LncRNA GACAT1 induces tongue squamous cell carcinoma migration and proliferation via miR‐149

Abstract Recent studies have observed that lncRNAs (long non‐coding RNAs) are involved in the progression of various tumours including tongue squamous cell carcinoma (TSCC). Recently, a new lnRNA, GACAT1, has been firstly identified in gastric cancer. However, its potential role in TSCC remains unknown. In this reference, we observed that GACAT1 was overexpressed in TSCC samples and cell lines. Of 25 TSCC specimens, GACAT1 expression was overexpressed in 18 patients (18/25, 72%) compared to non‐tumour specimens. Ectopic expression of GACAT1 induced cell growth and migration and promoted epithelial to mesenchymal transition in TSCC. In addition, ectopic expression of GACAT1 decreased miR‐149 expression in SCC1 cell. We observed that miR‐149 expression was down‐regulated in TSCC cell lines. Moreover, we observed that GACAT1 expression was negatively correlated with miR‐149 expression. GACAT1 overexpression induced TSCC cell growth and migration via regulating miR‐149 expression. These data provided that GACAT1 played an oncogenic role in the progression of TSCC partly through modulating miR‐149 expression.

However, Shi et al 30 reported that GACAT1 was overexpressed in gastric cancer samples and GACAT1 overexpression induced gastric cancer cell growth, migration and invasion. However, its role in TSCC development and the underlying molecular mechanisms has not been studied.
In the current study, our data observed that GACAT1 was overexpressed in TSCC samples and cell lines. Ectopic expression of GACAT1 induced cell growth and migration and promoted epithelial to mesenchymal transition (EMT) in TSCC.

| Quantitative RT-PCR
Total RNA of non-tumour and TSCC samples, cells were purchased with TRIzol kit (Invitrogen) following to instructions. Quantitative RT-PCR analysis was carried out to detect GACAT1, miR-149 and ki-67 expression using SYBR Green on the system of 7500 Real-Time PCR (Applied Biosystems). U6 was utilized as control for miR-149, and GAPDH was carried out as control for ki-67 and GACAT1. The

| Cell proliferation and migration assay
Cell growth rates were determined with MTT analysis (Sigma). Cell was kept in the 96-well dish, and 10 μL was added to each well. After incubation for about 2 hours, the absorbance value at the 490 nm was read on the plate reader. For cell migration, wound scratch analysis was carried out. Pipette tip was utilized to create the cell wound.
These treated cells were kept in the FBS-free medium. Image of the wound was taken at the 0 and 48 hours.

| Statistical analysis
Results were indicated as mean ± standard deviation. Student's t test was utilized to analysis the significance difference about these difference groups. The statistical analysis was carried out by SPSS P < .05 was defined as significance.

| GACAT1 induced TSCC cell growth and migration
To further investigate the function role of GACAT1 in TSCC development, we induced GACAT1 expression in SCC1 cell through transfection with pcDNA-GACAT1 ( Figure 3A). Ectopic expression of GACAT1 enhanced SCC1 cell growth by using MTT assay ( Figure 3B). Overexpression of GACAT1 increased ki-67 expression in SCC1 cell ( Figure 3C). By using wound-healing analysis, we observed that GACAT1 overexpression promoted SCC1 cell migration ( Figure 3D,E).

| GACAT1 promoted epithelial to mesenchymal transition in TSCC cell
Next, we observed that ectopic expression of GACAT1 induced the mesenchymal makers such as N-cadherin, Vimentin and Snail expression in the SCC1 cell ( Figure 4A). Moreover, we indicated that elevated expression of GACAT1 decreased epithelial maker Ecadherin expression ( Figure 4B). These data suggested that GACAT1 overexpression induced EMT transition in TSCC.

| GACAT1 suppressed miR-149 expression in TSCC cell
Ectopic expression of GACAT1 decreased miR-149 expression in SCC1 cell ( Figure 5A). We observed that the expression of miR-149 was significantly up-regulated in the SCC1 cell via transfection with miR-149 mimic ( Figure 5B). Elevated expression of miR-149 suppressed the GACAT1 expression in SCC1 cell ( Figure 5C).

| GACAT1 induced TSCC cell growth and migration via regulating miR-149
We next induced the expression of miR-149 through transfection with miR-149 mimic in the GACAT1-overexpressing SCC1 cell. We observed that ectopic expression of miR-149 decreased cell proliferation in the GACAT1-overexpressing SCC1 cell ( Figure 6A).

