Long non-coding RNA LINC01207 promotes cell proliferation and migration but suppresses apoptosis and autophagy in oral squamous cell carcinoma by the microRNA-1301-3p/lactate dehydrogenase isoform A axis

ABSTRACT Long noncoding RNAs (lncRNAs) have been reported to participate in the progression of various cancers, including oral squamous cell carcinoma (OSCC). This study aims to find out whether lncRNA LINC01207 regulates the progression of OSCC. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was conducted to evaluate gene expression in OSCC cells and tissues. Cell viability, proliferation, migration, apoptosis, and autophagy were detected using Cell Counting Kit-8 (CCK-8), colony formation, Transwell assays, flow cytometry, and western blot analysis. Luciferase reporter and RNA immunoprecipitation (RIP) assays were conducted to assess the interactions among genes. We found that LINC01207 was overexpressed in OSCC cells and tissues. LINC01207 silencing inhibited OSCC cell proliferation and migration but promoted apoptosis and autophagy, and LINC01207 overexpression had an opposite result. LINC01207 interacted with microRNA-1301-3p (miR-1301-3p) while lactate dehydrogenase isoform A (LHDA) was targeted by miR1301-3p. Effects caused by LINC01207 downregulation on OSCC cells were reversed by overexpression of LDHA. Overall, LINC01207 promotes OSCC progression via the miR-1301-3p/LDHA axis


Introduction
Oral squamous cell carcinoma (OSCC), ranking the sixth most common cancer worldwide, accounts for more than 90% of head and neck cancer [1,2]. OSCC is prone to distant metastasis, such as lung metastasis, bone metastasis, lymphatic metastasis, and blood metastasis [3]. There are over 300, 000 new cases of OSCC each year worldwide, and more than 140,000 patients die of OSCC each year [4,5]. Although great progress has been made to treat OSCC over the last years, the 5-year survival rate of OSCC patients has remained approximately 50% without any significant progress [6]. Hence, it is urgent to investigate novel clinical biomarkers that potentially function as targets for diagnosis or therapy of OSCC.
Autophagy is defined as the degradation of components via the autophagosome and lysosome [7,8]. The soluble autophagosome light chain 3 (LC3I) is the autophagosome related form (LC3II) during the autophagosome elongation process [9]. LC3II is degraded along with other components of autophagosome after fusion with lysosome. Therefore, the production of LC3II or the transformation of LC3 is regarded as indicator of autophagy activity [10]. Autophagy-related gene 7 (ATG7) composes part of the conjugation system involved in the redistribution of LC3II to the phagophore. The conjugation system consisting of ATG12-ATG5-ATG16 is pivotal for autophagosome elongation [11]. Evidence shows that autophagy is a major event in tumor progression [12].
Long noncoding RNAs (lncRNAs) consist of a subset of noncoding RNA transcripts that are more than 200 nucleotides in length [13][14][15]. LncRNAs play vital roles in gene regulation, genomic stability, survival, proliferation, and migration of cancer cells [16]. Studies have demonstrated that lncRNAs are closely related to the occurrence and progression of OSCC. For example, LINC00958 promotes OSCC cell proliferation, induces cell death and reduces autophagy [17]. LncRNA SNHG20 regulates OSCC cell migration, invasion, and proliferation via the microRNA-19b-3p/RAB14 axis [18]. Given that OSCC is the most common type of head and neck cancer, we selected several lncRNAs that were reported to exert important effects in head and neck cancer for the purpose of investigating the role of lncRNA in OSCC. We successfully screened out LINC01207 as the most significantly upregulated lncRNA in OSCC tissues. LINC01207, located in the genomic 4q32 locus, is reported to be upregulated in some cancers, and its downregulation could inhibit tumor growth and promote apoptosis [19,20]. The ceRNA (competitive endogenous RNA) mechanism is a common post-transcriptional mechanism in which lncRNAs act as the endogenous sponge of microRNAs (miRNAs) to free the inhibition of miRNAs on message RNAs (mRNAs). It has been widely reported that lncRNAs regulate OSCC progression via the ceRNA pattern [21,22]. Additionally, LINC01207 promotes head and neck squamous cell carcinoma cell proliferation and stemness characteristics by acting as a ceRNA for miR-5047 [23]. LINC01207 contributes to prostate cancer progression by downregulating miR-1972 and upregulating LIM and SH3 protein 1 [24]. Therefore, we inferred that LINC01207 may function as a ceRNA in OSCC.
Lactate dehydrogenase A (LDHA) is a major molecular mediator of the Warburg effect and plays a critical role in the metabolism of tumor cells [25,26]. It has been reported that elevated expression of LDHA is a hallmark of many tumors and is associated with the clinicopathological features and survival outcomes of patients [27,28]. Inhibition of LDHA typically results in accelerated oxygen consumption, reduced cell malignant transformation and markedly delayed tumor formation, indicating the underlying role of LDHA in tumor initiation or maintenance [29].
In the present study, we are aimed at exploring the biological role of LINC01207 in OSCC. Due to its higher expression in tumor tissues than in normal tissues, we hypothesized that LINC01207 may play an oncogenic role in OSCC. Subsequently, a series of molecular experiments were performed to test whether LINC01207 acts as a ceRNA in OSCC. Our study may provide a promising therapeutic target for the treatment of OSCC.

