Abstract
目的
舌鳞状细胞癌(tongue squamous cell carcinoma，TSCC)是口腔颌面部的常见癌症，严重危害人们的生命健康。核内不均一核糖核蛋白A2/B1(heterogeneous nuclear ribonucleoprotein A2/B1，hnRNP A2/B1)是一种RNA结合蛋白，可调控多种基因的表达，参与多种癌症的发生和发展。本研究旨在探究hnRNP A2/B1对TSCC进展的表达及作用。
方法
基于基因表达综合(Gene Expression Omnibus，GEO)数据库中口腔鳞状细胞癌(oral squamous cell carcinoma，OSCC)相关芯片GSE146483、GSE85195分析hnRNP A2/B1在OSCC及正常口腔黏膜细胞、组织中的差异表达情况，基于TSCC相关芯片GSE4676分析hnRNP A2/B1表达与TSCC患者无病生存期的相关性。收集2021年7月至12月于湖南省肿瘤医院就诊的30例TSCC患者的癌及癌旁组织样本，采用real-time RT-PCR和蛋白质印迹法验证TSCC患者样本中hnRNP A2/B1 的mRNA和蛋白质表达情况。以人TSCC细胞Tca-8113为研究对象，分别转染hnRNP A2/B1空载敲减质粒(sh-NC组)、敲减质粒(sh-hnRNP A2/B1组)、空载过表达质粒(OE-NC组)和过表达质粒(OE-hnRNP A2/B1组)。采用蛋白质印迹法检测hnRNP A2/B1敲减或过表达效率，细胞计数试剂盒-8(cell counting kit-8，CCK-8)检测Tca-8113细胞增殖活性，流式细胞术检测Tca-8113细胞凋亡率。
结果
基于GEO数据库中的OSCC相关芯片GSE146483、GSE85195分析发现：hnRNP A2/B1在OSCC及正常口腔黏膜细胞、组织中均存在差异表达(均P<0.01)，同时对TSCC相关芯片GSE4676的分析证实hnRNP A2/B1表达与TSCC患者无病生存期呈负相关(P=0.006）。Real-time RT-PCR和蛋白质印迹法结果显示：与癌旁组织样本相比，TSCC组织样本中hnRNP A2/B1的mRNA和蛋白质相对表达水平均显著上调(均P<0.01)。蛋白质印迹法结果显示：Tca-8113细胞在转染敲减或过表达hnRNP A2/B1质粒后，细胞内的hnRNP A2/B1表达水平被显著抑制或促进(均P<0.01)。CCK-8及流式细胞术结果显示：抑制Tca-8113细胞中hnRNP A2/B1的表达可以降低细胞增殖活性(P<0.05)，增高细胞凋亡率(P<0.01)；促进Tca-8113细胞中hnRNP A2/B1的表达可以增加细胞增殖活性(P<0.05)，降低细胞凋亡率(P<0.01)。
结论
HnRNP A2/B1是调控TSCC细胞增殖和凋亡水平的关键因子，通过抑制hnRNP A2/B1的表达可以降低TSCC细胞的增殖活性，促进TSCC细胞的凋亡。
Keywords: 舌鳞状细胞癌, 核内不均一核糖核蛋白A2/B1, 增殖, 凋亡
Abstract
Objective
Tongue squamous cell carcinoma (TSCC) is a common cancer in the oral and maxillofacial region, which seriously endangers people’s life and health.Heterogeneous nuclear ribonucleoprotein A2/B1(hnRNP A2/B1) is an RNA-binding protein that regulates the expression of a variety of genes and participates in the occurrence and development of a variety of cancers. This study aims to investigate the role of hnRNP A2/B1 in TSCC progression.
