraformaldehyde and stained with 0.1% crystal viole. The inverted microscope was used to count the number of cells in five randomly selected fields. Cell migration ability was assessed by wound healing assay. 5 × 10⁵ cells (per well) in stable growth were seeded into a 6-well plate and incubated to 80% confluence. Vertical scratches in the central area of monolayer adherent cells per well were generated using 100 µL pipette tip. Images of wound of same observed location at different time points were captured by the microscope and wound healing extent were evaluated by Image J.

In vivo experiment

To evaluate the tumorigenic capacity of the cells, animal experiments were carried out with the approval of the Animal Ethics Committee. 4–5 weeks old, 15–20 g weight Balb/c male nude mice were provided by Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animal feeding and experiment were performed in the Second Affiliated Hospital of the Harbin Medical University. 1×10⁶ (100µL) cells of transfected and untransfected by lentiviral were respectively injected subcutaneously into nude mice which divided randomly into scramble group and shKCNQ1OT1-1 group. Tumor formation was observed one week after subcutaneous injection. Afterwards, tumour volumes measured twice a week until mice were euthanized and tumors were surgically removed. A portion of tumors obtained were used for subsequent immunohistochemistry analysis.

Immunohistochemistry (IHC)

Paraffin-embedded LSCC and non-tumor tissues, as well as xenograft tumors were cutted into 4-µm slices. The antigen retrieval was performed after samples were removed from paraffin for deparaffinized and rehydrated. The expression levels of Ki-67, MMP-2, MMP-9, HOXA9 and ALKBH5 were measured by immunohistochemical staining with antibodies against Ki67(1:1000) (Abcam, UK), MMP-2 (1:250)(Abcam, UK), MMP-9 (1:1000)(Abcam, UK), HOXA9 (1:500)(PL Laboratories), ALKBH5(1:500)(Abcam, UK). After incubation with primary antibody at 4 ℃ overnight, slices were incubated with secondary antibody for 30 min at room temperature. Slices were then stained with diaminobenzidine and counterstained with hematoxylin. Thereafter, images of IHC staining were obtained a microscope.

RNA immunoprecipitation (RIP)

The Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore Sigma, 17–700) is used for RNA immunoprecipitation. According to the manufacturer’s instructions, RIP lysis buffer was prepared to treat cells. The cell lysates were incubated with protein antibodies and normal rabbit IgG overnight at 4°C. The RNA-protein/antibody complexes were then immunoprecipitated with protein A/G magnetic beads. RNA is extracted from the precipitated complex for qRT-PCR.

RNA pull-down assay and Western blot analysis

RNA pull-down assay was performed to detect the interaction between KCNQ1OT1 and ALKBH5. In simple terms, biotin (Bio)-labeled LncRNA KCNQ1OT1 were used to incubate with total proteins from AMC-HN-8 and TU-212 cell lysates. The complexes formed bound to Streptavidin-coupled Dynabeads, after which Western blot analysis was carried out to verify the enriched protains after elution and recovery. After the proteins was extracted and the protein concentration was measured, the proteins were separated electrophoretically in SDS-PAGE gels and transferred to PVDF membranes. Subsequently, the primary and secondary antibodies were incubated with PVDF membranes, respectively, according to conventional methods. Finally, Chemiluminescence detection reagent (ECL)(Solaibao, Beijing, China) was used for detection. All antibodies used in Western blot analysis, unless specially stated, were purchased from Aibokang (Shanghai) Trading Co., Ltd.

RNA stability assays

In order to test whether YTHDF2 can regulate the expression of KCNQ1OT1 by affecting the stability of KCNQ1OT1, we used medium containing actinomycin D (a9415, Sigma, USA, 5µg/mL) to culture LSCC cells,and then detected the half-life of KCNQ1OT1 transcripts. Use the same method to detect the effect of ALKBH5 knockdown on the half-life of HOXA9.

Luciferase Reporter assay

AMC-HN-8 and TU-212 cells were inoculated into 12-well plates at 50% density. Luciferase reporter gene plasmid and control plasmid were co-transfected into cells after reaching 70% cell confluency. Following the co-transfection for 36h, the Dual-Luciferase Reporter System (Promega, USA) was used to detect the luciferase activities in cell lysates. The experiment was performed in triplicate.