Tissue samples and cell lines
The OSCC samples (n = 30) and adjacent normal tissues (n = 30) were obtained from OSCC patients at Nanjing Stomatological Hospital Medical School of Nanjing University (Jiangsu, China) and then stored at −80°C. No other anticancer treatment was given to patients before the surgery. All patients provided written informed consent. This study was approved by the Ethics Committee of Nanjing Stomatological Hospital Medical School of Nanjing University (Jiangsu, China).
The OSCC cell lines, containing HSC-3 and HSC-4 cells, and human normal oral keratinocytes (NOK) cells were purchased from the Cell Bank of Chinese Academy of Science (Shanghai, China), and were incubated in Dulbecco's Modified Eagle Medium (Gibco, Thermo Fisher Scientific, Inc., USA) added with 10% fetal bovine serum and 1% streptomycin (Sigma, St Louis, MO, USA). All the cells were maintained in a humidified atmosphere at 37°C containing 5% CO 2 .

Colony formation assay
After the abovementioned transfection, HSC-3 and HSC-4 cells were seeded in 6-well plates (500 cells/well). After 14 days of incubation, the colonies were then fixed with 4% paraformaldehyde for 30 min and then incubated with 0.5% crystal violet (Beyotime, Shanghai, China) for 1 h at room temperature. And finally, the number of colonies with more than 50 cells was counted [33].

Cell migration assay
Cell migration was examined using Transwell inserts (pore size, 8.0 µm; Corning, USA) as previously described [34]. Briefly, 2 × 10 5 cells/well cells in 200 µl serum-free medium were suspended in the upper chamber, and the lower chamber was filled with 700 µl medium containing 15% fetal bovine serum. After 24 h at 37°C in an atmosphere containing 5% CO 2 , the membranes in the lower chamber were fixed in 4% paraformaldehyde at room temperature and stained with crystal violet for 20 min. After washing, images of the cells on the membranes were captured under a light microscope in five randomly selected fields per sample.

Flow cytometry
Annexin V-fluoresceine isothiocyanate (FITC)/ Prodium Iodide (PI) double-labeled staining kit (BD Biosciences, San Jose, CA, USA) was used in this assay. The procedure was performed as previously described [35]. The cells (2 × 10 5 /well) in 6-well plates were collected, washed twice with cold phosphate buffered saline, and resuspended in 1 × binding buffer. Subsequently, cells were stained with 10 μl Annexin V-FITC for 15 min and 5 µl PI for 10 min in the dark at room temperature. Cells were examined using the FACSCanto II flow cytometer (BD Biosciences). Analysis of flow cytometry data was performed using FlowJo version X.10.0.7-1 (FlowJo, LLC).

Western blot
Western blot was performed using standard and established protocol as previously published [36]. Proteins were extracted from OSCC cells using radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China). Protein concentration was quantified using a bicinchoninic acid assay kit (Beyotime).

RNA immunoprecipitation (RIP) assay
RIP was performed using a Magna RNA-binding protein immunoprecipitation kit (EMD Millipore, Billerica, MA, USA) [39]. At 90% confluence, cells were centrifuged at 4°C for 5 min at 1,000 × g, washed with pre-cooled phosphate buffered saline and lysed with radioimmunoprecipitation assay lysis buffer. Subsequently, the lysates were incubated for 10 min at 4°C with human Ago2 antibody (ab186733; Abcam; 5 µg) conjugated on magnetic beads, with IgG antibody (ab172730; Abcam; 5 µg) as the control group. Samples were treated with Proteinase K for 30 min at 55°C with gentle agitation. Immunoprecipitated RNA was isolated using TRIzol. Co-precipitated RNAs were purified, identified, and analyzed with RT-qPCR.

