乳酸上调非小细胞肺癌细胞中总胆固醇水平的机制研究

黄登亮; 张耀刚; 侯静; 田美媛; 江源; 孙莉; 李志琴; 童思贤; 郑凯曼; 姜军

doi:10.3969/j.issn.2095-5227.2023.03.008

摘要:

背景胆固醇与非小细胞肺癌发生相关并增强非小细胞肺癌细胞耐药性，研究显示乳酸能通过降低pH使SREBP2活化进而上调细胞内胆固醇水平。

目的探索乳酸调节非小细胞肺癌细胞内总胆固醇水平的其他分子机制。

方法分析TCGA数据库中非小细胞肺癌乳酸脱氢酶A(lactate dehydrogenase A，LDHA)与胆固醇合成代谢酶基因表达水平的相关性。将人肺支气管上皮细胞BEAS-2B和人非小细胞肺癌细胞H1299分为对照组(正常培养)和乳酸处理组(在培养基中添加乳酸)，将人非小细胞肺癌细胞A549分为对照组(正常培养)、乳酸处理组(在培养基中添加乳酸)和盐酸处理组(在培养基中添加盐酸)。利用总胆固醇测定试剂盒测定细胞内总胆固醇水平，qPCR测定细胞内胆固醇合成代谢酶基因的mRNA水平。利用染色质免疫共沉淀分析对照组和乳酸处理组A549细胞中胆固醇合成代谢酶基因启动子区组蛋白乳酸化水平。

结果 Wilcoxon秩和检验分析TCGA数据结果显示，LDHA高表达的非小细胞肺癌细胞中胆固醇合成代谢酶基因HMGCR、ACAT1、ACAT2、SQLE和FDFT1表达水平升高(P＜0.05)。独立样本t检验分析对照组和乳酸处理组细胞内总胆固醇水平及胆固醇合成代谢酶基因的mRNA水平，结果显示，与对照组细胞相比，乳酸处理组BEAS-2B、A549和H1299细胞内总胆固醇水平升高(P＜0.01)，且乳酸处理组BEAS-2B和A549细胞中HMGCR、FDPS、GGPS1和SQLE基因mRNA水平也升高(P＜0.01)。与对照组相比，乳酸和盐酸处理组A549细胞内总胆固醇水平均升高(P＜0.05)，且HMGCR和FDFT1基因mRNA水平也升高(P＜0.01)，但盐酸的作用显著弱于乳酸(P＜0.01)。染色质免疫共沉淀结果显示，乳酸处理组A549细胞中HMGCR、SQLE和GGPS1基因启动子区组蛋白乳酸化水平有升高趋势；与对照组相比，乳酸处理组A549细胞内总胆固醇水平升高(P＜0.01)，而乳酸脱氢酶抑制剂草氨酸钠能逆转乳酸上调A549细胞内总胆固醇水平(P＜0.01)。

结论乳酸能上调非小细胞肺癌细胞内总胆固醇水平，该过程可能的分子机制为组蛋白乳酸化修饰促进胆固醇合成代谢酶基因表达，且乳酸通过转化为乙酰辅酶A参与胆固醇合成。

Abstract:

Background Cholesterol is associated with non-small cell lung cancer tumorigenesis and promotes drug resistance of non-small cell lung cancer cell lines, and it is reported that lactate induces an increased intracellular cholesterol level via activated SREBP2 by low pH.

Objective To explore possible regulatory mechanism of cholesterol level by lactate.

Methods The correlation of expression levels between LDHA and cholesterol biosynthesis-related genes in human non-small cell lung cancer from TCGA was analyzed. The cultured human bronchial epithelial BEAS-2B cells and human non-small cell lung cancer H1299 cells were divided into control group (cultured with normal medium) and lactate group (cultured with medium containing lactate). Human non-small cell lung cancer A549 cells were divided into control group, lactate group and hydrochloric acid group (cultured with medium containing hydrochloric acid). The intracellular total cholesterol level was measured using total cholesterol test kit, and mRNA levels of cholesterol biosynthesis-related genes were examined with qPCR. In control and lactate-treated A549 cells, chromatin immunoprecipitation was used to analyze histone lactylation level within the promoters of genes involved in cholesterol biosynthesis.

Results Wilcoxon rank sum test analysis of TCGA data of human non-small cell lung cancer indicated that there was a positive correlation between LDHA expression level and expression levels of cholesterol biosynthesis-related genes including HMGCR, ACAT1, ACAT2, SQLE and FDFT1 (P<0.05). Compared to control group, BEAS-2B, A549 and H1299 cells treated with lactate had an increased intracellular total cholesterol level (P<0.01), and mRNA levels of HMGCR, FDPS, GGPS1 and SQLE were also up-regulated in BEAS-2B and A549 cells treated with lactate (P<0.01). Compared to control group, A549 cells treated with lactate or hydrochloric acid presented an increase in intracellular total cholesterol level (P<0.05) and mRNA levels of HMGCR and FDFT1 (P<0.01). However, the cholesterol level in lactate-treated A549 cells was much higher than that in hydrochloric acid-treated A549 cells (P<0.01). Higher histone lactylation level within HMGCR, SQLE and GGPS1 promoters was observed in lactate-treated A549 cells compared to control A549 cells. In addition, inhibition of LDHA by sodium oxamate reversed lactate-induced total cholesterol in A549 cells (P<0.01).

Conclusion Lactate triggers an increase in human non-small cell lung cancer cells. The possible underlying molecular mechanism may be histone lactylation stimulating expression of cholesterol biosynthesis-related genes, and lactate is also metabolized to generate acetyl coenzyme A to synthesize cholesterol.

乳酸上调非小细胞肺癌细胞中总胆固醇水平的机制研究

Exploration for mechanism of lactate-upregulated total cholesterol in non-small cell lung cancer cell