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Med One 2016;1(1):4; DOI:10.20900/mo.20160004


Direct evidence for the involvement of TP53 in the proliferation, migration and invasion of gastric carcinoma cells

Ying Li1 , Shaojun Liu2 * , Pei Zhang3

1Departmentof Endoscopy center,Shaanxi provincial people’s hospital, 710068, China;

2Department of Digestion Medicine, The Third Affiliated Hospital of Xiang Ya School of Medicine, Central South University, Changsha 410078, China;

3Department of Radiation Oncology, University of Maryland School of Medicine, Baltimore, MD21221.

Correspondence: Shaojun Liu

Published: 2/25/2016 7:01:13 AM


Background:Abnormal expression of p53 has been observed in gastric carcinoma cells, but whether p53 could be a target for the biological therapy of gastric carcinoma has not been validated.

Methods: TP53 was silenced by siRNA and overexpressed by vector. mRNA expression was measured by RT-PCR, and protein expression was measured by Western blot. Cell proliferation was analyzed using the CCK-8 method. Cell invasion and migration were analyzed using Transwell champer and wound healing assay, respectively.

Results:TP53 siRNA significantly silenced TP53 expression and pEGFP-TP53 vector overexpressed p53 protein. Silencing TP53 expression significantly increased cell proliferation, invasion, and migration in BGC-823 cells. In contrast, overexpression of p53 significantly inhibited cell proliferation, invasion, and migration in BGC-823 cells.

Conclusion: Overexpression of p53 could be a useful strategy for target inhibition of gastric carcinoma cell growth and metastasis.


Gastric cancer is one of the most common malignant tumors of the digestive tract, and its mortality rate ranks second among all cancers [1-3]. Technological advances in medical imaging technologies and therapeutic advances in chemoradiotherapy, biological target therapy, and cellular immunotherapy have brought some improvements to gastric carcinoma prognosis. However, the prognosis of gastric cancer remains poor, which may be associated with our poor understanding of the pathogenic mechanisms of gastric cancer [4,5].

Gastric cancer is a multifactorial disease. Overexpression of oncogenes and lack of expression of tumor suppressor genes are the main molecular mechanisms involved in the tumor growth, therapeutic resistance, and metastasis in gastric cancer. For example, overexpression of some genes, such as HER-2 , VEGFR-2, EGFR, VEGF-A, mTOR, and c-MET/HGF, has been widely reported in gastric cancer patients and targeting inhibition of these genes are currently being applied as treatment options in gastric cancer [6]. In contrast, tumor suppressor genes, such as CDKN2A/p16, CDH1/E-cadherin, RUNX3, and MLH1, were often reported to be inactivated by promoter methylation, mutations, or chromosomal losses [7]. In gastric cancer, genomic aberrations in TP53, PIK3CA, ErbB2, ErbB3, ARID1A and KRAS gene are frequently encountered[8]

Mutation of the TP53 gene is one of the most common anomalies in human cancer, which leads to the loss of TP53 gene expression or p53 functions [9]. TP53 gene mutations are observed in approximately 50-77% of advanced gastric cancers [10, 11]. Downregulation of the TP53 gene expression can significantly enhance cell proliferation, migration, and invasion of gastric cancer cells [12]. However, the effects of p53 protein in the malignancy of gastric cancer cells are rarely validated by directly silencing or artificially expressing p53 protein in a gastric carcinoma cell line.

This study investigated the roles of p53 in the proliferation, invasion, and migration of gastric cancer cells by silencing and overexpressing p53 expression in gastric carcinoma BGC-823 cells.



