Long non-coding RNA BBOX1-antisense RNA 1 enhances cell proliferation and migration and suppresses apoptosis in oral squamous cell carcinoma via the miR-3940-3p/laminin subunit gamma 2 axis

ABSTRACT Long non-coding RNAs (lncRNAs) play an essential role in oral squamous cell carcinoma (OSCC). We aimed to demonstrate the effects of lncRNA gamma-butyrobetaine hydroxylase 1 (BBOX1)-antisense RNA 1 (AS1) in OSCC and its regulatory mechanisms. The levels of BBOX1-AS1, microRNA (miR)-3940-3p, and laminin subunit gamma 2 (LAMC2) in OSCC were determined using reverse transcription-quantitative polymerase chain reaction. The correlations among BBOX1-AS1, miR-3940-3p, and LAMC2 were validated using luciferase, pull-down, and RNA immunoprecipitation assays. Cell proliferation, migration, and apoptosis were examined. BBOX1-AS1 and LAMC2 were notably overexpressed in OSCC, while miR-3940-3p showed the opposite trend. BBOX-1-AS1 silencing reduced the cell proliferation and migration, while promoting apoptosis. Mechanistically, BBOX1-AS1 modulates LAMC2 expression by competitively binding to miR-3940-3p. miR-3940-3p inhibition alleviated the inhibitory effects of BBOX1-AS1 deficiency on OSCC development. LAMC2 knockdown reversed these changes. Our results revealed that BBOX1-AS1 promotes the malignant phenotype of OSCC cells via the upregulation of LAMC2 expression by targeting miR-3940-3p, indicating that BBOX1-AS1 may be a novel target for OSCC intervention.


Introduction
Oral squamous cell carcinoma (OSCC) is a severe malignancy that mainly affects the head and neck [1]. According to the global cancer statistics of 2019, the 5-year survival rate for patients with OSCC was only approximately 50%. Targeting drugs show good efficacy against most tumors because of their high selectivity and low toxicity [2,3]. The use of targeted therapy has led to the development of novel approaches for the treatment of OSCC in patients with advanced cancer. Therefore, the identification of prognostic biomarkers and novel targets for targeted therapy is of great significance to understand the pathogenesis of OSCC.
Recently, a transcriptome investigation revealed that long non-coding RNAs (lncRNAs) are putative players in the tumorigenesis of most tumors [4]. LncRNA SNHG1 expression levels are elevated in OSCC cells and promote tumor cell growth by downregulating the expression of fucosyltransferase 8 by targeting microRNA (miR)-186 [5]. MYCinduced lncRNA enhances the invasion of OSCC cells by modulating the Wnt/β-catenin pathway [6]. LncRNA gamma-butyrobetaine hydroxylase 1 (BBOX1)-antisense RNA 1 (AS1) exists as an oncogene in most tumor cells. Abnormally high expression levels of BBOX1-AS1 are observed in most cancers, such as lung [7], cervical [8], and esophageal cancers [9], and BBOX1-AS1 is strongly linked to the overall survival of patients with these cancers, which suggests that targeting BBOX1-AS1 may be used as a therapeutic approach for cancer treatment. However, the regulatory effects of BBOX1-AS1 on OSCC development have not yet been elucidated. miRNAs consist of 18-22 nt endogenous noncoding RNA molecules [10]. miRNAs play significant roles in various physiological and pathological processes by regulating the mRNA expression, and aberrant miRNA expression is closely related to the initiation and progression of cancer [11]. Fu et al. found that miR-155 acts as a tumor promoter in the proliferation of OSCC cells by regulating cyclin dependent kinase inhibitor 1B [12]. Moreover, Rastogi et al. demonstrated that the downregulation of miR-377 expression facilitates OSCC cell migration by targeting HDAC9 [13]. BBOX1-AS1 acts as an miR-3940-3p sponge that participates in the regulation of gastric cancer progression [14]. However, the relationship between miR-3940-3p and BBOX1 in OSCC has not yet been studied.
Laminin subunit gamma 2 (LAMC2), a laminin extracellular matrix protein, is a cross-shaped heterotrimer composed of one heavy (α) and two light (β and γ) chains. High expression of LAMC2 promotes tumor metastasis via the epithelial-mesenchymal transition and epidermal growth factor receptor pathways [15]. In addition, abnormal LAMC2 expression in various cancers, including colorectal cancer [16], bladder cancer [17], and lung adenocarcinoma [18], is closely correlated with poor prognosis. These findings indicate that LAMC2 may serve as a potential tumor marker. Only a few studies have been conducted on LAMC2 expression in OSCC; therefore, the upstream regulatory mechanisms of LAMC2 as a target gene in OSCC need to be elucidated in future studies.
To study the functions of lncRNA BBOX1-AS1 and its underlying mechanisms in OSCC, we constructed an lncRNA-associated competing endogenous RNA (ceRNA) network in OSCC and verified that BBOX1-AS1 regulated OSCC development by sponging miR-3940-3p and regulating LAMC2 expression. Our results provide an effective diagnostic marker for OSCC.