Statistical analysis

The experiment data were analysed using SPSS version 17.0 software and presented as mean ± SD. All graphs were drawn with GraphPad Prism 5 and 8 software. P-value was calculated by the Student’s t-tests and P<0.05 was suggested statistically significant.

Overview of the m6A-LncRNA expression profiles in LSCC

Using the method of Epitranscriptomic Microarray, we evaluated the m6A-LncRNA expression profiles in three pairs of LSCC and non-tumor tissues. Differentially m6A-methylated LncRNAs based on "m6A methylation level" and "m6A quantity" passing fold change and statistical significance cutoffs were identified and compiled. Hierarchical clustering heatmap analysis was performed for differentially m6A-methylated LncRNAs. Cluster analysis arranges samples according to the similarity of m6A methylation level or quantity and the closeness of the relationship as shown in the dendrogram on top of the heatmaps. Results of hierarchical clustering heatmap show that there was a significant m6A-methylated LncRNAs expression profile between the samples (Fig. 1A). In order to compare the difference of m6A modification between the LSCC and non-tumor tissues, the fold change (FC) and the statistical significance (p-value) of the difference were calculated for each transcript. The default thresholds are |FC| ≥1.2 and p-values ≤ 0.05. Volcanic map showed the significant difference m6A-methylated LncRNA in LSCC and non-tumor tissues (Fig. 1B). The microarray data showed the significant differential m6A modification of the 42 LncRNAs. 33 LncRNAs showed high m6A-methylation and 9 LncRNAs showed low m6A-methylation (Fig. 1C and Table 1).

Table 1

The top 18 differently m6A-methylated lncRNAs.
GeneSymbol	Fold change	Regulation	Chromsome	Lenth	P-value
AC004562.1	1.629	up	chr17	665	0.039
RP11-426A6.5	1.451	up	chr9	1117	0.003
LINC00469	1.434	up	chr17	472	0.024
LOC729683	1.412	up	chr17	2348	0.036
CTA-293F17.1	1.395	up	chr7	737	0.023
LOC100131347	1.357	up	chr17	2305	0.042
RPL7	1.346	up	chr8	534	0.032
LOC105374727	1.340	up	chr5	431	0.020
CHST12	1.335	up	chr7	2067	0.007
KCNQ1OT1	1.732	down	chr11	91671	0.048
SPATA6L	1.676	down	chr9	3435	0.044
RP11-150O12.5	1.604	down	chr8	847	0.047
MECP2	1.346	down	chrX	342	0.002
AE000661.50	1.274	down	chr14	457	0.012
SELO	1.270	down	chr22	1626	0.018
RP11-661A12.7	1.249	down	chr8	431	0.046
ATP7A	1.242	down	chrX	5549	0.028
AP000688.8	1.2	down	chr21	1048	0.004

