Long non-coding RNA CTC-338M12.4 inhibition on activation of JAK/STAT signaling pathway via miRNA-27a-5p makes cell cycle arrest, cell proliferation and migration inhibition in tongue squamous cell carcinoma
10.3760/cma.j.cn115355-20231026-00165
- VernacularTitle:lncRNA CTC-338M12.4抑制miRNA-27a-5p对JAK/STAT信号通路的激活使舌鳞状细胞癌细胞周期阻滞、细胞增殖和迁移受抑制
- Author:
Xin PENG
1
;
Jin WANG
;
Yan XIONG
;
Xiaoquan LUO
;
Hui GUO
;
Jianwei PENG
Author Information
1. 湖北省第三人民医院口腔科，武汉 430071
- Keywords:
Tongue neoplasms;
Carcinoma, squamous cell;
RNA, long non-coding;
Janus kinases;
STAT transcription factors;
Signal transduction;
CTC-338M12.4;
miRNA-27a-5p
- From:
Cancer Research and Clinic
2024;36(11):801-807
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the expression level of long non-coding RNA (lncRNA) CTC-338M12.4 in tongue squamous cell carcinoma tissues, and its effects on the cell cycle, cell proliferation, and migration of tongue squamous cell carcinoma cells in vitro as well as its molecular mechanisms.Methods:The Gene Expression Omnibus (GEO) database was used to obtain the lncRNA series data set GSE139869, and the differential expression of CTC-338M12.4 in tongue squamous cell carcinoma tissues and adjacent tissues was analyzed. The transcriptional expression levels of CTC-338M12.4 in human immortalized oral keratinocytes (HOK) and tongue squamous cell carcinoma cell lines HN13, TSCCa, CAL-27, Tca8113, SCC15 were detected by using reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR). CAL-27 cells with the lowest expression level of CTC-338M12.4 were selected and were divided into the control group (co-transfected with vectors containing negative sequence) and CTC-338M12.4 group (co-transfected with CTC-338M12.4 overexpression vectors). The proliferation ability of CAL-27 cells in each group was detected by using cell colony formation assay, and the cell cycle distribution of CAL-27 cells was detected by using flow cytometry. The migration ability of CAL-27 cells was detected by using scratch test. The lncACTdb database was used to predict the complementary binding sites between CTC-338M12.4 and miRNA-27a-5p (miR-27a-5p), and dual-luciferase reporter gene assay was used to verify. The expression level of miR-27a-5p in CAL-27 cells of all groups was detected by using qRT-PCR. The protein expression level of related factors on JAK/STAT signaling pathway in CAL-27 cells of all groups was detected by using Western blot.Results:Analysis of GEO database data showed that transcriptional level CTC-338M12.4 in tongue squamous cell carcinoma tissues was lower than that in adjacent tissues, and the difference was statistically significant ( P < 0.01). Transcriptional level CTC-338M12.4 in tongue squamous cell carcinoma HNl3, TSCCa, CAL-27, Tca8113, and SCC15 cells was lower than that in HOK cells, and the differences were statistically significant (all P < 0.05). The transcriptional level relative expression level of CTC-338M12.4 in CAL-27 cells in the CTC-338M12.4 group was higher than that in the control group, and the difference was statistically significant ( P < 0.01). Cell colony formation assay showed that the colong number of CAL-27 cell in the CTC-338M12.4 group was less than that in the control group [(51±10) vs. (114±21)], and the difference was statistically significant ( t = 2.71, P = 0.035). Flow cytometry showed that the proportion of G 0/G 1 phase cells in CAL-27 cells in the CTC-338M12.4 group was higher than that in the control group [(64±3)% vs. (43±4)%], and the difference was statistically significant ( t = 4.87, P = 0.003). The scratch test showed that when scratching, the scratch width of both groups was similar ( P > 0.05); after scratch for 25 h, the scratch width of CAL-27 cells in the CTC-338M12.4 group was wider than that in the control group [(133±15) μm vs. (64±10) μm], and the difference was statistically significant ( t = 3.78, P = 0.009). The dual luciferase reporter gene assay showed that the relative luciferase activity of CAL-27 cells co-transfected with wild-type CTC-338M12.4 sequence and miR-27a-5p sequence was lower than that of CAL-27 cells co-transfected with wild-type CTC-338M12.4 sequence and miR-27a-5p irrelevant sequence, and the difference was statistically significant ( P < 0.001). The relative expression level of transcriptional level miR-27a-5p of CAL-27 cells in the CTC-338M12.4 group was lower than that in the control group, and the difference was statistically significant ( P = 0.003). Western blot showed that the protein expression levels of JAK/STAT signaling pathway p-JAK, p-STAT, p-Raf, p-ERK, and p-mTOR were lower than those in the control group. Conclusions:The level of lncRNA CTC-338M12.4 is low in tongue squamous cell carcinoma. CTC-338M12.4 mediates the inactivation of JAK/STAT signaling pathway via inhibiting miR-27a-5p expression in tongue squamous cell carcinoma CAL-27 cells, thereby leading to cell cycle arrest and inhibiting the cell proliferation and migration of CAL-27 cells.
