LRRC4通过LRR结构域抑制脑胶质母细胞瘤U251细胞的生长和侵袭

Suppression of Growth and Invasion Phenotypes in Glioblastoma U251 Cells by LRR Domain of LRRC4

LRRC4基因是候选的脑胶质瘤抑制基因,其细胞外区域含有一个保守的LRR和一个IgC2结构域.本研究结合生物信息学分析,通过一步PCR法构建了不同结构域缺失的突变体(pcΔLRR,pcΔIgC2或pcΔTm).并将全长LRRC4(pcLRRC4)和各突变体转染至U251细胞,构建了稳定表达的U251细胞系.通过MTT,软琼脂集落形成及Transwell体外侵袭模型检测发现,全长LRRC4能够抑制U251细胞的生长和侵袭;LRR结构域缺失的突变体不再抑制U251细胞的生长和侵袭;而IgC2或Tm区缺失的突变体仍然可以抑制U251细胞的生长和侵袭.该结果表明,LRRC4抑制U251细胞的生长和侵袭依赖于它的LRR结构域,而不是IgC2或Tm结构域.

LRRC4 was originally isolated following position cloning strategy and 5 ＇-RACE. LRRC4 is regarded as a candidate tumor suppressor gene and mapped to human chromosome 7q31-32. Analysis of LRRC4 amino acid sequence using SMART software indicated the presence of a conserved extracellular LRR （Leucine-rich repeat） domain and an IgC2 domain. In this study, deletion mutant vectors, pcALRR, pcΔIgC2 or pcΔTm（transmembrane domain）, were constructed through One-step PCR strategy. The LRRC4 mutants and the full length wildtype were transfected into U251 cells by liposome transfection, and stable expression cell lines were prepared. We found that the full length LRRC4 inhibited U251 cell proliferation by MTY, soft agar clony formation assay and blocked cell invasion by transwell invasion assay, whereas the LRR domain deletion mutant abolished these anti-proliferation and anti-invasion effects. However, the mutants of IgC2 or Tm deletion behaved similarly to LRRC4 in cell proliferation and invasion assays. We conclude that the LRR domain in LRRC4 is responsible for the growth and invasion suppression in U251 cells.