LncRNA TM4SF19-AS1 Promotes the Proliferation, Migration and Invasion of Laryngeal Squamous Cell Carcinoma by Targeting miR-153-3p/ITGAV Axis

doi:10.21203/rs.3.rs-720370/v1

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LncRNA TM4SF19-AS1 Promotes the Proliferation, Migration and Invasion of Laryngeal Squamous Cell Carcinoma by Targeting miR-153-3p/ITGAV Axis

https://doi.org/10.21203/rs.3.rs-720370/v1

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Background: LncRNA plays an important role in the gene regulatory network and can affect the progress of tumors. LncRNA TM4SF19-AS1 has been reported may associate with the occurrence and development of head and neck squamous cell carcinoma.

Methods: LncRNA TM4SF19-AS1 expression in laryngeal squamous cell carcinoma (LSCC) tissue samples was evaluated in TCGA database, and its expression in LSCC tissues and cells was further determined via qRT-PCR. CCK-8, EdU, wound healing and transwell assays were performed to access the cell biological behaviors of TM4SF19-AS1. The downstream regulatory mechanism of TM4SF19-AS1 regulating gene expression was further detected by WGCNA, subcellular location prediction, western blot and dual-luciferase reporter assay.

Results: The expression of TM4SF19-AS1 was upregulated in LSCC tissues and positively correlated with tumor-node-metastasis (TNM) stage and lymph node metastasis in LSCC patients. Knockdown of TM4SF19-AS1 suppressed the proliferation, migration and invasion of LSCC cells. Mechanistically, TM4SF19-AS1 acted as a competing endogenous RNA (ceRNA) that directly bound to miR-153-3p, and ITGAV was the direct target of miR-153-3p.

Conclusions: LncRNA TM4SF19-AS1 promotes the proliferation, migration and invasion of laryngeal carcinoma by targeting miR-153-3p/ITGAV axis, suggesting that TM4SF19-AS1 could be a potential diagnostic biomarker and an effective target for the treatment for LSCC.

Surgery

Oncology

laryngeal carcinoma

TM4SF19-AS1

miR-153-3p

ITGAV

In 2018, the global incidence of laryngeal cancer (LC) was about 1%, and 1% of cancer related deaths were caused by LC [1]. LSCC is the most common pathological type of LC. Like most tumors, LC occurs due to the interaction of somatic mutations with various environmental factors [2]. The risk factors of LC included tobacco-like products and alcohol abuse and both had a linear relationship with the development of LC [3]. Although the incidence rate of LC has declined due to the reduction in exposure to tobacco products, the 5-year survival rate dropped only from 66–63% over past 40 years [4, 5]. Therefore, it is of great importance to identify highly specific molecular biomarkers and clarify the tumorigenesis mechanism of LSCC.

LncRNA is classified as non-coding transcripts with more than 200 nucleotides, accounting for about 80% of non-coding RNA, and their expression largely depends on the tissue and cellular environment [6]. According to the different positions of lncRNA in the genome, it can be divided into: sense, antisense, intergenic, bidirectional and intron [7]. Recently, studies have found that lncRNA plays an important role in gene regulatory networks by controlling the structure and transcription of the nucleus, as well as regulating mRNA stability, translation and post-translational modification processes in the cytoplasm [8]. More studies have been paid on the mechanism of ceRNA. In human cancer, the PTEN-PTENP1 regulatory axis was reported as the first ceRNA network [9]. Additionally, lncRNA was also considered as one of the ceRNA, which release mRNA by sponging miRNA in the cytoplasm [10]. For example, lncRNA HOTAIR functioned as a competing endogenous RNA that could regulate the proliferation, migration and invasion of gastric carcinoma via the miR-331-3p/HER2 axis [11]. Besides, lncRNA has high tissue specificity and may become the targets for cancer therapy [12].

