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

Effect of long non-coding RNA GHET1 on autophagy and drug resistance to cisplatin in head and neck squamous cell carcinoma

目的探讨长链非编码RNA(lncRNA)GHET1对头颈部鳞状细胞癌(HNSCC)细胞自噬和顺铂耐药性的影响。方法从癌症基因组图谱(TCGA)数据库下载504例HNSCC患者肿瘤样本及43例配对癌旁组织(距离肿瘤原发灶边缘>2 cm)转录组测序(RNA-seq)数据及相应临床信息,数据更新时间为2022年8月;使用R软件分析肿瘤组织与癌旁组织、43例HNSCC配对样本中GHET1表达的差异。收集2019年1月至2023年6月福建省肿瘤医院32例中晚期可手术HNSCC患者资料,比较14例对化疗不敏感者和18例对化疗敏感者GHET1表达的差异;选择人HNSCC细胞株FaDu、Cal27,建立HNSCC顺铂耐药细胞株FaDu-DDP-R、Cal27-DDP-R;各细胞分别转染针对GHET1的siRNA(相应的siRNA组),对照组转染阴性对照siNC。实时荧光定量聚合酶链反应(qRT-PCR)检测GHET1及Beclin-1 mRNA的相对表达量;蛋白质印迹法检测Beclin-1、LC3-Ⅰ/Ⅱ、p63、GAPDH蛋白表达情况;利用自噬抑制剂3-甲基腺苷(3-MA)处理FaDu-DDP-R、Cal27-DDP-R细胞株,比较耐药细胞株与3-MA作用组耐药细胞株顺铂的半数抑制浓度(IC_(50))、GHET1、Beclin-1 mRNA及自噬相关蛋白表达的差异。结果TCGA数据库中504例HNSCC组织GHET1相对表达量高于43例癌旁组织,差异有统计学意义(Z=2.57,P<0.05);43例HNSCC组织GHET1相对表达量高于配对癌旁组织,差异有统计学意义(t=3.24,P=0.002)。临床18例化疗敏感组及14例化疗不敏感组HNSCC中,GHET1相对表达量分别为1.01±0.12和2.05±0.26,化疗不敏感组GHET1表达高于化疗敏感组(t=15.45,P<0.001)。FaDu、FaDu-DDP-R细胞株的IC_(50)分别为(35.6±1.9)μmol/L、(86.2±2.7)μmol/L,差异有统计学意义(t=26.64,P<0.001);Cal27、Cal27-DDP-R细胞株的IC_(50)分别为(68.8±8.9)μmol/L、(115.0±8.2)μmol/L,差异有统计学意义(t=6.60,P<0.01)。FaDu、FaDu-DDP-R细胞株GHET1相对表达量分别为1.00±0.10、3.57±0.07(t=33.85,P<0.001),FaDu-DDP-R细胞株GHET1相对表达量高于FaDu细胞株;Cal27、Cal27-DDP-R细胞株GHET1相对表达量分别为1.00±0.08、2.06±0.11(t=13.25,P<0.001),Cal27-DDP-R细胞株GHET1相对表达量高于Cal27细胞株。蛋白质印迹法检测结果显示,自噬相关蛋白Beclin-1、LC3-Ⅰ、LC3-Ⅱ、p63在FaDu-DDP-R、Cal27-DDP-R细胞株中的相对表达量分别高于FaDu、Cal27细胞株(均P<0.05)。qRT-PCR检测结果显示,对照组及siGHET1组FaDu-DDP-R细胞株GHET1的相对表达量分别为1.00±0.12、0.20±0.06(t=10.52,P<0.001);对照组及siGHET1组Cal27-DDP-R细胞株GHET1的相对表达量分别为1.00±0.09、0.51±0.03(t=8.90,P<0.001);两细胞株敲低组GHET1相对表达量均低于相应的耐药细胞株。对照组及siGHET1组FaDu-DDP-R细胞株Beclin-1 mRNA相对表达量分别为1.00±0.09、0.60±0.07(t=6.08,P<0.01),对照组及siGHET1组Cal27-DDP-R细胞株Beclin-1 mRNA的相对表达量分别为1.00±0.14、0.65±0.04(t=4.31,P<0.05),耐药细胞株敲低GHET1后Beclin-1 mRNA相对表达量均低于相应耐药细胞株。蛋白质印迹法检测结果显示,Beclin-1、LC3-Ⅰ、LC3-Ⅱ、p63在耐药细胞株敲低组中的相对表达水平均低于相应的耐药细胞组。siGHET1组耐药细胞株的顺铂IC_(50)均较相应耐药株低(均P<0.05),3-MA作用组耐药细胞株均较相应耐药细胞株的顺铂IC_(50)降低(均P<0.05)。结论lncRNA GHET1能够通过激活HNSCC细胞自噬,诱导其对顺铂化疗的耐受性。

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(IC_(50)),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).IC_(50) 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 IC_(50) 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-Ⅱand 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-Ⅱand p63 in the knockdown group of drug-resistant cell lines were lower than those in the corresponding drug-resistant cells group.The cisplatin IC_(50) 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 IC_(50) 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.