Clinical specimens
Fifty OSCC and paracancerous tissues were acquired from the patients hospitalized at the Department of Stomatology of the Sixth Hospital of Wuhan, Affiliated Hospital of Jianghan University (Wuhan, China). The tumor tissue was identified as OSCC and the tumor-adjacent tissue was identified as normal tissue by pathologists. Written informed consent was obtained from each patient before enrollment in this study. The use of clinical tissues was approved by the Hospital Research Ethics Committee.

Cell culture
Normal human oral keratinocyte (NHOK) cells and four OSCC cell lines (SAS, OECM1, OC3, and HSC3) were purchased from BeNa Culture Collection (Beijing, China). All cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum (ThermoFisher, USA) and incubated at 37°C with 5% CO 2 . After 2-3 stable generations, the cells were used for subsequent experiments.

Reverse transcription-quantitative PCR (RT-qPCR)
TRIzol reagent (Sigma-Aldrich, USA) was used to extract the total RNA from tissues and cells. Next, reverse transcription of RNA samples was performed to synthesize cDNA using the TaqMan MicroRNA Reverse Transcription Kit (Sigma-Aldrich, USA), according to the manufacturer's instructions. For PCR amplification, a Light Cycler instrument (Bio-Rad, Hercules, USA) was used to prepare the reaction system, followed by detection using the SYBR Green PCR kit (Enzynomics, Korea) and SYBR Premix Ex Taq II kit (Takara, USA). Relative expression was calculated using the 2 −ΔΔ Ct method, and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and uracil 6 [19]. Primer sequences are listed in Table 1.

Cell proliferation assay
Cells (2 × 10 4 cells/well) were maintained in a 96well plate and cultured for different incubation periods (24,48, and 72 h). Next, 10 μL of the cell counting kit (CCK)-8 solution (Abcam, USA) was added to the cells for 1 h [20]. Finally, the absorbance at 450 nm was measured using a microplate reader (Syngene, USA) to analyze the relative cell viability.

BrdU proliferation assay
After transfection, the cells (1 × 10 4 cells per well) were maintained in a 96-well plate overnight. The cells were then exposed to BrdU labeling reagent for 12 h, and it was followed by an assay using the ELISA BrdU kit (11,647,229,001; Roche, USA). The optical density of the cells at 450 nm was measured at different time-points using a microplate reader (Syngene, USA).

Transwell assay
A transwell assay was performed to detect the cell migration, as described previously [21]. Transfected cells (2 × 10 4 cells/mL) were grown in 100 μL of serum-free RPMI medium and added to a matrigelcoated transwell upper chamber (8 μm; BD Biosciences, USA). Then, 600 μL of complete RPMI  medium was added to the lower chamber. Subsequently, 4% formaldehyde was used to fix the migrated cells and 0.5% crystal violet was used to stain the cells. Finally, the number of cells was counted using an inverted microscope (Nikon, Tokyo, Japan).

Fluorescence in situ hybridization (FISH)
The LINC01207 FISH probe was designed and synthesized by RiboBio (Guangzhou, China). Briefly, the sections were deparaffinized, dehydrated in 100% ethanol, and dried. The slides were baked in a 60°C oven for 60 min and then dewaxed in xylene for 15 min. Then, 10 μL of hybridization buffer was added to the glass sample, and 1 μL probe was added to the hybridization buffer, placed in 46°C incubator, and hybridized for 1.5 h. After hybridization, the slides were washed with saline-sodium citrate buffer and mounted in an anti-fade solution containing DAPI. Images were obtained using a confocal fluorescence microscope (Leica, Germany) [22].

