Functional and mechanistic of TCF3 regulating linc00152 on carcinogenic effects in Burkitt's lymphoma
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摘要:
目的 探讨TCF3通过linc00152调控Daudi和Raji细胞增殖和凋亡中的功能和机制。 方法 采用实时定量聚合酶链反应(qPCR)检测linc00152的干扰效率；利用CCK8、Western blotting和流式细胞术检测linc00152对Daudi和Raji细胞增殖和凋亡的影响；ChIP-PCR检测TCF3与linc00152的启动子区结合；qPCR和Western blotting检测TCF3的干扰效率；qPCR检测TCF3对linc00152表达水平的影响。 结果 在Daudi和Raji细胞中siRNA能成功干扰linc00152的表达；CCK8结果显示，干扰linc00152能抑制Daudi和Raji细胞增殖，Western blotting和流式结果显示干扰linc00152能促进细胞凋亡(Daudi: 35.30±2.84 vs. 6.95±0.82; Raji: 33.17±2.18 vs. 6.47±0.38)；ChIP-PCR结果显示，TCF3能与linc00152的启动子结合；qPCR和Western blotting结果显示，TCF3的mRNA和蛋白水平均能被siRNA成功干扰；在Daudi和Raji细胞中，si-TCF3能降低linc00152的表达量(Daudi: 0.509±0.048 vs. 0.906± 0.146;Raji: 0.502±0.052 vs. 0.966±0.103)。 结论 linc00152能促进Daudi和Raji细胞增殖，抑制细胞凋亡，可能与TCF3对linc00152的调控有关。 Abstract:Objective To investigate the function and mechanism of TCF3 in regulating proliferation and apoptosis of Daudi and Raji cells through linc00152. Methods The interference efficiency of linc00152 was detected by real-time quantitative polymerase chain reaction (qPCR). CCK8, Western blotting, and flow cytometry were used to detect the effects of linc00152 on the proliferation and apoptosis of Daudi and Raji cells. ChIP-PCR detected the binding of TCF3 to the promoter region of linc00152. qPCR and Western blotting were used to detect the interference efficiency of TCF3. qPCR was used to detect the effect of TCF3 on the expression level of linc00152. Results siRNA successfully interfered with the expression of linc00152 in Daudi and Raji cells. CCK8 results showed that interference of linc00152 could inhibit the proliferation of Daudi and Raji cells, while Western blotting and flow cytometry results showed that interference of linc00152 could promote cell apoptosis (Daudi: 35.30±2.84 vs. 6.95±0.82; Raji: 33.17±2.18 vs. 6.47±0.38). ChIP-PCR results showed that TCF3 could bind to the promoter of linc00152. The results of qPCR and Western blotting showed that both the mRNA and protein levels of TCF3 could be successfully interfered by siRNA. In Daudi and Raji cells, si-TCF3 could reduce the expression of linc00152 (Daudi: 0.509±0.048 vs. 0.906± 0.146; Raji: 0.502±0.052 vs. 0.966±0.103). Conclusion Linc00152 can promote the proliferation of Daudi and Raji cells and inhibit apoptosis, which may be related to the regulation of linc00152 by TCF3. -
Key words:
- Burkitt lymphoma /
- Linc00152 /
- TCF3 /
- Proliferation /
- Apoptosis
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图 1 qPCR检测linc00152的干扰效率
注：与Si-NC组比较，aP < 0.05。
Figure 1. Quantification of linc00152 interference efficiency using qPCR
图 2 干扰linc00152对Daudi和Raji细胞增殖和凋亡的影响
Figure 2. Effect of linc00152 interference on proliferation and apoptosis in Daudi and Raji cells
图 3 ChIP检测TCF3结合在linc00152的启动子区
Figure 3. Detection of TCF3 binding in the promoter region of linc00152 using ChIP
图 4 qPCR和WB检测TCF3的干扰效率
Figure 4. Assessment of TCF3 interference efficiency using qPCR and WB analysis
图 5 qPCR检测干扰TCF3对linc00152表达的影响
Figure 5. qPCR-based detection of linc00152 expression modulation upon TCF3 interference
表 1 qPCR引物序列
Table 1. The primer sequences of qPCR
基因名称 引物序列(5'-3') 片段长度(bp) GAPDH 上游引物 TCAAGAAGGTGGTGAAGCAGG 115 下游引物 TCAAAGGTGGAGGAGTGGGT linc00152 上游引物 GCTCCTGGCACAGTCTTTTC 207 下游引物 AGGTTGGAATGTGGATGGAG TCF3 上游引物 AGCCTCTCTTCATCCACATTCC 260 下游引物 ACCTTCTTGGGCTGCGTGTCTA TCF3结合位点-1 上游引物 CTACTCTGAACTGCCTTGAAAA 204 下游引物 AGAGAGACCCAAACACTGACCC TCF3结合位点-2 上游引物 TGAGCCTGTAGCACATTACAAA 164 下游引物 AGAGAGACCCAAACACTGACCC TCF3结合位点-3 上游引物 CATGAGCCTGTAGCACATTACAAA 260 下游引物 CTCCCGACATTCTCTTCATACTTG 
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表 2 干扰linc00152后Daudi和Raji细胞凋亡率和相关蛋白表达水平比较(x±s)
Table 2. Comparative analysis of apoptosis rates and related protein expression levels in Daudi and Raji cells upon linc00152 interference (x±s)
组别 n Daudi细胞 Raji细胞 cl-Caspase-3/
GAPDHBax/GAPDH 细胞凋亡率
(%)cl-Caspase-3/
GAPDHBax/GAPDH 细胞凋亡率
(%)Control组 3 0.39±0.04 0.55±0.05 6.19±0.67 0.32±0.02 0.40±0.01 6.21±0.47 si-NC组 3 0.42±0.03 0.54±0.05 6.95±0.82 0.37±0.06 0.39±0.07 6.47±0.38 si-linc00152组 3 0.73±0.12ab 0.90±0.03ab 35.30±2.84ab 0.60±0.03ab 0.88±0.03ab 33.17±2.18ab F值 18.870 64.119 269.682 40.959 119.644 422.006 P值 0.003 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 注：与Control组比较，aP＜0.05；与si-NC组比较，bP＜0.05。
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