LncRNA KCNQ1OT1 was low m6A-methylation and up-regulated in LSCC

For the remaining samples of the m6A-LncRNA Epitranscriptomic Microarray, we evaluated the level of m6A modification in KCNQ1OT1, SPATA6L, RP11-150O12.5 and MECP2 by MeRIP-qPCR. Compared with non-tumor tissues, only KCNQ1OT1 was significantly low m6A-methylation in LSCC (Fig. 2A). The results of KCNQ1OT1 revealed strong consistency among the MeRIP-qPCR results and the microarray data. Then, we further expanded the LSCC sample to detect KCNQ1OT1 methylation level in 86 pairs of LSCC and non-tumor tissues. The results of MeRIP-qPCR showed that KCNQ1OT1 was significantly low m6A-methylation in LSCC compared with that in non-tumor tissues (Fig. 2B). Therefore, we chose KCNQ1OT1 for further study. Based on the sequence of KCNQ1OT1 (Additional file 2: Table S2), we used online prediction software RNA coding potential assessment tool (CPAT) (http://lilab.research.bcm.edu/cpat/index.php) to predict the potential protein-coding capacity of lncRNA KCNQ1OT1 and the result showed KCNQ1OT1 is a non-coding RNA (Additional file 3: Figure S1). Based on the analysis of TCGA database, it was found that KCNQ1OT1 was overexpression in head and neck squamous cell carcinoma (HNSCC), and its expression level was related to tumor grade (Fig. 2C-D). However, the expression of KCNQ1OT1 in LSCC has not been reported, therefore our team further explored the expression of KCNQ1OT1 in LSCC. Then KCNQ1OT1 expression was examined in the remaining samples of the m6A-LncRNA Epitranscriptomic Microarray. The results indicated that the expression of KCNQ1OT1 was remarkable up-regulated in the remaining samples (Fig. 2E). Next, we verified KCNQ1OT1 up-regulation in an expanded LSCC sample cohort consisting of 86 pairs of LSCC and non-tumor tissues by qRT-PCR. qRT-PCR was used to determine the expression percentage of KCNQ1OT1 in the cytoplasm and nucleus, and it was found that KCNQ1OT1 was mainly located in the nucleus of TU212 cells (Additional file 4: Figure S2). ISH was performed in an expanded LSCC sample cohort of 86 pairs of LSCC and corresponding non-tuomor tissues to verify the level of KCNQ1OT1 was significantly increased in LSCC (Fig. 2F). Consistently, we demonstrated that KCNQ1OT1 expression was significantly increased in LSCC. In this sample cohort, KCNQ1OT1 was significantly up-regulated in 42% (36 / 86) of LSCC patients (Fig. 2G-2H). According to the correlation between clinicopathological status and KCNQ1OT1 expression in LSCC patients, it was found that the expression of KCNQ1OT1 was correlated with differentiation and lymph node status (Table 2). In order to further evaluate the clinical significance of high KCNQ1OT1 expression in LSCC, we compared the difference of survival rate between patients with high and low expression levels of KCNQ1OT1 survival curve (86 patients, 5 patients were lost to follow-up). The results showed that high-expression of KCNQ1OT1 was significantly associated with poor prognosis at 60 months (Logrank p = 0.047, Fig. 2I). The expression of KCNQ1OT1 was detected in LSCC cell lines (TU212 and AMC-HN8), and human oral keratinocytes (HOK) was used as negative control. The results showed that the expression of KCNQ1OT1 in LSCC cell lines was significantly higher than that in HOK (Fig. 2J). In conclusion, KCNQ1OT1 is highly expressed and low m6A-methylation in LSCC, suggesting that KCNQ1OT1 may play an important role in LSCC.

Table 2

Clinical Characteristics of 86 LSCC patients according to KCNQ1OT1 expression levels.
Characteristics	n	KCNQ1OT1 expression		*P
Characteristics	n	Low expression	High expression	*P
Gender
Male	62	15	47	0.741
Female	24	5	19	0.741
Age(years)
< 60	40	10	30	0.721
≥ 60	46	10	36	0.721
T classification
T1-2	68	17	51	0.457
T3-4	18	3	15	0.457
Differentiation
Well to moderate	66	19	47	0.027*
Poor	20	1	19	0.027*
Lymph node metastasis
Negative	57	18	39	0.01*
Positive	29	2	27	0.01*
Primary location
Supraglottic	42	9	33	0.695
Glottic	44	11	33	0.695
Smoking
Yes	45	17	28	< 0.001*
No	41	3	38	< 0.001*
Family history of cancer
Yes	54	13	41	0.816
No	32	7	25	0.816

LncRNA KCNQ1OT1 depletion inhibits cell proliferation, migration and invasion of LSCC Cells

In order to investigate the regulatory function of KCNQ1OT1 in LSCC, we knockdown the expression of KCNQ1OT1 in TU212 and AMC-HN-8 cells. The expression of KCNQ1OT1 in TU212 and AMC-HN-8 cells stably transfected with shKCNQ1OT1 was significantly reduced, indicating that the transfection efficiency was relatively high. Next, shKCNQ1OT1-1 was selected for further experiments, based on its more effective inhibition (Fig. 3A). Since proliferation is one of the most malignant features of LSCC, we first evaluated the cell proliferation after KCNQ1OT1 knockdown. CCK-8 and Edu results showed that the growth of LSCC cells was significantly inhibited after KCNQ1OT1 was silenced (Fig. 3B and C). Cell staining with crystal violet showed that TU212 and AMC-HN-8 cell clone numbers were significantly reduced in the shKCNQ1OT1-1 group (Fig. 3D), which showed that KCNQ1OT1 knockdown inhibited cell proliferation. Furthermore, KCNQ1OT1 knockdown greatly inhibited wound closure and invasive abilities (Fig. 3E and 3F). In conclusion, these results indicate that KCNQ1OT1 promotes the growth of LSCC in vitro.

