本文選題：口腔鱗狀細(xì)胞癌 + PRSS8��； 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:口腔鱗狀細(xì)胞癌(oral squamous cell carcinoma,OSCC)是世界范圍內(nèi)最常見的口腔頜面部惡性腫瘤之一。近年來(lái)大樣本的流行病學(xué)研究表明,在中國(guó),OSCC發(fā)病率呈逐年上升趨勢(shì),且發(fā)病年齡日趨年輕化,發(fā)病率在3.6/10-8.0/10萬(wàn)之間。因其具有惡性程度高,易發(fā)生淋巴結(jié)轉(zhuǎn)移,預(yù)后差等特點(diǎn),導(dǎo)致OSCC的5年生存率在50%左右。目前對(duì)于OSCC早期診斷及預(yù)后的研究較缺乏,當(dāng)前的研究認(rèn)為在OSCC發(fā)生發(fā)展中的基因變化因素主要包括基因突變與表觀遺傳修飾異常。DNA甲基化的研究可以使我們更好的了解OSCC發(fā)生機(jī)制,為OSCC的早期診斷及預(yù)后提供新的理論基礎(chǔ)�，F(xiàn)階段研究表明,基因突變與表觀遺傳修飾異常是影響OSCC發(fā)生的重要因素,表觀遺傳修飾是一種可遺傳、可逆轉(zhuǎn)的生物學(xué)行為,主要包括DNA核苷酸胞嘧啶的甲基化修飾、組蛋白修飾、非編碼RNA調(diào)控等。其中,DNA甲基化的研究成為表觀遺傳學(xué)的研究熱點(diǎn)。PRSS8基因又稱為人類的前列腺蛋白(Prostasin)或CAP-1,位于染色體16p11.2上,不僅能維持機(jī)體的正常生理功能,而且參與生長(zhǎng)因子的調(diào)節(jié)、細(xì)胞的遷移多種炎癥、表皮生長(zhǎng)、腫瘤的發(fā)生等過(guò)程。近年來(lái)研究發(fā)現(xiàn)食管鱗癌、結(jié)直腸癌等癌組織中存在PRSS8基因甲基化狀態(tài)及PRSS8mRNA表達(dá)異常,PRSS8基因的異常改變有可能參與了癌癥的發(fā)生。蛋白酪氨酸磷酸酶非受體型22(PTPN22)基因位于染色體1p13.3-13.1上并參與上皮粘附。TCGA數(shù)據(jù)庫(kù)顯示PTPN22甲基化水平在腫瘤組織與正常組織之間有顯著差異。然而,盡管PTPN22基因在癌癥發(fā)生中有潛在重要性,但是關(guān)于它與腫瘤的報(bào)道很少。目前尚未發(fā)現(xiàn)PRSS8及PTPN22在OSCC中的表觀遺傳學(xué)變化相關(guān)報(bào)道。本研究以52例OSCC術(shù)后標(biāo)本(河北醫(yī)科大學(xué)第四醫(yī)院口腔科)為研究對(duì)象,通過(guò)分析OSCC組織和相應(yīng)正常黏膜組織中PRSS8及PTPN22基因的啟動(dòng)子區(qū)甲基化狀態(tài)及其mRNA的表達(dá)水平,并結(jié)合患者的臨床病理特征進(jìn)行統(tǒng)計(jì)分析,旨在揭示OSCC的發(fā)病機(jī)制,為OSCC的早期發(fā)現(xiàn)與預(yù)后的評(píng)估提供實(shí)驗(yàn)理論依據(jù)。方法:1應(yīng)用甲基化特異性聚合酶鏈反應(yīng)(Methylation Specific PCR,MSP)檢測(cè)52例經(jīng)術(shù)后病理診斷為OSCC的癌組織及相應(yīng)正常黏膜組織中PRSS8及PTPN22基因啟動(dòng)子區(qū)甲基化狀態(tài)。2應(yīng)用逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(Reverse Transcriptase-PCR,RT-PCR)技術(shù)檢測(cè)52例術(shù)后經(jīng)病理診斷為OSCC的標(biāo)本的癌組織及相應(yīng)正常黏膜組織中PRSS8及PTPN22的mRNA相對(duì)表達(dá)量。3應(yīng)用統(tǒng)計(jì)軟件SPSS21.0對(duì)實(shí)驗(yàn)結(jié)果進(jìn)行統(tǒng)計(jì)學(xué)分析。結(jié)果:1 OSCC和正常組織中PRSS8及PTPN22基因的甲基化狀態(tài)52例OSCC組織中PRSS8及PTPN22基因啟動(dòng)子甲基化率分別為57.7%(30/52),51.9%(27/52),52例正常組織甲基化率為34.6%(18/52),30.8%(16/52),經(jīng)統(tǒng)計(jì)學(xué)分析,OSCC組織PRSS8及PTPN22基因啟動(dòng)子甲基化率均明顯高于相應(yīng)正常組織,其差異都具有統(tǒng)計(jì)學(xué)意義(χ~2=5.571,P=0.018;χ~2=4.798,P=0.029)。(Table 1,2)(Fig.1,2)2 OSCC和正常組織中PRSS8及PTPN22基因mRNA的表達(dá)情況52例標(biāo)本中OSCC組織和正常組織PRSS8mRNA的表達(dá)量均符合正態(tài)分析,PRSS8mRNA的相對(duì)表達(dá)量為0.44±0.12,相應(yīng)正常組織的表達(dá)量為0.71±0.13,采用配對(duì)t檢驗(yàn),PRSS8基因mRNA的相對(duì)表達(dá)量在OSCC組織中明顯低于相應(yīng)正常組織,其差異具有統(tǒng)計(jì)學(xué)意義(t=-10.620,P=0.000)。(Table 3)(Fig.3)PTPN22 mRNA的相對(duì)表達(dá)量為0.44±0.20,相應(yīng)正常組織的表達(dá)量為0.63±0.20,PTPN22基因mRNA的相對(duì)表達(dá)量在OSCC組織中明顯低于相應(yīng)正常組織,其差異具有統(tǒng)計(jì)學(xué)意義(t=-4.647,P=0.000)。(Table 4)(Fig.4)3 PRSS8及PTPN22基因甲基化組與非甲基化組中mRNA相對(duì)表達(dá)量的比較在52例OSCC組織中,甲基化組PRSS8mRNA的相對(duì)表達(dá)量為0.43±0.12,非甲基化組為0.52±0.11,利用獨(dú)立樣本t檢驗(yàn)進(jìn)行分析,結(jié)果顯示甲基化組與非甲基化組PRSS8mRNA相對(duì)表達(dá)量之間的差異具有統(tǒng)計(jì)學(xué)意義(t=-2.880,P=0.006)。(Table 5)PTPN22mRNA在甲基化組相對(duì)表達(dá)量為0.47±0.18,在非甲基化組相對(duì)表達(dá)量為0.56±0.08,甲基化組與非甲基化組中PTPN22mRNA相對(duì)表達(dá)量之間的差異有統(tǒng)計(jì)學(xué)意義(t=-2.171,P=0.035)。