Methods
The differential expression of hnRNP A2/B1 in oral squamous cell carcinoma (OSCC) and normal oral mucosa cells and tissues was analyzed based on the gene expression profiles of GSE146483 and GSE85195 in the Gene Expression Omnibus （GEO) database. The correlation between hnRNP A2/B1 expression and disease-free survival of TSCC patients was analyzed based on TSCC related chip of GSE4676. TSCC cancer and paracancerous tissue samples of 30 patients were collected in Hunan Cancer Hospital from July to December 2021. Real-time RT-PCR and Western blotting were used to verify the mRNA and protein expression of hnRNP A2/B1 in TSCC patients’samples, respectively. Human TSCC Tca-8113 cells were transfected with hnRNP A2/B1 empty vector (a sh-NC group), knockdown plasmid (a sh-hnRNP A2/B1 group), empty vector overexpression plasmid (an OE-NC group) and overexpression plasmid (an OE-hnRNP A2/B1 group), respectively. The knockdown or overexpression efficiency of hnRNP A2/B1 was detected by Western blotting. The proliferation activity of Tca-8113 cells was detected by cell counting kit-8 (CCK-8), and the apoptosis rate of Tca-8113 cells was detected by flow cytometry.
Results
Based on the analysis of OSCC-related chips of GSE146483 and GSE85195 in the GEO database, it was found that hnRNP A2/B1 was differentially expressed in the OSCC and normal oral mucosa cells and tissues (all P<0.01). Meanwhile, the analysis of TSCC related chip GSE4676 confirmed that the expression of hnRNP A2/B1 was negatively correlated with the disease-free survival of TSCC patients (P=0.006). The results of real-time RT-PCR and Western blotting showed that the relative expression levels of hnRNP A2/B1 mRNA and protein in TSCC tissues were significantly up-regulated compared with those in adjacent tissues (all P<0.01). The results of Western blotting showed that the expression level of hnRNP A2/B1 in Tca-8113 cells was significantly inhibited or promoted after knockdown or overexpression of hnRNP A2/B1 (all P<0.01). The results of CCK-8 and flow cytometry showed that inhibition of hnRNP A2/B1 expression in Tca-8113 cells reduced cell proliferation activity (P<0.05) and increased cell apoptic rate (P<0.01). Overexpression of hnRNP A2/B1 in Tca-8113 cells significantly increased cell proliferation (P<0.05) and decreased cell apoptosis (P<0.01).
