摘要
目的利用全外显子测序寻找与卵巢癌发生发展相关的分子靶标,并进行体外细胞实验验证其可能影响卵巢癌发生的机制。方法本研究通过对8对浆液性卵巢癌组织进行全外显子测序,验证实验进一步对另外43个浆液性卵巢癌组织和156位正常女性的外周血标本进行目的基因测序。在体外实验中用Lipofectamine 3000做载体,将含CREBBP基因L1850fs和P1373S突变的质粒转染人浆液性卵巢癌细胞株A2780,以含CREBBP野生型的质粒和空载体为对照,采用Western blot法检测CREBBP的L1850fs和P1373S突变对环磷腺苷效应元件(CREB)的结合蛋白(CBP)蛋白表达的影响;CCK-8检查L1850fs和P1373S突变对细胞增殖的影响;Transwell小室试验检测L1850fs和P1373S突变对细胞迁移的影响;划痕试验检测L1850fs和P1373S突变对细胞侵袭的影响。结果通过全外显子测序发现了浆液性卵巢癌的2个高频突变位点——CREBBP的L1850fs(c.5548dupC)和P1373S(c.4117C>T)。L1850f是移码突变,P1373S是非同义序列。在51例浆液性卵巢癌病例中(全外显子测序8例,目的基因测序43例),CREBBP基因的突变率为41.2%,而正常女性外周血中突变率为21.8%,差异有统计学意义(χ^(2)=7.4,P=0.007)。Western blot结果显示,L1850fs突变能降低卵巢癌细胞中其相应CBP蛋白的表达,而P1373S突变对蛋白表达无影响。CCK-8分析表明,突变型CREBBP可促进卵巢癌细胞增殖。Transwell小室试验结果显示L1850fs和P1373S突变可促进卵巢癌细胞迁移。划痕试验分析表明,CREBBP基因突变可提高卵巢癌细胞侵袭能力。结论本实验通过全外显子测序发现了浆液性卵巢癌相关的两个点突变,CREBBP L1850fs和P1373S。进一步的细胞实验表明该突变可以通过影响CBP蛋白促进卵巢癌细胞增殖、侵袭、转移等,提示CREBBP L1850fs和P1373S可能成为卵巢癌诊断及治疗的新靶点。
Objective To find molecular targets related to the occurrence and development of ovarian cancer induced by whole-exome sequencing and to verify the mechanism in vitro cell experiments.Methods 8 pairs of serous ovarian cancer tissues were sequenced using whole-exome,and another 43 serous ovarian cancer tissues and 156 peripheral blood samples of normal women were performed target gene sequencing.In the in vitro experiment,the plasmids containing the CREBBP gene L1850fs and P1373S mutations were transfected into human serous ovarian cancer cell line A2780 with Lipofectamine 3000,and the plasmids containing wild-type CREBBP and the empty vector were used as controls.CRP protein expression after CREBBP L1850fs and P1373S mutated was evaluated by Western blot;cell proliferation of L1850fs and P1373S mutation was detected by CCK-8 assay;cell migration of L1850fs and P1373S mutation was detected by Transwell chamber test;cells invasion of L1850fs and P1373S mutation was detected by scratch test.Results Two high-frequency mutation sites of CREBBP gene on L1850fs(c.5548dupC)and P1373S(c.4117C>T)in serous ovarian cancer tissues were discovered using whole exome sequencing.L1850f was a frameshift mutation,and P1373S was a non-synonymous sequence.In 51 cases of serous ovarian cancer(8 cases of whole exome sequencing,43 cases of target gene sequencing),the mutation rate of CREBBPgene was 41.2%,while the mutation rate of normal female peripheral blood was 21.8%,the difference was statistically significant(χ^(2)=7.4,P=0.007).The results of western blotting showed that the L1850fs mutation could reduce the expression of CBP in ovarian cancer cell,while the P1373S mutation had no significant effect on CBP protein expression.CCK-8 analysis showed that mutant CREBBP could promote the proliferation of ovarian cancer cells.Transwell chamber test showed that L1850fs and P1373S mutation could promote the migration of ovarian cancer cells.Scratch test analysis showed that L1850fs and P1373S mutation could increase the invasion ability of ovarian cancer cells.Conclusion In this experiment,two point mutations of CREBBP in L1850fs and P1373S were found in serous ovarian cancer using whole exome sequencing.Further cell experiments showed that the two mutation could promote the proliferation,invasion and metastasis of ovarian cancer cells by affecting the CBP protein,suggesting that CREBBP L1850fs and P1373S might become new targets for the diagnosis and treatment of ovarian cancer.
作者
苏丹
郭晓霞
岳军
梅劼
Su Dan;Guo Xiaoxia;Yue Jun;Mei Jie(Dept of Gynecology and Obstetrics, Sichuan Academy of Medical Sciences & Sichuan Provincial People′s Hospital, Chengdu 610072)
出处
《安徽医科大学学报》
CAS
北大核心
2022年第5期695-701,共7页
Acta Universitatis Medicinalis Anhui
基金
四川省科技厅项目(编号:2015SZ0243)
四川省卫计委项目(编号:17PJ240)
成都市科技局项目(编号:2019-YF05-00244-SN)。
