利用深度測(cè)序技術(shù)篩選膀胱癌突變基因及對(duì)HECW1基因突變的驗(yàn)證
發(fā)布時(shí)間：2018-11-23 17:28
【摘要】：目的：利用外顯子組深度測(cè)序技術(shù)發(fā)現(xiàn)和篩選膀胱移行細(xì)胞癌(Transitional CellCarcinoma Of Bladder, TCCB)中的致病候選基因，并通過(guò)Sanger測(cè)序技術(shù)進(jìn)行驗(yàn)證，為膀胱癌病因?qū)W研究提供一定的理論依據(jù)，并探尋理想的腫瘤診斷標(biāo)志物和有效的治療靶點(diǎn)。 方法： 第一部分，取蘇州大學(xué)附屬第一醫(yī)院2012年4月手術(shù)治療的4例膀胱移行細(xì)胞癌患者癌及癌旁組織，其中2例為非肌層浸潤(rùn)性（非浸潤(rùn)組），2例為肌層浸潤(rùn)性（浸潤(rùn)組）。采用SOLID深度基因測(cè)序的方法進(jìn)行全外顯子組測(cè)序，尋找突變基因，并通過(guò)特定的篩選條件進(jìn)行過(guò)濾，選出其中發(fā)生錯(cuò)義突變的基因。 第二部分，取蘇州大學(xué)附屬第一醫(yī)院病理科提供的2011年9月至2012年9月膀胱移行細(xì)胞癌術(shù)后臘片標(biāo)本84例，其中浸潤(rùn)性54例，非浸潤(rùn)性30例，及2013年3月至5月本院手術(shù)治療的膀胱移行細(xì)胞癌患者腫瘤組織15例，其中浸潤(rùn)性7例，非浸潤(rùn)性8例和10例癌旁組織作為對(duì)照，將此25例組織制作成蠟塊。使用OMEGA試劑盒法提取癌及癌旁組織中基因組DNA，經(jīng)聚合酶鏈反應(yīng)（Polymerase ChainReaction，PCR）擴(kuò)增后，應(yīng)用Sanger測(cè)序技術(shù)驗(yàn)證深度測(cè)序中的突變結(jié)果。取制作的25例蠟塊，采用免疫組化Envision二步法，觀察HECW1蛋白在癌組織和癌旁組織中的表達(dá)差異。 結(jié)果： 第一部分：深度測(cè)序結(jié)果數(shù)據(jù)質(zhì)量滿意，，發(fā)現(xiàn)眾多突變基因，篩選出其中10個(gè)出現(xiàn)錯(cuò)義突變的基因，其中非浸潤(rùn)組4個(gè)，為IVL,RGPD5,ZNF79,SRRM5；浸潤(rùn)組3個(gè)，為CPNE7,RBMXL3,ACSM2A；兩組共有3個(gè)，為HECW1,ZNF273,TCHH。 第二部分：99例膀胱癌組織中發(fā)現(xiàn)6例HECW1基因突變，比例為6.06%，均為點(diǎn)突變，位于第11號(hào)外顯子，且與深度測(cè)序突變位點(diǎn)相同或在附近區(qū)域。其中，38例非浸潤(rùn)性膀胱癌組織DNA中，1例同義突變，比例為2.63%；61例浸潤(rùn)性膀胱癌組織DNA中，4例錯(cuò)義突變和1例無(wú)義突變（即突變?yōu)榻K止密碼子），比例為8.20%，兩者突變比例無(wú)統(tǒng)計(jì)學(xué)意義（P0.05），對(duì)照組10例未發(fā)現(xiàn)突變。免疫組化中，15例腫瘤組織9例陽(yáng)性，陽(yáng)性率60.0%，10例對(duì)照組1例陽(yáng)性，陽(yáng)性率10%，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 結(jié)論：（1）深度測(cè)序技術(shù)，特別是全外顯子組測(cè)序，可以作為泌尿系腫瘤病因?qū)W研究中行之有效的實(shí)驗(yàn)手段。（2）Sanger測(cè)序是檢驗(yàn)深度測(cè)序結(jié)果的金標(biāo)準(zhǔn)。（3）HECW1可能是與膀胱癌發(fā)生發(fā)展有關(guān)的候選基因。（4）HECW1蛋白在膀胱癌組織中的表達(dá)高于癌旁組織。（5）HECW1基因突變?cè)诎螂装┲械淖饔脵C(jī)制及是否影響蛋白的表達(dá)和功能還有待深入研究。
[Abstract]:Objective: to identify and screen candidate genes for (Transitional CellCarcinoma Of Bladder, TCCB) in bladder transitional cell carcinoma (TCC) by using exon group deep sequencing technique, and to provide a theoretical basis for the etiological study of bladder transitional cell carcinoma (TCC). And to explore the ideal tumor diagnosis markers and effective therapeutic targets. Methods: in the first part, 4 patients with bladder transitional cell carcinoma (TCC) were treated by surgery in the first affiliated Hospital of Suzhou University in April 2012, and 2 of them were non-myometrial infiltrating (non-invasive group). 2 cases were myometrial infiltration (infiltrating group). The SOLID deep gene sequencing method was used to sequence the whole exon group to search for the mutant gene, and the missense mutation gene was selected by specific screening conditions. In the second part, 84 specimens of transitional cell carcinoma of bladder were collected from Department of Pathology of the first affiliated Hospital of Suzhou University from September 2011 to September 2012. Among them, 54 cases were invasive and 30 cases were non-invasive. From March to May 2013, 15 cases of bladder transitional cell carcinoma (TCC) were treated by operation in our hospital, including 7 cases of infiltrating, 8 cases of non-invasive and 10 cases of paracancerous tissue as control. Genomic DNA, extracted from cancer and adjacent tissues was amplified by polymerase chain reaction (Polymerase ChainReaction,PCR) using OMEGA kit method. The mutation results in deep sequencing were verified by Sanger sequencing technique. The expression of HECW1 protein in cancer tissues and paracancerous tissues was observed by immunohistochemical Envision two-step method. Results: the first part: the quality of deep sequencing data was satisfactory, many mutation genes were found, and 10 of them had missense mutation, including 4 non-invasive genes, which were IVL,RGPD5,ZNF79,SRRM5;. Infiltration group 3, CPNE7,RBMXL3,ACSM2A; group 3, HECW1,ZNF273,TCHH. The second part: 6 cases (6.06%) of HECW1 gene mutations were found in 99 cases of bladder cancer, all of them were point mutations, located in exon 11, and were the same as or in the vicinity of deep sequencing mutation. Of 38 cases of non-invasive bladder cancer, 1 case had synonymous mutation (2.63%). In 61 cases of invasive bladder cancer DNA, 4 missense mutations and 1 nonsense mutation (i.e. mutation as termination codon) were found in 4 cases (8.20%). There was no significant difference between the two mutations (P0.05), while no mutation was found in 10 cases in the control group. In immunohistochemical staining, 9 cases were positive in 15 cases of tumor tissue, and 1 case was positive in 10 cases of control group. The difference was statistically significant (P0.05). Conclusion: (1) Deep sequencing, especially total exon sequencing, (2) Sanger sequencing is the gold standard for testing the results of deep sequencing. (3) HECW1 may be a candidate gene related to the occurrence and development of bladder cancer. The expression of HECW1 protein in bladder cancer tissues is higher than that in paracancerous tissues. (5) the mechanism of HECW1 gene mutation in bladder cancer and whether it affects the expression and function of HECW1 protein remains to be further studied.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.14
本文編號(hào)：2352228
[Abstract]:Objective: to identify and screen candidate genes for (Transitional CellCarcinoma Of Bladder, TCCB) in bladder transitional cell carcinoma (TCC) by using exon group deep sequencing technique, and to provide a theoretical basis for the etiological study of bladder transitional cell carcinoma (TCC). And to explore the ideal tumor diagnosis markers and effective therapeutic targets. Methods: in the first part, 4 patients with bladder transitional cell carcinoma (TCC) were treated by surgery in the first affiliated Hospital of Suzhou University in April 2012, and 2 of them were non-myometrial infiltrating (non-invasive group). 2 cases were myometrial infiltration (infiltrating group). The SOLID deep gene sequencing method was used to sequence the whole exon group to search for the mutant gene, and the missense mutation gene was selected by specific screening conditions. In the second part, 84 specimens of transitional cell carcinoma of bladder were collected from Department of Pathology of the first affiliated Hospital of Suzhou University from September 2011 to September 2012. Among them, 54 cases were invasive and 30 cases were non-invasive. From March to May 2013, 15 cases of bladder transitional cell carcinoma (TCC) were treated by operation in our hospital, including 7 cases of infiltrating, 8 cases of non-invasive and 10 cases of paracancerous tissue as control. Genomic DNA, extracted from cancer and adjacent tissues was amplified by polymerase chain reaction (Polymerase ChainReaction,PCR) using OMEGA kit method. The mutation results in deep sequencing were verified by Sanger sequencing technique. The expression of HECW1 protein in cancer tissues and paracancerous tissues was observed by immunohistochemical Envision two-step method. Results: the first part: the quality of deep sequencing data was satisfactory, many mutation genes were found, and 10 of them had missense mutation, including 4 non-invasive genes, which were IVL,RGPD5,ZNF79,SRRM5;. Infiltration group 3, CPNE7,RBMXL3,ACSM2A; group 3, HECW1,ZNF273,TCHH. The second part: 6 cases (6.06%) of HECW1 gene mutations were found in 99 cases of bladder cancer, all of them were point mutations, located in exon 11, and were the same as or in the vicinity of deep sequencing mutation. Of 38 cases of non-invasive bladder cancer, 1 case had synonymous mutation (2.63%). In 61 cases of invasive bladder cancer DNA, 4 missense mutations and 1 nonsense mutation (i.e. mutation as termination codon) were found in 4 cases (8.20%). There was no significant difference between the two mutations (P0.05), while no mutation was found in 10 cases in the control group. In immunohistochemical staining, 9 cases were positive in 15 cases of tumor tissue, and 1 case was positive in 10 cases of control group. The difference was statistically significant (P0.05). Conclusion: (1) Deep sequencing, especially total exon sequencing, (2) Sanger sequencing is the gold standard for testing the results of deep sequencing. (3) HECW1 may be a candidate gene related to the occurrence and development of bladder cancer. The expression of HECW1 protein in bladder cancer tissues is higher than that in paracancerous tissues. (5) the mechanism of HECW1 gene mutation in bladder cancer and whether it affects the expression and function of HECW1 protein remains to be further studied.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.14
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本文編號(hào)：2352228
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