支链氨基酸分解代谢在肺癌细胞中的功能

doi:10.3969/j.issn.1674-8115.2021.07.003

摘要/Abstract

摘要：

目的·探索支链氨基酸（branched-chain amino acid，BCAA）分解代谢在肺癌细胞中的功能和作用机制。方法·采用瞬时转染法分别向非小细胞肺癌细胞H1299、A549和HCC827转染含无义序列的小干扰RNA（small interfering negative control，siNC）、支链酮酸脱氢酶激酶（branched-chain keto acid dehydrogenase kinase，BCKDK）基因的小干扰RNA（small interfering BCKDK，siBCKDK），并采用蛋白质印迹法（Western blotting）检测siNC组和siBCKDK组中BCKDK表达水平、支链酮酸脱氢酶亚基E1α（branched-chain keto acid dehydrogenase e1，α polypeptide，BCKDE1α）及其磷酸化水平。采用CCK-8法检测上述2组细胞的增殖活力。用含0、100、200 μmol/L的BCKDK小分子抑制剂BT2（3，6-二氯-2-苯并噻吩羧酸）培养液分别培养上述3种细胞，采用Western blotting检测3种浓度BT2组细胞中BCKDE1α及其磷酸化水平。用CCK-8法检测0 μmol/L BT2组和200 μmol/L BT2组细胞的增殖活力。采用台盼蓝染色计算H1299和A549细胞中siNC组和siBCKDK组、0 μmol/L BT2组和200 μmol/L BT2组的细胞活率；并用碘化丙啶染色检测细胞周期，而后行Western blotting检测细胞周期依赖性蛋白激酶抑制因子1A（cyclin-dependent kinase inhibitor 1A，P21）的表达水平。结果·在H1299、A549、HCC827细胞中，与siNC组相比，siBCKDK组的BCKDK表达下调，BCKDE1α磷酸化水平下调，且细胞的增殖活力减弱（均P=0.000）。与0 μmol/L BT2组相比，100 μmol/L BT2组和200 μmol/L BT2组的上述3种细胞中BCKDE1α磷酸化水平下调，且200 μmol/L BT2组下调更明显；200 μmol/L BT2组较0 μmol/L BT2组的细胞增殖活力减弱（均P=0.000）。在H1299、A549细胞中，分别与siNC组、0 μmol/L BT2组相比，siBCKDK组、200 μmol/L BT2组的细胞活率均无差异，但发生了G0/G1期细胞周期阻滞（均P<0.05），且该2组的P21表达水平升高。结论·siBCKDK和BT2能够促进BCKDE1α去磷酸化，加快BCAA分解代谢，可能通过上调P21、阻滞细胞周期来抑制肺癌细胞的增殖。

关键词: 支链氨基酸, 肺癌, 细胞增殖

Abstract:

Objective·To explore the function and mechanism of branched-chain amino acid (BCAA) catabolism in lung cancer cells.

Methods·Small interfering negative control (siNC) and small interfering branched-chain keto acid dehydrogenase kinase (siBCKDK) were transfected into non-small cell lung cancer cells H1299, A549 and HCC827 by instantaneous transfection. The expression of BCKDK, branched-chain keto acid dehydrogenase e1, α polypeptide (BCKDE1α) and its phosphorylation in siNC group and siBCKDK group were detected by Western blotting. The proliferation activity of the above two groups of cells was detected by CCK-8 assay. The above three kinds of cells were cultured in culture medium containing 0, 100, 200 μmol/L BT2 (3, 6-dichlorobenzo[b]thiophene-2-carboxylic acid), respectively. The expression of BCKDE1α and its phosphorylation were detected by Western blotting. The proliferation activity of 0 μmol/L BT2 group and 200 μmol/L BT2 group was detected by CCK-8 assay. The cell viability of siNC group and siBCKDK group, 0 μmol/L BT2 group and 200 μmol/L BT2 group in H1299 and A549 cells was calculated by trypan blue staining. Cell number at different phases of cell cycle was detected by propidium iodide staining. Cyclin-dependent kinase inhibitor 1A (P21) expression was detected by Western blotting.