LncRNA TM4SF19-AS1 has been reported may associate with the prognostic of head and neck squamous cell carcinoma [13]. Nonetheless, the expression and function of TM4SF19-AS1 in LSCC are still unclear. In this study, we verified that TM4SF19-AS1 was highly expressed in LSCC tissues and positively correlated with TNM stage and lymph node metastasis. Also, TM4SF19-AS1 regulated cell proliferation, migration and invasion of LSCC by sponging miR-153-3p and targeting ITGAV.

LSCC datasets and clinical samples

The fragments per kilobase of per million (FPKM) of LSCC transcriptome, RNA counts data and corresponding clinical data from 123 LSCC patients were downloaded from the TCGA database. Frozen fresh tumor tissues and corresponding adjacent normal tissues were collected from 36 patients with LSCC (median age, 63.39 years; range, 39-80 years), who underwent surgical resection between March 2015 and May 2018 at the Second Hospital of Hebei Medical University. None of the patients received chemotherapy, radiotherapy or other anticancer treatments before surgery. The final diagnosis of patients were independently diagnosed by two pathologists. In addition, the clinicopathological characteristics of the patients analyzed in the present study included details of age, smoking, drinking, TNM stage, lymph node metastasis and tumor differentiation. Written informed consent was obtained from each patient and the process was approved by the Ethics Committee of Hebei Medical University and the Second Hospital of Hebei Medical University.

Data preprocessing and differential expression analysis

The software “edgeR” package of R was used for profiling the expression of differentially expressed lncRNAs (DElncRNAs), miRNAs (DEmiRNAs) and mRNAs (DEmRNAs) in the TCGA database. The thresholds were set as |log2 foldchange(FC) | >1, with the p value< 0.05.

Weighted gene co-expression network analysis of TM4SF19-AS1 and DEmRNAs

TM4SF19-AS1 and DEmRNAs were used for WGCNA analysis to explore the relationship between co-expression modules and clinical factors. We removed the outliers in the sample, and constructed a weighted adjacency matrix of gene expression data by using a power function based on an appropriate soft threshold parameter β. Then, the adjacency matrix was converted into a topological overlap matrix (TOM), and average linkage hierarchical clustering was carried out based on the TOM-based dissimilarity. In this study, the number of genes in each module exceeded 30.

Subcellular location prediction and ROC curve analysis of TM4SF19-AS1

Different cellular localization of lncRNA has different physiological functions. The prediction of subcellular localization is of great significance for the following study. We used the online tool Lnclocator to predict the subcellular location of lncRNA [14]. Through R software, ROC curve analysis was used to evaluate lncRNA as a molecular marker for predicting patient prognosis.

Cell culture and transfection

Laryngeal cancer cell lines (TU177, TU686, TU212 and AMC-HN-8) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). TU177, TU686 and TU212 were cultured in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.), and supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA). AMC-HN-8 was cultured in DMEM medium (Invitrogen; Thermo Fisher Scientific, Inc.), and supplemented with 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.). All cells were cultured in a humidified chamber at 37˚C with5% CO₂.

For small interfering (si) RNA knockdown experiments, TM4SF19-AS1 siRNA (si-TM4SF19-AS-1 and si-TM4SF19-AS-2) and negative control (NC) were synthesized and purchased from Shanghai GenePharma Co., Ltd. Cells cultured in 6-well plates at 40% confluence (4x10⁵ cells. per plate) were transfected using Lipofectamine^®3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and were harvested after 48 h of incubation at 37˚C.

The miR-153-3p mimics, inhibitors and the corresponding NCs (all 40 pg/µl) were purchased from Shanghai GenePharm Co., Ltd, and transfected into laryngeal cancer cells (2x10⁵) using Lipofectamine^®3000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 8 h at 37˚C. Then, the culture medium was replaced with fresh DMEM supplemented with 10% FBS at 8 h post-transfection. Subsequent experiments were performed 24 h post-transfection.

