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?Many studies have found that the disorder of circadian rhythm-related factors is related to the recurrence and metastasis of nsclc, so identifying circadian rhythm-related factors is also currently required in clinical practice. The study found that hlf is significantly reduced in tissues of early relapse, which is significantly related to the early progression of nsclc in patients with distant metastasis. And upregulation of hlf can inhibit lung colonization, while silencing hlf can promote the metastasis of nsclc cells to the bone, liver and brain.

?Mechanism: Downregulation       Hlf   Promote non-anchored    Nsclc     Anaerobic metabolism of cells under low-nutrition conditions, activates the nf-κb/p65 signaling pathway through pparα and pparγ transport. Gene deletion and methylation help downregulate the expression of hlf                                                                                                                                                                                                 

?While multiple therapeutic strategies have good prospects for the treatment of primary non-small cell lung cancer, non-small cell lung cancer remains the main cause of cancer-related deaths due to its early recurrence and widespread metastasis potential.

?In terms of name, circadian rhythm-related molecules are composed of a series of clock genes and proteins, which play an important role in the regulation of the sleep cycle. More and more studies have reported that dysregulation of circadian rhythm-related regulators is related to tumor occurrence and metastasis through different mechanisms. In recent years, the role of circadian clock genes in nsclc      has received widespread attention. For example, in lung adenocarcinoma, the combined downregulation of cry2, bmal1 and rora                                                                                                                                                                                        

?By analyzing multiple circadian-related regulators from the cancer genomic map (TCGA) and our previous integration of array expression (AE-meta) RNA expression profiles, in the current study, we identified a circadian gene, hepatoleum leukemia factor (Hlf), a member of the family of proline and acid-rich alkaline leucine zipper transcription factor (parbzip) with significant reductions in early recurrence of NSCLC tissue, which is significantly associated with early progression and distant metastasis in patients.

?The nsclc    RNA sequencing dataset from tcga                                                                                                                                                                                                                                                 

?Our previous results of the integrated data spectrum based on affymetrixu133    plus2.0 chip (AE-meta) combined with TCGA analysis further showed that the expression of hlf                                                                                                                                                                                                                                   

Pictures? Real-time    PCR        Western   Blotting analysis showed that compared with the matched ant, mRNA                                                                                                                                                                                                                                   

?     western   blot   Analysis of pairing    nsclc    Comparison of protein levels and pairing    ant                                                                                          �

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?    1 year relapse     nsclc                                                                                                                                                                                                                                                 

?In addition, compared with non-recurrent nsclc                                                                                                                       �

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?Different subtypes     nsclc                                                                                                                                                                                                                                                   � Other subtypes of cell lung cancer, n=26) (Figure b). Compared with tcga(ant)(ant,=109,adc,=511,sqc,=502), hlf expression in sqc                                                                                                                                                                                                                                 �

?     Kaplan-meier survival analysis further showed that compared with patients with high hlf expression, nsclc expressed in low hlf expression, patients with poor progression-free survival (Figure 2f), which is consistent with the analysis results of tcga, ae-meta and k-mplotter (Figure 2g-i).

? Similarly, short progression-free survival was observed in patients with low hlf expression adc and sqc (Figure 2j-q).

The above results show that low expression of Hlf is related to the late clinical pathological characteristics and early progression of NSCLC.

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?In addition, the expression level of hlf     gradually declines with the increase of clinical grading and stage (Supplementary Fig. 1g–i) (Supplementary Fig. 1e,f).

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?We first divided the patients into different subgroups of grades i to iii and i to iv. As shown in Supplementary Figure 2a-f, under the same pathological grading, patients with low expression of hlf and high progression-free survival were short. Correspondingly, according to multiple independent data sets, including our samples (Supplementary Figures 2g and h), tcga (Supplementary Figures 2i and j), ae-meta (Supplementary Figures 2k and l) and k-m plotter (Supplementary Figures 2m?o), similar findings were also found in patients with nsclc at the same clinical stage.

?To sum up, our results show that low expression    hlf   is significantly positively correlated with early progression and advanced clinical pathological characteristics of patients with nsclc   .

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? Further research found that in patients with nsclc, hlf downregulation was positively correlated with short local recurrence-free survival and distant metastasis-free survival (Supplementary Figures 3a-d). Similarly, the local recurrence-free survival and distant metastasis-free survival were poor in patients with low expression of hlf and sqc. (Supplementary Figure 3i-l) (Supplementary Figure 3e-h). The above results show that low expression of hlf promotes early local recurrence and multiorgan distant metastasis.

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?To further determine the role of hlf                                                                                                                                                                                                                                                          �

?As shown in Supplementary Figures 4a and b, compared with non-cancerous immortalized lung bronchial epithelial cells, beas-2b and normal lung fibroblasts, wi-38, the mRNA and protein levels of hlf                                                                                                                                                                                                                              � nci-h520(h520) lung cancer cells, these cells express the lowest hlf level in all nsclc cell lines and endogenously downregulate the expression of nci-h460(h460) and nci-h1299(h1299) cells, both of which express the highest endogenous hlf level (Supplementary Figures 4c and d).

