口腔鳞癌中NKD1、NKD2基因mRNA表达及甲基化的研究
发布时间：2018-08-28 07:12
【摘要】：目的:口腔鳞状细胞癌(Oral Squamous Cell Carcinoma,OSCC)是口腔颌面部最常见恶性肿瘤之一,占全球恶性肿瘤的3%。据统计,我国每年口腔癌的新发病例约4.65万,发病年龄大多在40-60岁之间,男性相对女性较多。随着医疗水平的逐渐提高,以手术切除为主的综合序列治疗方法使口腔癌的预后有了明显改善,但患者的5年生存率仍停留在55%-60%。众所周知,OSCC的预后取决于临床分期,组织学分级,淋巴结转移等因素,但大多数患者就诊时已属晚期,因此口腔癌的早期诊断,早期治疗十分重要。研究者报道除基因变异、缺失等遗传因素外,DNA甲基化等表观遗传学的改变是肿瘤的发生的早期事件。临床应用表观遗传学标志物能够对口腔鳞癌做到早期诊断,对预后准确预测,进而改善患者的生存质量,提高患者的生存率。人源NKD基因家族包括NKD1(Naked cuticle homolog 1)和NKD2(Naked cuticle homolog 2)基因,NKDl、NKD2基因分别定位于染色体16q12.1及5p15.3上,研究发现NKD1、NKD2基因相关定位位点在肝癌、胃癌、乳腺癌等多种癌症中频繁的出现杂合性缺失(Loss of heterozygosity,LOH),包括乳腺癌、骨肉瘤在内的多种恶性肿瘤中均存在NKD1、NKD2基因m RNA表达水平异常及甲基化状态的改变。但是在口腔鳞癌的发生发展中,NKD1、NKD2基因的表达及甲基化状态尚无文献报道。因此本实验通过采用RT-PCR和MSP方法检测NKD1、NKD2基因的表达水平及甲基化状态,并分析其与OSCC各项临床病理特征之间的相关性,探讨NKD1、NKD2基因在OSCC的发生发展中的作用,为口腔鳞癌的早期诊断,早期治疗提供理论依据。方法:1标本均来源于2015年9月至2016年11月期间河北医科大学第四医院口腔科手术治疗的52例OSCC患者,以术中切除的OSCC组织作为实验组,以相对应癌旁2cm正常黏膜组织为对照组,实验组及对照组组织学诊断均经术后病理验证,标本于-80℃生物标本库中冻存。2采用反转录聚合酶链反应(RT-PCR,Reverse Transcription Polymerase Chain Reaction)检测52例OSCC及相对应正常组织内NKD1、NKD2基因m RNA表达量。3应用甲基化特异性聚合酶链反应(MSP,Methylation Specific Polymerase Chain Reaction)检测52例OSCC及相对应正常组织内NKD1、NKD2基因启动子区Cp G岛甲基化状态。4应用SPSS21.0软件进行统计学分析,基因m RNA表达差异比较应用配对t检验,成组t或近似t检验;两基因m RNA表达相关性应用直线相关分析;基因甲基化的比较应用Fisher确切概率法或四格表χ2检验;两基因间甲基化相关性应用Spearman等级秩和相关分析。当P0.05证明差异存在统计学意义。结果:1 NKD1基因m RNA相对表达量在OSCC组织中为0.50±0.12,低于其对应正常口腔黏膜组织0.66±0.18,二者差别有统计学意义(t=19.934,P0.001)。临床Ⅰ期+Ⅱ期OSCC组织中NKD1基因m RNA相对表达量是0.53±0.12,高于临床Ⅲ期+Ⅳ期0.44±0.10(t=-3.041,P=0.004);有淋巴结转移组NKD1 m RNA的相对表达量0.44±0.10,低于无淋巴结转移组0.52±0.12(t=2.469,P=0.017);NKD1基因m RNA的相对表达量与年龄、性别和吸烟与否无相关性(P0.05)。2 NKD2基因m RNA的相对表达量在OSCC组织中为0.42±0.10,低于其对应正常口腔黏膜0.62±0.12,二者差异有统计学意义(t=10.915,P0.001)。临床Ⅰ期+Ⅱ期OSCC组织中NKD2基因m RNA相对表达量为0.44±0.10,高于临床Ⅲ期+Ⅳ期中0.37±0.05(t=-3.739,P0.001);有淋巴结转移组0.37±0.07低于无淋巴结转移组0.45±0.10(t=2.873,P=0.006);吸烟组0.38±0.09低于不吸烟组0.45±0.08(t=-3.395,P=0.001);NKD2基因m RNA的相对表达量与性别及年龄无相关性(P0.05)。3 NKD1基因甲基化阳性表达率在OSCC组织中48.08%(25/52)高于相对应正常口腔黏膜组织26.92%(14/52),二者差异有统计学意义(χ2=4.964,P=0.026)。临床Ⅰ期+Ⅱ期OSCC组织中NKD1基因甲基化率为35.48%(11/31),低于Ⅲ期+Ⅳ期66.67(14/21)(χ2=4.877,P=0.027);NKD1甲基化率在有淋巴结转移组中70.59%(12/17)高于无淋巴结转移组37.14%(13/35)(χ2=5.127,P=0.024);NKD1基因甲基化率与年龄、性别及吸烟无关(P0.05)。4 NKD2基因甲基化阳性表达率在OSCC中44.23%(23/52)高于相应的正常口腔黏膜组织21.15%(11/52),两者差别有统计学意义(χ2=6.292,P=0.012)。临床Ⅰ期+Ⅱ期OSCC组织中NKD2基因甲基化率为29.03%(9/31),低于Ⅲ期+Ⅳ期66.67%(14/21)(χ2=7.188,P=0.007);有淋巴结转移组64.71%(11/17)高于无淋巴结转移组34.29%(12/35)(χ2=4.293,P=0.038);吸烟组57.14%(16/28)高于不吸烟组29.17%(7/24)(χ2=4.1,P=0.043)。NKD2甲基化阳性表达率与性别和年龄无关(P0.05)。5在OSCC中,NKD1甲基化组m RNA相对表达量0.44±0.11低于非甲基化组m RNA表达量0.54±0.11(t=-3.344,P=0.002)。NKD2基因甲基化组m RNA相对表达量0.36±0.06低于非甲基化组m RNA的相对量0.47±0.09(t=-4.934,P0.001)。6在OSCC中,NKD1、NKD2两基因均发生甲基化者共16例,均未发生甲基化者共20例,两基因甲基化状态在OSCC组织中呈正相关关系(r=0.038,P=0.005);NKD1、NKD2两基因m RNA相对表达量在OSCC组织中呈正相关关系(r=0.312,P=0.024)。结论:1 NKD1、NKD2基因m RNA相对表达下降或沉默可能参与OSCC的发生与发展。其中,NKD1基因异常表达与临床分期及淋巴结转移相关。NKD2基因异常表达与临床分期、淋巴结转移及吸烟有关。2 NKD1、NKD2基因启动子区Cp G岛高甲基化状态可能是OSCC发生的主要机制之一。其中,NKD1基因异常甲基化状态与临床分期及淋巴结转移有关。NKD2基因异常甲基化状态与临床分期、淋巴结转移及吸烟有关。3在OSCC中NKD1、NKD2基因高甲基化状态可能是致其m RNA表达下降或沉默的主要原因之一。4 NKD1与NKD2在OSCC的发生发展中可能存在协同作用。