Cell counting kit-8 (CCK-8) assay

Cell counting kit-8 (CCK-8) (Sangon Biotech, Inc.) assay was used to detect the proliferation of laryngeal cancer cells under different treatments. Briefly, the transfected cells concentration was adjusted to 2.5x10³cells/well and inoculated into 96 well plate at 37˚C. 10 μl CCK-8 was added to the wells at 24, 48 and 72 h post-inoculation. Before measuring the optical density (OD), the original culture medium of each well was replaced with a mixture of 10 μl CCK-8 and 100 μl of culture medium, and then the cells were incubated at 37˚C for another 2 h. The OD of each well was analyzed at a wavelength of 450 nm (620 nm as reference) using a microplate reader (BioTek, Inc.).

EdU incorporation assay

EdU (Beyotime, Shanghai, China) assay was also used to evaluate the cell proliferation ability. AMC-HN-8 cell was seeded in 6-well plates with slides and cultured for 24h. After transfection for 24 h, the cell proliferation was tested according to the EdU kit protocol. Then it was incubated with 10 μM EdU working solution for 2 h, fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 and incubated with DAPI solution to stain nuclei. Imagines were obtained with the fluorescence microscope (Olympus, Tokyo, Japan).

Wound healing assay

Following 48 h transfection, cells were cultured into 6-wellplates until 90% confluence. A vertical lines was drawn with a 200µl sterile micropipette tip against the ruler, and the cells were incubated in serum-free medium for an additional 24 h. Images of the wound were taken at 0 and 24 h respectively.

Transwell assay

A 24-well transwell chamber (8-µm pore size; BD Biosciences) was used for migration assays. After 48 h of transfection, 2.5x10⁴cells per well were plated into the upper chamber containing 200 µl serum-free medium. Then the upper chamber was placed in the lower chamber with 600 µl of medium containing 20% FBS. After 24 h of incubation, the cells on the upper surface of the upper chamber were removed, and the cells on the bottom side of the upper chamber were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Transwell chambers coated with 30 µg Matrigel (BD Biosciences, CA, USA) were used for invasion assays. The number of migratory cells was counted in five randomly selected regions in each well at 200x magnification using inverted microscope.

Bioinformatics prediction and dual-luciferase reporter assay

The web tools StarBase v3.0 was used to predict the potential binding sites of TM4SF19-AS1 and ITGAV transcripts on miR-153-3p [15]. PsiCHECK^TM-2 reporter plasmids (Promega, Madison, WI, USA) containing the wild type (WT) and mutated (MUT) sequences for the 3’ untranslated region (3’UTR) of TM4SF19-AS1 and ITGAV. The empty plasmid served as a control. After that, the constructed luciferase vectors were co-transfected with miR-153-3p mimics or miR-NC into HEK 293T cells. Following incubation for 48 h, the luciferase activity was tested using the Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s protocol. Firefly luciferase activity was normalized to Renilla luciferase activity.

Real-time quantitative reverse transcription PCR (qRT-PCR)

The total RNA in LSCC tissues and 4 cell lines were extracted using a Trizol kit (Solarbio). The concentration and purity of total RNA samples were determined by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The RNA was synthesized into complementary DNA (cDNA) according to the instructions of the FastQuant RT kit (KR106; Tiangen). qRT-PCR analysis was performed on ABI-7500 using Go Taq® qPCR Master Mix (Promega, Madison, WI). The primer sequences of these genes were as follows: TM4SF19-AS1 forward, 5ʹ- GGT CTG CAC AGG TCT GAC TTC-3ʹ and reverse, 5ʹ-CCC CAC CAT GCG TTA TGC TT TT-3ʹ; ITGAV forward, 5ʹ-GTT TCA GTG TGC ACC AGC AG-3ʹ and reverse, 5ʹ-AAG GCC ACT GAA GAT GGA GC-3ʹ; 18S forward, 5ʹ-CGC CGC TAG AGG TGA AAT TC-3ʹ and reverse, 5ʹ-CCA GTC GGC ATC GTT TAT GG-3ʹ.