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?The mouse tail vein model was used to study the lung colonization and growth ability of nsclc cells. As shown in Figures 3a-d and Supplementary Figure 4e, upregulation                                                                                                                                                                                                                                      

?It is worth noting that the alteration of hlf expression not only affects the formation of large lung nodules (defined as >50 tumor cells in the analyzed tumor section) (Figure 3b), but also affects the formation of small lung nodules (defined as 5-50 tumor cells in the analyzed tumor section) (Supplementary Figure 4e), which supports the view that hlf plays an important role in early colonization of tumor cells and subsequent lung intubation.

?Finally, compared with the end of the experiment, the corresponding high and low expression levels were further confirmed in the tumor tissues of mouse tumors injected with hlf and downregulated h460 cells (Supplementary Figure 4f).

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?Then, the mouse model of in vivo left ventricle inoculation was used to detect the effect of hlf on the distant metastasis ability of nsclc, overexpression of vector/nci-h1975, hlf    nci-h1975, vector/nci-h460 and hlfsh#1/nci-h460     hlfsh#1/nci-h460                                                                                                                                                                                                �

?6 weeks after inoculation of cells, the tibia, brain, and liver tissues of the mice were collected and stained. As shown in the figure. Figure 3f-i and Supplementary Figure 4g, we detected fewer metastatic tumors in the mouse group, including bone, brain and liver, and longer cumulative survival in the mouse group, including bone, brain and liver, and had a longer cumulative survival period.

?In contrast, silencing Hlf significantly enhances the formation of bone, brain and liver metastases and shortens survival (Figure 3f-i and Supplementary Figure 4h).

?So, these results show that low-expressing hlf nsclc cells have stronger local colonization and growth capabilities, as well as stronger distant metastasis capabilities.

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?k-8, colony formation and an apoptosis were further studied. The biological function of hlf in nsclc                                                                                                                                                                                                                                                

?Our results show that upregulating hlf                                                                                                                                                                                                                                                         �

?Low expression    Hlf   May promote the adaptation of cells to low-trophic states through the nutritional metabolic pathway, because even under normal oxygen-containing conditions, cancer cells are more inclined to metabolize through the anaerobic pathway.

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?However, upregulating hlf     inhibition of nsclc                                                                                                                                                                                                                                                �

?In fact, the imbalance of the circadian rhythm gene has led to the metabolic disorder of nsclc. hlf    It is widely involved in a variety of substance metabolism processes, including lipids and oxidative metabolism.

?Under low nutritional conditions composed of low serum (1%) and low glucose (1g/l) culture medium, hlf    overexpression inhibited

nsclc     The proliferation of cells, but increases the apoptotic potential and vice versa (Figure 4c-e).

?                                                                                                                              �

?Observation under low nutritional conditions    nsclc                                                                                                                                                                                                                                                   �

?Paraconically, upregulation of hlf         intracellular                                                                                                                                                                                                                                              �

?In contrast, silencing     hlf                                                                                                                                                                                                                                                    �

?In fact, even under normal oxygen content, cancer cells show changes in glucose metabolism characteristics and prefer anaerobic metabolism, which is called the "Waberg effect". To sum up, our results show that low expression                                                                                                                                                                                                                                      �

?To further determine the potential mechanism of inhibiting nsclc growth and metastasis under low nutritional conditions, we transfected luciferase reporter plasmids of multiple signaling pathways into nsclc cells. As shown in Figure 5a, upregulation of hlf significantly enhanced nsclc cells ppar signaling activity and inhibited nf-κb signaling activity; on the contrary, silencing hlf    hlf                                                                                                                                                                                                     �

?Gene set enrichment analysis (gsea) was performed based on the expression of hlf in tcga                                                                                                                                                                                                                                                  

? Importantly, some evidence shows that hlf    involves in translocation and activation of ppar                                                                                                                                                                                                                                               �

?First, the expression levels of pparα, pparβ/δ and pparγ in 10 pairs of nsclc were detected. As in Supplementary Figures 6g and h, pparα expression was significantly downregulated in 4/10 pairs of tissues, pparγ was expressed in 8/10 pairs, and pparβ/δ was upregulated in 8/10 pairs. It has been widely reported that pparα and pparγ play a role in tumor suppression signal in      nsclc, and it was reported that pparβ/δ play a carcinogenic effect.

?     western   blot     analysis consistently showed that the upregulation of hlf                                                                                                                                                                                                                                        �

?We used the pparα agonist fenofibrate, the pparγ agonist pioglitazone and the nf-κb signal inhibitor ly2409881 to further study the significance of the signal axis in the functional role of hlf in nsclc cells.

?Our results show that in hlf     silent cells, fenofibrate and pioglitazone significantly upregulated the activity of ppar                                                                                                        �

?However, fenofibrate, pioglitazone and ly2409881    The difference decreased the activity of nf-κb signal in silenced cells.

? Importantly, fenofibrate, pioglitazone and ly2409881 weakened hlf downregulated the stimulating effect of cells on non-anchored growth and anti-hypoxia ability. In contrast, fenofibrate, pioglitazone and ly2409881 reversed the pro-proliferation (clonal growth) and anti-apoptotic effects of hlf downregulation under low nutritional conditions on nsclc.
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