RNA immunoprecipitation (RIP) assay
Using the RIP assay kit (Millipore, USA), as previously described [24], the cells from each group were lysed with RIP buffer. Antibodies against argonaute 2 (AGO2) and immunoglobulin G (IgG) were incubated with magnetic beads to prepare the antibody-coated beads. Supernatants of cell lysates were incubated with AGO2 or IgG antibody-coated magnetic beads for 4 h at 4°C.
RNA complexes on the beads were isolated and used for RT-qPCR analysis.

RNA Pull-down assay
RNA pull-down assay was performed as previously described [25]. SAS and OC3 cells were incubated with biotinylated-miR-3940-3p and biotinylated-NC for 48 h and lysed with RNase. M-280 streptavidin magnetic beads (Sigma, USA) were incubated with the cell lysate. The beads were washed twice with a low-salt wash buffer. RNA-binding proteins were purified, and their relative expression was used for RT-qPCR analysis.

Statistical analysis
IBM SPSS software (version 19.0) was used for statistical analysis, and GraphPad Prism (version 8) was used for mapping. The data obtained from at least three independent experiments are represented as the mean ± SD. The correlations among BBOX1-AS1, miR-3940-3p, and LAMC2 expression levels in OSCC tissues were assessed using Pearson's analysis. Differences between groups were compared using the Student's t-test or analysis of variance, in which a P-value < 0.05 was considered to be statistically significant.

Results
In this study, we investigated the effects of lncRNA BBOX1-AS1 on OSCC and its regulatory mechanisms. Our data revealed significant upregulation of BBOX1-AS1 and LAMC2 expression levels, and downregulation of miR-3940-3p expression in OSCC. Moreover, we found that BBOX1-AS1 facilitated the proliferation and migration, while suppressing the apoptosis of OSCC cells by upregulating LAMC2 expression by targeting miR-3940-3p. Overall, our findings demonstrate the role of the BBOX1-AS1/miR-3940-3p/LAMC2 axis in OSCC and suggest that these molecules may be used as novel targets for OSCC therapy.

LAMC2 is potentially regulated by BBOX1-AS1/ miR-3940-3p and affects oral cancer progression
BBOX1-AS1 has two downstream miRNA targets, miR-3940-3p and miR-361-3p. The latter has been reported to be a potential oncogene in oral cancer [27], whereas the former has not yet been studied in oral cancer. In addition, we detected significantly downregulated miR-3940-3p levels in oral cancer cells (Figure 1(a)). By intersecting the predicted mRNAs of miR-3940-3p using the starBase algorithm and the differentially expressed mRNAs in oral cancer by analyzing GSE19089 with adjusted P < 0.05 and logFC ≥ 1.5, LAMC2 was identified (Figure 1(b)). LAMC2 was found to be overexpressed in head and neck squamous cell carcinoma (Figure 1(c), data obtained from GEPIA database). The overall survival analysis showed that a higher level of LAMC2 predicted a poorer survival outcome (Figure 1d, data obtained from http://kmplot.com/analysis [28]). In addition, LAMC2 is known to enhance OSCC progression [27,29,30]. However, whether LAMC2 is involved in ceRNA and regulates oral cancer progression remain unclear.

miR-3940-3p is a target gene of BBOX1-AS1
To further study the regulatory role of BBOX1-AS1 in OSCC progression, we first detected the subcellular localization of BBOX1-AS1 using FISH assay, which showed that BBOX1-AS1 was mainly located in the cytoplasm of SAS and OC3 cells (Figure 3(a)), indicating that BBOX1-AS1 could act as a ceRNA via the spongy adsorption of miRNA. We subsequently analyzed the potential target genes of BBOX1-AS1 using starBase data. After a series of screenings, miR-3940-3p was selected as the target gene (Figure 3(b)). Next, luciferase assay results showed that the miR-3940-3p mimic considerably weakened the luciferase activity in OSCC cells transfected with the WT reporter vector of BBOX1-AS1, while luciferase activity was unaffected in cells transfected with the MUT reporter vector (Figure 3(c)). RIP results showed that large amounts of BBOX1-AS1 and miR-3940-3p were enriched by anti-AGO2 relative to anti-IgG antibodies (Figure 3(d)). These results demonstrate the relationship between BBOX1-AS1 and miR-3940-3p. Spearman's correlation analysis showed that BBOX1-AS1 expression was negatively correlated with miR-3940-3p expression (Figure 3(e)). In addition, miR-3940-3p expression levels were significantly decreased in all four OSCC cell lines (figure 3(f)), and BBOX1-AS1 expression was negatively correlated with miR-3940-3p expression (Figure 3(g)). In general, miR-3940-3p was determined to be a target gene of BBOX1-AS1.