KCNQ1OT1 promotes LSCC growth in vivo

In order to verify the effect of KCNQ1OT1 on LSCC, we constructed a xenograft tumor model in nude mice. The KCNQ1OT1-knockdown group and the negative control group were implanted subcutaneously on the right side of nude mice respectively. Compared with the negative control group, the tumor volume of KCNQ1OT1-knockdown group was significantly reduced (Fig. 4A). In similar, the tumor weight of KCNQ1OT1-knockdown group was significantly lower than that of negative control group (Fig. 4B). Ki-67 expression in vivo is one of the key indicators of cell growth, the expression of Ki-67 in each group was further detected by immunohistochemistry (IHC). The results showed that the expression of Ki-67 was significantly decreased in KCNQ1OT1-knockdown group (Fig. 4C). Matrix Metalloproteinase 2 (MMP-2) and Matrix Metalloproteinase 9 (MMP-9) indicate the ability of invasion and metastasis, so we simultaneously detected them in transplanted tumor tissues. The results showed that the immunohistochemical signals of MMP-2 and MMP-9 in KCNQ1OT1-knockdown group were much lower than those in negative control group (Fig. 4D). Overall, KCNQ1OT1 promotes the growth and metastasis of LSCC in vivo. These results strongly suggest that KCNQ1OT1 may be an oncogene of LSCC.

The expression of ALKBH5 was positively correlated with that of KCNQ1OT1 in LSCC

Meanwhile, ALKBH5 is a well-characterized RNA m6A demethylase. The data from GEPIA showed that the expression level of ALKBH5 in HNSCC is higher than in normal tissue (Fig. 5A). Using HOK cell as negative control, the expression of ALKBH5 in LSCC cell lines (TU212 and AMC-HN8) was detected. The results showed that the expression of ALKBH5 in LSCC cells was significantly higher than that in HOK cells (Fig. 5B). Then the expression of ALKBH5 in 86 pairs of LSCC and non-tumor tissues was detected by qRT-PCR. The results showed that ALKBH5 was highly expressed in LSCC (Fig. 5C). The protein expression levels of ALKBH5 in 86 paired cancerous and adjacent noncancerous tissues were detected by IHC. As shown in Fig. 5D, ALKBH5 exhibited nuclear localization, and high level of ALKBH5 is observed in LSCC compared with non-tumor tissue. These results suggest that ALKBH5 is overexpressed in LSCC. We then detected a linear correlation between the expression of ALKBH5 and KCNQ1OT1 in 86 pairs of LSCC. Notably, ALKBH5 expression was positively correlated with the KCNQ1OT1 expression in 86 pairs of LSCC (Fig. 5E).

ALKBH5 mediates expression of KCNQ1OT1 in an m6A-YTHDF2-dependent manner

ALKBH5 is a demethyltransferase that dynamically regulates m6A modification. Therefore, we speculate whether the low m6A-methylation of KCNQ1OT1 in LSCC is due to the binding with ALKBH5. The online software RIPseq was used to predict the combination of KCNQ1OT1 and ALKBH5. The results showed that random forest (RF) classifier was 0.85 and support vector machine (SVM) classifier was 0.95 (Fig. 6A). In the performance evaluation experiment, the prediction of probability greater than 0.5 is considered to be "positive", which means that the corresponding RNA and protein are likely to interact. The interaction between KCNQ1OT1 and ALKBH5 is further verified by RIP assays and RNA pull-down analysis (Fig. 6B and C). LSCC cell lines TU212 and AMC-HN-8 were transfected with ALKBH5 lentiviral vector, and the transfection efficiency was confirmed by qRT-PCR (Fig. 6D). This raises the question whether ALKBH5 regulates KCNQ1OT1 expression through m6A methylation-dependent manner. To test this hypothesis, we first knockdown ALKBH5 in TU212 and AMC-HN8 cells resulted in decreased expression of KCNQ1OT1(Fig. 6E). MeRIP-qPCR was used to confirm that ALKBH5 mediated m6A demethylation of KCNQ1OT1. As expected, ALKBH5 knockdown significantly increased the m6A level of KCNQ1OT1, which confirmed that KCNQ1OT1 transcripts were functionally important substrates of ALKBH5 (Fig. 6F). Treatment with a global methylation inhibitor (DAA) led to the upregulation of KCNQ1OT1 levels in TU212 and AMC-HN8 cells (Fig. 6G). Then, we predicted three m6A recognition sites with very high confidence in KCNQ1OT1 sequence based on SRAMP (Fig. 6H). Then, three m6A recognition sites of KCNQ1OT were mutated (GAC to GCC). When ALKBH5 was knockdown, KCNQ1OT1-M1 and KCNQ1OT-M2 decreased significantly, indicating that m6A mainly regulates KCNQ1OT1 expression in LSCC cells through M3 region, although M1 and M2 may also be recognized to some extent (Fig. 6I). The results show that most of m6A is enriched in M3 region of KCNQ1OT1.

