DataSheet1_Associations of serum cystatin C concentrations with total mortality and mortality of 12 site-specific cancers.docx

Purpose:Cystatin C (CysC), beyond its biomarker role of renal function, has been implicated in various physical and pathological activities. However, the impact of serum CysC on cancer mortality in a general population remains unknown. We aimed to examine the associations of serum CysC concentrations with total mortality and mortality of 12 site-specific cancers.Methods:We included 241,008 participants of the UK Biobank cohort with CysC measurements who had normal creatinine-based estimated glomerular filtration rates and were free of cancer and renal diseases at baseline (2006–2010). Death information was obtained from the National Health Service death records through 28 February 2021. Multivariable Cox proportional hazards models were used to compute hazard ratios (HR) per one standard deviation increase in log-transformed CysC concentrations and 95% confidence intervals (95% CI) for mortality.Results:Over a median follow-up of 12.1 (interquartile range, 11.3–12.8) years, 5,744 cancer deaths occurred. We observed a positive association between serum CysC concentrations and total cancer mortality (HR = 1.16, 95% CI: 1.12–1.20). Specifically, participants with higher serum CysC concentrations had increased mortality due to lung cancer (HR = 1.12, 95% CI: 1.05–1.20), blood cancer (HR = 1.29, 95% CI: 1.16–1.44), brain cancer (HR = 1.19, 95% CI: 1.04–1.36), esophageal cancer (HR = 1.20, 95% CI: 1.05–1.37), breast cancer (HR = 1.18, 95% CI: 1.03–1.36), and liver cancer (HR = 1.49, 95% CI: 1.31–1.69).Conclusion:Our findings indicate that higher CysC concentrations are associated with increased mortality due to lung, blood, brain, esophageal, breast, and liver cancers. Future studies are necessary to clarify underlying mechanisms.</p

Overexpression of Nuclear Apoptosis-Inducing Factor 1 Altered the Proteomic Profile of Human Gastric Cancer Cell MKN45 and Induced Cell Cycle Arrest at G1/S Phase

<div><p>Nuclear apoptosis-inducing factor 1 (NAIF1) was previously reported to induce apoptosis. Moreover, the expression of NAIF1 was significantly down-regulated in human gastric cancer tissues compared to adjacent normal tissues. However, the mechanism by which the NAIF1 gene induces apoptosis is not fully understood. Our results show that NAIF1 was minimally expressed in all the tested gastric cancer cell lines. Our data also demonstrates that NAIF1 is localized in the nuclei of cells as detected by monitoring the green fluorescence of NAIF1-GFP fusion protein using fluorescent confocal microscopy. Next, a comparative proteomic approach was used to identify the differential expression of proteins between gastric cancer cell lines MKN45/NAIF1 (−) and MKN45/NAIF1 (+). We found five proteins (proteasome 26S subunit 2, proteasome 26S subunit 13, NADH dehydrogenase Fe-S protein 1, chaperonin containing TCP1 subunit 3 and thioredoxin reductase 1) that were up-regulated and three proteins (ribonuclease inhibitor 1, 14-3-3 protein epsilon isoform and apolipoprotein A-I binding protein) that were down-regulated in the MKN45 cells overexpressing NAIF1. We also discovered that NAIF1 could induce cell cycle arrest at G1/S phase by altering the expression of cell cycle proteins cyclinD1, cdc2 and p21. The differentially expressed proteins identified here are related to various cellular programs involving cell cycle, apoptosis, and signal transduction regulation and suggest that NAIF1 may be a tumor suppressor in gastric cancer. Our research provides evidence that elucidates the role of how NAIF1 functions in gastric cancer.</p></div

Verification of the 2-DE results.

<p>(A) Verification of the 2-DE results by real-time qPCR. *Significant difference (<i>P</i><0.05) **Significant difference (<i>P</i><0.001). (B) Verification of the 2-DE results by western blotting and the relative volume intensity of differential expressed proteins, using β-actin as control. *Significant difference (<i>P</i><0.05).</p

Information on indentified proteins.

<p>AC: accession number.</p><p>The “+” and “−” indicated the up-regulated and down-regulated proteins in MKN45 cells overexpressing NAIF1 compared with control, respectively.</p

Proteomic analysis of MKN45 cells overexpressing NAIF1.

<p>(A) Representative 2-DE maps of MKN45 cells transfected with control plasmid or NAIF1. (B) and (C) Megascopic pictures and relative volume intensity of differential expressed proteins. (B) Represents the up-regulated protein points while (C) represents the down-regulated proteins.</p

NAIF1 induced cell cycle arrest at G1/S phase.

<p>(A) and (B) columns show the different cell cycle distribution between cells transfected with NAIF1 and with the GFP vector 24 h or 48 h after transfection in (A) BGC823 cells and (B) MKN45 cells. To ensure the right population, GFP-positive population was analyzed. *Significant difference (<i>P</i><0.05). (C) Western blotting shows the change in cell cycle regulating proteins associated with NAIF1 induced G1/S phase cell cycle arrest. β-actin was used as the control. Quantified data are shown as the mean ± SD (n = 3); Significant difference from the NAIF1 overexpressing group, Student's t test, *P<0.05.</p

Primer information of real-time Q PCR.

<p>Primer information of real-time Q PCR.</p