Conclusion
HnRNP A2/B1 is a key factor regulating the proliferation and apoptosis of TSCC cells. Inhibition of hnRNP A2/B1 expression can reduce the proliferation activity of TSCC cells and promote the apoptosis of TSCC cells.
Keywords: tongue squamous cell carcinoma, heterogeneous nuclear ribonucleoprotein A2/B1, proliferation, apoptosis
舌鳞状细胞癌(tongue squamous cell carcinoma，TSCC)是口腔颌面部常见的癌症，其发病人数约占口腔癌总发病人数的1/3[1]。TSCC具有生长速度快、淋巴结易转移和恶性程度高的特点[2-3]，且目前尚无特异的生物标志物。TSCC的病死率较高，患者5年生存率仅有32%~54%[4-5]，严重危害人们的生命安全。
核内不均一核糖核蛋白A2/B1(heterogeneous nuclear ribonucleoprotein A2/B1，hnRNP A2/B1)是一种RNA结合蛋白，可作为N 6-甲基腺嘌呤(N 6-methyladenosine，m6A)甲基化修饰过程中的阅读器参与调控多种基因的表达[6]。已有研究证实，hnRNP A2/B1在多种癌症中的表达紊乱，并参与骨肉瘤[7]、前列腺癌[8]和大肠癌[9]等癌症的发生和发展。但hnRNP A2/B1与TSCC的关系尚未明确，因此，本研究旨在从细胞水平探究hnRNP A2/B1在TSCC的表达及作用，为TSCC的预防和治疗提供参考。
1. 材料与方法
1.1. 材料
TSCC的癌及癌旁组织样本由湖南省肿瘤医院提供，人TSCC细胞Tca-8113购自中国科学院典型培养物保藏委员会细胞库，RPMI 1640培养基、胎牛血清均购自武汉普诺赛生命科技有限公司，磷酸盐缓冲液、胰蛋白酶、TRIzol、Annexin V 细胞凋亡检测试剂盒、ECL显影液均购自赛默飞世尔科技(中国)有限公司，BeyoRTTM II cDNA合成试剂盒、BeyoFastTM SYBR Green qPCR Mix试剂盒、二喹啉甲酸(bicinchonininc acid，BCA)蛋白质检测试剂盒、细胞计数试剂盒-8(cell counting kit-8，CCK-8)均购自上海碧云天生物技术有限公司，PCR引物购自湖南擎科生物技术有限公司，放射免疫沉淀分析(radio-immunoprecipitation assay，RIPA)裂解液、hnRNP A2/B1、甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase，GAPDH)一抗和相应二抗均购自武汉三鹰生物技术有限公司，LipofectamineTM 2000转染试剂盒、hnRNP A2/B1敲减及过表达载体均购自上海吉玛制药技术有限公司。微量核酸仪(NanoDrop 2000)、PCR仪、电泳仪、转膜仪、酶标仪、流式细胞仪均为赛默飞世尔科技(中国)有限公司产品，细胞培养箱为宾德环境试验设备(上海)有限公司产品，成像仪为美国Alpha公司产品。
1.2. 方法
1.2.1. 生物信息分析
基于基因表达综合(Gene Expression Omnibus，GEO)数据库中口腔鳞状细胞癌(oral squamous cell carcinoma，OSCC)相关芯片GSE146483(OSCC细胞与人类正常口腔黏膜上皮细胞全基因组表达谱)及GSE85195(OSCC组织及正常组织的RNA表达谱)分析hnRNP A2/B1在OSCC及正常口腔黏膜细胞、组织中的差异表达情况，基于TSCC组织相关芯片GSE4676分析hnRNP A2/B1表达与TSCC患者无病生存期的相关性。从GPL17077获取GSE146483注释信息，GPL6480获取GSE85195注释信息，GPL2891获取GSE4676的注释信息，获得差异倍数变化(fold change，FC)上调(logFC>1，P<0.05)和下调(logFC<-1，P<0.05)的基因。
1.2.2. 临床组织标本的收集