The next question we needed to investigate was how m6A affects KCNQ1OT1 expression. Published studies have shown that m6A "reader" proteins selectively recognize and mediate the degradation of m6A-containing RNA [25]. In order to detect which reader protein is responsible for ALKBH5 mediated KCNQ1OT1 upregulation, we used YTHDF1, YTHDF2, YTHDF3 and YTHDC2 antibodies to carry out RIP analysis. The results showed that among YTHDF1, YTHDF2, YTHDF3 and YTHDC2, only YTHDF2 was significantly associate with KCNQ1OT1(Fig. 6J). Furthermore, the interaction between KCNQ1OT1 and YTHDF2 was enhanced after silencing ALKBH5 expression in TU212 and AMC-HN8 cells (Fig. 6K), while ALKBH5 overexpression in HOK cells inhibited this interaction (Fig. 6L). In order to detect whether YTHDF2 is involved in ALKBH5-mediated KCNQ1OT1 upregulation, we further knockdown YTHDF2 in LSCC cells to detect the change of KCNQ1OT1 expression. The results had identified that knockdown of ALKBH5 decreased the expression of KCNQ1OT1 and increased the m6A-methylation level. YTHDF2 and ALKBH5 knockdown at the same time rescued the decrease of KCNQ1OT1 expression (Fig. 6M). we further investigate whether YTHDF2 regulates KCNQ1OT1 expression by regulating its stability. We used the transcription inhibitor actinomycin D to detect the half-life of KCNQ1OT1 transcripts in each group, respectively. Indeed, the half-life of KCNQ1OT1 in both cells with ALKBH5 knockdown was reversed after deletion of YTHDF2 expression (Fig. 6N). Therefore, ALKBH5 mediates KCNQ1OT1 expression via an m6A-YTHDF2-dependent manner.

KCNQ1OT1 directly interacts with HOXA9 in LSCC

Starbase 3.0 was used to predict mRNA as a target for KCNQ1OT1. The predicted results showed that there was a direct binding site between KCNQ1OT1 and HOXA9, and the potential complementary binding sites of HOXA9 and KCNQ1OT1 were shown in Fig. 7A. Previous studies have shown that HOXA9 was significantly up-regulated in LSCC tissues compared to control tissues [26–27]. The analysis of TCGA database showed that HOXA9 was highly expressed in HNSCC, and its expression level was related to tumor grade and lymph node metastasis status (Additional file 5: Figure S3A-C). qRT-PCR verified that HOXA9 was overexpression in LSCC tissues compared with non-tumor tissues in 86 pairs of LSCC sample cohort, and its expression is positively correlated with the expression level of KCNQ1OT1 (Additional file 6: Figure S4A-B). We further analyzed the relationship between HOXA9 expression and survival rate in 86 pairs of LSCC sample cohort, and found that the high expression of HOXA9 was significantly associated with poor prognosis (Additional file 6: Figure S4C). Using HOK cell as a negative control group, it was found that the expression of HOXA9 in LSCC cell lines (TU212 and AMC-HN-8) was significantly higher than that in HOK (Additional file 6: Figure S4D). IHC was used to detect the expression of HOXA9 in 86 pairs of LSCC and non-tumor tissues, HOXA9 was expressed significantly higher in LSCC tissues and mainly located in the nucleus (Additional file 7: Figure S5). Next, we knocked down and over-expressed the expression of HOXA9 in LSCC cell lines (TU212 and AMC-HN-8), and indicated the transfection efficiency by qRT-PCR (Additional file 8: Figure S6A-B). CCK-8, EDU, colony formation, transwell and wound healing assays further demonstrated that the silencing of HOXA9 inhibited LSCC cells proliferation, migration and invasion ability (Additional file 9: Figure S7A-E). qRT-PCR was used to detect the differential expression level of HOXA9 after KCNQ1OT1 knockdown in LSCC cells. The results showed that the expression of HOXA9 decreased significantly after KCNQ1OT1 knockdown (Fig. 7B). Luciferase reporter assays was used to further verify that HOXA9 is the target of KCNQ1OT1. The results showed that luciferase activity increased after transfection with wild-type vector. However, the luciferase activity of the mutant type vector was not affected (Fig. 7C). Culturing KCNQ1OT1 knockdown LSCC cell line TU212 and AMC-HN-8 with medium containing the transcription inhibitor actinomycin D to block mRNA synthesis, and the results indicated that HOXA9 mRNA attenuation rate was faster in KCNQ1OT1 knockdown LSCC cells (Additional file 10: Figure S8). The inhibition of ALKBH5 could partially neutralize the effect of over-expressed HOXA9 on HOXA9 protein expression in LSCC cell line TU212 (Fig. 7D),while ALKBH5 overexpression could partially neutralize the effect of HOXA9 inhibition on the expression of HOXA9 protein in LSCC cell line AMC-HN-8 (Fig. 7E). In addition, CCK-8, Edu, colony formation, transwell and wound healing assays confirmed that the ectopic expression of HOXA9 promoted the progress of LSCC, and partially rescued the decline of LSCC cell proliferation and invasion after ALKBH5 knockdown (Fig. 7F, G, H, I, J).

