Hong Z Q, Cui Y S, Tian Y P, Wu Y N, Zheng X, Feng Y, Sun G G
School of Public Health, North China University of Science and Technology, Tangshan 063210, China.
Hebei Key Laboratory of Precision Medicine for Medical and Industrial Integration, Tangshan 063000, China.
Zhonghua Zhong Liu Za Zhi. 2024 Sep 23;46(9):889-903. doi: 10.3760/cma.j.cn112152-20231024-00226.
To further explore the role and mechanism of hsa_circ_0001776 and mir-1265 in lung squamous carcinoma by verifying the expression level of hsa_circ_0001776 in plasma, tissues, and cells of lung squamous carcinoma. Plasma was collected from patients with lung squamous carcinoma treated at Tangshan People's Hospital and healthy individuals from 2020 to 2022. Lung squamous carcinoma tissue microarrays purchased from Shanghai Xinchao Biotechnology Company in 2022. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of hsa_circ_0001776 in lung squamous carcinoma plasma, tissues, and cells, and fluorescence in situ hybridization was used to verify the expression of hsa_circ_0001776 in lung squamous carcinoma. The localization of hsa_circ_0001776 in NCI-H1703 was verified by fluorescence in situ hybridization. The lung squamous carcinoma cells NCI-H1703 and NCI-H226 were cultured and divided into the circ-negative control (NC) group, hsa_circ_0001776 overexpression group, miR-NC group, miR-1265 mimic group, hsa_circ_0001776+miR-NC group, and hsa_circ_0001776+miR-1265 mimic group.The cell proliferation, motility and apoptosis were detected by the cell counting kit-8 (CCK-8) method, clone formation, Transwell invasion and migration, and scratch assay, and flow cytometry, respectively. The downstream of hsa_circ_0001776 was predicted by circular RNA interactome website, and the interaction between hsa_circ_0001776, miR-1265 was further determined by dual luciferase reporter gene assay, and nude mice subcutaneous tumorigenesis assay detected the growth of transplanted tumors. Fluorescence in situ hybridization results showed that the fluorescence intensity of hsa_circ_0001776 in lung squamous carcinoma tissues was lower than that in paracancerous tissues, and the fluorescence intensity of miR-1265 in lung squamous carcinoma tissues was higher than that in paracancerous tissues (both ＜0.05). The expression level of hsa_circ_0001776 in the plasma of lung squamous carcinoma patients was lower than that in the plasma of healthy people, and the expression level of miR-1265 was higher than that in the plasma of healthy people (both ＜0.05). The expression levels of hsa_circ_0001776 in lung squamous carcinoma cells NCI-H1703, NCI-H226 and SK-MES-1 were lower than that in bronchial epithelial cells BEAS-2B (all ＜0.05), and the relative expression levels of miR-1265 in NCI-H1703 and NCI-H226 were higher than that