收集2021年7月至12月于湖南省肿瘤医院就诊的30例TSCC患者的癌及癌旁组织样本，其中男性患者21例，女性患者9例；年龄≤60岁患者11例，>60岁患者19例；处于TNM分期I、Ⅱ期的样本共20例，Ⅲ、Ⅳ期的样本10例；病理分化程度检测显示高分化样本17例，中、低分化样本13例。癌旁组织样本取距癌组织>2 cm处样本。所有入组患者均未患有其他肿瘤、未经过放射治疗(以下简称“放疗”)及化学治疗(以下简称“化疗”)，且具有完整临床病理资料。本研究已通过湖南省肿瘤医院伦理委员会批准(审批号：KYJJ-2021-096)。
1.2.3. 细胞培养
人TSCC细胞Tca-8113采用含有10%胎牛血清的RPMI 1640培养基，置于37 ℃，5% CO2，97%湿度的培养箱中培养。待细胞生长融合度约达80%时，用磷酸盐缓冲液清洗、胰蛋白酶消化后，进行传代培养。
1.2.4. Real-time RT-PCR
用TRIzol提取总RNA，微量核酸仪检测提取出RNA的纯度和浓度，BeyoRTTM II cDNA合成试剂盒将RNA反转录为cDNA，并以cDNA为模板用BeyoFastTM SYBR Green qPCR Mix试剂盒进行PCR扩增，hnRNP A2/B1正向引物为5'-ATTGATGGGAGA-GTAGTTGAGCC-3'，反向引物为5'-ATTCCGCCAA-CAAACAGCTT-3'；GAPDH正向引物为5'-ACAGC-CTCAAGATCATCAGC-3'，反向引物为5'-GGTCAT-GAGTCCTTCCACGAT-3'。反应条件：首先95 ℃ 2 min；然后行40个循环，每个循环由95 ℃下15 s，60 ℃下30 s，72 ℃下30 s组成；最后72 ℃ 5 min。以GAPDH为内参基因，采用2-ΔΔCt计算hnRNP A2/B1的相对表达水平。
1.2.5. 蛋白质印迹法
取剪碎的组织样本或待检测的细胞，加入RIPA裂解液，提取组织或细胞中的总蛋白质，采用BCA法测定蛋白质的浓度；取适量蛋白质溶液，置于100 ℃煮沸5 min，对蛋白质进行变性处理，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，随后转膜，在室温下用5%脱脂乳封闭2 h后，分别加入hnRNP A2/B1 (1꞉500)和GAPDH(1꞉500)一抗，在4 ℃下孵育过夜，用TBST洗涤3次，然后用二抗(1꞉3 000)在37 ℃下孵育1 h，洗膜后加入ECL显影液，避光显影后用Alpha View系统对蛋白质条带进行成像，并用Image J软件进行定量分析。
1.2.6. 细胞转染
取生长状态良好且处于对数生长期的Tca-8113细胞，以每孔1×105个的密度接种于6孔板中；当细胞生长融合度约达60 %时，分别转染空载敲减质粒(sh-NC组)、hnRNP A2/B1敲减质粒(sh-hnRNP A2/B1组)、空载过表达质粒(OE-NC组)和hnRNP A2/B1过表达质粒(OE-hnRNP A2/B1组)。具体转染过程参照LipofectamineTM 2000转染试剂盒说明书。转染24 h后更换培养基，继续培养48 h。以不进行任何处理、正常培养的Tca-8113细胞为对照组，进行后续实验。
1.2.7. 细胞活性检测
取生长状态良好且处于对数生长期的Tca-8113细胞，以每孔4×103个的密度接种至96孔板中，置于细胞培养箱中分别培养12、24、48和72 h；培养结束后每孔加入新鲜配制的10 μL CCK-8溶液，置于培养箱中继续培养4 h，然后用酶标仪检测波长为 450 nm处的吸光度值。
1.2.8. 细胞凋亡检测
采用膜联蛋白V(Annexin V)-异硫氰酸荧光素(fluorescein isothiocyanate，FITC)/碘化丙啶(propidium iodide，PI)双染法检测转染后Tca-8113细胞的凋亡情况。以每孔1×105个细胞的密度将细胞接种于6孔板中，当细胞生长至融合度达80%时，改用无血清培养基培养细胞24 h，按照前述方法分组转染，转染后48 h，用胰蛋白酶消化细胞，制备成细胞悬液，离心后重悬细胞，加入Annexin V-FITC和PI各5 μL混匀，避光。
1.3. 统计学处理
采用GraphPad Prism 8软件对实验数据进行统计分析；数据以均数±标准差( ±s)表示；2组间比较采用独立样本t检验，多组间比较采用单因素方差分析。P<0.05表示差异有统计学意义。
2. 结 果
2.1. HnRNP A2/B1在TSCC样本中高表达
基于GEO数据库中的OSCC相关芯片GSE146483、GSE85195分析发现，hnRNP A2/B1在OSCC及正常口腔黏膜细胞、组织中的存在差异表达(P<0.01，图1A、1B)，同时对TSCC相关芯片GSE4676的分析证实hnRNP A2/B1表达水平与TSCC患者无病生存期呈负相关(P=0.006，图1C)。 Real-time RT-PCR和蛋白质印迹法结果显示：与癌旁组织样本相比，TSCC组织样本中hnRNP A2/B1的mRNA和蛋白质相对表达水平均显著上调(均P<0.01，图1D、1E)。
图1.
