Wu Ming-Hua, Li Xiao-Ling, Huang Chen, Tang Yun-Lian, Zhang Zu-Ping, Li Gui-Yuan
Cancer Research Institute, Central South University, Changsha, China.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2007 Apr;32(2):231-4.
To examine the expression absence of LRRC4 gene in glioblastoma cell lines.
RT-PCR and Northern blot were used to detect the expression of LRRC4 gene in 6 glioblastomas cells lines. Polymerase chain reaction and DNA sequencing were used to screen the LRRC4 gene mutation, while bioinformation assay was used to search for the reason of LRRC4 gene absence in U251 cell lines.
The expression of LRRC4 was absent in 6 malignant glioma cell lines (U251, U87, BT325, SF126, SF767 and M17), which were examined by Northern-blot and RT-PCR assay. All sequencing of PCR products from gDNA of SF126, SF767, and M17 cell lines contained the point mutation at the same position ( LRRC4 geneT977A) (3/5), which was a synonymous mutation. However, PCR products from gDNA of U251 and U87 cell lines (2/5) were not obtained. The expression absence of LRRC4 was ascribed to the loss of homozygosity of 7q32-ter in U251 cell lines.
The expression of LRRC4 gene is absent in glioblastoma cell lines, and it offers the important experiment proof for LRRC4 to act as a new candidate of brain tumor suppressor gene from glioma. The loss of homozygosity of 7q32-ter contributed to the expression absence of LRRC4 in U251 cell lines.
检测胶质母细胞瘤细胞系中LRRC4基因的表达缺失情况。
采用逆转录聚合酶链反应(RT-PCR)和Northern印迹法检测6种胶质母细胞瘤细胞系中LRRC4基因的表达。利用聚合酶链反应和DNA测序筛选LRRC4基因突变,同时采用生物信息学分析探究U251细胞系中LRRC4基因缺失的原因。
通过Northern印迹法和RT-PCR检测发现,6种恶性胶质瘤细胞系(U251、U87、BT325、SF126、SF767和M17)均无LRRC4表达。SF126、SF767和M17细胞系基因组DNA的PCR产物测序结果显示,在同一位置(LRRC4基因T977A)存在点突变(3/5),此为同义突变。然而,未获得U251和U87细胞系基因组DNA的PCR产物(2/5)。U251细胞系中LRRC4表达缺失归因于7q32-ter的纯合性缺失。
胶质母细胞瘤细胞系中不存在LRRC4基因表达,这为LRRC4作为胶质瘤脑肿瘤抑制基因的新候选者提供了重要实验证据。7q32-ter的纯合性缺失导致U251细胞系中LRRC4表达缺失。