Potential antisense oligonucleotide targeting LINC00673 on proliferation and apoptosis in tongue squamous cell carcinoma

Abstract

Objectives

The incidence of tongue cancer, characterized by an aggressive nature, is rising annually in both Indonesia and globally. A molecular factor related to human tongue carcinoma is the high expression of long non-coding RNA 673 (LINC00673), which is associated with poor survival rates and larger tumor sizes. To address the challenge of poor outcomes, antisense therapy is a targeted method designed to degrade LINC00673 through the activity of RNAase-H1. Therefore, this study evaluated the potential of LINC00673 antisense oligonucleotide inhibiting proliferation and inducing apoptosis in human tongue cancer cells (H357).

Methods

H357 cells were transfected with LINC00673 antisense, sense, and scramble control oligonucleotide (SCO). Cell proliferation was assessed using the methylthiazol tetrazolium assay and apoptosis based on acridine orange–ethidium bromide staining.

Results

The results showed that H357 cells treated with antisense oligonucleotide exhibited significantly reduced proliferation compared with those treated using the sense oligonucleotide, SCO, transfection only, and negative control groups at the same time points and concentrations. Apoptosis analysis showed that the cells treated with antisense oligonucleotide exhibited prominent orange-red fluorescence and apoptotic morphological changes, whereas cells in the control groups predominantly retained green fluorescence, showing their viability.

Conclusions

LINC00673 antisense oligonucleotide treatment significantly inhibited proliferation and induced apoptosis in human tongue cancer cells.

Introduction

Cancer is the second leading cause of death globally, with 19 million new cases and 10 million cancer-related deaths reported in 2022.¹ According to the Republic of Indonesia's Basic Health Research (RISKESDAS), the prevalence of cancer, including oral, increased from 1.4 to 1.79 per 1000 people between 2013 and 2018.^2,3 GLOBOCAN data also indicate that a steady annual increase occurred from 354,000 cases in 2018 to 378,000 in 2020,^4,5 and 389,000 in 2022.¹ Indonesia ranked 10th globally for the highest incidence of oral cancer in 2022, accounting for approximately 6500 cases.⁶ Tongue cancer is the most frequent subtype of oral squamous cell carcinoma (OSCC), responsible for over 50 % of cases.7, 8, 9, 10 This subtype is an aggressive form of OSCC, with a survival rate of less than 50 %. The poor prognosis is attributed to its location in vascular and lymphatic tissue, which facilitates early metastasis to cervical lymph nodes.^11,12

Previous studies have shown that long non-coding RNA (LncRNA), i.e., RNA transcripts consisting of more than 200 nucleotides, plays a significant role in cancer. Abnormal LncRNA expression is often observed in cancer and is implicated in carcinogenesis, although the precise mechanism involved remains unclear.¹³ Dysregulation of LINC00673 has been reported to contribute to the progression of oral tongue squamous cell carcinoma (OTSCC).^14,15

Antisense oligonucleotide (ASO) is a strategy used to directly target LncRNA. This strategy employs a chemically modified single-stranded oligonucleotide designed to bind selectively to target RNA and form RNA–ASO complexes through Watson–Crick base pairing. These complexes recruit the RNase H enzyme, leading to degradation of the target RNA and disruption of its biological function.^16,17 Furthermore, the single-stranded structure improves intracellular uptake and enhances the binding specificity to the target RNA.¹⁸ In clinical settings, ASO exhibits a long half-life, efficient cellular uptake, and strong inhibitory activity.¹⁹

The role of LINC00673 in tongue cancer cells is primarily associated with invasion and migration.¹⁵ However, studies of another cancer suggest potential roles in proliferation and apoptosis.^19,20 Therefore, the present study was conducted to investigate the potential effects of ASO LINC00673 on proliferation and apoptosis in human tongue cancer cells.

Materials and Methods

Cell culture

Human tongue squamous cell carcinoma cells (H357, catalogue number: 06092004) were obtained from The European Collection of Authenticated Cell Cultures. Cells were cultured with Dulbecco's modified Eagle medium (Sigma, St. Louis, MO, USA) supplemented with 10 % fetal calf serum (Moregate BioTech, Bulimba, Australia), 100 μg/mL streptomycin, and 100 U/mL penicillin (Invitrogen Corp., Carlsbad, CA, USA). The cultured cells were incubated at 37 °C with 95 % humidity and 5 % CO₂.