HnRNP A2/B1在TSCC样本中高表达
Figure 1 HnRNP A2/B1 is highly expressed in TSCC samples
A-C: Microarray-based analysis of hnRNP A2/B1 expression levels in OSCC cells (A) and clinical tissues (B) samples, and the correlation between hnRNP A2/B1 expression levels and disease-free survival in TSCC (C); D and E: Real-time RT-PCR and Western blotting detection of hnRNP A2/B1 mRNA (D) and protein (E) relative expression levels in clinical TSCC tissue samples and paracancerous tissue samples (n=30). **P<0.01. GSE146483: OSCC cells related chips in GEO database; GSE85195: OSCC tissues related chips in GEO database; GSE4676: TSCC tissues related chips in GEO database; GEO: Gene Expression Omnibus; HnRNP A2/B1: Heterogeneous nuclear ribonucleoprotein A2/B1; HNOMEK: Human normal oral mucosal epithelial keratinocytes; OSCC: Oral squamous cell carcinoma; PT: Paracancerous tissue; TSCC: Tongue squamous cell carcinoma; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.
2.2. HnRNP A2/B1低表达抑制TSCC细胞增殖，促进TSCC细胞凋亡
蛋白质印迹法结果显示：sh-hnRNP A2/B1组细胞内的hnRNP A2/B1表达水平显著低于sh-NC组(P<0.01，图2A)。CCK-8结果显示：sh-hnRNP A2/B1组Tca-8113细胞的增殖活性明显低于sh-NC组(P<0.05，图2B)。流式细胞术结果显示：sh-hnRNP A2/B1组Tca-8113细胞的凋亡率显著高于sh-NC组(P<0.01，图2C)。
图2.
HnRNP A2/B1 低表达抑制TSCC细胞增殖，促进TSCC细胞凋亡
Figure 2 Low expression of hnRNP A2/B1 inhibited TSCC cell proliferation and promoted TSCC cell apoptosis
A: Interference efficiency of hnRNP A2/B1 was detected by western bloting. B: Effect of knockdown of hnRNP A2/B1 on the proliferation activity of Tca-8113 cells was detected by CCK-8. C and D: Effect of low expression of hnRNP A2/B1 on the apoptic of Tca-8113 cells was detected by flow cytometry (D), and the apoptosis rate was calculated (C). *P<0.05, **P<0.01. HnRNP A2/B1: Heterogeneous nuclear ribonucleoprotein A2/B1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; NC: Negative control; TSCC: Tongue squamous cell carcinoma; PI: Propidium iodide; FITC: Fluorescein isothiocyanate; H: Height.
2.3. HnRNP A2/B1过表达促进TSCC细胞增殖，抑制TSCC细胞凋亡
蛋白质印迹法结果显示：OE-hnRNP A2/B1组细胞内的hnRNP A2/B1表达水平显著高于OE-NC组(P<0.01，图3A)。CCK-8结果显示：OE-hnRNP A2/B1组Tca-8113细胞的增殖活性明显高于OE-NC组(P<0.05，图3B)。流式细胞术结果显示：OE-hnRNP A2/B1组Tca-8113细胞的凋亡率显著低于OE-NC组(P<0.01，图3C)。
图3.
HnRNP A2/B1过表达促进TSCC细胞增殖，抑制TSCC细胞凋亡
Figure 3 Overexpression of hnRNP A2/B1 promotes TSCC cell proliferation and inhibits TSCC cell apoptosis
A: Transfection efficiency of hnRNP A2/B1 overexpression vector was detected by Western bloting. B: Effect of hnRNP A2/B1 overexpression on the proliferation activity of Tca-8113 cells was detected by CCK-8. C and D: Effect of hnRNP A2/B1 overexpression on the apoptosis of Tca-8113 cells was detected by flow cytometry (D), and the apoptic rate was calculated (C). *P<0.05, **P<0.01. HnRNP A2/B1: Heterogeneous nuclear ribonucleoprotein A2/B1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; OE: Overexpression; NC: Negative control; TSCC: Tongue squamous cell carcinoma; PI: Propidium iodide; FITC: Fluorescein isothiocyanate; H: Height.