| D ISCUSS I ON
In this study, we observed that GACAT1 was overexpressed in TSCC samples and cell lines. Of 25 TSCC specimens, GACAT1 was overexpressed in 18 patients (18/25, 72%) compared to F I G U R E 1 GACAT1 was overexpressed in TSCC samples and cell lines. A, The GACAT1 expression was detected by using qRT-PCR analysis. B, Of 25 TSCC specimens, GACAT1 expression was overexpressed in 18 patients (18/25, 72%) compared to non-tumour specimens. C, The GACAT1 expression in TSCC cell lines (Tca8113, SCC1, SCC-4 and SCC-15) and one non-tumour specimen was detected by qRT-PCR assay. ***P < .001 via sponging miR-875-3p. However, its role and mechanism in TSCC development have not been studied. We firstly studied the expression level of GACAT1 in TSCC samples and cell lines. We proved that expression of GACAT1 was increased in TSCC specimens and cells.
Furthermore, we indicated that ectopic expression of GACAT1 induced TSCC cell growth and migration and promoted EMT in TSCC F I G U R E 3 GACAT1 induced TSCC cell growth and migration. A, The expression of GACAT1 was determined by qRT-PCR assay. B, Ectopic expression of GACAT1 enhanced SCC1 cell growth by using MTT assay. C, Overexpression of GACAT1 increased the ki-67 expression in the SCC1 cell. D, GACAT1 overexpression promoted the SCC1 cell migration by using wound-healing analysis. E, The relative wound closure was shown. **P < .01 and ***P < .001 F I G U R E 4 GACAT1 promoted epithelial to mesenchymal (EMT) transition in TSCC cell. A, Ectopic expression of GACAT1 induced the mesenchymal makers such as N-cadherin, Vimentin and Snail expression in the SCC1 cell. B, Elevated expression of GACAT1 decreased epithelial maker E-cadherin expression. *P < .05 and **P < .01 cell. However, more TSCC samples are needed in our further work.
Thus, these data indicated that GACAT1 may act as one oncogene in tumorigenicity of TSCC cell and provided one potential therapeutic strategy for TSCC.
Increasing studies have suggested that lncRNAs function as ceRNA to modulate tumour progression. [36][37][38] For instance, Ren and colleagues demonstrated that lncRNA CRNDE increased TSCC cell invasion and growth via down-regulating miR-384 F I G U R E 5 GACAT1 suppressed miR-149 expression in TSCC cell. A, Ectopic expression of GACAT1 decreased miR-149 expression in SCC1 cell. B, Expression of miR-149 was significantly up-regulated in the SCC1 cell via transfection with miR-149 mimic. C, Elevated expression of miR-149 suppressed the GACAT1 expression in SCC1 cell. **P < .01 and ***P < .001 F I G U R E 6 GACAT1 induced TSCC cell growth and migration via miR-149. A, The cell proliferation was determined by using MTT assay. B, The mRNA expression of ki-67 was measured by using qRT-PCR analysis. C, The SCC1 cell migration in the different groups was measured by using wound-healing analysis. D, The relative wound closure was shown. *P < .05; **P < .01 and ***P < .001 expression. 39 Zhang et al 40 showed that KCNQ1OT1 modulated cisplatin resistance and proliferation through regulating miR-211-5p in tongue cancer. Ma et al 41 reported that lncRNA GIHCG enhanced TSCC cell proliferation, cycle and migration via regulating miR-429. Zuo and colleagues found that lncRNA CASC15 induced TSCC progression via modulating miR-33a-5p expression. 42 In addition, previous report showed that GACAT1 overexpression induced gastric cancer cell proliferation, migration and invasion through modulating miR-149 expression. 30 In line with this, we found that ectopic expression of GACAT1 decreased miR-149 expression in SCC1 cell. Moreover, GACAT1 overexpression induced TSCC cell growth and migration via regulating miR-149 expression. Previous reports showed that miR-149 played critical roles in tumour development. MiR-149-5p suppressed medullary thyroid carcinoma cell invasion and proliferation via targeting GIT1. 43 Moreover, miR-149 inhibited growth and induced sensitivity of ovarian tumour cell to cisplatin through targeting XIAP. 44 MiR-149 sensitizes oesophageal tumour cell to the cisplatin via regulating DNA polymerase β expression. 45 Thus, our results suggested that GACAT1 promoted TSCC progression via modulating miR-149 and its target genes.
Together, we described that GACAT1 was overexpressed in TSCC samples and cell lines. GACAT1 overexpression induced TSCC cell growth and migration via regulating miR-149 expression. These data implied that GACAT1 played an oncogenic role in the progression of TSCC partly through modulating miR-149 expression.

ACK N OWLED G EM ENTS
We thank Ying Hu, Tao Jiang and Lixia Zhang (Jinan Stomatological Hospital, China) for their assistance.

CO N FLI C T O F I NTE R E S T
There is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data are not shared.