节律分子Timeless调控糖代谢重编程促进肝癌细胞生长

Timeless promotes the proliferation of hepatocellular carcinoma cell by reprogramming of glucose metabolism

目的探讨节律分子Timeless对肝癌细胞生长的影响及其分子机制。方法采用肝癌公共数据信息分析Timeless在肝癌及癌旁组织中的表达；在高表达Timeless的肝癌细胞株MHCC97H和低表达Timeless的肝癌细胞株MHCC97L中，分别用RNA干扰和真核表达质粒方法沉默或上调肝癌细胞中Timeless的表达后，采用实时荧光定量PCR和Westernblot检测干扰和过表达Timeless的效果以及Timeless下游分子的表达。采用葡萄糖摄取实验、乳酸检测实验、细胞外液pI-I检测实验、细胞氧耗实验和MTS实验，分析Timeless对肝癌细胞糖酵解、氧化磷酸化和细胞生长的影响。结果肝癌组织中TimelesS的表达高于癌旁组织（P〈0．05）。敲低Timeless后．Timeless干扰组1和Timeless干扰组2细胞葡萄糖相对摄取水平分别为0．510±0．119和0．508±0．099，与对照组比较，差异均有统计学意义（均P〈0．05）：Timeless过表达组细胞葡萄糖相对摄取水平为1．953±0．324，与空质粒组比较，差异有统计学意义（P〈0．05）。Timeless干扰组1和Timeless干扰组2细胞乳酸产生相对水平分别为0．579±0．096和0．550±0．120，与对照组比较．差异有统计学意义（P〈0．05）：Timeless过表达组细胞乳酸产生相对水平为1．463±0．179，与空质粒组比较，差异有统计学意义（P〈O．05）。Timeless干扰组1和Timeless干扰组2细胞外pH值为7．390＋0．035和7．370±0．060，与对照组比较，差异有统计学意义（P〈0．05）；Timeless过表达组细胞外pH值为7．130±0．031，与空质粒组比较，差异有统计学意义（P〈0．05）。Timeless干扰组1和Timeless干扰组2细胞耗氧率分别为3．686±0．389和3．955±0．431，与对照组（1．690±0．297）比较，差异有统计学意义（P〈0．05）；Timeless过表达组细胞耗氧率为1．302＋0．336，与空质粒组（3．185±0．262）比较，差异有统计学意义（P〈0．05）。Timeless抑制p53的表达。与Timeless＋p53对照组比较，Timeless＋p53干扰组细胞葡萄糖相对摄取水平、乳酸产生相对水平、细胞外培养液pH和细胞氧耗率差异均无统计学意义（均P〉0．05）；与Timeless＋p53空质粒组比较，Timeless＋p53过表达组细胞葡萄糖相对摄取水平、乳酸产生相对水平、细胞外培养液pH和细胞氧耗率差异均无统计学意义（均P〉0．05）。Timeless通过抑制p53的表达促进肝癌细胞生长。结论Timeless通过抑制p53的表达促进细胞糖酵解和抑制氧化磷酸化，进而促进肝癌细胞生长。

Objective To explore the function and molecular mechanism of Timeless in promoting hepatocellular carcinoma （ HCC ） growth. Methods The expression of Timeless in HCC and paraeancer tissues were analyzed by using the public data of HCC. Timeless was overexpressed in MHCC97L cells and silenced in MHCC97H cells, respectively, and the expression of Timeless and its downstream molecules were detected by real-time PCR and western blot. The effects of Timeless on cell glycolysis, oxidative phosphorylation and proliferation were detected by the glucose uptake experiment, lactic acid detection experiment, the extracellular fluid pH detection experiment, cell oxygen consmnption test and cell viability assay, respectively. Results The level of Timeless in HCC tissue was significantly higher than that of paracancer tissue （P 〈 0.05 ）. The relative cellular glucose uptake levels in the groups of Timeless knockdown, including siTimeless-1 and siTimeless-2 group were 0. 510±0. 119 and 0. 508±0. 099, respectively, significantly different from that of control group （P〈0.05） ; The relative cellular uptake level of Timeless overexpressed group was 1.953±0.324, significantly different from that of vector transfected group （P〈0.05）. The relative levels of lactic acid production in the siTimeless-1 and siTimeless-2 group were 0.579±0.096 and 0.550±0.120, respectively, significantly different from that of control group （P〈0.05） ; The relative production level of lactic acid in the Timeless overexpressed group was 1. 463±0. 179, significantly different that of vector transfected group （P〈0.05）. The extracellular pH values of siTimeless-1 and siTimeless-2 group were 7.390±0.035 and 7.370±0.060, respectively, significantly different from that of control group （P〈0.05） ; the extracellular pH value of Timeless overexpressed group was 7.130±0.031, significantly different than vector transfected group （P〈0.05）. Oxygen consumption rate of siTimeless-1 and siTimeless-2 group were 3.686±0.389 and 3.955±0.431, respectively, significantly higher than 1.690±0.297 of control group （P〈0.05） ; Oxygen consumption rate of Timeless overexprcssed group was 1.302±0.336, significantly lower than 3.185±0.262 of vector transfected group （P〈0.05） Timeless inhibited the expression of p53. The cell glucose uptake, lactic acid production, the pH of extracellular culture medium and cell oxygen consumption of control group were not significantly different from that of Timeless and p53 co-silenced group [ （si-Timeless＋sip53） group] （P〉0.05） ; the glucose uptake, the production of lactic acid, the pH of the extracellular culture medium and the oxygen consumption of Timeless co-transfected with p53 （Timeless＋p53） group were not significantly different from those of vector transfected group （P〉0.05）. Timeless promoted the proliferation of HCC cells through inhibiting the expression of p53. Conclusion Timeless promotes reprogramming of glucose metabolism and proliferation of HCC cells by inhibiting the p53- dependent signaling pathway.