in human bronchial epithelial cells BEAS -2B (all ＜0.05). The expression of hsa_circ_0001776 was correlated with age, lymph node metastasis, clinical stage, and tumor stage in patients with lung squamous carcinoma (all ＜0.05). Fluorescence in situ hybridization results showed that hsa_circ_0001776 was mainly expressed in the cytoplasm. The results of dual-luciferase reporter assay showed complementary binding of miR-1265 to hsa_circ_0001776. The absorbance values of the hsa_circ_0001776 overexpression group in NCI-H1703 and NCI-H226 cells were lower than that of the circ-NC group (＜0.05). The number of cell clones in the hsa_circ_0001776 overexpressed group was (52±3) and (53±4), the number of migrating cells was (476±17) and (113±7), the number of invading cells was (100±2) and (184±2), and the cell migration rate was (25.00±4.36)% and (36.02±5.55)%, which were lower than those of the circ-NC group [(104±4) and (106±2), (783±29) and (517±16), (657±45) and (473±9), (48.95±8.69)% and (48.70±1.57)%, all ＜0.05]. The apoptosis rates in the overexpression hsa_circ_0001776 group were (24.77±2.303)% and (19.67±1.16)%, respectively, both higher than those in the circ-NC group [(11.83±1.15)% and (9.50±0.66)%, respectively, both ＜0.05]. MiR-1265 mimic group had a higher apoptotic rate in the NCI-H1703 and NCI-H226 than those of the miR-NC groups (＜0.05). miR-1265 mimic group had (56±13) and (51±8) cell clones, (556±13) and (405±6) migrating cells, (486±6) and (359±7) invading cells, cell migration rates of (68.56±5.51)%, (81.74±8.04)%, were higher than those of miR-NC group [(31±4) and (21±8), (154±19) and (186±5), (227±6) and (176±7), (25.83±4.26)% and (53.12±4.14) %, all ＜0.05]. The apoptotic rates in the miR-1265 mimic group were (11.83±2.55)% and (17.50±1.05)%, respectively, which were lower than those in the miR-NC group [(32.67±4.44)% and (39.90±2.88)%, respectively, both <0.05]. The absorbance values of NCI-H1703 and NCI-H226 in the overexpression of hsa_circ_0001776+miR-1265 mimic group were higher than those of the overexpression of hsa_circ_0001776+miR-NC group (<0.05). The overexpression of hsa_circ_0001776+miR-1265 mimic group had (128±15) and (133±8) cell clones, (623±10) and (310±7) migrating cells, (643±16) and (420±7) invading cells, (66.39±4.46)% cell migration rate and (68.60±3.53)%, were higher than those of the hsa_circ_0001776+miR-NC group [(86±7) and (80±16), (380±11) and (115±5), (152±7) and (94±4), respectively, (31.41±5.91)% and (30.94±0.67)%, all ＜0.05]. The apoptotic rates in the overexpression of hsa_circ_0001776+miR-1265 mimic group were (19.27±0.15)% and (11.53±0.75)%, respectively, both lower than those in the overexpression of hsa_circ_0001776+miR-NC group [(27.77±1.29)% and (18.43±0.71)%, both ＜0.05]. The results of the subcutaneous tumorigenesis assay in nude mice showed that the volume of tumors in the overexpression of hsa_circ_0001776 group was lower than that in the circ-NC group (＜0.05). hsa_circ_0001776 is downregulated in lung squamous cell carcinoma, and hsa_circ_0001776 can inhibit the development of lung squamous cell carcinoma by targeting miR-1265.
