摘要
目的探讨环状RNA(circular RNA,circRNA)BICD2(circ-BICD2)对口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)谷氨酰胺代谢、细胞增殖、迁移侵袭、凋亡的影响和可能机制。方法纳入2016年1月至2018年1月就诊于郑州大学第一附属医院口腔科,手术切除的35例OSCC患者的肿瘤组织及其癌旁正常组织样本。通过实时定量PCR(real-time quantitative PCR,RT-qPCR)和蛋白质印迹法检测OSCC组织与癌旁组织、OSCC细胞(CAL27、SCC9、SCC15)与正常上皮细胞HIOEC细胞中circ-BICD2、miR-296-5p和转录球蛋白2(transgelin2,TAGLN2)的表达水平。生物信息学方法、双荧光素酶报告实验、RNA结合免疫沉淀反应(RNA binding protein immunoprecipitation,RIP)实验检测circ-BICD2与miR-296-5p、miR-296-5p与TAGLN2的靶向关系。在CAL27和SCC9细胞中分别转染circ-BICD2小干扰RNAsi-circ-BICD2#1(敲低1组)、si-circ-BICD2#2(敲低2组)及阴性对照si-NC(siRNA空白组);在敲低1组细胞中分别转染miR-296-5p抑制物anti-miR-296-5p(抑制物组)及其阴性对照anti-miR-NC(抑制物对照组)。在CAL27和SCC9细胞中转染miR-296-5p模拟物(miR过表达组)及其阴性对照miR-NC(miR空白组)。在miR过表达组细胞中转染TAGLN2(共转染组)及空白质粒pcDNA(共转染对照组)。运用细胞计数试剂盒(cell counting kit 8,CCK-8)、集落形成实验、流式细胞术、划痕愈合实验、Transwell实验等检测OSCC生物学行为。同时检测谷氨酰胺(glutamine,gln)消耗量、α-酮戊二酸(α-ketoglutarate,α-KG)产生量和ATP浓度观察其对细胞代谢的影响。蛋白质印迹法检测各组CAL27和SCC9细胞中的细胞周期素D1(cyclinD1)和谷氨酰胺水解酶(glutamine hydrolase,GLS1)蛋白表达量。取4周龄清洁级BALB/c雌性裸鼠12只,于腋下皮肤分别注射转染sh-circ-BICD2(敲低组)和sh-NC(对照组)后的SCC9细胞单细胞悬液,建立移植瘤模型,每组6只。注射32 d时断颈处死裸鼠并切除肿瘤,检测肿瘤质量及两组的circ-BICD2、miR-296-5p及TAGLN2的表达水平。结果OSCC组织中circ-BICD2、TAGLN2表达量(分别为2.54±0.71和1.86±0.15)显著高于正常组织(分别为1.00±0.35和1.00±0.11),miR-296-5p表达量(0.56±0.23)显著低于正常组织(1.00±0.32)(P<0.05)。敲低1组CAL27和SCC9细胞活力、克隆形成数、迁移率、侵袭细胞数、S期细胞比例、gln消耗量、α-KG产生量、ATP浓度、cyclinD1和GLS1蛋白表达均显著低于siRNA空白组,细胞凋亡率、G0/G1期细胞比例显著高于siRNA空白组(P<0.05)。与抑制物对照组相比,抑制物组CAL27和SCC9细胞miR-296-5p表达显著降低,细胞活力、克隆形成数、迁移率、侵袭细胞数、S期细胞比例、gln消耗量、α-KG产生量、ATP浓度、cyclinD1和GLS1蛋白表达均显著升高,细胞凋亡率、G0-G1期细胞比例显著降低(P<0.05)。与miR空白组相比,miR过表达组CAL27和SCC9细胞TAGLN2 mRNA、蛋白表达量均显著降低。与共转染对照组比较,共转染组CAL27和SCC9细胞TAGLN2 mRNA、蛋白表达量均显著升高(P<0.05)。动物实验结果显示,与对照组相比,敲低组裸鼠的肿瘤体积、质量、circ-BICD2及TAGLN2的表达水平均显著降低,miR-296-5p表达水平显著升高(P<0.05)。结论OSCC组织和细胞中circ-BICD2均为高表达,干扰circ-BICD2可抑制OSCC细胞增殖、迁移侵袭、谷氨酰胺代谢以及肿瘤生长,促进细胞凋亡,其机制与调控miR-296-5p/TAGLN2分子轴有关。
Objective To investigate the effects of circular RNA(circRNA)BICD2(circ-BICD2)on glutamine metabolism,cell proliferation,migration,invasion and apoptosis of oral squamous cell carcinoma(OSCC)cells and to further explore the possible mechanism.Methods OSCC cells were purchased from Shanghai Institute of Cellular Cells,Chinese Academy of Sciences.Samples of OSCC tissues and corresponding adjacent tissues were collected from 35 OSCC patients who underwent surgical resection at the First Affiliated Hospital of Zhengzhou University from January 2016 to January 2018.Real-time quantitative PCR(RT-qPCR)and western blot were performed to detect the expression levels of circ-BICD2,miR-296-5p and transgelin2(TAGLN2)in OSCC tissues and cells.Bioinformatics,dual luciferase report experiment and RNA immunoprecipitation(RIP)experiment were used to determine the targeting relationship between circ-BICD2 and miR-296-5p,miR-296-5p and TAGLN2.According to different transfection oligonucleotides or plasmids,CAL27 and SCC9 cells were divided into si-NC group(transfection si-NC),si-circ-BICD2 group(transfection si-circ-BICD2),si-circ-BICD2+anti-miR-NC group(transfected with si-circ-BICD2 and anti-miR-NC),si-circ-BICD2+anti-miR-296-5p group(transfected with si-circ-BICD2 and anti-miR-296-5p),miR-NC group(transfected with miR-NC),miR-296-5p group(transfected with miR-296-5p mimic),miR-296-5p+pcDNA(transfected with