The classification of head and neck tumors includes tumors of nasal cavities, paranasal sinuses, oral cavity and larynx, nasopharynx, oropharynx, and hypopharynx [28]. More than 90% of head and neck tumors are squamous cell carcinoma [29], therefore they are classified as head and neck squamous cell carcinoma (HNSC) [30]. Laryngeal squamous cell carcinoma (LSCC) is one of the largest subtypes [28–29]. It is necessary to study the pathogenesis of LSCC and identify the risk factors of LSCC mortality. Increasing evidences show that lncRNAs play an important role in the development of tumor and have been proved to be important regulators of signal pathways in the carcinogenesis [31]. N6-methyladenosine (m6A) is a prevalent internal modification of mRNAs, which regulates gene expression by modulating RNA processing, localization, translation. In this study, we used the Epitranscriptomic Microarray to detect the differential expression of m6A-LncRNA in in three pairs of LSCC and non-tumor tissues. The microarray data showed that there were 42 lncRNAs significant differences in m6A modification (|FC| ≥1.2 and p-values ≤ 0.05). They include 33 high m6A-methylation lncRNAs and 9 low m6A-methylation lncRNAs, respectively. Next, we used the remaining samples to carry out MeRIP-qPCR experiments on KCNQ1OT1, SPATA6L, RP11-150O12.5 and MECP2. The results showed that only KCNQ1OT1 showed significantly low m6A-methylation in LSCC samples compared with non-tumor tissues. LncRNA KCNQ1 overlapping transcript 1 (KCNQ1OT1) is located at the human KCNQ1 locus, with a length of 91 kb. Recent study confirms that KCNQ1OT1 plays essential roles in tumor proliferation, metabolism, epithelial–mesenchymal transition (EMT) and growth by acting as competing endogenous RNA (ceRNA) [32–33]. It has been proved that KCNQ1OT1 can promote cell proliferation and autophagy and inhibit cell apoptosis by regulating mir-204-5p/ATG3 axis, which provides a promising target for non-small cell lung cancer (NSCLC) treatment [34]. However, the mechanism of KCNQ1OT1 affecting the development of laryngeal cancer has not been studied. Therefore, KCNQ1OT1 was selected for further study in this research. Further investigation found an interesting phenomenon that KCNQ1OT1 shows low m6A-methylation and high expression in LSCC compared with non-tumor tissues. Based on clinical data, in vivo and in vitro experiments, it is confirmed that the expression level of KCNQ1OT1 is closely related to the development and prognosis of LSCC. In LSCC, the relationship between KCNQ1OT1 low m6A-methylation and its high expression needs further study.

