稳定沉默钙网蛋白表达通过下调VEGF、MMP2/9的表达抑制SNK6细胞侵袭能力

Silencing Calreticulin Expression Inhibits Invasion Ability of SNK6 Cells in Vitro via Down-Regulating Expression of VEGF and MMP2/9

目的:探索稳定下调钙网蛋白(calreticulin,CALR)对NK/T细胞淋巴瘤细胞SNK6细胞侵袭性的影响及其可能机制。方法:设计靶向人钙网蛋白的特异性短发卡RNA(short hairpin RNA,shRNA)序列,构建重组慢病毒载体;三质粒系统包装慢病毒颗粒,感染SNK6细胞,嘌呤霉素抗性筛选稳定下调钙网蛋白表白的SNK6细胞。应用CCK8法测定细胞增殖活力,Transwell小室测定细胞侵袭。Western blot检测SNK6细胞CALR表达水平,荧光定量PCR检测SNK6细胞CALR、MMP2、MMP9和VEGF的转录水平。结果:成功构建重组慢病毒载体pLKO.1-puro-shCALR,并包装慢病毒,筛选出稳定下调钙网蛋白的SNK6细胞株。稳定下调SNK6细胞钙网蛋白表达后,SNK6侵袭性减弱,MMP2、MMP9和VEGF的转录水平降低。结论:稳定下调SNK6细胞钙网蛋白的表达可使SNK6细胞侵袭性减弱,其机制可能与降低MMP2、MMP9和VEGF的转录水平有关。

Objective:To investigate the effect of steadily down-regulating the expression of calreticulin(CALR)on the invasion of natural killer/T-cell lymphoma SNK6 cells,and explore its possible mechanism.Methods:The sequences of specific short hairpin RNA(shRNA)targeting on human CALR were designed,and were inserted into pLKO.1-puro lentivirus vector,and the reconbinant lentivirus vector was obtained;the lentivirus particles were backed by three-plasmid system and transfected into SNK6 cells,the SNK6 cells stably down-regulating the CALR expression were sercened by puromytain,the CALR-silencing effect was verified by real-time PCR and Western blot.CCK-8 assay was used to evaluate the cell viability,The transwell invasion assays was used to analyse invasion of SNK6 cells.The mRNA expression of Calreticulin,MMP2,MMP9 and VEGF was determined by real time PCR,the protein expression of Calreticulin and GAPDH was analyzed by Western blot.Results:The recombinant lentiviral vector pLKO.1-puroshCALR was successfully constructed,packed into the lentivirus,then the SNK6 cells stably down-regulating Calreticulin expression was obtained.When Calreticulin was down-rengulated in SNK6 cells,the proliferation rate was reduced and the invasion ability was decreased;the mRNA levels of VEGF and MMP-2/9 also were reduced.Conclution:The stable down-regnlation of CALR expression in SNK6 cells can attenuate the imvasiveness of SNK6 cells,which maybe related with transcriptional decrease of MMP2,MMP9 and VEGF.