【摘要】：目的:口腔鱗狀細胞癌(Oral Squamous Cell Carcinoma,OSCC)是口腔頜面部最常見惡性腫瘤之一,占全球惡性腫瘤的3%。據(jù)統(tǒng)計,我國每年口腔癌的新發(fā)病例約4.65萬,發(fā)病年齡大多在40-60歲之間,男性相對女性較多。隨著醫(yī)療水平的逐漸提高,以手術切除為主的綜合序列治療方法使口腔癌的預后有了明顯改善,但患者的5年生存率仍停留在55%-60%。眾所周知,OSCC的預后取決于臨床分期,組織學分級,淋巴結轉移等因素,但大多數(shù)患者就診時已屬晚期,因此口腔癌的早期診斷,早期治療十分重要。研究者報道除基因變異、缺失等遺傳因素外,DNA甲基化等表觀遺傳學的改變是腫瘤的發(fā)生的早期事件。臨床應用表觀遺傳學標志物能夠?qū)谇击[癌做到早期診斷,對預后準確預測,進而改善患者的生存質(zhì)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基因分別定位于染色體16q12.1及5p15.3上,研究發(fā)現(xiàn)NKD1、NKD2基因相關定位位點在肝癌、胃癌、乳腺癌等多種癌癥中頻繁的出現(xiàn)雜合性缺失(Loss of heterozygosity,LOH),包括乳腺癌、骨肉瘤在內(nèi)的多種惡性腫瘤中均存在NKD1、NKD2基因m RNA表達水平異常及甲基化狀態(tài)的改變。但是在口腔鱗癌的發(fā)生發(fā)展中,NKD1、NKD2基因的表達及甲基化狀態(tài)尚無文獻報道。因此本實驗通過采用RT-PCR和MSP方法檢測NKD1、NKD2基因的表達水平及甲基化狀態(tài),并分析其與OSCC各項臨床病理特征之間的相關性,探討NKD1、NKD2基因在OSCC的發(fā)生發(fā)展中的作用,為口腔鱗癌的早期診斷,早期治療提供理論依據(jù)。方法:1標本均來源于2015年9月至2016年11月期間河北醫(yī)科大學第四醫(yī)院口腔科手術治療的52例OSCC患者,以術中切除的OSCC組織作為實驗組,以相對應癌旁2cm正常黏膜組織為對照組,實驗組及對照組組織學診斷均經(jīng)術后病理驗證,標本于-80℃生物標本庫中凍存。2采用反轉錄聚合酶鏈反應(RT-PCR,Reverse Transcription Polymerase Chain Reaction)檢測52例OSCC及相對應正常組織內(nèi)NKD1、NKD2基因m RNA表達量。3應用甲基化特異性聚合酶鏈反應(MSP,Methylation Specific Polymerase Chain Reaction)檢測52例OSCC及相對應正常組織內(nèi)NKD1、NKD2基因啟動子區(qū)Cp G島甲基化狀態(tài)。4應用SPSS21.0軟件進行統(tǒng)計學分析,基因m RNA表達差異比較應用配對t檢驗,成組t或近似t檢驗;兩基因m RNA表達相關性應用直線相關分析;基因甲基化的比較應用Fisher確切概率法或四格表χ2檢驗;兩基因間甲基化相關性應用Spearman等級秩和相關分析。當P0.05證明差異存在統(tǒng)計學意義。結果:1 NKD1基因m RNA相對表達量在OSCC組織中為0.50±0.12,低于其對應正�？谇火つそM織0.66±0.18,二者差別有統(tǒng)計學意義(t=19.934,P0.001)。臨床Ⅰ期+Ⅱ期OSCC組織中NKD1基因m RNA相對表達量是0.53±0.12,高于臨床Ⅲ期+Ⅳ期0.44±0.10(t=-3.041,P=0.004);有淋巴結轉移組NKD1 m RNA的相對表達量0.44±0.10,低于無淋巴結轉移組0.52±0.12(t=2.469,P=0.017);NKD1基因m RNA的相對表達量與年齡、性別和吸煙與否無相關性(P0.05)。2 NKD2基因m RNA的相對表達量在OSCC組織中為0.42±0.10,低于其對應正�？