LINC01296 promotes cancer stemness traits in oral carcinomas by sponging miR-143

Abstract

Background/purpose

Emerging evidence has shown that various failures in cancer therapy, such as drug resistance, metastasis, and cancer relapse are attributed to cancer stem cells (CSCs). Also, growing attention has been paid to the regulation of non-coding RNAs in cancer stemness. Here, we aimed to investigate the contribution of LINC01296 in the modulation of oral CSCs.

Materials and methods

The phenotypic assays including migration, invasion, and colony-forming abilities were carried out in CSCs of two types of oral cancer cells (SAS and GNM) following the knockdown of LINC01296. In addition, the percentage of cells expressing stemness marker, ALDH1, and drug resistance marker, ABCG2, was examined as well as the self-renewal capacity after silencing of LINC01296. Moreover, a luciferase reporter was used to validate the direct interaction between LINC01296 and miR-143.

Results

Our results showed that LINC01296 was significantly overexpressed in oral cancer tissues and positively correlated with stemness markers. The phenotypic and flow cytometry assays demonstrated that suppression of LINC01296 reduced the aggressiveness, cancer stemness features, and colony-forming and self-renewal abilities in oral CSCs. Furthermore, we demonstrated that LINC01296 may enhance cancer stemness features through suppression of the effect of miR-143.

Conclusion

Silencing of LINC01296 may be a promising direction for oral cancer therapy by reducing cancer stemness via regulation of miR-143.

Introduction

Oral cancer is one of the most prevalent cancers worldwide with around 377,713 new diagnoses and over 177,750 deaths estimated in 2020.¹ It includes cancers that occur from the lip, tongue, mouth, and oropharynx collectively. Although these cancers are twice as common in men as in women, it has been noted that the incidence rates among females were slightly increased in certain regions.² Despite the accessibility of the oral cavity to direct examination and advances in surgery and radio/chemotherapy, the prognosis of oral cancer is still unsatisfactory. One major obstacle to conventional cancer therapies is the existence of cancer stem cells (CSCs), which are implicated in drug resistance, cancer recurrence, and metastasis.³ Multiple markers have been employed to identify oral CSCs, including CD44,⁴ aldehyde dehydrogenase 1 (ALDH1),⁵ Sox2,⁶ and ATP binding cassette subfamily G member 2 (ABCG2).⁷ It is important to target CSCs and understand the key regulator of cancer stemness in order to develop effective cancer management.

Recent studies have suggested that non-coding RNAs may emerge as crucial modulators of cancer stemness.^8,9 Non-coding RNAs include a heterogeneous group of RNA molecules that are not translated into functional proteins but possess various biological functions. Numerous non-coding RNAs have been discovered, such as short non-coding RNAs (e.g. microRNAs; ∼22 nucleotides) or long non-coding RNAs (lncRNAs; >200 nucleotides). It has been known that microRNAs modulate the stability of their target genes by pairing with complementary sites within the 3′ untranslated region (3′-UTR), resulting in degradation of the target transcript by cleavage or deadenylation.¹⁰ Aside from serving as a scaffold or guide,¹¹ increasing evidence has suggested that lncRNAs can regulate gene expression by titrating microRNAs with their microRNAs response elements (MREs).¹² These lncRNAs are also known as competing endogenous RNA (ceRNAs) or miRNA sponges as they can sequester the distribution of miRNAs and inhibit their suppressive effect on target mRNAs. Various studies have shown that long non-coding RNA LINC01296 is overexpressed in oral cancer tissues and can enhance the aggressiveness of cancer cells.13, 14, 15 Nevertheless, the significance of LINC01296 in the regulation of oral cancer stemness has not been investigated.

Herein, we aimed to examine the functional role of LINC01296 in numerous phenotypes of oral CSCs, such as migration, invasion, colony-forming, and self-renewal capacities. Also, the expression levels of ALDH1 and ABCG2 were assessed. Moreover, we validate the direct interaction between one putative interacting microRNA of LINC01296 and its implication in cancer stemness.

