LUCAT1 as an oncogene in tongue squamous cell carcinoma by targeting miR‐375 expression

Abstract Emerging studies suggested that lncRNAs play a crucial molecular role in cancer development and progression. LncRNA LUCAT1 has been proved as oncogenic molecular in lung cancer, glioma, osteosarcoma, renal carcinoma and oesophageal squamous cell carcinoma. However, its roles and function mechanisms in tongue squamous cell carcinoma (TSCC) are still unknown. We showed that the expression of LUCAT1 was up‐regulated in the TSCC cells and tissues and the higher LUCAT1 expression was associated with the poor overall survival (OS). Knockdown expression of LUCAT1 suppressed TSCC cell proliferation, cycle and migration. In addition, we demonstrated that miR‐375 overexpression inhibited the luciferase activity of LUCAT1 wild‐type and knockdown LUCAT1 promoted the miR‐375 expression in TSCC cell. Furthermore, we indicated that miR‐375 expression was down‐regulated in the TSCC cell lines and tissues and the lower expression of miR‐375 was associated with poor OS. The expression of miR‐375 was inversely correlated with LUCAT1 expression in the TSCC tissues. Knockdown LUCAT1 promoted TSCC cell proliferation, cell cycle and migration partly through regulating miR‐375 expression. In summary, this study suggested the tumorigenic effect of lncRNA LUCAT1 in TSCC cells by targeting miR‐375 expression.

studies also found that lncRNAs play crucial roles in a lot of cell biological processes such as cell development, growth, apoptosis, invasion and migration. [24][25][26][27][28] Recently, a novel lncRNA LUCAT1 was reported to be up-regulated in several tumours such as lung tumour, glioma, osteosarcoma, renal carcinoma and oesophageal squamous cell carcinoma. [29][30][31][32][33] For example, Sun et al 33  We indicated that LUCAT1 expression was up-regulated in the TSCC cell lines and tissues and the higher LUCAT1 expression was associated with the poor overall survival. Knockdown expression of LUCAT1 suppressed TSCC cell proliferation, cell cycle and migration.

| Tissues, cell transfection and culture
Fresh TSCC samples and matched non-cancerous samples were collected from TSCC cases in Jinan Stomatological Hospital, China. All samples were immediately frozen in the liquid nitrogen after surgery.
Written informed consent was obtained from each TSCC patient, and

| Quantitative RT-PCR
The isolation of RNA from samples or cells was extracted by using TRIzol Kit (Invitrogen) following the instruction. The expression of LUCAT1 and miR-375 was measured using the SYBR Green Kit (Takara, Dalian, China) on the Q5 Real-Time PCR System (Bio-Rad, Berkely, CA). The relative expression level of LUCAT1 and miR-375 was determined using the 2-DDCT method. The primers utilized in this study were shown as fol-

| Statistical analysis
Data were listed as the mean ± SD (standard deviation). One-way ANOVA or Student's t test (two-tailed) was used to detect the data with SPSS 18.0. P value < 0.05 was accepted as statistically significant.

| LUCAT1 expression was up-regulated in TSCC cell lines and tissues
To explore the clinical importance of lncRNA LUCAT1 in TSCC, we measured the LUCAT1 expression in TSCC cell lines and samples using qRT-PCR. We firstly revealed that the expression of LUCAT1 was up-regulated in the TSCC cell lines (SCC4, UM1, Cal27 and SCC1) compared with normal keratinocyte cell (NHOK) ( Figure 1A).
Next, we showed that the LUCAT1 expression was higher in the TSCC tissues than in the adjacent normal tissues ( Figure 1B). In addition, we found that the expression of LUCAT1 was up-regulated in 29 patients (29/40, 72.5%) compared with adjacent normal tissues ( Figure 1C). Furthermore, we indicated that the higher LUCAT1 expression was associated with the poor overall survival (OS) among the TSCC cohort by using Kaplan-Meier survival analysis ( Figure 1D). Moreover, inhibition expression of LUCAT1 decreased the S phases in the SCC1 cell ( Figure 2C). Wound scratch assay revealed that LUCAT1 silencing decreased the SCC1 cell migration ( Figure 2D).