Statistical analysis
Data were analyzed using SPSS 19.0 software (SPSS Inco, Chicago, IL, USA) and are presented as the mean ± standard deviation. One-way or two-way analysis of variance and student's t-test were utilized to compare the differences between groups. Linear correlation analysis was performed using Spearman's correlation coefficient. Data were statistically significance when p < 0.05.

Results
In this study, we investigated the biological role and studied the molecular mechanisms of LINC01207 in OSCC. Our data showed a significant upregulation of LINC01207 in OSCC tissues and cell lines. LINC01207 overexpression promoted the malignant phenotypes of OSCC cells, and inhibited cell apoptosis and autophagy. LINC01207 upregulated LDHA expression by acting as a ceRNA for miR1301-3p. Overall, our study demonstrated a functional role of the LINC01207/miR-1301-3p/LDHA axis in OSCC, implying that targeting these molecules could be a novel approach in OSCC treatment.

LINC01207 is overexpressed in OSCC tissues and cells
Given that OSCC is the most common type of head and neck cancer, we selected several lncRNAs (LINC00520, LINC00461, LINC00460, LINC00052, LINC00467, and LINC01207) that were reported to exert important effects in head and neck cancer to investigate the role of lncRNA in OSCC. RT-qPCR presented that LINC01207 expression was significantly upregulated in OSCC tumor tissues compared to that in adjacent normal tissues, while the other lncRNAs showed no significant changes (Figure 1  (a)). Similarly, the higher level of LINC01207 in HSC-3 and HSC-4 cells than in NOK cells was also detected by RT-qPCR (Figure 1(b)). Therefore, we chose LINC01207 for the study.

LINC01207 silencing inhibits OSCC cell growth and promotes apoptosis and autophagy
To verify the function of LINC01207 in OSCC, the following assays were performed. After transfecting sh-NC or sh-LINC01207#1/2 into OSCC cells, as presented by RT-qPCR, LINC01207 expression was significantly downregulated in OSCC cells (Figure 2(a)). The CCK-8 assay demonstrated that after LINC01207 knockdown, the viability in HSC-3 and HSC-4 was inhibited (Figure 2(b)). Consistently, the number of colonies was decreased after transfection of sh-LINC01207#1/2 (Figure 2(c)). Transwell assay was used to examine the impact of LINC01207 in OSCC cell migration, and the results showed that the migration of HSC-3 and HSC-4 cells was significantly suppressed by LINC01207 knockdown (Figure 2(d)). Flow cytometry analysis showed that the apoptotic rate was increased in cells transfected with sh-LINC01207#1/2, which indicated that LINC01207 knockdown promotes OSCC cell apoptosis. (Figure 2(e)). We further performed western blot to test the change of autophagy. As shown, the protein expression of ATG5 and LC3-II was upregulated by LINC01207 silencing while that of P62 was downregulated, suggesting LINC01207 silencing promotes cell autophagy in  was upregulated by pcDNA3.1-LINC01207 (Figure 3(a)). Experiments demonstrated that overexpression of LINC01207 significantly facilitated OSCC cell viability, proliferation, migration, and inhibited apoptosis and autophagy (Figure 3(b-f)). Therefore, LINC01207 acts as an oncogenic gene in OSCC.

LDHA overexpression reverses the effects of LINC01207 knockdown in OSCC
Since the ceRNA mechanism among LINC01207, miR-1301-3p and LDHA was verified, the rescue assays were in need. RT-qPCR and western blot analysis revealed that LDHA was strongly overexpressed by pcDNA3.1-LDHA in HSC-3 cells (Figure 6(a-b)). CCK-8 and colony formation  assays showed that the cell viability and proliferation suppressed by LINC01207 silencing were rescued by LDHA overexpression (Figure 6(c-d)). LINC01207 depletion-mediated suppressive effects on HSC-3 cell migration were reduced after LDHA was overexpressed (Figure 6(e)). Flow cytometry analysis presented that LINC01207 silencing promoted cell apoptosis while LDHA overexpression reversed the results (figure 6(f)). Moreover, the protein expression of ATG5 and LC3-II was upregulated by LINC01207 silencing while was downregulated after LDHA overexpression, and the that of P62 showed an opposite result (Figure 6(g)).