通过检测人源环状RNA（hsa_circ_0001776）在肺鳞癌患者血浆、组织及细胞中的表达水平，进一步探究hsa_circ_0001776和微小RNA-1265（mir-1265）在肺鳞癌中的作用及机制。收集2020年至2022年在唐山市人民医院接受治疗的肺鳞癌患者及健康个体的血浆。2022年购买上海新朝生物技术公司的肺鳞癌组织芯片。采用实时定量聚合酶链反应（qRT-PCR）检测hsa_circ_0001776在肺鳞癌血浆、组织及细胞中的表达，并用荧光原位杂交技术验证hsa_circ_0001776在肺鳞癌中的表达。通过荧光原位杂交技术验证hsa_circ_0001776在NCI-H1703细胞中的定位。培养肺鳞癌细胞NCI-H1703和NCI-H226，并分为环状RNA阴性对照组（circ-NC组）、hsa_circ_0001776过表达组、微小RNA阴性对照组（miR-NC组）、mir-1265模拟物组、hsa_circ_0001776 + miR-NC组和hsa_circ_0001776 + mir-1265模拟物组。分别采用细胞计数试剂盒-8（CCK-8）法、克隆形成实验、Transwell侵袭和迁移实验、划痕实验以及流式细胞术检测细胞增殖、迁移和凋亡情况。通过环状RNA相互作用组网站预测hsa_circ_0001776的下游靶点，并用双荧光素酶报告基因实验进一步确定hsa_circ_0001776与mir-1265之间的相互作用，通过裸鼠皮下成瘤实验检测移植瘤的生长情况。荧光原位杂交结果显示，hsa_circ_0001776在肺鳞癌组织中的荧光强度低于癌旁组织，mir-1265在肺鳞癌组织中的荧光强度高于癌旁组织（均P＜0.05）。肺鳞癌患者血浆中hsa_circ_0001776的表达水平低于健康人血浆，mir-1265的表达水平高于健康人血浆（均P＜0.05）。hsa_circ_0001776在肺鳞癌细胞NCI-H1703、NCI-H226和SK-MES-1中的表达水平低于支气管上皮细胞BEAS-2B（均P＜0.05），NCI-H1703和NCI-H226中mir-1265的相对表达水平高于人支气管上皮细胞BEAS -2B（均P＜0.05）。hsa_circ_0001776的表达与肺鳞癌患者的年龄、淋巴结转移、临床分期及肿瘤分期相关（均P＜0.05）。荧光原位杂交结果显示，hsa_circ_0001776主要表达于细胞质中。双荧光素酶报告基因实验结果显示，mir-1265与hsa_circ_0001776存在互补结合。hsa_circ_0001776过表达组NCI-H1703和NCI-H226细胞的吸光度值低于circ-NC组（P＜0.05）。hsa_circ_0001776过表达组的细胞克隆数分别为（52±3）和（53±4），迁移细胞数分别为（476±17）和（113±7），侵袭细胞数分别为（100±2）和（184±2），细胞迁移率分别为（25.00±4.36）%和（36.02±5.55）%，均低于circ-NC组[（104±4）和（106±2），（783±29）和（517±16），（657±45）和（473±9），（48.95±8.69）%和（48.70±1.57）%，均P＜0.05]。hsa_circ_0001776过表达组的凋亡率分别为（24.77±2.303）%和（19.67±1.16）%，均高于circ-NC组[（11.83±1.15）%和（9.50±0.66）%，均P＜0.05]。mir-1265模拟物组NCI-H1703和NCI-H226细胞的凋亡率高于miR-NC组（P＜0.05）。mir-1265模拟物组的细胞克隆数分别为（56±13）和（51±8），迁移细胞数分别为（556±13）和（405±6），侵袭细胞数分别为（486±6）和（359±7），细胞迁移率分别为（68.56±5.51）%、（81.74±8.04）%，均高于miR-NC组[（31±4）和（21±8），（154±19）和（186±5），（227±6）和（176±7），（25.83±4.26）%和（53.12±4.14）%，均P＜0.05]。mir-1265模拟物组的凋亡率分别为（11.83±2.55）%和（17.50±1.05）%，均低于miR-NC组[（32.67±4.44）%和（39.90±2.88）%，均P＜0.05]。hsa_circ_0001776 + mir-1265模拟物组NCI-H1703和NCI-H226细胞的吸光度值高于hsa_circ_0001776 + miR-NC组（P＜0.05）。hsa_circ_0001776 + mir-1265模拟物组的细胞克隆数分别为（128±15）和（133±8），迁移细胞数分别为（623±10）和（310±7），侵袭细胞数分别为（643±16）和（420±7），细胞迁移率分别为（66.39±4.46）%和（68.60±3.53）%，均高于hsa_circ_0001776 + miR-NC组[（86±7）和（80±16），（380±11）和（115±5），（152±7）和（94±4），（31.41±5.91）%和（30.94±0.67）%，均P＜0.05]。hsa_circ_0001776 + mir-1265模拟物组的凋亡率分别为（19.27±0.15）%和（11.53±0.75）%，均低于hsa_circ_0001776 + miR-NC组[（27.77±1.29）%和（18.43±0.71）%，均P＜0.05]。裸鼠皮下成瘤实验结果显示，hsa_circ_0001776过表达组的肿瘤体积低于circ-NC组（P＜0.05）。hsa_circ_0001776在肺鳞状细胞癌中表达下调，且hsa_circ_0001776可通过靶向mir-1265抑制肺鳞状细胞癌的发展。
