Objective: To investigate the effect of Caveolin-1on the proliferation and migration ability in gastric carcinoma cells MGC-803 and its mechanism. Methods: Plasmid DNA pcDNA3.1-Cav1 was extracted by extracted kits, and transfected Cav-1 gene sequences were found. The expression levels of Cav-1 protein were detected by Western blot. And the proliferation was analyzed by CCK8 assay. The effect of Cav-1 on migration was detected by wound healing. The expression levels of BMI-1 protein were detected by western blot. Results: 1) Western Blot showed that the expression levels of Cav-1 were higher in MGC-803/Cav-1 than control group, P < 0.05. 2) CCK8 showed, that the proliferation of MGC-803/Cav-1 was inhibited, but P > 0.05. Wound healing showed, that the migration ability of MGC-803/Cav-1 fell off, P < 0.05. 3) Western Blot showed that the expression levels of BMI-1 were lower in MGC-803/Cav-1 than in the control group, P < 0.05. Conclusion: Caveolin-1 can inhibit the proliferation and migration ability of gastric carcinoma cells and its mechanism may relate to BMI-1.
Gastric cancer (GC) is a fast-growing malignant tumor in the digestive system, which is the third most common cause of cancer-related deaths. Despite the improvements in treatment and early detection reduced the incidence of GC, >1 million new cases were estimated worldwide in 2018, while China is one of the countries with the highest incidence of GC [
RPMI 1640 and 10% foetal bovine serum were purchased from GIBCO Company (USA), penicillin and streptomycin were purchased from north China Pharmaceutical Co., Shijiazhuang, Hebei, China. Human GC cell lines MGC-803 was purchased from the Biotechnology Development Co. Ltd (Shanghai, China), and was maintained in a 37˚C incubator with 5% CO2. QIAGEN extracted kits was obtained from QIAGEN Co. Ltd (Germany). Antibodies against Caveolin-1 and BMI-1 were purchased from Santa Cruz Co. Ltd (USA).
Human GC cell lines MGC-803 were cultured in RPMI-1640 supplemented with 10% foetal bovine serum, 100 IU/mL penicillin, and 100 IU/mL streptomycin, maintained in an incubator at 37˚C and 5% CO2.
As described previously, Human GC cell lines MGC-803 were prepared. The cell line was maintained in DMEM medium, and seeded in 35-mm culture plates. The cell line was divided into the control group (without treatment), and MGC-803/Cav-1 groups (transfected with pcDNA3.1-Cav-1). The cells were in the logarithmic growth phase the next day, were transfected in experiments, and washed once with cold PBS. The pcDNA3.1-Cav-1 plasmid was extracted by QIAGEN plasmid extraction kit. The prepared cells of MGC-803/Cav-1 groups were transfected according to the specific steps referred to in kit instructions. After 6 h, the culture medium was changed to DMEM containing 10% FBS (without penicillin and streptomycin). After 48 h, 1500 lg/mL of G418 (Amersco, Inc., Solon, OH, USA) was added, and resistance screening was performed. The culture medium was changed once every 2 days. CAV-1 overexpression was verified using western blot analysis.
The cell line was maintained in DMEM medium, and seeded in 35-mm culture plates. After 48 h, the DMEM was removed, and the cells were washed once with cold PBS and cultured in DMEM without FBS for 4 h. RIPA lysis buffer was used for lysis cells and total protein of the cells was extracted. BCA assay was used to detect the concentrations of the total protein. Protein samples were separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% nonfat dry milk for 2 h at room temperature, incubated with primary antibodies, rabbit anti-Caveolin-1 (1:2000), and GAPDH (1:1000), overnight at 4C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:4000) for 1.5 h at room temperature. The membranes were washed with TBST three times and then ECL (Beyotime, Haimen, Jiangsu, China) color and exposed in the darkroom. After being scanned, the stripes’ width and grey value were analyzed by the Image J image analysis system (national Institutes of Health, Bethesda, MD, USA). Each experiment was repeated three times.