[Abstract]:Objective: Oral Squamous Cell Carcinoma (OSCC) is one of the most common malignant tumors in the oral and maxillofacial region, accounting for 3% of the global malignant tumors. It is well known that the prognosis of OSCC depends on clinical stage, histological grading, lymph node metastasis and other factors, but most patients are in advanced stage. Therefore, early diagnosis and early treatment of oral cancer are ten. Researchers report that in addition to genetic factors such as gene mutation and deletion, epigenetic changes such as DNA methylation are early events in tumorigenesis. Clinical application of epigenetic markers can make early diagnosis of oral squamous cell carcinoma, predict prognosis accurately, improve the quality of life of patients, and improve the survival rate of patients. The NKD gene family includes NKD1 (Naked cuticle homolog 1) and NKD2 (Naked cuticle homolog 2) genes. NKDl and NKD2 genes are located on chromosomes 16q12.1 and 5p15.3, respectively. Studies have found that NKD1 and NKD2 related loci frequently occur loss of heterozygosity (LOH) in liver cancer, gastric cancer, breast cancer and other cancers. The expression of NKD1 and NKD2 m RNA was abnormal and methylation status was changed in many malignant tumors including breast cancer and osteosarcoma. However, the expression and methylation status of NKD1 and NKD2 genes were not reported in oral squamous cell carcinoma. To explore the role of NKD1 and NKD2 genes in the development of OSCC, and to provide theoretical basis for early diagnosis and treatment of oral squamous cell carcinoma. Methods: 1 Specimens were collected from the 4th Hospital of Hebei Medical University from September 2015 to November 2016. Fifty-two patients with OSCC who underwent surgical treatment in stomatology department were divided into experimental group and control group. The tissues of OSCC resected during operation were taken as control group and 2 cm normal mucosa adjacent to cancer as control group. Histological diagnosis of the experimental group and control group were confirmed by pathology after operation. The specimens were frozen in the biology specimen bank at - 80 C. 2 The specimens were frozen in reverse transcription polymerase chain reaction (RT-PCR, Reverse Transcrip). Methylation-specific polymerase chain reaction (MSP) was used to detect the methylation status of NKD1 and NKD2 gene promoter Cp G islands in 52 OSCC and corresponding normal tissues. SS21.0 software for statistical analysis, comparison of gene m RNA expression differences using paired t test, group t or approximate t test; two gene m RNA expression correlation using linear correlation analysis; comparison of gene methylation using Fisher exact probability method or quadruple table_2 test; methylation correlation between the two genes using Spearman rank and correlation score Results: The relative expression of NKD1 m RNA in OSCC tissues was 0.50+0.12, which was lower than 0.66+0.18 in corresponding normal oral mucosa tissues. The difference was statistically significant (t=19.934, P 0.001). The relative expression of NKD1 m RNA in clinical stage I+II OSCC tissues was 0.53+0.12, which was higher than that in clinical stage I+II OSCC