br Fig ANGPTL is overexpressed in AFP producing gastric
Fig. 1. ANGPTL6 is overexpressed in AFP-producing gastric cancer cell lines. (A) The RNA expression of ANGPTL1-8 in six gastric cancer cell lines (GCIY, FU97, MKN1, SGC-7901, MGC-803, AGS). (B) The protein level of ANGPTL6 in AFPGC is much higher than that in common gastric cancer. The data are expressed as the mean ± standard deviation (SD) and are representative of three independent experiments. *P < 0.05; **P < 0.01 compared with control.
basement. After 24 h incubation and fixation with formaldehyde, the Okadaic acid that did not migrate through the holes and removed by cotton swabs. Subsequently, the cells inside the filter membrane were sub-jected to 0.1% crystal violet for 20 min. The cells that penetrated the lower membrane were enumerated. Cells in five random fields re-present as the average number of cells/field of each membrane. In each case, triplicate independent experiments were conducted for each group.
2.8. Cell apoptosis assay
An Annexin V-FITC/PI Apoptosis Kit (BD Biosciences, San Jose, CA, USA) was used to investigate cell apoptosis. Briefly, a total of 1 × 105 cells were harvested for 24 h, washed using PBS and resuspended in 100 μl binding buﬀer at room temperature. Annexin V-FITC (5 μl) was then added and the cell sample was incubated in the dark for 5 min before incubation for another 15 min in the dark with 5 μl PI. The fluorescence intensity analysis was calculated by flow cytometry (FACS Calibur, BD Biosciences). Details are subject to the manufacturer’s in-structions.
2.9. The Kaplan- Meier plotter
ANGPTL6 was entered into an online database, the Kaplan-Meier Plotter (www.kmplot.com). To obtain a Kaplan-Meier plot to assess the prognostic value of ANGPTL6 RNA levels. A total of 882 clinical gastric cancer patients were included to analyze overall survival (OS). Patients were separated into two groups by best cutoﬀ (high vs. low expression) and evaluated via a Kapan-Meier survival curve. Hazard rations (HRs) with 95% confidence intervals (95% CIs) and log rank P value were used to assess statistical significance.
2.10. Immunohistochemistry analysis
All specimens were fixed in 4% formalin and embedded in paraﬃn before IHC analysis. All procedures were performed according to our previous reports . The staining of tumor sections was performed by three independent researchers under blinded methods. Five microscope fields were selected randomly for investigation to determine the posi-tive staining area and staining intensity of tumor cells.
2.11. In vivo tumor growth inhibition experiments
Ten five-week-old male nude mice (4–6 weeks old, Chinese Academy of Science) were maintained under specific pathogen-free conditions and given treatment in accordance with institutional guidelines. Each mouse was independently injected subcutaneously (5 × 10^6 GCIY cells or GCIY-shANGPLT6 per site) into the right flanks. Tumor volume was calculated by the equation: tumor volume = 1/ 2(length × width^2). Xenograft samples were collected, fixed in for-malin and embedded in paraﬃn. The Committee approved all animal work.
2.12. Statistical analysis
Statistical analysis was performed as described previously . In comparisons of ANGPTL1-8 expression levels, statistical significance was evaluated using rank sum test. All data are presented as the mean ± standard error of the mean (SEM) of three independent ex-periments. Student’s t-test was used to compare the mean value of any two groups. SPSS (version 23.0) was used for the statistical analysis. P < 0.05 was considered statistically significant.
3.1. Overexpression of ANGPTL6 in AFPGC cell lines compared to that of common gastric cell lines
The clinical features of AFPGC are similar to those of hepatocellular carcinoma (HCC). It is well known that HCC is a typical hypervascular solid tumor. The invasive tumor cells exhibit vasculogenic mimicry, simulating the angiogenesis network, which plays a vital role in the malignancy of HCC. Therefore, we hypothesized that AFPGC secretes certain molecules that promote angiogenesis mimicking HCC. To in-vestigate whether angiopoietin-like proteins (ANPGTLs) respond to gastric cancer progression, we first screened all eight members of the ANGPTL family and examined their endogenous mRNA expression le-vels of in six human gastric cancer cell lines by qRT-PCR. Strikingly, the results demonstrated that the mRNA expression of ANGPTL6 was abundant in all ANGPTL family members, while the expression of the other seven members was nearly undetectable in all gastric cancer cell lines (Fig. 1A). Furthermore, the mRNA expression of ANGPTL6 in two AFPGC cell lines (GCIY and FU97) was much higher than that in four common gastric cancer cell lines (MKN1, SGC-7901, MGC-803, AGS) (Fig. 1A). Western blot analysis confirmed that the endogenous protein level of ANGPTL6 in AFPGC cell lines was much higher than that in CGC cell lines (Fig. 1B).