Materials and methods

Oral cancer cell lines and OSCC tissues

The OSCC cell lines SAS and GNM were cultivated as previously described.^16,17 OSCC (T) and normal paired noncancerous matched tissues (N) specimens were collected with written informed consents and all protocols were approved by The Institutional Review Board in Chung Shan Medical University Hospital, and assess the expression of LINC01296, CD44, and Sox2.

Quantitative real-time PCR (qRT–PCR) analysis for expression detection

Total RNA were prepared from cells using Trizol reagent according to the manufacturer's protocol (Invitrogen Life Technologies, Carlsbad, CA, USA). qRT–PCRs of mRNAs were reverse-transcribed using the Superscript III first-strand synthesis system (Invitrogen Life Technologies). qRT-PCR reactions on resulting cDNAs were performed on an ABI StepOne™ Real-Time PCR Systems (Applied Biosystems, Waltham, MA, USA).¹⁸

Lentiviral-mediated RNAi for silencing LINC01296

The pLV-RNAi vector was purchased from Biosettia Inc. (Biosettia, San Diego, CA, USA). The method of cloning the double-stranded shRNA sequence was follow the manufacturer's protocol. Oligonucleotide sequence of lentiviral vectors expressing shRNA that targets human LINC01296 was synthesized and cloned into pLVRNAi to generate a lentiviral expression vector.¹⁹ The target sequences for LINC01296 were listed as follows: Sh-LINC01296-1: 5′-AAAAGGTGGTTTCCACAAGAAAATTGGATCCAATTTTCTTGTGGAAACCACC-3′; Sh-LINC01296-2: 5′-AAAAGGAGGATGGTTTCAACATATTGGATCCAATATGTTGAAACCATCCTCC-3′.

Migration, invasion, and anchorage-independent growth

These assays have been well-established in our laboratory and all procedures were follow the previously described protocol.¹⁹

Sphere-forming assay

For the self-renewal assay, single cells were obtained from accurtase treated primary spheres and the cell density of passage was 1000 cells/ml in the DMEM/F-12 supplemented with N2 (R&D Systems, Minneapolis, MN, USA), 10 ng/mL epidermal growth factor (EGF, Invitrogen Life Technologies), 10 ng/mL basic fibroblast growth factor (bFGF, Invitrogen Life Technologies), and penicillin/streptomycin at 10³ live cells/low-attachment six-well plate (Corning, somerville, MA, USA).²⁰

Flow cytometry analysis

Cells were stained with anti-ABCG2 conjugated to phycoerythrin (Miltenyi Biotech., Auburn, CA, USA) according to the manufacturer's instructions. Red (>650 nm) fluorescence emission from 10,000 cells illuminated with blue (488 nm) excitation light will be measured with a FACSCalibur (Becton Dickinson, New York City, NJ. USA) followed by CellQuest software.²¹

Statistical analysis

Statistical Package of Social Sciences software (version 13.0) (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. Data from at least triplicate analysis were shown as mean ± SD. Student's t test was used to determine the statistical significance of the differences between experimental groups; P values less than 0.05 will be considered statistically significant.

Results

As shown in Fig. 1A, the expression of LINC01296 was markedly elevated in tumor tissues compared to normal oral specimens by qRT-PCR analysis. To explore the implication of LINC01296 in cancer stemness, we examine the relationship between LINC01296 and several stemness-associated factors. As expected, the expression level of LINC01296 was positively associated with CD44 (Fig. 1B) and Sox2 (Fig. 1C) in oral cancer samples.

Figure 1.

LINC01296 is upregulated in oral cancer tissues and positively correlated with stemness marker. (A) The relative expression of LINC01296 was elevated in tumor tissues (T) compared with normal oral specimens by qRT-PCR analysis. ∗p < 0.01 compared to normal specimens (N); the expression of LINC01296 was positively associated with CD44 (B) and Sox2 (C) in oral cancer samples.