tissues. The relative expression of NKD1 m RNA in patients with lymph node metastasis was 0.44 (- 3.041, P = 0.004), 0.44 (- 0.10) lower than that in patients without lymph node metastasis (0.52 (- 0.12) (t = 2.469, P = 0.017), and the relative expression of NKD1 m RNA was not correlated with age, sex and smoking (P 0.05). The relative expression of NKD2 gene m RNA in clinical stage I + II OSCC tissues was 0.44 + 0.10, higher than that in clinical stage III + IV stage 0.37 + 0.05 (t = - 3.739, P 0.001), and 0.37 + 0.07 in lymph node metastasis group was lower than that in non-lymph node metastasis group (0.45 + 0.001). 10 (t = 2.873, P = 0.006); 0.38 (+ 0.09) in smoking group was lower than 0.45 (+ 3.395, P = 0.001) in non-smoking group; the relative expression of NKD2 gene m RNA was not correlated with sex and age (P 0.05). 3 NKD1 gene methylation positive expression rate in OSCC tissues was 48.08% (25/52) higher than that in normal oral mucosa tissues (26.92% (14/52). The methylation rate of NKD1 gene was 35.48% (11/31), lower than 66.67 (14/21) in stage III + IV (2 = 4.877, P = 0.027), and 70.59% (12/17) in patients with lymph node metastasis was higher than 37.14% (13/35) in patients without lymph node metastasis (2 = 5.127, P = 0.024). The positive expression rate of NKD2 gene methylation in OSCC was 44.23% (23/52) higher than that in corresponding normal oral mucosa (21.15% (11/52). The difference was statistically significant (2 = 6.292, P = 0.012). The methylation rate of NKD2 gene in stage I + II OSCC was 29.03% (9/31), which was lower than that in stage III + IV (66.67% (14/21) (2 = 7.012). 188, P = 0.007; 64.71% (11/17) in lymph node metastasis group was higher than 34.29% (12/35) in non-lymph node metastasis group (2 = 4.293, P = 0.038); 57.14% (16/28) in smoking group was higher than 29.17% (7/24) in non-smoking group (2 = 4.1, P = 0.043). The positive expression rate of NKD2 methylation was not related to sex and age (P In non-methylation group, the expression of M RNA was 0.54 (-3.344, P = 0.002). In methylation group, the relative expression of M RNA was 0.36 (-0.06) lower than that in non-methylation group, the relative expression of M RNA was 0.47 (-4.934, P 0.001). In OSCC, the methylation of NKD1 and NKD2 genes occurred in 16 cases, and no methylation occurred in 20 cases. There was a positive correlation (r = 0.038, P = 0.005) in OSCC tissues, and a positive correlation (r = 0.312, P = 0.024) between the relative expression of NKD1 and NKD2 m RNA in OSCC tissues. The abnormal expression of NKD2 gene is related to clinical stage, lymph node metastasis and smoking. 2 NKD1, hypermethylation of Cp G island in the promoter region of NKD2 gene may be one of the main mechanisms of OSCC. Metastasis and smoking are related. 3 The hypermethylation of NKD1 and NKD2 may be one of the main reasons for the decrease or silence of M RNA expression in OSCC. 4 NKD1 and NKD2 may play a synergistic role in the occurrence and development of OSCC.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.8
本文编号：2208670
