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目的:探索肝脏激酶B1(liver kinase B1,LKB1或serine-threonine kinase 11,STK11)基因协同二甲双胍对人宫颈癌HeLa细胞增殖与凋亡的影响及其可能的机制。方法:构建含有LKB1基因的重组质粒LKB1-pEGFP-n1并转染HeLa细胞,MTT检测LKB1基因对经二甲双胍处理的HeLa细胞增殖的影响,流式细胞术检测对细胞周期和凋亡的影响,Western blotting检测对LKB1-AMPK信号通路相关蛋白AMPK、ACC和Rb磷酸化水平的影响。结果:LKB1-pEGFP-n1和空载体pEGFP-n1成功转染HeLa细胞,LKB1-pEGFP-n1转染细胞内稳定表达LKB1。二甲双胍处理LKB1-pEGFP-n1转染细胞的IC50显著低于pEGFP-n1转染细胞[(2.9±0.4)vs(7.8±1.3)mmol/L;t=-6.9921,P=0.002 2]及野生型HeLa细胞[(2.9±0.4)vs(9.6±1.5)mmol/L;t=-7.527 1,P=0.001 7]。经二甲双胍处理后,LKB1-pEGFP-n1转染细胞周期阻滞于G1期,而pEGFP-n1转染和野生型细胞周期无显著变化。二甲双胍作用剂量为15 mmol/L时,LKB1-pEGFP-n1转染细胞凋亡率较pEGFP-n1转染及野生型HeLa细胞显著增多[(28.6±2.3)%vs(9.6±1.6)%、(17.8±1.9)%,均P<0.05]。LKB1-pEGFP-n1转染细胞内AMPKα和ACC的磷酸化水平较pEGFP-n1转染和野生型细胞升高,Rb磷酸化水平降低。结论:LKB1能够协同二甲双胍影响HeLa细胞的增殖与凋亡,其可能通过LKB1-AMPK信号通路发挥作用。
Objective: To explore the effect of liver kinase B1 (LKB1 or serine-threonine kinase 11, STK11) gene and metformin on the proliferation and apoptosis of human cervical cancer HeLa cells and its possible mechanism. Methods: The recombinant plasmid LKB1-pEGFP-n1 containing LKB1 gene was constructed and transfected into HeLa cells. The effect of LKB1 gene on the proliferation of HeLa cells treated with metformin was examined by MTT assay. The cell cycle and apoptosis were detected by flow cytometry. blotting was used to detect the phosphorylation of AMPK, ACC and Rb in LKB1-AMPK signaling pathway. Results: HeLa cells were successfully transfected with LKB1-pEGFP-n1 and empty vector pEGFP-n1, and LKB1 was stably expressed in LKB1-pEGFP-n1 transfected cells. The IC50 of metformin-treated LKB1-pEGFP-n1 transfected cells was significantly lower than that of pEGFP-n1 transfected cells [(2.9 ± 0.4 vs 7.8 ± 1.3) mmol / L; t = -6.9921, P = 0.002 2] HeLa cells [(2.9 ± 0.4) vs (9.6 ± 1.5) mmol / L; t = -7.527 1, P = 0.001 7]. After treatment with metformin, the cell cycle of LKB1-pEGFP-n1 transfection was blocked in G1 phase, but there was no significant change in pEGFP-n1 transfection and wild-type cell cycle. Compared with pEGFP-n1 transfected and wild-type HeLa cells, the apoptotic rate of LKB1-pEGFP-n1 transfected cells was significantly increased [(28.6 ± 2.3)% vs (9.6 ± 1.6)%] at a dose of 15 mmol / 17.8 ± 1.9)%, all P <0.05]. Phosphorylation of AMPKα and ACC in LKB1-pEGFP-n1 transfected cells was higher than that in pEGFP-n1 transfected and wild-type cells, and the level of Rb phosphorylation was decreased. Conclusion: LKB1 can affect the proliferation and apoptosis of HeLa cells in combination with metformin, which may play a role through the LKB1-AMPK signaling pathway.