Discussion
In recent years, emerging evidence has revealed that lncRNAs are involved in OSCC progression [40,41]. Moreover, LINC01207 was shown to be an oncogenic gene in tumor progression. LINC01207 downregulation led to the promotion of cell apoptosis and the inhibition of proliferation in gastric cancer [20]. LINC01207 inhibits prostate cancer cell apoptosis and promotes proliferation [24]. In our study, we discovered the overexpression of LINC01207 in OSCC tissues and cells, implying its possible involvement in OSCC progression. We knocked down the expression of LINC01207 in OSCC cells to examine its biological role. Our results revealed that LINC01207 silencing inhibited OSCC cell (c-g) after transfecting sh-LINC01207#1 or cotransfecting sh-LINC01207#1 with pcDNA3.1-LDHA in HSC-3 cells, the cell viability was assessed by CCK-8 assay, proliferation was evaluated by colony formation assay, migration was examined by transwell assay, apoptosis was detected by flow cytometry assay, and the protein levels of autophagy-related proteins were presented by western blot. *P < 0.05, **P < 0.01. proliferation and migration but promoted cell apoptosis, and LINC01207 overexpression showed an opposite result. Additionally, a previous study demonstrated that LINC01207 silencing promotes autophagy in pancreatic cancer by increasing LC3II and beclin-1 protein expression while decreasing P62 expression [42]. Similarly, in our study, the inhibitory function of LINC01207 on OSCC cell autophagy was discovered. Therefore, these results suggested that LINC01207 promotes the progression of OSCC in vitro. Animal studies in the future are needed to further confirm whether LINC01207 exerts oncogenic effects in OSCC.
Accumulating evidence suggests that lncRNAs may act as ceRNAs for particular miRNAs to modulate the target genes of the miRNAs, including GC [43,44]. In this study, we assessed the subcellular localization of LINC01207 and observed that majority of LINC01207 were located in the cytoplasm of OSCC cells, which suggested that LINC01207 may function as a ceRNA for sponging miRNA. Thus, we predicted a miRNA that can interact with LINC01207 in OSCC cells and found that LINC01207 has a binding site for miR-1301-3p. MiR-1301-3p has been reported to be downregulated in many tumors and function as a tumor suppressor. Xu G et al. found in their study that miR-1301-3p upregulation significantly suppresses colorectal cancer cell growth [45]. The inhibitory effects of miR-1301-3p on breast cancer cell proliferation were found [46]. MiR-1301-3p is downregulated in papillary thyroid cancer and inhibits tumor growth [47]. In this study, we also found that miR-1301-3p was significantly downregulated in OSCC cells, and its expression was negatively correlated to LINC01207 expression. Thus, LINC01207 may play a role in OSCC by sponging miR-1301-3p.
Generally, as a ceRNA, the function of lncRNAs depends on the miRNA targets. LDHA was selected as a direct target of miR-1301-3p through bioinformatics analysis and luciferase reporter assay. LDHA has a key role in tumor cell metabolism and adaptation to unfavorable environmental or cellular conditions [48]. LDHA is overexpressed in prostate cancer and induces a favorable microenvironment for tumor progression [49]. With the deepening of research, our study revealed that LDHA was overexpressed in OSCC cells.
Additionally, rescue experiments revealed that LDHA overexpression could rescue the effects caused by knockdown of LINC01207 in OSCC. Moreover, it was reported that LDHA promotes tumorigenesis by facilitating glycolysis and epithelial-mesenchymal transition in OSCC [50], implying the oncogenic property of LDHA in OSCC. Thus, these results demonstrated that LINC01207 acts as a sponge for miR-1301-3p and upregulates the expression of its endogenous target LDHA in OSCC. However, our study has some limitations. The interactions between cytokines in cancer cells are complex, and whether LINC01207 regulates other potential mechanisms will be examined. Additionally, the sample size will be increased to verify the associations between LINC01207 and the prognosis of patients with OSCC.

Conclusion
Overall, we demonstrated that LINC01207 is upregulated in OSCC tissues and cells. LINC01207 upregulates LDHA expression to promote OSCC cell proliferation, migration, and inhibit apoptosis and autophagy by acting as a ceRNA that sponges miR-1301-3p. Our results provide inspiration for further understanding of the mechanisms of OSCC. In the future work, we will explore the mechanisms of LINC01207 upregulation in OSCC, the correlations between LINC01207 and other miRNAs or proteins, which will further deepen our understanding of the pathogenesis of LINC01207 and make LINC01207 a potential novel diagnostic and therapeutic target for OSCC.

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

Funding
This work was supported by Nanjing Medical Science and Technique Development Foundation (No. QXR17175).