miR-296-5p mimic and pcDNA)and miR-296-5p+TAGLN2 group(transfected with miR-296-5p mimic and pcDNA-TAGLN2).Cell counting kit 8(CCK-8),colony formation experiment,flow cytometry,Scratch healing test and Transwell experiment were applied to detect OSCC cell viability,number of colonies,cycle distribution,apoptosis rate,migration rate and invasive cell numbers.Glutamine(gln)consumption,α-ketoglutaric acid(α-KG)production and adenosine triphosphate(ATP)concentration were also detected by the kit.The expression of cyclinD1 and Glutamine hydrolase(GLS1)proteins of CAL27 and SCC9 cells in each of the groups were detected by western blot.Twelve four-week-old clean BALB/c female nude mice were injected with a single-cell suspension of SCC9 cells into the axillary skin to establish a transplanted tumor model.Twelve transplanted tumor model mice were divided into sh-circ-BICD2 group and sh-NC group.Mice were sacrificed by cervical dislocation at 32 days after injection,the tumors were removed and the tumor weight was tested.Results The expressions of circ-BICD2(2.54±0.74)and TAGLN2(1.86±0.15)were increased(P<0.05),while the expression of MiR-296-5p was decreased in OSCC tissues(P<0.05).Cell viability,clone formation numbers,migration rate,invasive cell numbers,S phase cell ratio,glu consumption,α-KG production,ATP concentration,expression of cyclinD1 and GLS1 proteins of OSCC cells were significantly reduced after interference with circ-BICD2 expression.The apoptosis rate,the proportion of cells in G0-G1 phase and the expression of miR-296-5p were significantly increased(P<0.05).Inhibiting the expression of miR-296-5p coulld reverse the effect of interfering with circ-BICD2 on OSCC cell proliferation,migration,invasion,apoptosis and glutamine metabolism(P<0.05).After overexpression of miR-296-5p,cell viability,clone formation red number,migration rate,number of invasive cells,S phase cell ratio,glu consumption,α-KG production,ATP concentration and expressions of cyclinD1,GLS1 and TAGLN2 proteins in OSCC cells were significant decrease.The rate of apoptosis and the proportion of cells in G0-G1 phase were significantly increased(P<0.05).Compared with overexpression of miR-296-5p,cell viability,clone formation red number,migration rate,number of invasive cells,S phase cell ratio,glu consumption,α-KG production,ATP concentration,cyclinD1,GLS1 and TAGLN2 proteins in OSCC cells after overexpression of miR-296-5p and TAGLN2 were significantly increased,and the apoptosis rate and the proportion of cells in the G0-G1 phase were significantly decreased(P<0.05).Compared with the sh-NC group,the tumor weights of mice in the sh-circ-BICD2 group were significantly reduced(P<0.05).Conclusions Circ-BICD2 was highly expressed in OSCC cells.Interfering with circ-BICD2 could inhibit the proliferation,migration and invasion of OSCC cells,glutamine metabolism and tumor growth and promote cell apoptosis,which might be relate to the regulation of miR-296-5p/TAGLN2 molecular axis.
作者
张艳靖
朱青青
周海霞
王海业
周弘
Zhang Yanjing;Zhu Qingqing;Zhou Haixia;Wang Haiye;Zhou Hong(Department of Oral and Maxillofacial Surgery,The First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)
出处
《中华口腔医学杂志》
CAS
CSCD
北大核心
2021年第11期1098-1108,共11页
Chinese Journal of Stomatology