MGC-803/Cav-1 and control group cells were seeded at a concentration of 5000 per well into a 96-well plate and routinely incubated for 36 h at 37˚C. The white well was used for zero adjustments. 10 µl CCK-8 reagent was added into every well at 0, 24, 48, 72 hours, the plates were maintained at 37˚C 2 h later, then the absorbance of cells was measured at 450 nm using a microplate reader. Each group of cells was set with 5 auxiliary holes and repeated three times. Cell proliferation rates were estimated according to the following formula:
proliferation rate (%) = optical density (OD) value in experimental group/OD value in normal control group × 100%.
MGC-803/Cav-1 and control group cells were seeded at a density of 10,000 per well into a 24-well plate and routinely incubated for 36 h at 37˚C. A 10-µl pipette tip was used to scratch the cell monolayer once cells had reached 90% confluence. The floating cells were washed by PBS for two times. Subsequently, the cells were cultured in DMEM without FBS. Finally, the cells were imaged using an inverted microscope (Olympus, Japan) at 0, 24 and 48 h after wounding. The width of the scratch distance between the two phases was measured to estimate the migration of cells and can reflect the difference in the cell migration ability in different groups. The recovered wound area (%) at the indicated time point
(24/48 h) was calculated according to the following formula:
[(wound width at 0 h) − (wound width at 24/48 h)]/2/wound width at 0 h × 100%.
SPSS19.0 statistical software was used for data analysis. Data are presented as the mean ± SEM. Data were compared between two groups by Student’s t-test. And a value of P < 0.05 was considered statistically significant.
Expression of Caveolin-1 was detected in Human GC cell lines MGC-803, MGC-803/Cav-1 group, and the control group. Western blot results showed that the expression of Caveolin-1 was high in the MGC-803/Cav-1 group (7.31 ± 0.38) than that in control group (1.99 ± 0.06) (
To evaluate the effects of Caveolin-1 over-expression on the abilities of proliferation in MGC-803 cell lines, cell proliferation rates were estimated by CCK-8 assay. As shown in
with the control group at 24, 48, and 72 h, particularly obvious at 72 h. There were no significant differences at any time point.
We deduced the Caveolin-1 gene may inhibit cell proliferation.
The results of the wound healing assay also provided consistent conclusions same as with the CCK8 assay. In MGC-803/Cav-1 group, the recovered wound area at 0, 24, 48 h were (3.09 ± 0.41, 8.86 ± 1.83, 19.40 ± 3.09) separately, lower than that in the control group (4.15 ± 0.67, 13.47 ± 3.51, 35.63 ± 7.22), as showed in
Western blot is used to detect the expression of BMI-1 in MGC-803/Cav-1 group and control group. The expression of BMI-1 was found significantly decreased in the MGC-803/Cav-1 group (2.95 ± 0.16) compared with the control group (7.35 ± 0.15) (
Gastric cancer (GC) is an often seen malignant tumor with a high incidence and mortality in China. Worldwide, GC is the third most common cause of cancer-related mortality, >1 million new cases were estimated worldwide in 2018 [
As a component of membrane caveolae, identified in the 1950s [
The mechanism of Caveolin-1 acting as a tumor suppressor is controversial. Caveolin-1 may inhibit the activity of the cyclinD1 gene promoter, and then inhibit the MAPK pathway and the phosphorylation of the Src tyrosine kinase [
In conclusion, our findings demonstrated that Caveolin-1 inhibited the proliferation and invasion of GC cells line through the BMI1 gene. We hope that our findings could provide new insight into the treatment of gastric cancer. Although the relationship between Caveolin-1 and BMI-1 and the role of Caveolin-1 are needed to be further research in gastric cancer.
CHENGDE Science and Technology Research and Development Program. NO. 202109A055.
The authors declare no conflicts of interest regarding the publication of this paper.
Wang, J. and Liu, S.S. (2022) Caveolin-1 Inhibits Proliferation and Migration of Gastric Cancer Cell via Inactivating BMI-1. Open Journal of Pathology, 12, 80-87. https://doi.org/10.4236/ojpathology.2022.123010