ASO experiments

ASOs targeting LINC00673 (Gene ID: 100499467) were synthesized by Cusabio Technologies (TX, USA), and each sequence was modified with phosphorothioate backbones. The ASOs were designed by identifying secondary structures of the target RNA that allowed efficient hybridization, such as terminal ends, internal loops, hairpins, and bulges. The selected sequences had a G/C content of 40–60 % and binding energy of ≤ −8 kcal/mol. The entire design process was performed using the sFold platform (https://sfold.wadsworth.org). To confirm target specificity, the designed sequences were validated using Nucleotide BLAST (https://blast.ncbi.nlm.nih.gov).21, 22, 23 In this study, an ASO with a sequence of 5′-TGGCACCTCTTTCTTGTCCT-3′ was used to target LINC00673, and comparisons were made with two control oligonucleotides: a sense oligonucleotide (SO) with the sequence of 5′-AGGACAAGAAAGAGGTGCCA-3′ and a scramble control oligonucleotide (SCO) with the sequence of 5′-CCCTCTCTTACGTCTGTTT-3′. Cells were transfected with oligonucleotides using SuperKine™ Lipo3.0 (Abkine, USA) according to the manufacturer's instructions.

Cell proliferation assay

Cell proliferation was assessed using the methylthiazol tetrazolium (MTT) assay. H357 cells were seeded in 96-well plates at a density of 10⁴ cells per well and divided into several groups, before treatment with ASO, SO, and SCO at concentrations of 1.56, 3.13, 6.25, and 12.5 μM. The negative control in this study received no oligonucleotide or transfection reagent. In addition, the transfection control was only treated with the transfection reagent. Cells were incubated for 24 and 48 h after treatment, with five replicates for each group. Subsequently, cell proliferation was measured based on optical density (OD) readings obtained using a microplate reader at a wavelength of 590 nm. The OD values were converted into cell viability percentages using the following formula:

$$\%\text{Cell}\text{viability}=\frac{\text{OD}\text{treatment}-\text{OD}\text{blank}}{\text{OD}\text{control}-\text{OD}\text{blank}}\times 100\%$$

where OD treatment is the absorbance of treated cells, OD control is the absorbance of the control negative group, and OD blank is the absorbance of the medium without cells.

Dual staining apoptosis assay

Apoptosis was evaluated using a double-staining method with acridine orange (AO) and ethidium bromide (EB). H357 cells were cultured in 24-well plates at a density of 2 × 10⁴ cells per well, with coverslips placed at the bottom of each well. Cells were treated with ASO, SO, or SCO at concentrations of 6.25 and 12.5 μM. The negative control group was not treated with oligonucleotides or transfection reagent, and cells were incubated for 24 h. The culture medium was removed before staining and wells were washed with 1 × phosphate-buffered saline. Coverslips were then mounted on object slides, before staining the cells with 10 μL of AO–EB staining solution and incubating for 15 min. AO penetrated all of the cell samples and stained nuclei with green fluorescence. However, EB only penetrated into samples with compromised membranes and orange-red colored fluorescence was emitted. Viable cells appeared green, whereas apoptotic cell exhibited green-yellow to orange-red fluorescence, depending on the stage of apoptosis. Four replicates were analyzed for each treatment group. Fluorescence images were analyzed using ImageJ software.

Statistical analysis

Statistical analyses were performed using SPSS (version 20, IBM, NY, USA). Before statistical testing, homogeneity of variance and normality were assessed to determine whether parametric or nonparametric tests should be applied. The MTT cell proliferation data were not normally distributed or homogeneous (P < 0.05). Therefore, nonparametric tests (Kruskal–Wallis and Mann–Whitney U) were used with Bonferroni correction. A corrected P-value of <0.0167 was considered to indicate a statistically significant difference. The apoptosis data were normally distributed and homogeneous (P > 0.05). Subsequently, one-way analysis of variance and independent t-tests were applied to analyze the apoptosis percentages, with statistical significance set at P < 0.05.

Results

The proliferation assay results showed that after incubation for 24 h, the cell viability percentages were lower for cells treated using ASO LINC00673 (Figure 1), SO LINC00673 (Figure 2), and SCO LINC00673 (Figure 3) compared with the negative control groups (P < 0.0167). After 48 h, cell proliferation only decreased significantly in the ASO LINC00673 group (Figure 1) compared with the control negative group across all concentrations (P < 0.0167). By contrast, no significant reductions in cell proliferation were found in the SO and SCO LINC00673 groups compared with the negative control (P > 0.0167). Treatment using ASO LINC00673 significantly reduced the cell viability percentage (P < 0.0167) compared with the SO and SCO LINC00673 treatments at the same concentration and after the same incubation times. Significant differences were not observed between the SO and SCO groups (P > 0.0167), except on day 2 at concentrations of 1.56 and 12.5 μM, where the cell proliferation percentage was higher after treatment using SO LINC00673 than SCO LINC00673. In addition, there were no significant differences in cell proliferation after treatment with ASO LINC00673 at any concentration after both 24 h and 48 h (P > 0.05).

