Page 90 - TD-3-1

Tumor Discovery MMP-1 as a potential biomarker for gastric cancer

cancer-related deaths in Asia. Gastric cancer encompasses CTTTTTT-3’ and sh2:5’-GCGTGTG ACAGTAAGCTA
1
two main classifications: early and advanced stages. In AC CT T CAAGAGAGGT TAGCT TACT GT CA
early gastric cancer (EGC), lesions are confined to the CACGCTTTTTT-3’. Other reagents employed in this study
mucosal and submucosal layers of the gastric wall. EGC included: 1640 medium (Hyclone, USA); fetal bovine serum
patients typically exhibit a 5-year survival rate exceeding (BI, Zhejiang Sorfa Life Science Research Co., Ltd., China);
90%, whereas, for advanced gastric cancer patients, Lipofectamine 3000 (Thermo Fisher Scientific, USA);
this rate drops below 30%. The incidence rate of gastric Penicillin-Streptomycin solution (Shanghai Beyotime
cancer increases progressively with age, with a median Biotechnology Co., Ltd., China); trypsin ((Shanghai
age of 70 years at diagnosis, and the prognosis is usually Beyotime Biotechnology Co., Ltd., China); SDS-PAGE
poor. Approximately 10% of gastric cancers are detected assay kit (Shanghai Beyotime Biotechnology Co., Ltd.,
in patients aged 45 years or younger. Therefore, early China); ECL assay kit ((Shanghai Beyotime Biotechnology
diagnosis of gastric cancer plays a vital role in improving Co., Ltd., China); RIPA lysis buffer (Shanghai Beyotime
patient prognosis and reducing mortality, which are keys Biotechnology Co., Ltd., China); BCA assay kit (Shanghai
to improving survival rates. 2,3 Beyotime Biotechnology Co., Ltd., China); CCK8 assay kit
Matrix metalloproteinases (MMPs), produced by (MCE, China); and an EDU assay kit (Guangzhou RiboBio
both tumor cells and normal cells, are also known as Co., Ltd., China).
interstitial collagenases. Among the MMPs, matrix 2.2. Cell culture, cell transfection, and grouping
metalloproteinase-1 (MMP-1) is one of the most
commonly expressed types. The overexpression of The 1640 culture medium, consisting of 10% fetal bovine
MMP-1 is correlated with metastasis, inflammation, and serum and 1% double antibodies, was used to culture
4,5
tumor invasion, highlighting its significant role in the NCI-N87 human gastric cancer cells (hereafter referred
pathogenesis of gastric cancer. A comprehensive study to as N87) under culture conditions of 5% CO at 37°C.
6,7
2
involving 1,377 cases and 1,543 controls showed that the During the logarithmic growth phase, good-condition
MMP1-1607 1G > 2G polymorphism was associated with N87 cells were cultured into 6-well plates and transfected
a significantly elevated risk of gastric cancer. Furthermore, when they reached an approximately 70% growth rate. The
8
in tumors, MMP-1 overexpression has been reported to be MMP-1-overexpression plasmid (Ad-MMP-1), two RNA-
correlated with increased invasive and migratory capacities interference plasmids, and corresponding controls were
of hepatocellular carcinoma cells. 9 transfected using Lipofectamine 3000, and the cells were
further cultured.
In the present study, gene sequencing revealed that
the enhanced expression of MMP-1 is correlated with 2.3. Cell proliferation
the malignant properties of gastric cells in both early Transfected N87 cells were inoculated into 96-well plates at
and advanced gastric cancer. Furthermore, an analysis a density of 5000 cells/well, with each well containing 100
of 20 pairs of EGC and paracancerous tissue samples µL of medium. Six replicate wells were prepared for each
demonstrated that elevated MMP-1 expression levels experiment to ensure experimental accuracy.
exhibited high sensitivity and specificity in identifying
EGC. Therefore, MMP-1 holds promise as a potential 2.3.1. CCK8 assays
diagnostic marker for EGC and plays a regulatory role in After incubation for 24, 48, and 72 h, 10 µL of CCK8
gastric cancer cells.
reagent was added to each well and incubated for an hour.
2. Materials and methods The optical density values were measured at 450 nm using
a multifunction microplate reader, and the data were
2.1. Materials subsequently analyzed.

The NCI-N87 human gastric cancer cell line utilized in
the present research was purchased from the Shanghai 2.3.2. EdU assays
Cell Bank of the Chinese Academy of Sciences, China. After 24 h of incubation, the cells were treated with the
The human-derived MMP-1 gene shRNA downregulation EdU reagent and incubated for an additional 2 h for
vector, overexpression vector, and pLent-U6-GFP-Puro labeling. Subsequently, the cells were fixed with 4%
negative control plasmid with a nonsense sequence paraformaldehyde and treated with 0.5% Triton X-100,
were constructed by Shandong WZ Biosciences Inc., followed by Apollo dye for 30 min. Cells were washed three
China. The specific shRNA sequences used were as times with PBS and then stained with Hoechst nuclear dye
follows: sh1: 5’-GCTAGCTCAGGATGACATTGATT at a ratio of 1:100 for 10 min. After washing, cells were
CAA GA GAT CAAT GT CAT C CT GA GCTA G imaged and counted for further analysis.

Volume 3 Issue 1 (2024) 2 https://doi.org/10.36922/td.1973