(Table6)4 PRSS8和PTPN22基因啟動(dòng)子甲基化率與患者臨床參數(shù)之間的關(guān)系男性組PRSS8和PTPN22基因啟動(dòng)子甲基化率分別為53.3%(16/30),46.7%(14/30),女性組甲基化率分別為63.6%(14/22),59.1%(13/22),其差異均無(wú)統(tǒng)計(jì)學(xué)意義(χ~2=0.552,P=0.458),(χ~2=0.785,P=0.376);年齡≥60歲組甲基化率分別為62.5%(20/32),56.3%(18/32),年齡60歲組患者甲基化率分別為50.0%(10/20),45.0%(9/20),其差異均無(wú)統(tǒng)計(jì)學(xué)意義(χ~2=0.788,P=0.375),(χ~2=0.624,P=0.430);飲酒組甲基化率分別為59.3%(16/27),51.9%(14/27),不飲酒組甲基化率分別為56.0%(14/25),52.0%(13/25),其差異均無(wú)統(tǒng)計(jì)學(xué)意義(χ~2=0.056,P=0.812);(χ~2=0.000,P=0.991);吸煙組甲基化率分別為51.7%(15/29),48.3%(14/29),不吸煙組甲基化率分別為65.2%(15/23),56.5%(13/23),其差異均無(wú)統(tǒng)計(jì)學(xué)意義(χ~2=0.957,P=0.328),(χ~2=0.349,P=0.554);臨床Ⅰ期+Ⅱ期組甲基化率分別為38.1%(8/21),28.6%(6/21),臨床Ⅲ期+Ⅳ期組甲基化率分別為71.0%(22/31),67.7%(21/31),其差異均有統(tǒng)計(jì)學(xué)意義(χ~2=5.543,P=0.019),(χ~2=66.589,P=0.000);淋巴結(jié)轉(zhuǎn)移組甲基化率分別為73.9%(17/23),65.2%(13/23),無(wú)淋巴結(jié)轉(zhuǎn)移組甲基化率分別為44.8%(13/29),41.4%(12/29),差異均有統(tǒng)計(jì)學(xué)意義(χ~2=4.446,P=0.035),(χ~2=2.920,P=0.047)。高中分化組甲基化率分別為47.1%(16/34),41.2%(14/34),低分化組甲基化率分別為77.8%(14/18),72.2%(13/18),差異均具有統(tǒng)計(jì)學(xué)意義(χ~2=4.550,P=0.033),(χ~2=4.544,P=0.033)。(Table 7,8)5 OSCC中PRSS8和PTPN22基因mRNA相對(duì)表達(dá)水平及與臨床參數(shù)之間的關(guān)系男性組患者PRSS8和PTPN22mRNA的相對(duì)表達(dá)量分別為0.44±0.13,0.41±0.19,女性組的相對(duì)表達(dá)量分別為0.46±0.11,0.49±0.21,差異均無(wú)統(tǒng)計(jì)學(xué)意義(t=-0.586,P=0.560),(t=-1.481,P=0.145);年齡≥60歲組的相對(duì)表達(dá)量分別為0.45±0.13,0.44±0.20,年齡60歲組相對(duì)表達(dá)量分別為0.43±0.10,0.46±0.20,差異均無(wú)統(tǒng)計(jì)學(xué)意義(t=-0.481,P=0.633),(t=-0.451,P=0.654);飲酒組的相對(duì)表達(dá)量分別為0.43±0.11,0.47±0.22,不飲酒組的相對(duì)表達(dá)量分別為0.47±0.13,0.42±0.17,差異均無(wú)統(tǒng)計(jì)學(xué)意義(t=-1.197,P=0.237),(t=1.016,P=0.315);吸煙組相對(duì)表達(dá)量分別為0.44±0.11,0.48±0.22,不吸煙組相對(duì)表達(dá)量分別為0.45±0.13,0.40±0.18,差異均無(wú)統(tǒng)計(jì)學(xué)意義(t=-475,P=0.637),(t=1.323,P=0.192);根據(jù)TNM分期,進(jìn)行臨床分期,其中Ⅰ+Ⅱ期相對(duì)表達(dá)量分別為0.49±0.11,0.56±0.12,Ⅲ+Ⅳ期分別為0.41±0.12,0.46±0.20,均具有顯著差異,差異有統(tǒng)計(jì)學(xué)意義(t=2.335,P=0.024),(t=2.076,P=0.043)。淋巴結(jié)轉(zhuǎn)移組分別為0.42±0.11,0.44±0.17,無(wú)淋巴結(jié)轉(zhuǎn)移組分別為0.49±0.13,0.55±0.11,差異有統(tǒng)計(jì)學(xué)意義(t=-2.119,P=0.039),(t=-2.688,P=0.010)。高中分化組相對(duì)表達(dá)量分別為0.48±0.12,0.57±0.11,低分化組相對(duì)表達(dá)量分別為0.39±0.11,0.49±0.12,具有統(tǒng)計(jì)學(xué)意義(t=2.736,P=0.009),(t=2.188,P=0.033)。(Table9,10)結(jié)論:1 PRSS8和PTPN22基因在OSCC組織中的mRNA相對(duì)表達(dá)量明顯低于其相應(yīng)的正常黏膜組織,提示PRSS8和PTPN22基因在OSCC中可能主要起抑癌基因的作用。2 OSCC組織中PRSS8和PTPN22基因啟動(dòng)子區(qū)甲基化率明顯高于其相應(yīng)的正常黏膜組織,提示PRSS8和PTPN22基因甲基化可能是OSCC發(fā)生的機(jī)制之一。3 PRSS8和PTPN22啟動(dòng)子區(qū)甲基化率與患者性別、年齡等因素?zé)o關(guān),與分化程度、淋巴結(jié)轉(zhuǎn)移、病理分級(jí)等有關(guān),提示PRSS8和PTPN22基因啟動(dòng)子區(qū)甲基化可能與OSCC的預(yù)后有關(guān)。
[Abstract]:Objective: oral squamous cell carcinoma (OSCC) is one of the most common oral and maxillofacial malignant tumors in the world. In recent years, the epidemiological study of large samples shows that the incidence of OSCC is increasing year by year in China, and the age of the disease is becoming younger and younger. The incidence of OSCC is between 3.6/10-8.0/10 million. High degree of sex, easy to occur lymph node metastasis, poor prognosis and so on, resulting in the 5 year survival rate of about 50% of OSCC. At present, the research on early diagnosis and prognosis of OSCC is lack. The current research suggests that the gene change factors in the development of