Role of the Long Intergenic Non-Protein-Coding RNA 1278/miR-185-5p/Cystatin SN Axis in Laryngeal Cancer Cells

Laryngeal cancer accounts for 25%–30% of tumors in the head and neck. Cystatin SN (CST1) was revealed to show upregulated expression in this cancer, while its functions and upstream pathway remain unknown and need investigation. The current study was designed to solve this problem. We designed short hairpin RNAs targeting CST1 for the loss-of-function assays to probe the influences of CST1 in laryngeal cancer cell proliferation and motility. The upstream competitive endogenous RNA pattern of CST1 was searched using bioinformatics analysis and confirmed by luciferase reporter assays. The experimental results demonstrated that CST1 is a tumor facilitator in laryngeal cancer by stimulating cellular proliferative, migrative, and invasive abilities. CST1 is regulated by the long intergenic non-protein-coding RNA 1278 (LINC01278)/miR-185-5p axis. LINC01278 knockdown and miR-185-5p overexpression exert the same functions as CST1 knockdown to repress cancer cell proliferation, migration, and invasion. In conclusion, LINC01278 plays an oncogenic role in laryngeal cancer by suppressing miR-185-5p to enhance CST1 expression, which enriches the molecular mechanism for the carcinogenesis of laryngeal cancer.


Introduction
Laryngeal cancer takes up 25%-30% of tumors in the head and neck [1]. Because of industrialization, aging, and high smoking rates, its incidence has increased [2]. Although there were advances in radiation, surgery, and chemotherapy for treatment of laryngeal cancer, the outcomes of patients with advanced cancer are poor [3]. The molecular mechanisms underlying laryngeal cancer tumorigenesis are not fully revealed. Lacking effective biomarkers hinders the diagnosis and therapy of laryngeal cancer. Thus, to explore the molecular mechanism for the tumorigenesis of laryngeal cancer possesses great importance.
Based on GSE84957, it was found that cystatin SN (CST1) is upregulated in 30 laryngeal cancer specimens compared with the adjacent nonneoplastic tissues by 4.256fold changes [4]. CST1 is a member of the cystatin superfamily that suppresses the proteolytic activities of cysteine proteases [5]. In recent years, CST1 was found to be involved in the progression of various cancers, for instance, CST1 increases cell migrative and invasive capacities in gastric cancer [6]. It enhances cancer stem cell properties in papillary thyroid carcinoma and increases cell motility [7]. In colorectal cancer, CST1 mediates autophagy and oxidative stress response to control cell death [8]. These studies support the oncogenic role of CST1, while its functions in laryngeal cancer remain unclear. We hypothesized that CST1 can promote malignant phenotypes of laryngeal cancer cells and designed loss-of-function assays to explore it.
The competitive endogenous RNA (ceRNA) pattern is a classically posttranscriptionally regulatory mechanism in laryngeal cancer [9,10]. Long noncoding RNAs (lncRNAs) with the length of more than 200 nucleotides and micro-RNAs (miRNAs) with the length of approximate [18][19][20][21][22] nucleotides are the dominant members in the ceRNA pattern [11]. A ceRNA network hypothesis has been proposed, which suggested that miRNAs play an important role in the ceRNA network by binding mRNA and inhibiting mRNA expression [12]. According to the ceRNA theory, lncRNAs can act as miRNA sponges regulating expression of the target mRNA by ceRNA network [13]. The discovery of these interactions presented a new perspective on cancer therapy.
Although there are numerous studies on the functions of CST1 in various cancers [14,15], its upstream ceRNA pathway has not been studied. We explored the upstream lncRNA and miRNA for CST1 in laryngeal cancer cells and investigated the functions of these noncoding RNAs, which might offer new insight into the mechanism of laryngeal cancer tumorigenesis. were utilized in the present study. After proper resuscitation, cells were cultured in RPMI-1640 medium (GIBCO, USA) added with 12% fetal bovine serum in a humidified incubator at 37°C with 5% CO 2 . Every other day, the culture medium was changed.
2.6. Colony Formation Assay. In 6-well plates, transfected TU686 and TU177 cells with 1,000 cells per well were inoculated and cultured in a 60 mm dish at 37°C in 5% CO 2 for 2 weeks. Cell culture was discontinued once the colonies were visible to the human eye, which were then stained with 1% crystal violet for 20 min (500 μL/well, Beyotime, Shanghai, China). The colonies that contained more than 50 cells were counted using an inverted light microscope (Nikon ECLIPSE Ti, Japan).