Downregulation of LAMC2 counteracts the effects of miR-3940-3p knockdown on OSCC cells
Expression analysis showed that miR-3940-3p inhibitor enhanced LAMC2 expression, but this effect could be reversed by LAMC2 knockdown (Figure 6(a, b)). CCK-8 and BrdU experiments showed that downregulation of miR-3940-3p expression facilitated OSCC cell proliferation, which was abolished by LAMC2 silencing (Figure 6(c, d)). Transwell assay showed that LAMC2 knockdown could counteract the promoting effect of miR-3940-3p inhibitor on the migration of OSCC cells (Figure 6(e)). Additionally, Bcl-2 expression levels were increased, while those of Bax were decreased in the SAS and OC3 cells transfected with the miR-   3940-3p inhibitor. LAMC2 knockdown exerted the opposite effect (figure 6(f)). These results indicated that LAMC2 is a target of miR-3940-3p.

BBOX1-AS1 enhances the proliferation and migration, and suppresses the apoptosis of OSCC cells by regulating LAMC2 expression
RT-qPCR and western blotting analysis revealed that the expression levels of LAMC2 were decreased in BBOX1-AS1 knockdown SAS and OC3 cells (Figure 7(a, b)). Subsequently, functional analysis showed that LAMC2 overexpression reversed the decrease in cell viability and proliferation induced by the downregulation of BBOX1-AS1 expression (Figure 7(c, d)). Transwell assay showed that the inhibitory effect of BBOX1-AS1 knockdown on cell migration could be alleviated by LAMC2 overexpression (Figure 7(e)). Additionally, LAMC2 overexpression counteracted the BE downregulation of Bcl-2 expression and upregulation of Bax expression induced by BBOX1-AS1 knockdown (figure 7(f)).