Figure 1.

Percentage of H357 cell proliferation after treatment with antisense oligonucleotide at various concentrations (μM). Statistically significant differences were determined using Bonferroni correction for multiple comparisons per concentration (P < 0.0167). ∗ denotes a significant difference compared with the negative control (0 μM) on the same incubation day. Abbreviations: 24H: 24 h, 48H: 48 h.

Figure 2.

Percentage of H357 cell proliferation after treatment with sense oligonucleotide at various concentrations (μM). Statistically significant differences were determined using Bonferroni correction for multiple comparisons per concentration (P < 0.0167). ∗ denotes a significant difference compared with the negative control (0 μM) on the same incubation day. Abbreviations: 24H: 24 h, 48H: 48 h.

Figure 3.

Percentage of H357 cell proliferation after treatment with scramble control oligonucleotide at various concentrations (μM). Statistically significant differences were determined using Bonferroni correction for multiple comparisons per concentration (P < 0.0167). ∗ denotes a significant difference compared with the negative control (0 μM) on the same incubation day. Abbreviations: 24H: 24 h, 48H: 48 h.

After incubation for 24 h, only cells treated with transfection reagent exhibited significantly reduced proliferation compared with the negative control (P < 0.0167). Treatment using ASO LINC00673 at all test concentrations significantly reduced cell proliferation (P < 0.0167), whereas the results obtained using SO and SCO LINC00673 did not differ from the control. After incubation for 48 h, no significant differences in proliferation reduction were observed compared to the negative control (P > 0.0167). However, ASO LINC00673 continued to suppress cell proliferation compared with the transfection-only group, whereas SO and SCO LINC00673 had no inhibitory effects.

The dual AO–EB staining assay results showed that viable cells exhibited green fluorescence, whereas apoptotic cells produced yellow-green to orange-red fluorescence. The negative control group Figure 4A and those treated with SO LINC00673 6.25 μM Figure 4B, SO LINC00673 12.5 μM Figure 4C, SCO LINC00673 6.25 μM Figure 4D, and SCO LINC00673 12.5 μM Figure 4E predominantly contained viable cells with green fluorescence, and apoptosis was not observed. By contrast, cells treated with ASO LINC00673 exhibited distinct morphological changes associated with apoptosis. Treatment with ASO LINC00673 at 6.25 μM Figure 4F induced chromatin condensation within the nuclei (arrow). Treatment with ASO LINC00673 at 12.5 μM resulted in membrane blebbing (shown by arrows in Figure 4G), and orange-red staining indicated non-viable apoptotic cells. The percentages of apoptotic cells among the cells treated with ASO LINC00673 at 6.25 μM and 12.5 μM were 26 % and 35.8 %, respectively. Statistical analysis showed that the percentage of apoptosis was significantly higher in cells treated with ASO LINC00673 than SO and SCO LINC00673 at the same concentration (P < 0.05). In addition, the percentage of apoptosis was significantly higher after treatment with ASO LINC00673 at 12.5 μM compared with 6.25 μM (P < 0.05). According to these results, treatment using ASO LINC00673 led to greater inhibition of proliferation and apoptosis induction compared with the negative control and oligo treatment groups (SO and SCO LINC00673).

Figure 4.

Acridine orange–ethidium bromide staining results after treatment for 24 h. (A) Negative control; (B, C) SO at concentrations of 6.25 and 12.5 μM; (D, E) SCO at concentrations of 6.25 and 12.5 μM, showing viable cells with green nuclear staining. (F, G) Apoptotic morphological changes (yellow arrow) observed using ASO at concentrations of 6.25 and 12.5 μM, characterized by a change in color of nuclear staining to orange-red. Scale bar: 100 μm. Abbreviations: ASO, antisense oligonucleotide; SO, sense oligonucleotide; SCO, scramble control oligonucleotide.

Discussion

LINC00673 or steroid receptor RNA activator-like non-coding RNA 1 (SLNCR1) is an lncRNA on chromosome 17q24.3. Meta-analysis studies have shown that LINC00673 is upregulated in various cancers, and its high expression is associated with advanced tumor node metastasis (TNM) stage, poor overall survival, and higher incidence of metastasis.^24,25 In OTSCC, elevated LINC00673 expression has been observed in cell lines, tissue biopsies, and patient data sets. This high expression in OTSCC is correlated with increased tumor size, local invasion, and higher TNM stage. Poor prognosis was also reported in patients with high expression of LINC00673 in OTSCC, with lower overall survival and relapse-free survival.¹⁵

