| Background and ObjectiveTumor material energy metabolism is closely related to tumor progression.The correlation of altered cellular metabolism and biological behaviors of cancer has received increased attention as an important hallmark of cancer.The human ZAG gene assigned to the chromosome 7q22.1 and comprised four exons and three introns, ZAG is a multifaceted and secretory 42 kDa protein, ZAG functions as a lipid mobilizing factor in human adipocytes,cancer cells, and plays a key role in lipid mobilization a process that is regulated by FASN.Activation of FASN and mTOR has been found to be involved in a variety of tumors.However,it remains unclear related mechanisms of ZAG、FASN and Akt/mTOR signaling pathway.We investigated the relationship between ZAG and FASN in colorectal cancer cell,and also focused on the effects of Akt/mTOR signaling pathway regulation on the biological behavior of colorectal cancer cell. This work will provide the experimental and theoretical evidence for molecular diagnostic marker and targeted therapic target of colorectal cancer.Methods1. ZAG gene and ZAG protein were detected by RT-PCR and Western blot of four kinds of colorectal cancer cell lines (LoVo/HT-29/Caco-2/HCT-116).The target cell line was selected base on lower expression of ZAG.2. A recombinant overexpression plasmid named as pGCMV/EGFP/Neo/ZAG and control plasmid were transfected into human colorectal cancer LoVo cells which with the lower expression of ZAG, Expression of ZAG gene and ZAG protein were studied by RT-PCR and western blot analysis.3. Reverse transcription- polymerase chain reaction was used to measure the expression of FASN gene、mTOR gene、Akt gene in three LoVo cell groups,and also ZAG、FASN、t-mTOR、p-mTORser2448、t-Akt、p-Aktser473 protein were detected by western blot analysis.4. Cytoplasm ATP and the supernatant lactic acid were detected in order to research the relationship between cellular metabolism and biological behaviors of cancer.5. MTT and colony formation assays were used to check the proliferation potential of LoVo cells which overexpression ZAG. Cell cycle (propidium iodide stained) assayed was carried out by flow cytometry. Further, cell migration was investigated by the transwell analysis.6. A series concentration (0.01μM,0.1 μM, 1μM, 10μM, 100μM) of cerulenin and corresponding DMSO (v/v) were selected to intervene HT-29 and LoVo cells. Cell inhibitory concentration of 50% (IC50) was detected by drug toxicity test.7. We used fluorescence quantitative reverse transcription- polymerase chain reaction and western blot to detect the ZAG、FASN、t-mTOR、p-mTORser2448、t-Akt、p-Aktser473 levels in two kinds of colon cells treated with cerulenin, a potent FASN inhibitor.8. ATP and lactic acid were detected to investigate the variance of the energy metabolism in colorectal cancer HT-29 and LoVo cells use cerulenin IC50 respectively.9. Cell cytotoxicity assay was studied by cell counting kit-8(CCK8) assay. The capacity of cell proliferation and migration was investigated by clonogenic and invasion assay respectively. Analysis of apoptosis was detected by flow cytometry of LoVo and HT-29 cells.Results1. The expression of ZAG in four human colorectal cancer cells (LoVo, HT-29, Caco-2, and HCT116) was detected using RT-PCR and western blot. LoVo cells showed the lowest expression of ZAG gene (0.46±0.05) (P<0.05). And compared with HT-29, Caco-2, and HCT116 cells,LoVo cells also had the lowest expression of ZAG prtoein (0.43±0.05)(P<0.05), LoVo cells were selected for further experiments.2. The pGCMV/EGFP/Neo/ZAG plasmid and the control plasmid were transfected into LoVo cells separately. Green fluorescent signal was measured by fluorescence microscope in order to select stable transfection cell, the expression of ZAG gene in three groups was detected using RT-PCR. The rate of ZAG gene/GAPDH gene expression for experimental groups were 0.65±0.05, Compared with the other groups, the expression of ZAG protein was also higher (0.67±0.06) (P<0.05) in experimental group.3. RT-PCR and western blot results showed that the expression of mTOR gene(0.81±0.06)、Akt