3. 讨 论
TSCC是口腔颌面部发病率和病死率最高的癌症，近年来，其发病年龄向年轻化趋势发展。TSCC可导致患者说话、发音、咀嚼及吞咽障碍，对患者的日常生活质量和甚至生命安全造成严重影响。目前，临床上TSCC通常采用手术治疗为主，放化疗和靶向治疗为辅的综合治疗[10-11]，但是术后创伤、放化疗的不良反应会导致患者生活质量明显下降[12-14]。寻找TSCC早期生物标志物，可以为TSCC的早期筛查和治疗提供新方向。
HnRNP A2/B1是一种RNA结合蛋白，作为m6A修饰的调节因子参与多种癌症的发生与发展[6]。HnRNP A2/B1与肝细胞癌转移存在相关性，促进其降解可以有效抑制肝细胞癌转移[10]；hnRNP A2/B1的高表达和骨肉瘤较差的预后存在相关性，可作为骨肉瘤的新治疗靶点和预后生物标志物[8]；hnRNP A2/B1可以通过m6A修饰，上调腺苷三磷酸柠檬酸裂解酶和乙酰辅酶a羧化酶1表达，促进食管癌进展[15]。在本研究中，通过对TSCC芯片进行分析发现hnRNP A2/B1在TSCC样本中的表达出现异常上调。因此推测，hnRNP A2/B1可能在TSCC进展中发挥重要作用。
HnRNP A2/B1参与调控包括癌细胞增殖和凋亡在内的多种癌症进展关键过程。在胰腺导管腺癌细胞中，hnRNP A2/B1可通过与鼠类肉瘤病毒癌基因(Kirsten rat sarcoma viral oncogene homolog，KRAS)相互作用调节其活性，进而影响KRAS依赖性胰腺导管腺癌细胞的增殖、迁移和凋亡[16]。HnRNP A2/B1通过与E1A结合蛋白P300协同作用增强环氧化酶-2的表达，并通过影响上皮细胞钙黏素的表达调节上皮-间充质转化，从而促进肺癌细胞的生长[17-18]。本研究发现：抑制TSCC细胞中hnRNP A2/B1表达可以降低TSCC细胞的增殖活性，促进其凋亡；而促进hnRNP A2/B1的表达则会增加TSCC细胞的增殖活性，抑制其凋亡。这些结果表明hnRNP A2/B1参与调控TSCC细胞的增殖和凋亡。
综上所述，hnRNP A2/B1是决定TSCC细胞增殖和凋亡水平的关键因子，在TSCC发生和发展过程中发挥作用。抑制hnRNP A2/B1的表达可以降低TSCC细胞的增殖活性，促进TSCC细胞的凋亡。明确hnRNP A2/B1在TSCC中的功能作用，对TSCC的预防和治疗具有重要意义。
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《中南大学学报(医学版)》原名《湖南医科大学学报》，创刊于1958年，为教育部主管、中南大学主办的医药卫生类综合性学术期刊。本刊已被美国医学文献分析和联机检索系统(MEDLINE, PubMed)、荷兰《医学文摘》(EM)、Scopus、WHO西太平洋地区医学索引(WPRIM)、中国科学引文数据库(核心库) (CSCD)，《WJCI报告》等国内外多家重要数据库或权威文摘期刊收录；是中国科技论文统计源期刊、中文核心期刊及中国期刊方阵“双效”期刊；为中国高校百佳科技期刊、中国精品科技期刊、RCCSE中国权威学术期刊(A+)和湖南省十佳科技期刊。
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基金资助
湖南省自然科学基金(2020JJ5333)；湖南省卫健委头颈外科临床重点专科建设项目(湘卫财务发〔2019〕4号)；长沙市自然科学基金(kq2208149)。
This work was supported by the Natural Science Foundation of Hunan Province (2020JJ5333), the Key Clinical Specialty Construction Project (Head & Neck Surgery) of Health Commission of Hunan Province (Xiangwei Finance〔2019〕NO.4), and the Natural Science Foundation of Changsha (kq2208149), China.
利益冲突声明
作者声称无任何利益冲突。
作者贡献
刘彦 研究选题和设计，实验操作，论文撰写。谌星 研究选题和设计，数据采集及统计学分析，论文修改。所有作者阅读并同意最终的文本。
原文网址
http://xbyxb.csu.edu.cn/xbwk/fileup/PDF/202305633.pdf
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