In mammals, m6A modification occurs through methyltransferase complex, which is mainly composed of METTL3, METTL14, WTAP and other components. This modification can be reversed by demethylase FTO and ALKBH5 [35, 36]. Chao et al. demonstrate that the m6A demethylase ALKBH5 affects the proliferation and invasion of lung adenocarcinoma cells under IH by downregulating m6A modification on FOXM1 mRNA and by promoting FOXM1 expression [37]. Other studies have revealed a new mechanism by which ALKBH5 methylation lncRNA KCNK15-AS1 inhibits the movement of pancreatic cancer, identifying a potential therapeutic target for pancreatic cancer [38]. Here, the results suggest that ALKBH5 is overexpression and positively correlated with that of KCNQ1OT1 in LSCC. ALKBH5 affects the m6A methylation level of KCNQ1OT1 by regulating specific sites on KCNQ1OT1. Mechanistically, ALKBH5 reduced the m6A methylation level of KCNQ1OT1, and subsequently decreases the binding level of KCNQ1OT1 in reader protein YTHDF2, which is an m6A reader protein that can lead to RNA degradation. It is further confirmed that there is a direct binding site between KCNQ1OT1 and HOXA9, and rescue experiments demonstrate that ALKBH5-KCNQ1OT1-HOXA9 axis affect tumor proliferation, invasion and metastasis. This means that ALKBH5-KCNQ1OT1-HOXA9 axis may be an effective target for the diagnosis and treatment of LSCC.

For the first time, we integrally analyzed differential expression of m6A-LncRNA in LSCC and non-tumor tissues, revealing a tight relationship between m6A methylation and the emergence of LSCC. We found a positive correlation between the expression of ALKBH5 and KCNQ1OT1 in LSCC. In addition, ALKBH5 increase can promote the development of LSCC by promoting KCNQ1OT1-HOXA9 signaling via a m6A-YTHDF2-dependent manner (Fig. 8). However, whether the combined expression of ALKBH5-KCNQ1OT1-HOXA9 can be used as a diagnosis and treatment biomarker of LSCC needs more sample data. In the future, our team will continue to explore the potential clinical significance of lncRNA methylation and its methylation modification in LSCC. At the same time, this study enriched the research on the pathogenesis of LSCC, and provided a new idea for the diagnosis and treatment of LSCC.

Overall, we proposed the role of ALKBH5-mediated m6A modification of LncRNA KCNQ1OT1 in the progression of LSCC. In terms of mechanism, our study found that ALKBH5 and YTHDF2 are involved in the methylation modification of LncRNA KCNQ1OT1 and its expression level in LSCC, and further promote the development of LSCC through KCNQ1OT1-HOXA9 signal transduction. The discovery of this mechanism may play an important role in the diagnosis and treatment of LSCC in the future.

ALKBH5: AlkB homolog 5, RNA demethylase; CCK-8: Cell Counting Kit-8; DMEM: Dulbecco’s modified Eagle’s medium; FBS: Foetal Bovine Serum; FTO: FTO alpha-ketoglutarate dependent dioxygenase; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; HNSC: Head and neck squamous cell carcinoma; IHC: Immunohistochemical; LSCC: Laryngeal squamous cell carcinoma; METTL14: Methyltransferase like 14; METTL3: Methyltransferase like 3; m6A: N6-methyladenosine; mRNA-seq: mRNA transcriptomic sequencing; OS: Overall Survival; PBS: Phosphate buffered saline; qRT-PCR: Quantitative real-time PCR; RIP: RNA immunoprecipitation; WTAP: WT1 associated protein; YTHDF1: YTH N6-methyladenosine RNA binding protein 1; YTHDF2: YTH N6-methyladenosine RNA binding protein 2 YTHDF3: YTH N6-methyladenosine RNA binding protein 3 KCNQ1OT1: opposite strand/antisense transcript 1; YTHDC1: YTH domain containing 1; HOXA9: homeobox A9; SPATA6L: spermatogenesis associated 6 like; MECP2: methyl-CpG binding protein 2; MeRIP-PCR: methylated RNA immunoprecipitation-PCR; ISH: Isolated systolic hypertension; EDU: a 5-ethynyl-20-deoxyuridine assay kit; HOK: human oral keratinocytes; MMP-2: Matrix Metalloproteinase 2; MMP-9: Matrix Metalloproteinase 9; DAA: 3-deazaadenosine; EMT: epithelial–mesenchymal transition; ceRNA: competing endogenous RNA; BSA: Bovine Serum Albumin

Ethics approval and consent to participate

It was reviewed and approved by the Ethical Review Committee of the Second Affiliated Hospital of Harbin Medical University, and the tissue samples were obtained with the patient's informed consent. The animal experiments was reviewed and approved by the Ethics Committee of Harbin Medical University.

Consent for publication

All authors have agreed to publish this manuscript.

Availability of data and material

The authors declare that the data in this article are available.