| LUCAT1 regulated the miR-375 expression in TSCC cell
Previous study showed that LUCAT1 could target the miR-375 expression. TSCC cell line SCC1 cell was transfected with  Figure 3A). Luciferase reporter assays showed that overexpression of miR-375 decreased the luciferase activity of LUCAT1 wild-type but not the LUCAT1 mutant type ( Figure 3B).

| miR-375 expression was down-regulated in TSCC cell lines and tissues
To explore the clinical importance of miR-375 in TSCC, we measured the miR-375 expression in TSCC cell lines and samples using qRT-PCR. We firstly revealed that the expression of miR-375 was down-regulated in the TSCC cell lines (SCC4, UM1, Cal27 and SCC1) compared with normal keratinocyte cell (NHOK) ( Figure 4A). Next, we showed that the miR-375 expression was lower in the TSCC tissues than in the adjacent normal tissues ( Figure 4B). In addition, we found that the expression of miR-375 was down-regulated in 28 patients (28/40, 70%) compared with adjacent normal tissues ( Figure 4C). Furthermore, we indicated that the lower miR-375 expression was associated with the poor OS among the TSCC cohort by using Kaplan-Meier survival analysis ( Figure 4D). We also demonstrated that expression of miR-375 was inversely correlated with LUCAT1 expression in the TSCC tissues ( Figure 4E).

| Knockdown expression of miR-375 promoted TSCC cell proliferation, cycle and migration
TSCC cell line SCC1 cell was transfected with miR-375 inhibitor (anti-miR-375) and negative control by using Lipofectamine 2000 ( Figure 5A). Knockdown expression of miR-375 enhanced the SCC1 cell proliferation by using MTT analysis ( Figure 5B).
Moreover, inhibition expression of miR-375 increased the S phases in the SCC1 cell ( Figure 5C). Wound scratch assay revealed that miR-375 silencing promoted the SCC1 cell migration ( Figure 5D and E).

| LUCAT1 interacts with miR-375 to regulate TSCC cell proliferation, cell cycle and migration
To further study the interaction between miR-375 and LUCAT1, functional rescue experiments were done. As shown in Figure 6A, MTT assay indicated that LUCAT1 silencing suppressed SCC1 cells growth, whereas miR-375 silencing partially rescued the reduction of growth. Moreover, knockdown expression of miR-375 partially reversed the cell cycle suppression of LUCAT1 knockdown ( Figure 6B). Furthermore, wound scratch assay revealed that LUCAT1 silencing suppressed SCC1 cell migration, whereas miR-375 knockdown partially rescued the reduction of cell migration ( Figure 6C and D).

| D ISCUSS I ON
TSCC is one of most common malignant cancers worldwide. 34,35 Cancer metastasis was the first cause of death in tumour patients, and searching novel biomarkers and elucidating the molecular mechanism of TSCC are the major topics of research on TSCC. 11,36,37 Dysregulated expression of lncRNAs was found in a lot of human cancers using RNA sequencing and was correlated with tumour progression, survival and tumorigenesis in TSCC. [38][39][40] We studied the role of a novel lncRNA LUCAT1 in the development of TSCC.
The lncRNA, LUCAT1, was reported to be up-regulated in several tumours such as lung tumour, glioma, osteosarcoma, renal carcinoma and ESCC. [29][30][31][32][33]41 For example, Sun et al 33  Growing evidence proved that a new regulatory mechanism exists between miRNAs and lncRNAs. 27,42,43 LncRNA could play as molecular sponges to miRNA, thereby inhibiting miRNA expression. 22 In addition, it has been shown that LUCAT1 increased the glioma cell invasion and proliferation through regulating miR-375 expression.
In this study, we showed that overexpression of miR-375 decreased the luciferase activity of LUCAT1 wild-type but not the LUCAT1 mu- In conclusion, our results reveal that LUCAT1 was up-regulated in the TSCC cell lines and tissues and the higher LUCAT1 expression was associated with the poor overall survival. Knockdown expression of LUCAT1 suppressed TSCC cell proliferation, cell cycle and migration via sponging miR-375 expression, providing a new insight into carcinogenesis of TSCC.

This study was supported by Medical Health Science and Technology
Project of Shandong Province(2018ws483) and "Science and Technology Development Project of Jinan" (No. 201907092).

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to declare.