Western blot analysis

The total cell protein was extracted using radioimmunoprecipitation assay (Beyotime Biotechnology, Shanghai, China) containing protease inhibitors (Roche, Guangzhou, China). Protein concentration was determined using a BCA kit (Pierce, Appleton, WI). Equal amounts of the protein samples were separated using 10% SDS-poly acrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto a polyvinylidene difluoride membrane (PVDF; Millipore, Billerica, MA, USA). The membranes were blocked with tris-buffered saline (TBS) containing 5% skimmed milk at 37˚C for 2 h. Then primary antibodies against ITGAV (dilution of 1:1000) and GAPDH (dilution of 1:1000) incubated with the membrane at 4˚C overnight. On the second day, the membrane was washed three times and subsequently incubated with secondary antibody marked by horseradish peroxidase for 1 hour at 37 ˚C. Finally, an enhanced chemiluminescence assay kit was used to observe the protein bands, and the Image J software was used to quantify the protein signal through densitometric analysis.

Statistical analyses

ROC curve analysis was performed in SPSS 22.0 to evaluate the diagnostic value of TM4SF19-AS1. GraphPad Prism 7 was used to analyze the expression correlation between lncRNAs, miRNAs and mRNAs. Two tailed Student's t-tests were used to compare test and control groups between cell lines or tissues. Independent-samples t-test was used to analyze the correlation between gene expression level and clinicopathological data. A p value less than 0.05 was considered a statistically significant difference. The rest of the statistical analysis and drawing were performed using the software package in the statistical software environment R software 4.0.0.

TM4SF19-AS1 expression was increased in LSCC tissues and positively correlated with clinical staging

Data in the TCGA database was used to identify whether TM4SF19-AS1 was involved in the development of LSCC. A total of 1014 DElncRNAs (743 up-regulated and 271 down-regulated), 162 DEmiRNAs (105 up-regulated and 57 down-regulated) and 4985 DEmRNAs (2917 up-regulated and 2068 down-regulated) were identified in the TCGA database. It was observed that TM4SF19-AS1 level was overexpressed in LSCC tissues compared with adjacent normal laryngeal tissues (Fig. 1a). ROC curve analysis was used to evaluate the sensitivity and specificity of TM4SF19-AS1 as a biomarker of LSCC. As shown in Fig. 1b, the area under the curve (AUC) of TM4SF19-AS1 was 0.896, suggesting the TM4SF19-AS1 might be used for the diagnosis of LSCC .

To further study its function, TM4SF19-AS1 and 2917 up-regulated DEmRNAs in the TCGA database were used to construct a WGCNA network to analyze the TM4SF19-AS1 co-expression module and clinical phenotypes. Firstly, we eliminated the abnormal sample (TCGA-CN-4735-01A) with a cutting height = 40,000. The scale-free network was constructed with the soft threshold power of 4, and the hierarchical clustering tree with 11 modules was identified (Fig. 1c). As a result, TM4SF19-AS1 and 457 mRNAs were identified in the turquoise module, which was positively correlated with clinical staging, T stage, smoking and negatively correlated with age at index (Fig. 1d). The relationship between gene significance (GS) and the module membership (MM) in the turquoise module were calculated and the result showed that MM in the turquoise module was significantly correlated with clinical stage (r = 0.4, p = 5E-19), pathologic T (r = 0.31, p = 1.2E-11), smoking (r = 0.24, p = 2E-07) and age at index (r = 0.3, p = 5.6E-11).

TM4SF19-AS1 was expressed at high levels and down-regulation of it inhibits cell proliferation, migration and invasion in LSCC

The expression of TM4SF19-AS1 in 36 paired LSCC tissues and corresponding normal tissues and 4 types of LSCC cell lines were determined by qRT-PCR. As illustrated in Fig. 2a, b, the TM4SF19-AS1 expression was significantly up-regulated in LSCC tissues compared with that of in the adjacent normal tissues, and among the 4 cell lines, TM4SF19-AS1 has the highest expression in the AMC-HN-8 cell line. Therefore, AMC-HN-8 was selected for the following studies. Additionally, the expression level of TM4SF19-AS1 was closely related to TNM stage and lymph node metastasis (Table 1), but had no correlation with other clinicopathological characteristics (age, smoking, drinking and tumor differentiation). Through WGCNA analysis, we knew that TM4SF19-AS1 had a significant correlation with clinical stage, speculating that it might be related to proliferation, migration and invasion. As confirmed by qRT-PCR, transfection of si-TM4SF19-AS1 significantly knocked down the expression of TM4SF19-AS1 in AMC-HN-8 cell line (Fig. 2c). Additionally, CCK-8 and EdU assays indicated that si-TM4SF19-AS1 dramatically decreased the proliferation of AMC-HN-8 cell line (Fig. 2d, e). At the same time, wound healing assays and transwell assays also showed that silencing TM4SF19-AS1 could reduce the migration and invasion ability of AMC-HN-8 cell line (Fig. 2f, g).