To determine the functional role of LINC01296 in cancer stemness, short hairpin RNA (shRNA)-mediated knockdown of LINC01296 in two types of oral CSCs (SAS and GNM; OCSCs) was conducted. The knockdown efficiency was confirmed by measuring the mRNA expression levels following transfection of the sh-Luc. and sh-RNA LINC01296 vectors into cells (Fig. 2A). One important feature of CSCs is that they still retain stem-cell functionality to form metastatic colonies after dissemination through epithelial-mesenchymal transition (EMT).²² Hence, we evaluated the migration and invasion capacities and showed that inhibition of LINC01296 markedly suppressed the aggressiveness of these two types of OCSCs (Fig. 2B and C). Moreover, we demonstrated that the colony-forming ability was downregulated as well (Fig. 2D).

Figure 2.

Silencing of LINC01296 downregulates the cell motility and colony formation of OCSCs derived from SAS and GNM cells. (A) Silencing efficiency of shRNA-mediated knockdown of LINC01296 in two types of OCSCs derived from SAS and GNM cells; (B) Migration, (C) invasion, and (D) colony forming abilities in cells transfected with sh-Luc or shRNA-LINC01296. ∗P < 0.05 compared to sh-Luc.

Subsequently, we analyzed the percentage of cells expressing the CSC marker, ALDH1, and we found that silencing LINC01296 significantly diminished the activity of ALDH1 in these two types of OCSCs derived from SAS and GNM cells (Fig. 3A). Likewise, we assessed the percentage of ABCG2-positive cells and showed that ABCG2 positivity was reduced after the knockdown of LINC01296 (Fig. 3B). Additionally, we demonstrated that the self-renewal capacity of these OCSCs was downregulated using the sphere-formation assay (Fig. 3C). Collectively, these results suggested that suppression of LINC01296 decreased the cancer stemness characteristics of OCSCs.

Figure 3.

Downregulation of LINC01296 inhibits the features of OCSCs. (A) ALDH1 activity, (B) ABCG2 positivity, (C) and sphere-forming ability of two types of OCSCs cells transfected with sh-Luc or shRNA-LINC01296. ∗P < 0.05 compared to sh-Luc.

LINC01296 has been reported to modulate cell migration, invasion, proliferation, and apoptosis of thyroid cancer cells via interacting with miR-143-3p.²³ In an effort to verify the direct relationship between LINC01296 and miR-143, the luciferase reporter assay was employed to validate the target site. As shown in Fig. 4A, the complementarity between the 3′ untranslated region (3′UTR) of LINC01296 and miR-143 was illustrated. Reporter plasmids containing either full-length (Wt-LINC01296) or mutated (mut-LINC01296) forms of the miR-143-binding region were constructed and co-transfected with miR-143 mimics into cells. The luciferase activity of Wt- LINC01296 vector was reduced when co-transfected with miR-143 mimics, whereas no significant change was found in the mut-LINC01296 vector in both SAS and GNM CSCs (Fig. 4B).

Figure 4.

MiR-143 is a direct target of LINC01296. (A) The sequences of the putative miR-143 binding sites in wild-type and mutant LINC01296 at the 3′-untranslated region (3′UTR); (B) Luciferase activity was decreased in cells co-transfected with WT-LINC01296 and miR-143 mimic. ∗P < 0.05 compared to with miR-Scr. Wild-type (wt); mutant (Mut).

Furthermore, we demonstrated that inhibition of miR-143 reversed the self-renewal ability in SAS-OCSCs transfected with sh-RNA LINC01296 (Fig. 5A). Similarly, downregulation of migration ability after silencing LINC01296 was reverted by suppression of miR-143 in SAS-OCSCs (Fig. 5B). Altogether, these findings indicated that LINC01296 contributed to these cancer stemness features through repression of miR-143.

Figure 5.

The inhibitory effects of silencing LINC01296 on self-renewal and migration abilities are reversed by inhibition of miR-143. Self-renewal (A) and migration (B) capacities of cells transfected with sh-LINC01296 with or without miR-143 inhibitors were evaluated. ∗P < 0.05 compared with sh-Luc group; #P < 0.05 compared with sh-LINC01296 group.