OSCC mainly include gene mutation and epigenetic modification of abnormal.DNA methylation. We have a better understanding of the OSCC mechanism and provide a new theoretical basis for the early diagnosis and prognosis of OSCC. Current studies have shown that gene mutation and epigenetic modification are an important factor affecting the occurrence of OSCC. Epigenetic modification is a genetic, reversible biological behavior, mainly including the methylation of the DNA nucleotides cytosine. Chemical modification, histone modification, non coded RNA regulation, among which, DNA methylation has become the focus of epigenetic research. The.PRSS8 gene, known as human prostate protein (Prostasin) or CAP-1, is located on chromosome 16p11.2. It not only maintains normal physiological functions of the body, but also participates in the regulation of growth factors and the migration of cells. In recent years, it has been found that the methylation status of PRSS8 gene and the abnormal PRSS8mRNA expression in the cancer tissues of the esophageal squamous cell carcinoma and colorectal cancer have been found, and the abnormal changes of the PRSS8 gene may be involved in the occurrence of cancer. The protein tyrosine phosphatase non receptor 22 (PTPN22) gene is located in the chromosome 1p13.3 -13.1 and involved in the epithelial adhesion.TCGA database show that there is a significant difference between the level of PTPN22 methylation in the tumor tissue and the normal tissue. However, although the PTPN22 gene is of potential importance in the occurrence of cancer, there are few reports about it and the tumor. The epigenetic changes associated with PRSS8 and PTPN22 in OSCC have not been found. In this study, 52 specimens of OSCC (Department of Stomatology, Hebei Medical University, Fourth Hospital of Hebei Medical University) were studied. The methylation status of the promoter region of the PRSS8 and PTPN22 genes in the OSCC tissues and the corresponding normal mucosa tissues and the expression level of mRNA were analyzed, and the clinicopathological features of the patients were statistically analyzed in order to reveal the hair of OSCC. The mechanism of the disease provides experimental theoretical basis for the assessment of early detection and prognosis of OSCC. Methods: 1 the methylation specific polymerase chain reaction (Methylation Specific PCR, MSP) was used to detect the reversal of the methylation status of PRSS8 and PTPN22 gene promoter regions in the cancerous tissues of OSCC and in the corresponding normal mucosal tissues after the pathological diagnosis of OSCC. Reverse Transcriptase-PCR (RT-PCR) technique was used to detect the mRNA relative expression of PRSS8 and PTPN22 in the specimens of 52 patients with OSCC after surgery and.3 application statistics software SPSS21.0 to analyze the results of the experimental data. Results: 1 OSCC and PRSS8 and PTPN22 in normal tissues. The methylation status of genes in 52 OSCC tissues was 57.7% (30/52), 51.9% (27/52) and 34.6% (18/52) and 30.8% (16/52) in 52 normal tissues, respectively. The methylation rates of PRSS8 and PTPN22 genes in OSCC tissues were