Table 1

Correlation of the expression of TM4SF19-AS1 with clinicopathologic data
		TM4SF19-AS1
Parameter	No.	Mean ± SD	P-value
Age
<60	11	3.195 ± 1.253	0.945
≥60	25	3.167 ± 1.058
Smoking
Positive	34	3.205 ± 1.119	0.505
Negative	2	2.660 ± 0.830
Drinking
Positive	24	3.229 ± 1.173	0.685
Negative	12	3.068 ± 0.987
TNM stage
Ⅰ+Ⅱ	11	2.055 ± 0.678	0.000
Ⅲ+Ⅳ	25	3.668 ± 0.868
Lymph node metastasis
Positive	11	4.429 ± 0.322	0.000
Negative	25	2.624 ± 0.832
Tumor differentiation
Well	16	3.362 ± 0.934	0.291^a
Moderate	16	2.940 ± 1.226	0.498^b
Poor	4	3.367 ± 1.332	0.994^c
^aCompared with moderately differentiated. ^bCompared with poorly differentiated. ^cCompared with well differentiated.

TM4SF19-AS1 negatively regulated miR-153-3p expression and bound miR-153-3p in AMC-HN-8 cells

The mechanism of lncRNA is related to cell location. Through the online tool Lnclocator, we predict that TM4SF19-AS1 is mainly located in the cytoplasm (Fig. 3a) and speculate that it may have a ceRNA mechanism. Then, StarBase 3.0 was used to predict the possible miRNA binding sites of TM4SF19-AS1, and there were five miRNAs (miR-153-3p, miR-19a-3p, miR-19b-3p, miR-485-5p and miR-6884-5p) that might target TM4SF19-AS1. Interestingly, only miR-153-3p expression was down-regulated in LSCC samples from the TCGA database (Fig. 3b). Subsequently, miR-153-3p mimic or miR-153-3p inhibitor was used to up-regulate or down-regulate the expression of miR-153-3p in laryngeal cancer cells (Fig. 3c). Moreover, qRT-PCR assays indicated that knockdown of TM4SF19-AS1 increased the expression of miR-153-3p in LSCC cell line (Fig. 3d), while the upregulation of miR-153-3p failed to cause changes in TM4SF19-AS1 levels (Fig. 3e). Additionally, we confirmed the interaction of TM4SF19-AS1 and miR-153-3p through dual-luciferase reporter assay. As shown in Fig. 3f, g, the group that cotransfected with miR153-3p mimic and TM4SF19-AS1-WT significantly reduced the luciferase activity (p < 0.01).

ITGAV was verified as a functional target of miR-153-3p in AMC-HN-8 cells

We also used Starbase3.0 to predict the targeted mRNA of miR-153-3p and obtained 3721 mRNAs. In order to increase the accuracy of the prediction results, the mRNA co-expressed with TM4SF19-AS1 in WGCNA was analyzed by GO and KEGG enrichment. For cell components (CC) enrichment, the TM4SF19-AS1-related mRNAs were mainly distributed in collagen-containing extracellular matrix, cell-substrate junction and focal adhesion (Fig. 4a). For Molecular function (MF) enrichment, the mRNAs were mainly related in extracellular matrix structural constituent, cell adhesion molecule binding and integrin binding (Fig. 4b). For Biological process (BP) enrichment, the mRNAs were classified into extracellular structure organization, extracellular matrix organization and cell − substrate adhesion (Fig. 4c). KEGG pathway analysis revealed 15 statistically significant signaling pathways of these mRNAs (Fig. 4d). Interestingly, the top 3 pathway were related to tumorigenesis and development. From the results of GO and KEGG, we speculated that the TM4SF19-AS1-related mRNAs may mediate the occurrence and metastasis of tumors.