The gastric carcinoma BGC-823 cell line was purchased from Sunbio Company (Anyang, South Korea). RPMI 1640 and fetal bovine serum were purchased from Gibco Company (Langley, OK, USA). Transwell chamber and Martrigel were purchased from BD Bioscience Company (Franklin Lakes, New Jersey, USA). The lipidosome cell transfection kit was purchased from Invitrogen (Grand Island, NY, USA). The anti-P53 and anti-β-actin antibody were purchased from Xinlebio (Shanghai, China). CCK-8 detection kit was purchased from Beyotime Institute (Shanghai, China). Trizol reagents and cDNA synthesis kit were purchased from TAKARA (Japan). The restriction enzyme and T4 DNA ligase were purchased from NEB. The plasmid extraction kit was purchased from Omega Bio-tek, Inc (Norcross, GA, USA). The cell protein extraction kit was purchased from Thermo Scientific Company (Waltham, MA USA).

SiRNA design and vector construction

The TP53 siRNA sequence was designed using a publically available online software ( The complementary siRNA sequences were synthesized and made ready to use by Invitrogen. To clone the TP53 gene into the pEGFP-N1 vector, the cDNA of the TP53 gene was amplified using the forward primer: 5`- CTCGAG GAACAGCTTTGAGGTG-3` with Xho I sequence in italics and reverse primer: 5`- GGATCC GTCTCTCCCAGGACAGGCACAAAC-3` with the BamHI sequence in italics. The PCR amplification was performed at 94°C, 3min, followed by 30 cycles at 94°C, 35s, 58.7°C, 35s, 68°C, 1.5 min. The amplified DNA fragment was purified from agarose gel, digested with XhoI and BamH I, ligated into pEGFP-N1 vector (digested with XhoI/BamHI), and transformed into DH5α competent cells. The clones were selected and verified by sequencing. The produced vector was designated pEGFP-TP53.

Cell culture and transfection

BGC-823 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. The cells were transformed with siRNA or pEGFP-TP53 plasmid using lipidosome cell transfection kit by following the user mannual.

RNA extraction and reverse transcription

Cells were harvested and centrifuged, and the cell pellets were used for RNA isolation with TRizol reagent by following the manufaturer's manual. 1 ug of RNA was used for cDNA reverse transcription using a cDNA synthesis kit. The synthesized cDNA was kept at -20°C.

Western blot

Total protein was extracted from cultured cells using a protein extraction kit by following the manufacturer's manual. The protein concentration was determined by BCA protein assay. The protein aliquots were kept at -80°C. Western blot was performed as previously described [13]. Briefly, 40 µg of protein was loaded for each lane and separated on 10% SDS-PAGE gels. After transferring and blocking, the membranes were incubated with anti-p53 or β-actin antibody overnight at 4°C, followed by secondary antibody for 1 hr at room temperature. The bands were visualized using ECl reagents and scanned for density analysis. The expression of β-actin was used as an internal control for sample loading.

Cell proliferation assay

Cells in 96-well plates were transfected with siRNA or plasmid and continuously incubated for 6 hrs, 12 hrs, 24 hrs, and 36 hrs. Ten µl of CCK8 solution was added into each well and the cells were continuously incubated for 3 hrs. The absorbance value was read at 450 nm in an ultraviolet spectrophotometer.

Cell invasion assay

Invasion assay was conducted in a 24-well Transwell chamber where the membranes were coated with matrigel. 200ul of (1×105 cells/ml) cell suspensions in serum free medium were spread onto the wells in the upper chamber and 1000 ul of complete cell medium containing 10% FBS were applied to the wells in the lower chamber and incubated for 12, 24 and 36 hrs at 37°C, 5% CO2. The membranes at in the lower chamber were removed and stained with 0.1% crystal violet dye for 15 min. The cells were counted under a Fluorescence Inversion Microscope.

The cell migration assay

The cell migration was analyzed using wound healing assay. Briefly, 5 × 105 cells were seeded in 6-well plates. After transfection, cells were cultured for 24h and then the scratch line was made using pipette tips. After rinsing with 1 × PBS for 3 times, cells were continuously cultured in serum-free medium for 24 hrs. After taking photos, the scratch width in photos was analyzed using Image J software

Data analysis

Data were analyzed using SPSS software and presented as mean ± standard error. Variance Analysis was used for statistical difference. A p< 0.05 was considered statistically different.


