Abstract: In order to investigate the effects of miR-148a and hexokinase 2 (HK2) gene on glycolytic metabolic pathway of human breast cancer cells and their possible mechanisms, real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of miR-148a in various breast cancer cell lines, and then breast cancer cell lines with relatively low expression of miR-148a were selected as research objects. Furthermore, the effect of miR-148a expression on glucose uptake, lactic acid production and cell proliferation index of breast cancer cells was observed to explore the effect of miR-148a on glucose metabolism ability of breast cancer cells. Subsequently, the targeted relationship between miR-148a and HK2 genes was predicted through the online database of TargetScan, and then verified through the double luciferase reporting experiment, Western blot and gene recovery experiment, so as to further clarify the mechanism of miR-148a and HK2 in the glycolytic metabolic pathway of breast cancer cells. Through qRT-PCR, the expression of miR-148a was found to be decreased in various breast cancer cell lines, especially in MDA-MB231 (P<0.000 1). Overexpression of miR-148a significantly decreased glucose uptake, lactic acid production and cell proliferation of MDA-MB231 cells (P<0.01). While inhibition of miR-148a expression significantly increased glucose uptake, lactic acid production and cell proliferation index of MDA-MB231 cells (P<0.01). Predicted by TargetScan online database, miR-148a had partial binding sites with the 3′ untranslated region (3′-UTR) of HK2 gene; and the double luciferase report experiment found that miR-148a was bound to the wild-type HK2 3′-UTR luciferase report vector and not to the mutant HK2 3'-UTR. Western blot analysis showed that overexpression of miR-148a significantly decreased the expression of HK2 protein in MDA-MB231 cells (P<0.000 1), while inhibition of miR-148a promoted the expression of HK2 protein significantly (P<0.05). Gene recovery experiment showed that overexpression of HK2 gene significantly increased glucose uptake, lactic acid production and cell proliferation index of MDA-MB231 breast cancer cells (P<0.01). The overexpressed miR-148a vector and the overexpressed HK2 vector were co-transfected into MDA-MB231 cells, and miR-148a reversed the increasing of glucose uptake and lactic acid production caused by HK2, and inhibited cell proliferation. Therefore, studies suggested that miR-148a could inhibit glycolytic metabolism and cell proliferation of breast cancer cells MDA-MB231 by targeted inhibition of HK2 gene expression.

Key words: miR-148a, breast cancer, glycolysis, hexokinase 2

摘要： 为了探讨miR-148a及己糖激酶2（hexokinase 2，HK2）基因对人乳腺癌细胞糖酵解代谢途径的影响和可能机制，利用实时荧光定量PCR（real-time fluorescent quantitative PCR，qRT-PCR）检测多种乳腺癌细胞系中miR-148a的表达量，从中筛选miR-148a表达量相对较低的乳腺癌细胞系作为研究对象。再通过观察miR-148a表达量的变化对乳腺癌细胞葡萄糖摄取量、乳酸生成量和细胞增殖指标的影响，以探究miR-148a对乳腺癌细胞糖代谢能力的影响。随后，通过TargetScan在线数据库预测miR-148a和HK2基因的靶向关系，再通过双荧光素酶报告实验、Western免疫印迹以及基因回复实验进行验证，以进一步明确miR-148a和HK2在乳腺癌细胞的糖酵解代谢途径中的作用机制。通过qRT-PCR发现miR-148a在多种乳腺癌细胞系表达降低，尤其是在乳腺癌细胞系MDA-MB231中表达量显著降低（P<0.000 1）。过表达miR-148a使MDA-MB231细胞的葡萄糖摄取量、乳酸生成量、细胞增殖指标均显著下降（P<0.01）；而抑制miR-148a表达使MDA-MB231细胞葡萄糖摄取量、乳酸生成量、细胞增殖指标均显著上升（P<0.01）。通过TargetScan在线数据库预测得出，miR-148a与HK2基因3′非编码区（3′-untranslated region，3′-UTR）具有部分结合位点；而双荧光素酶报告实验发现miR-148a与野生型HK2基因的3′-UTR荧光素酶报告载体结合，不与突变型HK2基因的3′-UTR结合。Western免疫印迹检测结果表明，过表达miR-148a使MDA-MB231细胞中HK2蛋白表达量显著下降（P＜0.000 1），而抑制miR-148a表达则促进HK2蛋白表达量显著上升（P<0.05）。基因回复实验显示，过表达HK2基因使MDA-MB231乳腺癌细胞的葡萄糖摄取量、乳酸生成量、细胞增殖指标显著上升（P＜0.01）；将过表达miR-148a载体与过表达HK2载体共转染MDA-MB231细胞，miR-148a逆转了HK2所致的葡萄糖摄取量增加和乳酸生成量上升，并抑制细胞增殖。因此，研究提示，miR-148a可通过靶向抑制HK2基因表达而抑制乳腺癌细胞MDA-MB231糖酵解代谢和细胞增殖。

关键词: miR-148a, 乳腺癌, 糖酵解, 己糖激酶2