Effect of long non-coding RNA GHET1 on autophagy and drug resistance to cisplatin in head and neck squamous cell carcinoma
10.3760/cma.j.cn115355-20231106-00177
- VernacularTitle:长链非编码RNA GHET1对头颈部鳞状细胞癌细胞自噬和顺铂耐药性的影响
- Author:
Shuping WU
1
;
Jianhong YU
;
Yu WU
;
Hui LIU
Author Information
1. 福建医科大学肿瘤临床医学院 福建省肿瘤医院头颈外科，福州 350014
- Keywords:
Head and neck neoplasms;
Carcinoma, squamous cell;
Long non-coding RNA;
Autophagy;
Drug tolerance;
Cisplatin
- From:
Cancer Research and Clinic
2024;36(7):481-487
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effect of long non-coding RNA (lncRNA) GHET1 on autophagy and drug resistance to cisplatin in head and neck squamous cell carcinoma (HNSCC).Methods:Transcriptome sequencing (RNA-seq) data and the related clinical information of tumor samples from 504 HNSCC patients and 43 matched paracancerous tissues (> 2 cm from the tumor primary site margin) were downloaded from the Cancer Genome Atlas (TCGA) database. The data was updated in August 2022. R software was used to analyze the differences of GHET1 expression in cancer tissues, paracancerous tissues and 43 HNSCC matched samples. The data of 32 middle and advanced HNSCC patients who were eligible for surgery in Fujian Cancer Hospital from January 2019 to June 2023 were collected, and the differences of GHET1 expression between 14 patients insensitive to chemotherapy and 18 sensitive to chemotherapy were compared. Human HNSCC cell lines FaDu and Cal27 were selected to establish cisplatin-resistant HNSCC cell lines FADU-DDP-R and CAL27-DDP-R. The cells were transfected with siRNA targeting GHET1 (corresponding siRNA group), and the control group was transfected with negative control siNC. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the relative expression level of GHET1 and Beclin-1mRNA. The expressions of Beclin-1, LC3-Ⅰ/Ⅱ, p63 and GAPDH protein were detected by using Western blot. The autophagy inhibitor 3-methyladenosine (3-MA) was used to treat FADU-DDP-R and CAL27-DDP-R cell lines; the half inhibitory concentration ( IC50), the expression differences of GHET1, Beclin-1 mRNA and autophagy related protein of cisplatin were compared between the drug-resistant cell lines and the drug-resistant cell lines of the 3-MA group. Results:In TCGA database, the relative expression level of GHET1 in 504 HNSCC tissues was higher than that in 43 paracancerous tissues, and the difference was statistically significant ( Z = 2.57, P < 0.05); the relative expression level of GHET1 in 43 HNSCC tissues was higher than that in the paired paracancerous tissues, and the difference was statistically significant ( t = 3.24, P = 0.002). The relative expression level of GHET1 in HNSCC of 18 patients in chemotherapy-sensitive group and 14 patients in chemotherapy-insensitive group was 1.01±0.12 and 2.05±0.26, respectively, and the expression of GHET1 in the chemotherapy-insensitive group was higher than that in the chemotherapy-sensitive group ( t = 15.45, P < 0.001). IC50 of FaDu and FADU-DDP-R cell lines was (35.6±1.9) μmol/L and (86.2±2.7) μmol/L, respectively, and the difference was statistically significant ( t = 26.64, P < 0.001). The IC50 of Cal27 and CAL27-DDP-R cell lines was (68.8±8.9) μmol/L and (115.0±8.2) μmol/L, respectively, and the difference was statistically significant ( t = 6.60, P < 0.01). The relative expression levels of GHET1 in FaDu and FADU-DDP-R cell lines were 1.00±0.10 and 3.57±0.07, respectively ( t = 33.85, P < 0.001), and the relative expression level of GHET1 in FADU-DDP-R cell lines was higher than that in FaDu cell lines. The relative expression levels of GHET1 in Cal27 and Cal27-DDP-R cell lines were 1.00±0.08 and 2.06±0.11, respectively ( t = 13.25, P < 0.001), and the relative expression level of GHET1 in Cal27-DDP-R cell lines was higher than that in Cal27 cell lines. Western blot showed that the relative expression levels of autophagy related proteins Beclin-1, LC3-Ⅰ, LC3-Ⅱ and p63 in FADU-DDP-R and CAL27-DDP-R cell lines were higher than those in FaDu and Cal27 cell lines (all P < 0.05). The results of qRT-PCR showed that the relative expression levels of GHET1 in FADU-DDP-R cell lines in the control group and siGHET1 group were 1.00±0.12 and 0.20±0.06, respectively ( t = 10.52, P < 0.001). The relative expression levels of GHET1 in CAL27-DDP-R cell lines in the control group and siGHET1 group were 1.00±0.09 and 0.51±0.03, respectively ( t = 8.90, P < 0.001). The relative expression level of GHET1 in the knockdown group of the 2 cell lines was lower than that of the corresponding drug-resistant cell lines. The relative expression levels of Beclin-1 mRNA in FADU-DDP-R cell line in the control group and siGHET1 group were 1.00±0.09 and 0.60±0.07, respectively ( t = 6.08, P < 0.01); the relative expression levels of Beclin-1 mRNA in Cal27-DDP-R cell lines in the control group and siGHET1 group were 1.00±0.14 and 0.65±0.04, respectively ( t = 4.31, P < 0.05); the relative expression levels of Beclin-1 mRNA in drug-resistant cell lines after knocking down GHET1 were lower than those in corresponding drug-resistant cell lines. Western blot showed that the relative expression levels of Beclin-1, LC3-Ⅰ, LC3-Ⅱ and p63 in the knockdown group of drug-resistant cell lines were lower than those in the corresponding drug-resistant cells group. The cisplatin IC50 of drug-resistant cell lines in siGHET1 group was lower than that of the corresponding drug-resistant cell lines (all P < 0.05), and the cisplatin IC50 of drug-resistant cell lines in 3-MA group was lower than that of the corresponding drug-resistant cell lines (all P < 0.05). Conclusions:LncRNA GHET1 could induce the resistance to cisplatin by activating the autophagy in HNSCC.