gene (0.78±0.06)and t-mTOR protein (0.97±0.13)、t-Akt protein (0.83±0.14)in experimental groups were not different from their expression in blank group and control group (P>0.05).However,the expression of FASN gene (0.55±0.08)、p-mTORser2448(0.53±0.08) and p-Aktser473(0.58±0.10) in experimental group was lower than other groups(P<0.05).4. The results revealed that ATP concentration of experimental group was 611.23 ± 97.37 mmol/grot,In addition,the lactic acid concentration of experimental group was 29.84±5.04 mmol/L ,the level of ATP and lactic have statistical signifcance than blank and control groups (P < 0.05).5. MTT, colony formation and transwell assays were used to detect the biological behavior of colorectal cancer LoVo cells after over-expression ZAG expression. The proliferation rate(0.79±0.08) of the experimental group was significantly lower than that in the other groups (P < 0.05). The colony formation rate of LoVo cells transfected of the experimental group was 4.38%±0.71%, which is lower than other groups. The number of migrating cells in the ZAG-overexpressed cells (64.33±8.02) was lower than other groups.The experimental group cells showed an increased percentage of cells in the G2 phase(29.47%±2.03%) (P<0.05).6. The rate of proliferation of HT29 cells (82.34 ± 5.38 %,74.78 ± 9.44 %,66.21 ± 4.37 %,60.16 ± 9.95 %,38.05 ± 8.26 %) and LoVo cells (83.45 ± 7.86 %,75.05 ± 9.11 %,63.93 ±4.48 %,45.59 ± 11.42 %,21.68±6.87 %) continues to decline with the increasing of cerulenin concentration. The IC50 of cerulenin of HT29 cells (1.93 μM) and LoVo cells(0.37 μM). And those two concentrations of cerulenin were selected for further experiments.7. RT-PCR showed that after treated with cerulenin, HT-29 and LoVo cells have lower ZAG(0.37±0.06 and 0.29±0.06 ) expression, but did not have different expression of mTOR gene mTOR (0.76±0.14 and 0.85±0.10) and Akt gene (0.64±0.06 and 0.78±0.08)(P>0.05). Western blot showed that experimental group cells have lower FASN(0.34±0.08 and 0.52±0.04)、ZAG(0.38±0.05 and 0.21±0.04) p-mTORser2448(0.42±0.03 and 0.44±0.01) and p-Aktser473(0.55±0.10 and 0.58±0.10) expression (P<0.05).However,the expression of t-mTOR(0.90±0.10 and 0.88±0.10) and t-Akt (0.80±0.16 and 0.85±0.11) of experimental group did not have statistically significant compared with the other two groups (P>0.05).8. ATP concentration of experimental group cells (HT29 and LoVo) were 449.60 ± 94.81 mmol/grot and 528.73 ± 37.43 mmol/grot,and those values were signifcantly lower than other group cells (P < 0.05). In addition, the lactic acid concentration of HT29 and LoVo cells were 24.20 ± 3.05 mmol/L and 29.78±2.10 mmol/L after inhibited FASN expression and also have statistical signifcance than other two groups (P < 0.05).9. The colony formation rate of experimental group cells (HT29 and LoVo) were 6.36%%±1.07% and 11.16%±1.46. The cell number per colonies of experimental group cells (HT29 and LoVo) cells was also decreased. In the transwell assay, the migration cell number of HT29 and LoVo cells treated with cerulenin was 80.33 ± 12.40 and 96.67± 16.27 that lower than other groups. Flow cytometry analysis results showed increased apoptotic rate(37.41%±8.48% and 31.28%±3.98%)was found in the experimental group of HT29 and LoVo cells, as compared to the other groups .Conclusions1. Increased expression of ZAG can suppress the activity of the Akt/mTOR signaling pathway and FASN,and then ZAG involved in the regulation of biological behaviors(cell proliferation, invasion, apoptosis and cell cycle) and energy metabolism.2. Inhibited FASN suppresses the regulation of biological behaviors (cell proliferation,invasion, apoptosis and cell cycle) and energy metabolism of colorectal cancer cells, and also suppresses Akt/mTOR signaling pathway and ZAG.3. Inhibited FASN of HT29 and LoVo cells by down-regulating ZAG and Akt/mTOR signaling pathway. Combined with the first part of the study, Akt/mTOR signaling pathway may participate in the regulation of ZAG and FASN expression in colorectal cancer cell lines. |
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