[Abstract]:Objective: Oral Squamous Cell Carcinoma (OSCC) is one of the most common malignant tumors in the oral and maxillofacial region, accounting for 3% of the global malignant tumors. It is well known that the prognosis of OSCC depends on clinical stage, histological grading, lymph node metastasis and other factors, but most patients are in advanced stage. Therefore, early diagnosis and early treatment of oral cancer are ten. Researchers report that in addition to genetic factors such as gene mutation and deletion, epigenetic changes such as DNA methylation are early events in tumorigenesis. Clinical application of epigenetic markers can make early diagnosis of oral squamous cell carcinoma, predict prognosis accurately, improve the quality of life of patients, and improve the survival rate of patients. The NKD gene family includes NKD1 (Naked cuticle homolog 1) and NKD2 (Naked cuticle homolog 2) genes. NKDl and NKD2 genes are located on chromosomes 16q12.1 and 5p15.3, respectively. Studies have found that NKD1 and NKD2 related loci frequently occur loss of heterozygosity (LOH) in liver cancer, gastric cancer, breast cancer and other cancers. The expression of NKD1 and NKD2 m RNA was abnormal and methylation status was changed in many malignant tumors including breast cancer and osteosarcoma. However, the expression and methylation status of NKD1 and NKD2 genes were not reported in oral squamous cell carcinoma. To explore the role of NKD1 and NKD2 genes in the development of OSCC, and to provide theoretical basis for early diagnosis and treatment of oral squamous cell carcinoma. Methods: 1 Specimens were collected from the 4th Hospital of Hebei Medical University from September 2015 to November 2016. Fifty-two patients with OSCC who underwent surgical treatment in stomatology department were divided into experimental group and control group. The tissues of OSCC resected during operation were taken as control group and 2 cm normal mucosa adjacent to cancer as control group. Histological diagnosis of the experimental group and control group were confirmed by pathology after operation. The specimens were frozen in the biology specimen bank at - 80 C. 2 The specimens were frozen in reverse transcription polymerase chain reaction (RT-PCR, Reverse Transcrip). Methylation-specific polymerase chain reaction (MSP) was used to detect the methylation status of NKD1 and NKD2 gene promoter Cp G islands in 52 OSCC and corresponding normal tissues. SS21.0 software for statistical analysis, comparison of gene m RNA expression differences using paired t test, group t or approximate t test; two gene m RNA expression correlation using linear correlation analysis; comparison of gene methylation using Fisher exact probability method or quadruple table_2 test; methylation correlation between the two genes using Spearman rank and correlation score Results: The relative expression of NKD1 m RNA in OSCC tissues was 0.50+0.12, which was lower than 0.66+0.18 in corresponding normal oral mucosa tissues. The difference was statistically significant (t=19.934, P 0.001). The relative expression of NKD1 m RNA in clinical stage I+II OSCC tissues was 0.53+0.12, which was higher than