谇火つ�0.62±0.12,二者差異有統(tǒng)計學意義(t=10.915,P0.001)。臨床Ⅰ期+Ⅱ期OSCC組織中NKD2基因m RNA相對表達量為0.44±0.10,高于臨床Ⅲ期+Ⅳ期中0.37±0.05(t=-3.739,P0.001);有淋巴結轉移組0.37±0.07低于無淋巴結轉移組0.45±0.10(t=2.873,P=0.006);吸煙組0.38±0.09低于不吸煙組0.45±0.08(t=-3.395,P=0.001);NKD2基因m RNA的相對表達量與性別及年齡無相關性(P0.05)。3 NKD1基因甲基化陽性表達率在OSCC組織中48.08%(25/52)高于相對應正常口腔黏膜組織26.92%(14/52),二者差異有統(tǒng)計學意義(χ2=4.964,P=0.026)。臨床Ⅰ期+Ⅱ期OSCC組織中NKD1基因甲基化率為35.48%(11/31),低于Ⅲ期+Ⅳ期66.67(14/21)(χ2=4.877,P=0.027);NKD1甲基化率在有淋巴結轉移組中70.59%(12/17)高于無淋巴結轉移組37.14%(13/35)(χ2=5.127,P=0.024);NKD1基因甲基化率與年齡、性別及吸煙無關(P0.05)。4 NKD2基因甲基化陽性表達率在OSCC中44.23%(23/52)高于相應的正常口腔黏膜組織21.15%(11/52),兩者差別有統(tǒng)計學意義(χ2=6.292,P=0.012)。臨床Ⅰ期+Ⅱ期OSCC組織中NKD2基因甲基化率為29.03%(9/31),低于Ⅲ期+Ⅳ期66.67%(14/21)(χ2=7.188,P=0.007);有淋巴結轉移組64.71%(11/17)高于無淋巴結轉移組34.29%(12/35)(χ2=4.293,P=0.038);吸煙組57.14%(16/28)高于不吸煙組29.17%(7/24)(χ2=4.1,P=0.043)。NKD2甲基化陽性表達率與性別和年齡無關(P0.05)。5在OSCC中,NKD1甲基化組m RNA相對表達量0.44±0.11低于非甲基化組m RNA表達量0.54±0.11(t=-3.344,P=0.002)。NKD2基因甲基化組m RNA相對表達量0.36±0.06低于非甲基化組m RNA的相對量0.47±0.09(t=-4.934,P0.001)。6在OSCC中,NKD1、NKD2兩基因均發(fā)生甲基化者共16例,均未發(fā)生甲基化者共20例,兩基因甲基化狀態(tài)在OSCC組織中呈正相關關系(r=0.038,P=0.005);NKD1、NKD2兩基因m RNA相對表達量在OSCC組織中呈正相關關系(r=0.312,P=0.024)。結論:1 NKD1、NKD2基因m RNA相對表達下降或沉默可能參與OSCC的發(fā)生與發(fā)展。其中,NKD1基因異常表達與臨床分期及淋巴結轉移相關。NKD2基因異常表達與臨床分期、淋巴結轉移及吸煙有關。2 NKD1、NKD2基因啟動子區(qū)Cp G島高甲基化狀態(tài)可能是OSCC發(fā)生的主要機制之一。其中,NKD1基因異常甲基化狀態(tài)與臨床分期及淋巴結轉移有關。NKD2基因異常甲基化狀態(tài)與臨床分期、淋巴結轉移及吸煙有關。3在OSCC中NKD1、NKD2基因高甲基化狀態(tài)可能是致其m RNA表達下降或沉默的主要原因之一。4 NKD1與NKD2在OSCC的發(fā)生發(fā)展中可能存在協(xié)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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.8

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