significantly higher than those of the corresponding normal tissues, and the differences were all of the differences. Statistical significance (x ~2=5.571, P=0.018, X ~2=4.798, P=0.029). (Table 1,2) (Fig.1,2) 2 OSCC and the expression of PRSS8 and PTPN22 gene mRNA in normal tissues. The expression of OSCC tissue and normal tissues in 52 specimens were both 0.44 + 0.12, and the expression of the corresponding normal tissues was 0.71 The relative expression of PRSS8 gene mRNA was significantly lower than that of the corresponding normal tissue in OSCC tissue by paired t test. The difference was statistically significant (t=-10.620, P=0.000). (Table 3), the relative expression of PTPN22 mRNA was 0.44 + 0.20, the expression of the corresponding normal fabric was 0.63 + 0.20, and the relative expression of PTPN22 gene mRNA The difference in CC tissue was significantly lower than that of the corresponding normal tissue (t=-4.647, P=0.000). (Table 4) (Fig.4) the relative expression of mRNA in the methylation group and the non methylation group was compared with the non methylation group in 52 cases of OSCC tissues, the relative expression of PRSS8mRNA in the methylation group was 0.43 + 0.12, and the non methylation group was 0.52 + 0.11. Analysis by independent sample t test showed that the difference between the relative expression of PRSS8mRNA in methylation group and non methylation group was statistically significant (t=-2.880, P=0.006). (Table 5) PTPN22mRNA in methylation group was 0.47 + 0.18, the expression amount was 0.56 + 0.08 in non methylation group, methylation group and non methylation group. The differences in the relative expression of PTPN22mRNA were statistically significant (t=-2.171, P=0.035). (Table6) the relationship between the methylation rate of 4 PRSS8 and PTPN22 gene promoters and the clinical parameters of the patients was 53.3% (16/30), 46.7% (14/30), and 63.6% (14/22) and 59.1% (1) in the female group, respectively. 3/22), the difference was not statistically significant (x ~2=0.552, P=0.458), (x ~2=0.785, P=0.376), the methylation rates of the age group were 62.5% (20/32), 56.3% (18/32) and 50% (10/20) and 45% (9/20) in the 60 year old group, respectively, and the difference was not statistically significant (x ~2=0.788, P=0.375), and the methylation of the drinking group. The rates of methylation were 59.3% (16/27) and 51.9% (14/27). The methylation rates of non drinking groups were 56% (14/25) and 52% (13/25). The differences were not statistically significant (x ~2=0.056, P=0.812), and the methylation rates of smoking group were 51.7% (15/29), 48.3% (14/29), 65.2% (15/23) and 56.5% (13/23), respectively, and the differences were not all Statistical significance (x ~2=0.957, P=0.328), (x ~2=0.349, P=0.554); the methylation rates in phase I + stage group were 38.1% (8/21), 28.6% (6/21), and 71% (22/31) and 67.7% (21/31) in phase III + IV group, respectively, with statistical significance (x ~2=5.543, P=0.019), (chi ~2=66.589, P=0.000), and lymph node metastasis group methylation rates. The methylation rates of 73.9% (17/23), 65.2% (13/23), no lymph node metastasis group