Among the predication results, ITGAV was involved in both cell adhesion and PI3K/Akt signaling pathways, which are the top two pathways in the KEGG functional analysis. ITGAV has been reported to be an oncogene in many tumors such as liver cancer, colon cancer and laryngeal cancer. Therefore, the ITGAV expression was detected by qRT-PCR and the result showed that ITGAV expression in laryngeal cancer tissues was significantly higher than that in adjacent normal tissues (Fig. 5a). Furthermore, western blot assays revealed that the expression of ITGAV in laryngeal cancer tissues was significantly higher than that in adjacent normal tissues (Fig. 5b). Importantly, the expression of ITGAV was decreased in AMC-HN-8 cells after transfection with miR-153-3p mimic and increased after transfection with miR-153-3p inhibitor (Fig. 5c). Then, a dual-luciferase reporter assay was performed to confirm the direct binding between ITGAV and miR-153-3p. As presented in Fig. 5d, e, miR-153-3p mimics significantly attenuated the activity of the luciferase plasmid containing the WT binding sites compared with that of the MUT control (p < 0.01).

Tm4sf19-as1/mir-153-3p/itgav Axis Regulated Lscc Metabolism And Tumorigenesis

To further explore the possible functions of TM4SF19-AS1/miR-153-3p/ITGAV axis in LSCC, TM4SF19-AS1 siRNAs and miR-153-3p inhibitor were cotransfected in LSCC cells. As shown in Fig. 6a, transfection of TM4SF19-AS1 siRNAs decreased the expression of ITGAV, and transfection of miR-153-3p inhibitors could up-regulate the expression of ITGAV. Subsequently, co-transfection of TM4SF19-AS1 siRNAs and miR-153-3p inhibitors did not significantly change the expression of ITGAV compared with si-NC and inhibitor NC. Morever, the EdU and transwell assays showed that transfection of TM4SF19-AS1 siRNAs could decrease the proliferation, migration and invasion of AMC-HN-8 cells, however, these effects were partially reversed by miR-153-3p inhibitor (Fig. 6b-e).

In our research, we first analyzed the TCGA database and found that the expression of TM4SF19-AS1 in LSCC is different. Then, through qRT-PCR experiments, we found that the expression of TM4SF19-AS1 was significantly upregulated in LSCC tissues compared with adjacent normal tissues. High TM4SF19-AS1 expression was associated with TNM stage and lymph node metastasis. TM4SF19-AS1 was predicted to be located in the cytoplasm, and we speculated that it may have a ceRNA mechanism. We demonstrated that TM4SF19-AS1 could sponge miR-153-3p, thus regulating the ITGAV expression levels. In addition, TM4SF19-AS1 knockdown could inhibit the proliferation, migration and invasion of LSCC cells, however, these effects were partially reversed by miR-153-3p inhibitor.

In the past few years, the research of long non-coding RNA such as HOTAIR, H19, NEAT1 and MALAT1 in malignant tumors has gradually attracted attention [16–19]. However, so far, there are relatively few studies on lncRNA in laryngeal squamous cell carcinoma. In our research, we confirmed for the first time that the expression of TM4SF19-AS1 in laryngeal cancer tissues was higher than that in adjacent normal tissues and regulated the proliferation, migration and invasion of laryngeal squamous cell carcinoma cells by targeting miR-153-3p/ITGAV axis.