Discussion

While a vast body of knowledge is available on the significance of CSCs in cancer treatment failure, the molecular mechanism underlying the regulation of these cells remains largely unknown. Hence, it is imperative to decipher the possible modulators of CSCs in an effort to develop a treatment to target these CSCs. Multiple contributors have been considered to implicate in the modulation of cancer stemness, such as cytokines in tumor microenvironment²⁴ or non-coding RNAs for post-transcriptional regulation.^8,9 In the present study, we demonstrated how LINC01296 affected the CSCs characteristics of two types of OCSCs, including cell motility, colony forming, self-renewal ability, the expression of stemness, and drug resistance markers. Moreover, we showed that LINC01296 exhibited its regulatory effect on OCSCs through sequestration of miR-143.

It has been revealed that when LINC01296 was overexpressed, it aggravated various types of cancers including gastric cancer,²⁵ breast cancer,²⁶ and cholangiocarcinoma.²⁷ Similarly, upregulated pattern of LINC01296 was observed in oral cancer,13, 14, 15 in which it promoted its aggressiveness.

In line with these results, we showed the increased expression of LINC01296 in tumor tissues. Most importantly, our data demonstrated that silencing of LINC01296 led to the amelioration of CSCs phenotypes and the expression of stemness markers. We also showed that there was a positive correlation between LINC01296 and two stemness markers, CD44 and Sox2. To date, research addressing the regulation of LINC01296 in CSCs is still limited. It has been shown that LINC01296 directly interacted with nucleolin,²⁸ which can maintain the stemness of neuroblastoma by binding to the promoter region of CD34.²⁹ Besides, several microRNAs have been identified as direct targets of LINC01296 in various types of cancers, such as miR-122²⁵ or miR-5095.²⁷ In the present study, we showed that LINC01296 directly harbored miR-143 and enhanced the self-renewal and migration abilities of OCSCs. In agreement with our result, a previous study also showed that LINC01296 increased cell migration, invasion, and proliferation of thyroid cancer cells via interacting with miR-143-3p.²³

MiR-143 has been observed to co-express with miR-145 at chromosome 5q33 in a variety of cell types and tissues.³⁰ MiR-143 has been widely described as a tumor suppressor as it is downregulated in various cancers, such as gastric cancer,³¹ bladder cancer,³² or colorectal cancer.³³ Nevertheless, upregulation of miR-143 has been shown to be correlated with poor survival of bladder cancer patients.³⁴ Furthermore, it has been demonstrated that the expression of miR-143 progressively increased during CSCs differentiation, which promoted prostate cancer cell metastasis.³⁵ As for oral cancer, it has been shown that miR-143 was downregulated in tumor tissues and cell lines.^36,37 Zhang et al. demonstrated that miR-143 blocked cell-cycle progression at the G1/S checkpoint in oral cancer cells via suppression of the murine double minute 2 (MDM2) gene.³⁶ Another study showed that miR-143 inhibited cell growth, invasion and glucose metabolism of oral cancer by targeting hexokinase 2 (HK2).³⁷ Likewise, oral cancer cells transfected with miR-143 mimic exhibited lower migration capacity along with downregulation of various metastatic genes (e.g. MMP9, c-Myc, ADAMTS, and CXCR4).³⁸ These studies showed that miR-143 reduced the migration ability in oral cancer cells, which was consistent with our finding. Besides, miR-143 has been demonstrated to repress CSCs properties of prostate cancer cells, including self-renewal property and the expression of several stemness markers.³⁹ We also showed that miR-143 mediated the LINC01296-induced self-renewal ability of OCSCs.

Taken together, our findings demonstrated the essential roles of LINC01296 in OCSCs suppression, including the regulation of CSC phenotypes and expression of cancer stemness markers. Additionally, we showed that LINC01296 enhanced CSCs features via direct suppression of miR-143. Approaches to target the LINC01296/miR-143 axis in OCSCs may lead to promising results in ameliorating OSCC.

Conflicts of interest

All authors have no conflicts of interest relevant to this article.