were 44.8% (13/29) and 41.4% (12/29). The difference was statistically significant (x ~2=2.920, P=0.047). The methylation rates of the high school group were 47.1% (16/ 34), 41.2% (14/34), and 41.2% (14/18), 72.2% (13/18), respectively, 72.2% (13/18), and 72.2% (13/18), respectively. Statistical significance (x ~2=4.550, P=0.033), (x ~2=4.544, P=0.033). (Table 7,8) the relative expression level of PRSS8 and PTPN22 gene mRNA in 5 OSCC and the relationship with the clinical parameters, the relative expression of PRSS8 and PTPN22mRNA in the male group was 0.44 + 0.19, and the relative expression of the female group was 0.46 + 0.21, respectively. The difference was not statistically significant (t=-0.586, P=0.560), (t=-1.481, P=0.145), the relative expression of age group was 0.45 + 0.13,0.44 0.20, and the relative expression of age 60 year old group was 0.43 + 0.10,0.46 + 0.20 respectively, the difference was not statistically significant (t=-0.481, P= 0.633), (t=-0.451, P=0.654), and the relative expression of alcohol drinking group was 0.43 + 0, respectively. The relative expression of.11,0.47 + 0.22 was 0.47 + 0.13,0.42 + 0.17, respectively, and the difference was not statistically significant (t=-1.197, P=0.237), (t=1.016, P=0.315), and the relative expression of smoking group was 0.44 + 0.11,0.48 + 0.22 respectively, and the relative expression of non smoking group was 0.45 + 0.13,0.40 + 0.18 respectively, and the difference was not statistically significant (T, P=0.637). (T, P=0.637) =1.323, P=0.192), according to TNM staging, clinical staging was carried out, in which the relative expression of stage I + II was 0.49 + 0.11,0.56 + 0.12 and 0.41 + 0.12,0.46 + 0.20 respectively. The difference was significant (t=2.335, P=0.024), (t=2.076, P=0.043). The lymph node metastasis group was 0.42 + 0.11,0.44 + 0.17, no lymph node. The transfer group was 0.49 + 0.13,0.55 + 0.11 respectively (t=-2.119, P=0.039), (t=-2.688, P=0.010). The relative expression of the high school division group was 0.48 + 0.12,0.57 + 0.11 respectively, and the relative expression of the low differentiation group was 0.39 + 0.11,0.49 + 0.12 respectively, which had statistical significance (t=2.736, P=0.009), (t=2.188, P=0.033). (Table9,10) conclusion: 1 The relative expression of the RSS8 and PTPN22 genes in the OSCC tissues was significantly lower than that of the corresponding normal mucosa, suggesting that the PRSS8 and PTPN22 genes may play a role in the tumor suppressor gene in OSCC. The methylation rate of the PRSS8 and PTPN22 gene promoter regions in.2 OSCC tissues is significantly higher than that of the corresponding normal mucosal tissue, suggesting PRSS8 and bases. Methylation may be one of the mechanisms of OSCC, one of the mechanisms of.3 PRSS8 and PTPN22 promoter methylation is related to the degree of differentiation, lymph node metastasis, and pathological classification. It suggests that the methylation of the promoter region of the PRSS8 and PTPN22 genes may be related to the preimplantation of OSCC.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.8

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