In the present study, the administration of AS LINC00673 decreased the OTSCC proliferation activity compared with the control groups. LINC00673 expression was not directly measured but the observed inhibition of proliferation was consistent with results obtained from studies where LINC00673 expression was suppressed. LINC00673 inhibition has been reported to suppress cell growth through diverse mechanisms. In papillary thyroid carcinoma, inhibition using siRNA LINC00673 caused a decrease in proliferation due to reduced levels of LINC00673 regulating the activity of p53.²⁶ In addition, in esophageal squamous cell carcinoma, LINC00673 inhibition decreased proliferation, induced cell cycle arrest, and also increased the activity of the proliferation inhibitory protein cyclin-dependent kinase inhibitor 2C.²⁰ LINC00673 is reported to be upregulated in breast cancer and accelerate progression through the inhibition of miR-515-5p, thereby inactivating the Hippo pathway to increase the activity of MARK4. Moreover, LINC00673 knockdown using ASO in this cancer led to reduced proliferation and cyclin-D1 expression.¹⁹

On the first day of the MTT assay, significant inhibition was observed in all treatment groups compared with the negative control group. To confirm the inhibitory effect of ASO, the inhibition effect in each group was also compared to that in the transfection-only group. On day 1, lower inhibition was observed in the ASO group, but there were no differences in inhibition in the SO and SCO treatment groups compared with the transfection-only group. On day 2, significantly inhibited proliferation was still observed in the ASO group, but the inhibition of proliferation was not greater in the SO and SCO treatment groups than the transfection-only group. These results are consistent with the cytotoxic effects of cationic liposomes, which can induce cell cycle arrest and apoptosis by activating apoptotic pathways and inflammatory responses.²⁷

The AO–EB double-staining assay detected apoptotic induction in the AS LINC00673 treatment group compared with the other groups. Similar observations were also reported in other cancers when LINC00673 was experimentally inhibited, such as breast cancer and non-small cell lung cancer (NSCLC). The BAX/BCL2 ratio increased in breast cancer, and poly(ADP-ribose) polymerase cleavage was elevated in NSCLC whereas the pro-caspase 3 levels decreased.^19,28

SO is often used as a control in ASO-mediated knockdown.²⁹ Some studies have shown that compared with antisense, sense transcripts induce the expression of tumor suppressor proteins such as P27^Kip1 and tuberous sclerotic complex 2, thereby enhancing the anti-tumor effect.^30,31 In LncRNA, SO inhibits the interaction of mRNA with LncRNA natural antisense transcript, triggering the loss of the transcript's function.³² In the present study, on day 2, SO LINC00673 (1.56 and 12.5 μM) resulted in higher cell proliferation than SCO, and thus its molecular role in regulating LINC00673 requires further study.

In this study, the inhibitory effect of ASO was compared with SCO, which served as a control. SCO had the same number of nucleotides as ASO, but the base sequence of SCO was randomized so it lacked a biological function in order to assess the inhibitory effects of ASO. Based on the results, the inhibitory effect of ASO on LINC00673 could be considered an on-target effect because ASO treatment was successful whereas SCO had no effect.³³

This study was an initial exploration of the effects of an ASO for LINC00673 on OTSCC. The results provide preliminary insights into the impacts of ASO on cell proliferation and apoptosis, but further investigations are needed to directly evaluate the LINC00673 expression levels, transfection efficiency, and biomolecular mechanisms involved. ASO was compared with several other treatments (negative control, transfection only, SO, and SCO) to minimize potential confounding effects.

Conclusion

In conclusion, ASO LINC00673 can potentially inhibit proliferation and induce apoptosis in human tongue cancer cells. Further studies are needed to examine how differences in LINC00673 RNA expression might affect the level of inhibition of proliferation and induction of apoptosis, as well as the underlying biomolecular mechanisms in OTSCC.

Ethical approval

The study protocol was approved by the Ethics Committee Faculty of Dentistry, Prof Soedomo Dental Hospital, Universitas Gadjah Mada (ethical clearance number: 116/UN1/KEP/FKG-UGM/EC/2024).

Authors contributions

S: Conceptualization, supervision, data analysis and interpretation, and writing – review and editing. J: Supervision, data analysis and interpretation, and writing – review and editing. K: Conceptualization, investigation, data analysis and interpretation, writing – original draft, and writing – review and editing.

All authors have critically reviewed and approved the final draft and are responsible for the content and similarity index of the manuscript.

Source of funding

This study was funded by the Ministry of Higher Education, Science, and Technology (Kemendiktisaintek) of the Republic of Indonesia through the PMDSU program under contract number (048/E5/PG.02.00.PL/2024; 2795/UN1/DITLIT/PT.01.03/2024).

Conflict of interest

The authors report no conflict of interest.