CBX2在鼻咽癌的表达及其对鼻咽癌细胞生物学行为的影响

Expression of CBX2 in nasopharyngeal carcinoma and its effect on biological behavior of nasopharyngeal carcinoma cells

目的 目前关于色素框同源蛋白2(CBX2)在鼻咽癌中的作用报道较少。文章旨在研究CBX2在鼻咽癌中的表达及其生物学功能。方法 采用实时荧光定量PCR检测41例鼻咽癌组织和35例鼻咽慢性黏膜炎组织标本的CBX2 mRNA表达水平。设计并合成CBX2-target的小干扰RNA序列:siNC:正义链,5′-UUCUCCGAACGUGUCACGUTT-3′;反义链,5′-ACGUGACACGUUCGGAGAATT-3′。CBX2-siRNA#1正义链,5′-GGAUGACAGUGACUUAGAUTT-3′;反义链,5′-AUCUAAGUCACUGUCAUCCTT-3′。CBX2-siRNA#2:正义链,5′-CCAGAUCCUGGUGGCCAAATT-3′;反义链,5′-UUUGGCCACCAGGAUCU GGTT-3′。小干扰RAN(siRNA)瞬时转染鼻咽癌SUNE1细胞株,分别采用CCK-8法和平板克隆形成实验、流式细胞检测技术及Transwell法分析CBX2敲低表达后SUNE1细胞增殖、迁移和侵袭能力的变化;Western blot检测相关通路蛋白表达水平。结果 CBX2在鼻咽癌细胞系及组织中的表达水平均显著高于正常鼻咽上皮细胞系及鼻咽炎症组织。CBX2-siRNA#1和#2两个小干扰片段(0.19±0.11, 0.27±0.06)相比较siNC(0.59±0.06)敲低了CBX2的表达(P<0.01)。敲低CBX2表达后,SUNE1细胞细胞增殖速度、克隆形成数目均显著降低(P<0.01)。并且siRNA#1、#2 CBX2敲低后细胞迁移(56.33±3.51, 156.33±4.73)和侵袭数目(61.33±5.86, 118.67±4.73)相比siNC (225.00±3.60、199.33±5.13)显著减少(P<0.01)。细胞周期也发生显著改变,与siNC相比,siRNA#1组细胞周期发生了S期阻滞,而siRNA#2组发生G0/G1期阻滞。Western blot结果显示,CBX2敲低表达组β-catenin及LRP6、Cyclin D1蛋白表达较siNC明显下降(P<0.01)。结论 CBX2在鼻咽癌中的表达水平升高,且CBX2表达可能通过改变Wnt/β-catenin信号通路蛋白表达影响SUNE1细胞的增殖、迁移和侵袭。

Objective At present, there are few reports on the role of chromobox homolog 2(CBX2) in nasopharyngeal carcinoma. The aim of this paper is to investigate the expression and biological function of CBX2 in nasopharyngeal carcinoma. MethodsThe expression of CBX2 mRNA in 41 nasopharyngeal carcinoma tissues and 35 nasopharyngeal chronic mucositis tissues was detected by real-time PCR. The small interfering RNA sequence of CBX2 target was designed and synthesized: siNC: sense, 5’UUCUCCGAACGUGUCACGUTT3’;antisense, 5’ACGUGACACGUUCGGAGAATT3’. CBX2-siRNA#1 sense, 5’GGAUGACAGUGACUUAGAUTT3’;antisense, 5’AUCUAAGUCACUGUCAUCCTT3’. CBX2-siRNA#2: sense, 5’CCAGAUCCUGGUGGCCAAATT3;antisense, 5’UUUGGCCACCAGGAUCUGGTT3’. Small interference RAN(siRNA) was transiently transfected into nasopharyngeal carcinoma SUNE1 cell line. CCK-8 assay, plate clone formation assay, flow cytometry and Transwell assay were used to analyze the proliferation, migration and invasion of SUNE1 cells after CBX2 knockdown expression. The protein expression levels of related pathways were detected by Western blot. Results The expression level of CBX2 in nasopharyngeal carcinoma cell lines and tissues was significantly higher than that in normal nasopharyngeal epithelial cell lines and nasopharyngitis tissues. CBX2 siRNA#1 and #2(0.19±0.11, 0.27±0.06) knockdown the expression of CBX2 compared with siNC(0.59±0.06)(P<0.01). After knockdown of CBX2 expression, the proliferation rate and clone formation number of SUNE1 cells were significantly decreased(P<0.01). After siRNA#1 and #2 CBX2 knockdown, cell migration(56.33±3.51, 156.33±4.73) and invasion number(61.33±5.86) were observed. Compared with siNC(225.00±3.60, 199.33±5.13), siNC(118.67±4.73) was significantly decreased(P<0.01). The cell cycle was also significantly altered. Compared with siNC, siRNA#1 cell cycle was arrested in S phase and siRNA#2 cell cycle was arrested in G0/G1 phase. Western blot results showed that the protein expressions of β-catenin, LRP6 and Cyclin D1 in CBX2 knockdown group were significantly decreased compared with siNC(P<0.01). Conclusion The expression level of CBX2 in nasopharyngeal carcinoma is increased, and CBX2 expression may affect the proliferation, migration and invasion of SUNE1 cells by changing the protein expression of Wnt/β-catenin signaling pathway.