that in clinical stage I+II OSCC tissues. The relative expression of NKD1 m RNA in patients with lymph node metastasis was 0.44 (- 3.041, P = 0.004), 0.44 (- 0.10) lower than that in patients without lymph node metastasis (0.52 (- 0.12) (t = 2.469, P = 0.017), and the relative expression of NKD1 m RNA was not correlated with age, sex and smoking (P 0.05). The relative expression of NKD2 gene m RNA in clinical stage I + II OSCC tissues was 0.44 + 0.10, higher than that in clinical stage III + IV stage 0.37 + 0.05 (t = - 3.739, P 0.001), and 0.37 + 0.07 in lymph node metastasis group was lower than that in non-lymph node metastasis group (0.45 + 0.001). 10 (t = 2.873, P = 0.006); 0.38 (+ 0.09) in smoking group was lower than 0.45 (+ 3.395, P = 0.001) in non-smoking group; the relative expression of NKD2 gene m RNA was not correlated with sex and age (P 0.05). 3 NKD1 gene methylation positive expression rate in OSCC tissues was 48.08% (25/52) higher than that in normal oral mucosa tissues (26.92% (14/52). The methylation rate of NKD1 gene was 35.48% (11/31), lower than 66.67 (14/21) in stage III + IV (2 = 4.877, P = 0.027), and 70.59% (12/17) in patients with lymph node metastasis was higher than 37.14% (13/35) in patients without lymph node metastasis (2 = 5.127, P = 0.024). The positive expression rate of NKD2 gene methylation in OSCC was 44.23% (23/52) higher than that in corresponding normal oral mucosa (21.15% (11/52). The difference was statistically significant (2 = 6.292, P = 0.012). The methylation rate of NKD2 gene in stage I + II OSCC was 29.03% (9/31), which was lower than that in stage III + IV (66.67% (14/21) (2 = 7.012). 188, P = 0.007; 64.71% (11/17) in lymph node metastasis group was higher than 34.29% (12/35) in non-lymph node metastasis group (2 = 4.293, P = 0.038); 57.14% (16/28) in smoking group was higher than 29.17% (7/24) in non-smoking group (2 = 4.1, P = 0.043). The positive expression rate of NKD2 methylation was not related to sex and age (P In non-methylation group, the expression of M RNA was 0.54 (-3.344, P = 0.002). In methylation group, the relative expression of M RNA was 0.36 (-0.06) lower than that in non-methylation group, the relative expression of M RNA was 0.47 (-4.934, P 0.001). In OSCC, the methylation of NKD1 and NKD2 genes occurred in 16 cases, and no methylation occurred in 20 cases. There was a positive correlation (r = 0.038, P = 0.005) in OSCC tissues, and a positive correlation (r = 0.312, P = 0.024) between the relative expression of NKD1 and NKD2 m RNA in OSCC tissues. The abnormal expression of NKD2 gene is related to clinical stage, lymph node metastasis and smoking. 2 NKD1, hypermethylation of Cp G island in the promoter region of NKD2 gene may be one of the main mechanisms of OSCC. Metastasis and smoking are related. 3 The hypermethylation of NKD1 and NKD2 may be one of the main reasons for the decrease or silence of M RNA expression in OSCC. 4 NKD1 and NKD2 may play a synergistic role in the occurrence and development of OSCC.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.8
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