Competing interests

The authors declare that they have no competing interests.

Funding

This study was supported by the National Natural Science Foundation of China (No. 81772874 and 81272965) and the fellowship of China Postdoctoral Science Foundation (No. 2020 M670926).

Authors' contributions

YS, PW and ML designed the study. YL, BY, XW, LT and QL performed cell experiments. YL, BY, JW and XK carried out animal experiments. YL and BY contributed to data analysis. YL wrote the manuscript. YS and QL participated in the revision of the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

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Additionalfile1TableS1.doc
Additional file 1: Table S1. Primers used in the study. (DOCX)
Additionalfile2TableS2.doc
Additional file 2: Table S2. The sequences of lncRNA KCNQ1OT1. (DOCX)
Additionalfile3FigureS1.tif
Additional file 3: Figure S1. The result of prediction of the protein coding potential of KCNQ1OT1 by using the online software RNA coding potential assessment tool (CPAT). (TIF)
Additionalfile4figureS2.tif
Additional file 4: Figure S2. qRT-PCR was performed to detect the expression level of KCNQ1OT1 in nucleus or cytoplasm of LSCC cell line TU212 and AMC-HN-8. GAPDH was used as a cytoplasmic marker and U6 was used as a nuclear marker. (TIF)
Additionalfile5figureS3.tif
Additional file 5: Figure S3. (A-C) Based on TCGA database, HOXA9 was overexpressed in HNSC and related to tumor grade and lymph node metastasis status. (TIF)
Additionalfile6figureS4.tif
Additional file 6: Figure S4. (A) qRT-PCR was used to verified the expression of HOXA9 in 86 pairs of LSCC and non-tuomor tissues. (B) HOXA9 expression was positively associated with the expression level of KCNQ1OT1 in 86 pairs of LSCC. (C) The mRNA levels of HOXA9 in HOK cells and LSCC cells was detected by qRT-PCR. (D) High expression of HOXA9 was significantly associated with poor prognosis. (TIF)
Additionalfile7figureS5.tif
Additional file 7: Figure S5. Representative IHC images of HOXA9 expression in 86 pairs of LSCC and non-tumor tissues under ×400 magnification. (TIF)
Additionalfile8figureS6.tif
Additional file 8: Figure S6. (A) qRT-PCR indicated the expression of HOXA9 after HOXA9 knockdown in LSCC cells. (B) qRT-PCR indicated the expression of HOXA9 after HOXA9 overexpression in LSCC cells. (TIF)
Additionalfile9figureS7.tif
Additional file 9: Figure S7. (A-E) CCK-8, Edu, colony formation, transwell and wound healing assays demonstrated that silencing of HOXA9 expression suppressed LSCC cells proliferation, migration and invasion ability. (TIF)
Additionalfile10figureS8.tif
Additional file 10: Figure S8. The half-life curve indicated the effect of KCNQ1OT1 knockdown on the half-life of HOXA9. (TIF)

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ALKBH5-Mediated m6A Modification of LncRNA KCNQ1OT1 Triggers the Development of LSCC via Upregulation of HOXA9

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Patients and specimens

Microarray analysis

Methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR)

Quantitative real-time PCR (qRT-PCR)

Subcellular Fractionation Location

In situ hybridization

Cell culture and Cell transfection

Cell Proliferation assay

Colony Formation assay

Migration and invasion assay

In vivo experiment

Immunohistochemistry (IHC)

RNA immunoprecipitation (RIP)

RNA pull-down assay and Western blot analysis

RNA stability assays

Luciferase Reporter assay

Statistical analysis

Results

Overview of the m6A-LncRNA expression profiles in LSCC

LncRNA KCNQ1OT1 was low m6A-methylation and up-regulated in LSCC

LncRNA KCNQ1OT1 depletion inhibits cell proliferation, migration and invasion of LSCC Cells

KCNQ1OT1 promotes LSCC growth in vivo

The expression of ALKBH5 was positively correlated with that of KCNQ1OT1 in LSCC

ALKBH5 mediates expression of KCNQ1OT1 in an m6A-YTHDF2-dependent manner

KCNQ1OT1 directly interacts with HOXA9 in LSCC

Discussion

Conclusion

Abbreviations

Declarations

Ethics approval and consent to participate

Consent for publication

Availability of data and material

Competing interests

Funding

Authors' contributions

Acknowledgements

References

Supplementary Files

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