Objective: To investigate the effect of LNK gene silencing and overexpression on the expression of STAT3 gene in human monocytic leukemia cells (THP-1).
Methods: THP-1 cells were cultured, and the lentivirus was used as a vector to silence and overexpres the LNK gene stably. After transfection for 72 hours, the GFP expression levels were observed by inverted fluorescence microscopy. The lentiviral transfection efficiencies were detected by flow cytometry. The effects of LNK silencing and overexpression were confirmed, and the expression of STAT3 mRNA was detected by RT-PCR. The protein levels of LNK and STAT3 were detected by Western blotting.
Results: The GFP expression level of THP-1 cells reached more than 85% after transfection with lentivirus for 72 hours, and the transfection efficiency of cells was above 99%. mRNA expressions levels of LNK and STAT3 in LNK silencing group were signifycantly lower than those in control group, while LNK and STAT3 mRNA levels in the LNK overexpression group was significantly higher than those in control group (P＜0.05). The protein expression levels of LNK and STAT3 in LNK silencing group were significantly lower than those in control group, while that in LNK overexpression group was significantly higher than that in control group (P＜0.05).
Conclusion: The THP-1 cell line with LNK gene silencing and overexpression has been successfully established. The LNK gene silencing resulted in decrease of STAT3 expression; LNK gene overexpression and leads to inereases of STAT3 expression indicating that LNK participates in the regulation of STAT3.
题目: LNK基因沉默和过表达对THP-1细胞STAT3表达影响的研究.
目的: 探讨LNK基因沉默和过表达对人单核细胞白血病细胞（THP-1）STAT3基因表达的影响.
方法: 培养THP-1细胞；以慢病毒为载体介导SiRNA、LNK（SH2B3）稳定沉默及过表达LNK基因；转染72 h后于倒置荧光显微镜下观察细胞的GFP表达量；应用流式细胞术检测慢病毒感染效率；RT-PCR验证LNK沉默及过表达效应，检测STAT3 mRNA表达量；Western blot检测LNK、STAT3蛋白水平.
结果: 慢病毒感染72 h THP-1细胞GFP表达量达85%以上，细胞感染效率在99%以上。LNK沉默组LNK及STAT3 mRNA表达量明显低于对照组，而LNK过表达组的LNK和STAT3 mRNA表达水平明显高于对照组（P＜0.05）。LNK沉默组LNK、STAT3蛋白水平明显低于对照组，而在LNK过表达组明显高于对照组（P＜0.05）.
结论: 成功建立了LNK基因沉默及过表达的THP-1细胞株。沉默LNK基因导致STAT3表达下调；过表达LNK基因，使STAT3表达上调；此结果提示LNK可能参与了STAT3的调控.