Transwell
Assay. An 8 μm pore polycarbonate membrane Boyden chamber was used to detect invasion (Corning, USA). Cells (2 × 10 5 ) were cultured in serum-free medium in the upper transwell chamber that was precoated with 50 μL of Matrigel. 0.5 mL of complete medium with 10% FBS was added into the bottom chamber. Incubation was performed at 37°C in 5% CO 2 for one day. Next, cells on the upper chamber were wiped off with a cotton swab. Cells invading onto the lower surface of the membrane were fixed with methanol and stained with 2% crystal violet for 10 min. Five random visual fields of each membrane were selected for counting the invaded cells under a microscope (BHNK-PH001, Nikon Corporation; magnification, 100x). Cell migration assay was done similar to the migration assay except that no Matrigel was used. Journal of Oncology of 20 nM. Following 48 h transfection, renilla luciferase activity was normalized to firefly luciferase activity. Relative luciferase activity was calculated the Dual-Luciferase Reporter Assay system (Promega).
2.9. Statistical Analysis. Data are expressed as mean ± standard deviation (SD) from three biological repeats and three technical repeats. For comparison among three or more independent groups, analysis of variance followed by Dunnett's post hoc test and Tukey's post hoc test was performed. When comparing two independent groups, independent t -test was performed. Data were processed and analyzed with SPSS version 22.0 (SPSS Inc., USA). P < 0:05 was considered to indicate a statistically significant difference.

CST1 Shows Upregulation in Laryngeal Cancer Cells.
Compared with HuLa-PC cells, CST1 exhibited higher expression in TU686, M4E, TU177, and TU138 cells at the mRNA and protein levels (Figures 1(a) and 1(b)). TU686 and TU177 cells were used for the subsequent assays due to the relatively higher expression of CST1 in the two cells.
Results of immunofluorescence staining assay showed that TU686 and TU177 cells contained more CST1 protein than HuLa-PC cells (Figure 1(c)).

Discussion
Understanding of the molecular mechanism of laryngeal cancer pathogenesis is beneficial to the treatment of this disease. CST1 has the potential to promote cancer cell proliferation and motility [14,17]. CST1 was identified as an upregulated gene in laryngeal cancer tissues based on online data. We confirmed its upregulation in laryngeal cancer cells. Results of biological experiments revealed the inhibitory influence of CST1 deficiency on the proliferative, migrative, and invasive properties of laryngeal cancer cells. Our experimental findings suggested that CST1 could act as an oncogene to mediate laryngeal cancer carcinogenesis. After confirming the role of CST1 in laryngeal cancer cells, the lncRNA-miRNA-CST1 interactions were investigated. miRNAs participate in multiple activities in cells through modulating protein-coding genes. In recent study, it has been found that the level of miR-185-5p was significantly reduced in laryngeal squamous cell carcinoma tissues and serves as a target gene for specific lncRNAs [18]. For example, Fan et al. [19] demonstrated that miR-185-5p reduces the gene expression of HBV of hepatoma carcinoma cells by inhibiting ELK1. It is also reported that miR-506 inhibits the growth of laryngeal cancer cells by the inhibition of YAP126 [20]. In our study, miR-185-5p was revealed as the regulatory factor for CST1 by bioinformatics analysis and its binding to CST1 3 ′ UTR was confirmed by our experimental results. miR-185-5p shows downregulation in laryngeal cancer, which is consistent with a previous study [18]. We further demonstrated that miR-185-5p elicits a negative effect on laryngeal cancer proliferation and motility by targeting CST1, suggesting the antioncogenic function of miR-185-5p in laryngeal cancer.
Growing evidence showed that lncRNAs play crucial roles in cancers through sponging miRNAs [21,22]. LINC01278, an oncogene in other cancers, has been found related to clinical staging, distant metastasis, and poor prognosis of patients [23]. It is found that LINC01278 is significantly downregulated in papillary thyroid carcinoma tissues and cell lines and exerts a suppressor function in tumor cells  9 Journal of Oncology [24]. Huang et al. have found that LINC01278 promotes the metastasis of hepatocellular carcinoma by targeting miR-1258-Smad2/3 [25]. Meanwhile, Qi et al. found that in osteosarcoma tissues, the expression of LINC01278 is enhanced, and it promotes the proliferation of osteosarcoma cells through miR-133a-3p/PTHR1 signal [26]. These two latest studies suggest that the role of LINC01278 in tumor cells may depend on the specific tumor types, tumor microenvironment, or downstream targets. Our study further supports the oncogenic role of LINC01278. We found that LINC01278 serves as the regulatory factor for miR-185-5p. It upregulates CST1 expression by binding with miR-185-5p to antagonize its suppressive effect on CST1. Compared with miR-185-5p, LINC01278 has the opposite effects in laryngeal cancer to facilitate cell proliferation and motility.
A major shortcoming of this research is lacking animal studies to further support the role of LINC01278/miR-185-5p/CST1 in laryngeal cancer at the preclinical level. Clinical data to reveal the expression levels of miR-185-5p and LINC01278 in patients with laryngeal cancer were needed. Moreover, apoptosis is an important factor associated with cancer progression. Our further work will focus on the effects of the LINC01278-mediated ceRNA pattern in the apoptosis or other forms of cell death in laryngeal cancer.
To sum up, LINC01278 is an oncogene in laryngeal cancer by contributing to cancer cell proliferative, migrative, and invasive properties through the miR-185-5p/CST1 axis, which sheds new insight into the ceRNA mechanism of laryngeal cancer pathogenesis.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.