Results·In H1299, A549 and HCC827 cells, compared with siNC group, expression of BCKDK and phosphorylation of BCKDE1α in siBCKDK group were down-regulated, and the proliferation activity was decreased (all P=0.000). Compared with 0 μmol/L BT2 group, the phosphorylation of BCKDE1α was down-regulated in H1299, A549 and HCC827 cells in both 100 μmol/L BT2 group and 200 μmol/L BT2 group, but more obviously in 200 μmol/L BT2 group. The cell proliferation activity in 200 μmol/L BT2 group was decreased compared with that in 0 μmol/L BT2 group (all P=0.000). There was no significant difference in the cell viability of H1299 and A549 cells between siBCKDK group and siNC group, 200 μmol/L BT2 group and 0 μmol/L BT2 group, but G0/G1 cell cycle arrest occurred (all P<0.05) with the expression of P21 increasing.

Conclusion·siBCKDK and BT2 can promote the dephosphorylation of BCKDE1α and accelerate the catabolism of BCAA, which may inhibit the proliferation of lung cancer cells by up-regulating P21 and blocking cell cycle.

Key words: branched-chain amino acids (BCAA), lung cancer, cell proliferation

中图分类号:

R363.2⁺1

贺艳琪, 迟锐, 陈梦萍, 陈思, 刘春良, 刘云霞, 孙海鹏. 支链氨基酸分解代谢在肺癌细胞中的功能[J]. 上海交通大学学报(医学版), 2021, 41(7): 858-864.

Yan-qi HE, Rui CHI, Meng-ping CHEN, Si CHEN, Chun-liang LIU, Yun-xia LIU, Hai-peng SUN. Function of branched-chain amino acid catabolism in lung cancer cells[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(7): 858-864.

图/表 7

图1 siBCKDK对NSCLC细胞中BCKDK蛋白表达及BCKDE1α磷酸化水平的影响Note： A?C. Expression levels of BCKDK protein and BCKDE1α phosphorylation in H1299 cells (A), A549 cells (B) and HCC827 cells (C) treated by siBCKDK.

Fig 1 Effect of siBCKDK on BCKDK protein expression and BCKDE1α phosphorylation in NSCLC cells

图2 siBCKDK对NSCLC细胞增殖能力的影响Note： A?C. Growth curves of H1299 cells (A), A549 cells (B) and HCC827 cells (C) after BCKDK was knocked down by siBCKDK. ①P=0.000, compared with the siNC group.

Fig 2 Effect of siBCKDK on proliferation of NSCLC cells

图3 BT2对NSCLC细胞中BCKDE1α磷酸化水平的影响Note： A?C. Expression of BCKDE1α phosphorylation in H1299 cells (A), A549 cells (B) and HCC827 cells (C) after BT2 treatment.

Fig 3 Effect of BT2 on phosphorylation of BCKDE1α in NSCLC cells

图4 BT2对NSCLC细胞增殖能力的影响Note： A?C. Growth curves of H1299 cells (A), A549 cells (B) and HCC827 cells (C) after BT2 treatment. ①P=0.000, compared with the 0 μmol·L-1 BT2 group.

Fig 4 Effect of BT2 on proliferation of NSCLC cells

图5 siBCKDK或BT2对NSCLC细胞活率的影响Note： A/B. Cell viability of H1299 cells treated with siBCKDK (A) or 200 μmol·L-1 BT2 (B). C/D. Cell viability of A549 cells treated with siBCKDK (C) or 200 μmol·L-1 BT2 (D). ①P=0.0811, ③P=0.477, compared with the siNC group; ②P=0.226, ④P=0.175, compared with the 0 μmol·L-1 BT2 group.

Fig 5 Effects of siBCKDK or BT2 on the survival rate of NSCLC cells

图6 siBCKDK或BT2对NSCLC细胞周期的影响Note： A/B. Cell percentage of H1299 cell cycle at different stages after BT2 (A) or siBCKDK (B) treatment. C/D. Cell percentage of A549 cell cycle at different stages after BT2 (C) or siBCKDK (D) treatment. ①P=0.000, ②P=0.031, compared with the 0 μmol·L-1 BT2 group; ③P=0.015, compared with the siNC group; ④P=0.008, ⑤P=0.030, compared with the 0 μmol·L-1 BT2 group; ⑥P=0.025, ⑦P=0.021, compared with the siNC group.

Fig 6 Effect of siBCKDK or BT2 on cell cycle of NSCLC cells

图7 siBCKDK或BT2对NSCLC细胞内P21表达水平的影响Note： A/B. Expression of P21 in H1299 cells treated with siBCKDK (A) or 200 μmol·L-1 BT2 (B). C/D. Expression of P21 in A549 cells treated with siBCKDK (C) or 200 μmol·L-1 BT2 (D).

Fig 7 Effects of siBCKDK or BT2 on expression of P21 in NSCLC cells

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