In all eukaryotics, miRNAs are a highly conserved non-coding RNA molecules [20]. Through repressing target genes, miRNAs play multiple roles in controlling cellular functions [21]. An increasing number of studies have shown that lncRNAs can act as competing endogenous RNAs, interacting with miRNAs, thereby isolating these molecules and reducing their regulatory effects on target mRNAs [22]. Using bioinformatics methods, we predicted a binding between miR-153-3p and TM4SF19-AS1. Previous studies revealed that miR-153-3p is down regulated in a variety of squamous cell carcinoma and is closely related to the occurrence and development of tumor. For instance, the expression of miR-153-3p in oral cancer tissues and cells is significantly reduced, and it is involved in regulating tumor proliferation [23]. Liu et al. disclosed that miR-153-3p is down-regulated in esophageal squamous cell carcinoma tissues, and LINC00152 facilitate the proliferation of esophageal squamous cell carcinoma cells via miR-153-3p/FYN axis [24]. Ma et al. revealed that miR-153-3p is down-regulated in cervical cancer cells, and upregulated circ_0005576 promotes cervical cancer progression via the miR-153/KIF20A axis [25]. miR-153-3p has also been reported to be involved in the biological processes of a variety of non-squamous cell malignancies, such as malignant melanoma [26], breast cancer [27], acute lymphoblastic leukemia [28], osteosarcoma [29], thyroid cancer [30], gastric cancer [31], and ovarian carcinoma [32]. We found that miR-153-3p expression was down-regulated in LSCC samples from the TCGA database. The expression of miR-153-3p in LSCC cells was negatively correlated with TM4SF19-AS1. The proliferation, migration and invasion changes caused by knocking down TM4SF19-AS1 can be restored by miR-153-3p inhibitor.

The product of ITGAV is a protein that belongs to the integrin alpha chain family. More and more studies have shown that this protein can regulate cancer progression. Blocking of integrin αVβ3 effectively inhibits the proliferation and angiogenesis of many types of cancer cells [33]. ITGAV contributes to the formation of actin stress fibers and plays an important role in the migration and invasion of hepatocellular carcinoma [34]. The expression of ITGAV in laryngeal and hypopharyngeal squamous cell carcinoma is significantly higher than that in normal tissues, the well-differentiated group is significantly lower than the medium and poorly differentiated groups, and the lymph node metastasis group is significantly higher than the non-lymph node metastasis group [35]. Our studies also demonstrated that the expression of ITGAV was highly in LSCC tissues.

In conclusion, our study elucidated that the expression of TM4SF19-AS1 was up-regulated in LSCC tissues and modulated the proliferation, migration and invasion of laryngeal cancer cells through miR-153-3p/ITGAV axis. These findings suggested that TM4SF19-AS1 might be a potential diagnostic marker for laryngeal cancer, and also become a promising target for the treatment of LSCC.

Ethics approval and consent to participate

All procedures performed in studies involving human participants were in accordance with the standards upheld by the Ethics Committee of Hebei Medical University and the Second Hospital of Hebei Medical University and with those of the 2008 Helsinki Declaration and its later amendments for ethical research involving human subjects. Written informed consent was obtained from each patient.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Funding

This work was supported by the grants from National Natural Science Foundation of China (81972553) and the Project of Clinical Medical Talent Training and Basic Project Research Funded by Government (Hebei finance society (2019) No.139)

Authors’ contributions

Baoshan Wang and Yudong Liu were responsible for the conception and design of the study. Yudong Liu, Yuexin Tian and Yanzhuo Zhang participated in the experiment. Yudong Liu and Xiaojuan Feng were responsible for acquisition and data analysis. Huan Cao helped to provide clinical samples and some experimental samples. Baoshan Wang and Yudong Liu drafted the manuscript. All authors read and approved the final version of the manuscript.

Acknowledgements

Not applicable.

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LncRNA TM4SF19-AS1 Promotes the Proliferation, Migration and Invasion of Laryngeal Squamous Cell Carcinoma by Targeting miR-153-3p/ITGAV Axis

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Abstract

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Introduction

Materials And Methods

Results

TM4SF19-AS1 expression was increased in LSCC tissues and positively correlated with clinical staging

Tm4sf19-as1/mir-153-3p/itgav Axis Regulated Lscc Metabolism And Tumorigenesis

Discussion

Conclusion

Declarations

References

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