结直肠癌干细胞中miR-520c-3p的表达及其对丙酮酸代谢的调控机制研究

背景与目的 研究显示，丙酮酸代谢的改变在结直肠癌的发生发展中起重要作用，而结直肠癌干细胞中microRNA（miRNA）表达异常可能与丙酮酸代谢密切相关。笔者前期通过TCGA数据库分析发现，miR-520c-3p在结直肠癌中表达升高，且与预后相关。然而，miR-520c-3p是否参与了丙酮酸代谢尚不清楚。因此，本研究探讨结直肠癌干细胞中miR-520c-3p的表达与丙酮酸代谢的关系。方法 选择人结肠癌细胞株，并从中分离纯化结直肠癌干细胞。检测过表达或敲低miR-520c-3p后，结直肠癌干细胞及结直肠癌细胞增殖能力、丙酮酸氧化水平、乳酸产量的变化。用_D-［U-¹³C］葡萄糖孵育细胞，质量同位素分析追踪葡萄糖衍生碳的命运。通过miRNA序列分析和遗传学手段分析和鉴定miR-520c-3p的功能底物。结果 过表达miR-520c-3p后，结直肠癌干细胞的增殖能力明显增强、丙酮酸氧化水平明显下降、乳酸产量明显升高（均P<0.05）；用_D-（U-¹³C）葡萄糖培养后，未标记的柠檬酸盐（m+0）明显增加，而高阶柠檬酸盐标记（m+1、m+4和m+5）明显减少（均P<0.05）。在敲低miR-520c-3p后，结直肠癌干细胞的上述情况呈反向变化（均P<0.05）。过表达或敲低miR-520c-3p对结直肠癌细胞的上述指标均无明显影响（均P>0.05）。miR-520c-3p可以靶向线粒体丙酮酸载体1（MPC1）mRNA 3''UTR（P<0.05）。过表达miR-520c-3p后，结直肠癌干细胞中MPC1的mRNA与蛋白水平均明显下降，敲低miR-520c-3p后则相反（均P<0.05）。TCGA数据库分析结果显示，低表达MPC1的结直肠癌患者预后较差（P<0.05）。敲低MPC1后，结直肠癌干细胞的丙酮酸氧化水平明显降低、乳酸产量明显增高、增殖能力均明显增强（均P<0.05）；用_D-［U-¹³C］葡萄糖培养后，未标记的柠檬酸盐（m+0）在敲低MPC1的结直肠癌干细胞中明显增加，而高阶柠檬酸盐标记（m+1、m+4和m+5）明显减少（均P<0.05）。同时敲低miR-520c-3p和MPC1后，结直肠癌干细胞丙酮酸氧化水平、乳酸产量、增殖能力均无明显变化（均P>0.05）。结论 结直肠癌中miR-520c-3p的高表达与较差的预后相关，其机制可能是miR-520c-3p靶向MPC1调控结直肠癌干细胞中的丙酮酸代谢水平，促进了结直肠癌干细胞的增殖。

Expression of mir-520c-3p and its regulatory mechanism on pyruvate metabolism in colorectal cancer stem cells

Background and Aims Studies have shown that changes in the metabolism of pyruvate play an important role in the occurrence and development of colorectal cancer, and abnormal expression of microRNAs (miRNAs) in colorectal cancer stem cells may be closely related to pyruvate metabolism. The author''s previous analysis of the TCGA database found that miR-520c-3p was upregulated in colorectal cancer and correlated with prognosis. However, it is not clear whether miR-520c-3p is involved in pyruvate metabolism. Therefore, this study explores the relationship between the expression of miR-520c-3p in colorectal cancer stem cells and pyruvate metabolism.Methods Human colon cancer cell lines were selected, and colorectal cancer stem cells were isolated and purified from them. Changes in the proliferation ability, pyruvate oxidation levels, and lactate production in colorectal cancer stem cells and colorectal cancer cells were detected after overexpression or knockdown of miR-520c-3p. Cells were incubated with _D-(U-¹³C) glucose, and mass isotopomer analysis was used to trace the fate of glucose-derived carbons. MiR-520c-3p functional substrates were analyzed and identified through miRNA sequence analysis and genetic approaches.Results After overexpression of miR-520c-3p, the proliferation ability of colorectal cancer stem cells significantly increased, while the levels of pyruvate oxidation significantly decreased and lactate production significantly increased (all P<0.05). When cultured with _D-(U-¹³C) glucose, the unlabeled citrate (m+0) in colorectal cancer stem cells significantly increased, while the higher-order citrate isotopomers (m+1, m+4, and m+5) significantly decreased (all P<0.05). Conversely, after knockdown of miR-520c-3p, the above-mentioned changes in colorectal cancer stem cells were reversed (all P<0.05). Overexpression or knockdown of miR-520c-3p had no significant effect on the above-mentioned indicators in colorectal cancer cells (all P>0.05). MiR-520c-3p could target the 3''UTR of mitochondrial pyruvate carrier 1 (MPC1) mRNA (P<0.05). After overexpression of miR-520c-3p, both mRNA and protein levels of MPC1 in colorectal cancer stem cells significantly decreased, while the opposite was observed after knockdown of miR-520c-3p (all P<0.05). Analysis of TCGA database showed that colorectal cancer patients with low MPC1 expression had poorer prognosis (P<0.05). Knockdown of MPC1 led to a significant decrease in pyruvate oxidation levels, significant increase in lactate production, and significant enhancement of proliferation ability in colorectal cancer stem cells (all P<0.05). When cultured with _D-(U-¹³C) glucose, the unlabeled citrate (m+0) significantly increased, while the higher-order citrate isotopomers (m+1, m+4, and m+5) significantly decreased in colorectal cancer stem cells with MPC1 knockdown (all P<0.05). Meanwhile, after knockdown of both miR-520c-3p and MPC1, there were no significant changes in pyruvate oxidation levels, lactate production, and proliferation ability in colorectal cancer stem cells (all P>0.05).Conclusions The high expression of miR-520c-3p in colorectal cancer is associated with poor prognosis, and its mechanism may be related to its regulation of pyruvate metabolism in colorectal cancer stem cells through targeting MPC1, promoting the proliferation of colorectal cancer stem cells.