Discussion
OSCC poses a serious threat to the overall survival of patients, and many patients are initially diagnosed with advanced OSCC [31]. Therefore, the search for novel key genes or biomarkers is necessary to facilitate the early diagnosis and better prognosis of patients with OSCC [32]. This study demonstrated that BBOX1-AS1 expression was elevated in OSCC. Functional in vitro experiments revealed that BBOX1-AS1 knockdown impaired OSCC cell growth, while promoting cell apoptosis.
In addition, a novel mechanism for BBOX1-AS1 function is that it modulates miR-3940-3p/LAMC2 signaling to exert tumor-promoting effects.
As a newly discovered lncRNA, lncRNA BBOX1-AS1 has shown tumor-promoting effects in a wide range of tumors [33]. For example, BBOX1-AS1 expression is elevated in cervical cancer, and the growth and migration of cervical cancer cells can be repressed by BBOX1-AS1 silencing [34]. In colorectal cancer, BBOX1-AS1 acts as an oncogene and effectively accelerates cell proliferation and metastasis [35]. In ovarian cancer, BBOX1-AS1 knockdown suppresses tumor progression by decreasing the viability of cells [36]. These results suggest that BBOX1-AS1 may play a tumor-promoting role in major tumors and accelerate tumor progression. Therefore, we speculated that BBOX1-AS1 may also function as an antitumor gene in oral cancer. Our study showed that BBOX1-AS1 was highly expressed in OSCC.
In vitro experiments showed that cell proliferation and migration were abolished, while apoptosis was promoted by BBOX1-AS1 silencing, indicating that BBOX1-AS1 may act as a pro-oncogenic factor to promote the progression of oral cancer.
Furthermore, lncRNAs not only directly participate in the regulation of gene expression, but also act as ceRNAs to interact with miRNAs, thereby regulating the levels of protein-coding target genes and affecting various physiological and pathological processes [37,38]. Lina et al. found that lncRNA CASC2 represses OSCC cell growth by sponging miR-21 [39]. Furthermore, lncRNA H1 enhances OSCC progression by silencing miR-138 [40]. miR-3940-3p introduced in our study belongs to a class of RNA molecules that have not yet been extensively studied. miR-3940-3p expression is higher in recurrent ovarian cancer than in primary ovarian cancer, and may be used as a biomarker for predicting recurrent ovarian cancer [41]. Interestingly, only one study exploring the specific biological function of miR-3940-3p indicated that miR-3940-3p expression levels were reduced in gastric cancer cells and that BBOX1-AS1 promoted gastric cancer cell progression by targeting miR-3940-3p [14]. Our data showed that miR-3940-3p expression levels are downregulated in OSCC cells. Luciferase and RIP assays confirmed that miR-3940-3p is the target gene of BBOX1-AS1, which is consistent with the above results. Moreover, BBOX1-AS1 expression was negatively correlated with miR-3940-3p expression. In rescue assays, miR-3940-3p knockdown significantly enhanced OSCC cell proliferation and migration, while promoting apoptosis. These changes were reversed by BBOX1-AS1 deficiency. Overall, BBOX1-AS1 promotes OSCC development by suppressing miR-3940-3p expression.
We further studied the downstream regulation pathway of BBOX1-AS1 in OSCC and constructed a complete lncRNA-miRNA-mRNA network. We analyzed the downstream target genes of miR-3940-3p in OSCC cells. LAMC2 is closely associated with various biological processes, including cell adhesion and tumor cell migration [42]. LAMC2 has been widely studied in oral cancer. Zhou et al. reported that LAMC2 is a crucial factor related to OSCC and that its silencing inhibits tumor growth in vivo [27]. Rong et al. demonstrated that miR-5580-3p inhibits OSCC tumorigenesis by downregulating the expression of LAMC2 [43]. Moreover, upregulation of LAMC2 is closely associated with the invasive phenotype of OSCC, suggesting that LAMC2 overexpression may aggravate the severity of OSCC [44]. In OSCC, LAMC2 functions as an oncogenic factor and promotes tumor cell growth. Ning et al. revealed that the lncRNA CASC9 promotes the malignancy of OSCC cells by sponging miR-545-3p to enhance LAMC2 expression [45]. However, the role of LAMC2 in the ceRNA network in the pathogenesis of OSCC remains to be elucidated. In our study, LAMC2 was predicted to be the target gene of miR-3940-3p using starBase data. Results of GEPIA database analysis revealed that LAMC2 expression was increased in OSCC, and upregulation of LAMC2 expression was highly related to unfavorable patient outcomes. Subsequently, LAMC2 was confirmed to be directly targeted by miR-3940-3p, and the expression levels of LAMC2 and miR-3940-3p were negatively correlated with each other. Next, LAMC2 knockdown partially alleviated the promoting effect of the miR-3940-3p inhibitor on BBOX1-AS1silenced OSCC cells. These data suggested that BBOX1-AS1 influences OSCC progression by modulating the miR-3940-3p/LAMC2 pathway.
This study has some limitations. First, more cellular experiments, including cell migration and invasion tests are needed to validate the effects of BBOX1-AS1 in OSCC by regulating the miR-3940-3p/LAMC2 axis. Second, in vivo experiments, such as xenograft tumor assays, must be carried out in follow-up studies. Finally, the correlation between BBOX1-AS1 and the clinicopathological characteristics of patients with OSCC requires further investigation in future studies.

Conclusion
In conclusion, our results showed that BBOX1-AS1 facilitates oral cancer cell proliferation and migration and suppresses apoptosis by upregulating LAMC2 expression by targeting miR-3940-3p. Based on these findings, BBOX1-AS1 may be used as a potential target for OSCC treatment.

Author contributions
CGZ conceived and designed the study. WS performed the experiments and data analysis. MC formed the methodology and did the investigation. WS wrote the paper. CGZ reviewed and edited the manuscript. All authors read and approved the manuscript.

Disclosure statement
No potential conflict of interest was reported by the author(s).