TP53 gene expression in BGC-823 cells

Transfection of TP53 siRNA was significantly downregulated (Fig. 1A), but transfection of pEGFP-TP53 plasmid was significanly increased (Fig. 1B), TP53 mRNA expression. pEGFP-TP53 transfection resulted in 2.78-fold increase in p53 protein expression(Fig. 1C, 1D). In contrast, transfection of TP53 siRNA resulted in 67% decrease in p53 protein expression (Fig. 1C, 1D). These findings suggested that the TP53 gene was successfully silenced or overexpressed

Fig. 1 TP53 gene expression. A) RT-PCR detection of TP53 mRNA expression after transfection of TP53 siRNA (left panel) and pEGFP-TP53 plasmid (right panel). **P< 0.01 between two groups. B) Western blot of P53 protein expression. Left panel: representative Western blots. Right panel: semi-quantatitive analysis of Western blots. *P< 0.05, **P< 0.01 vs. pEGFP-TP53.

Analyzing the effect of p53 on cell proliferation

Cell proliferation was analyzed using CCK-8 kit. TP53 siRNA transfection significantly increased proliferation in BGC-823 cells (P< 0.05). In contrast, pGEFP-TP53 plasmid transfection significantly decreased proliferation in BGC-823 cells (P < 0.05) (Fig. 2).

Fig. 2 Cell proliferation rates. Cells were transfected with TP53 siRNA or pEGFP-TP53 plasmid. *P< 0.05, **P< 0.01 vs. pEGFP-TP53

The effect of P53 expression on cell migration

Wound healing assay showed that TP53 siRNA transfection significantly increased cell migration (P< 0.05), pGEFP-TP53 plasmid transfection significantly decreased cell migration in BGC-823 cells (P < 0.05) (Fig. 3).

Fig. 3 The effect of p53 on cell migration. Migration was analyzed by wound healing assay.

The effect of TP53 expression on cells invasion

Transwell invasion assay showed that TP53 siRNA transfection significantly increased cell invasion (P< 0.05), but pGEFP-TP53 plasmid transfection significantly decreased cell invasion in BGC-823 cells in a time dependent manner (P< 0.05) (Fig. 4).

Fig. 4 The effect of p53 on cells invasion. A) Images of transwell champer assay. B) Cell counting of transwell chanper assay. *P< 0.05, **P< 0.01 vs. TP53 siRNA transfection at the same time point.


Despite the increasing understanding of the molecular mechanisms and the subsequent development in targeting therapy for gastric cancer, the prognosis of gastric cancer remains poor, particularly in patients with advanced disease. TP53 gene mutations were reported in as high as 77% of patients with metastatic tumors [11]. These mutations impair p53’s tumor suppressor effects, such as inhibiting proliferation, migration and invasion of gastric cancer cells [12]. However, there is no direct evidence regarding whether restoring TP53 gene function could inhibit the growth and metastasis of gastric tumor. This study demonstrated that silencing of TP53 gene expression significantly increased cell proliferation, invasion and migration in gastric carcinoma cells, whereas overexpression of p53 significantly inhibited cell proliferation, invasion, and migration.

The functions of p53 protein include controlling the cell cycle, inhibiting proliferation, inducing cell apoptosis, stimulating functional factor synthesis, and regulating cell metabolism, as well as taking part in DNA damage repair. [14]. Dysfunction of p53 is also one of the important forces driving the carcinogenesis and progression of gastric cancer. Various types of TP53 gene mutations not only damage its original tumor suppressor function, but also produce new functions that promote the development and progression of tumors. In this study, silencing of TP53 gene expression enhanced the malignant phenotype of human gastric adenocarcinoma cells. In contrast, expression of wild-type p53 protein restored the tumor suppressive function of p53 in gastric adenocarcinoma cells. This study indicates that p53 plays a very important role in regulating the proliferation, migration, and invasion of gastric carcinoma cells. Restoring TP53 gene expression can restore p53 tumor suppressive function and can be used as a treatment strategy.

In conclusion, overexpression of p53 could be a strategy for target inhibition of gastric carcinoma cell growth and metastasis.
















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