Regulatory role of programmed cell death 4 in precancerous gastric lesions and early gastric cancer

Background Programmed cell death 4 (PDCD4) as a newly identified tumor suppressor is involved in inhibiting tumorigenesis and tumor progression. Epithelial mesenchymal Transition (EMT) is also associated with tumorgenesis of gastric cancer. However, few studies have elucidated the role of PDCD4 in regulation of EMT during precancerous gastric lesions (PLGC) and early gastric cancer. Methods In this study, the expression of PDCD4 and EMT-associated proteins in PLGC and early gastric cancer tissues were detected by immunohistochemistry. The relationship between the expression of PDCD4 and EMT-associated proteins and clinical and pathological parameters were analyzed. The PDCD4 high-expressed SGC-7901 cell line was also used to evaluate the function of PDCD4 in vitro and in vivo. Results PDCD4 and EMT associated proteins expression were significantly altered in PLGC and early gastric cancer tissues. After transfection with the PDCD4 gene, the expression of E-cadherin was significantly increased, the expression of N-cadherin and Vimentin were also remarkably inhibited. PDCD4 high-expressed also inhibited tumor growth and pathological features in nude mice xenograft model. Conclusion This study elucidates a regulatory role of PDCD4 in PLGC and early gastric cancer and provide insights into EMT-associated target which may provide novel therapeutic recourse.

Background Gastric cancer is often described via a sequence of events, a stepwise progression from non-active gastritis (NAG), chronic active gastritis (CAG), precursor lesions of gastric cancer (PLGC) and gastric cancer [1]. Although many researchers have studied the molecular mechanisms of carcinogenesis and tumor progression of gastric cancer, the pathogenesis has remained largely unknown [2][3]. Thus, it is very important to investigate the mechanisms of gastric cancer initiation and progression and find an effective preventive method for gastric cancer [4].
Programmed cell death protein 4 (PDCD4) is a novel tumor suppressor protein involved in programmed cell death [5]. Its association with cancer progression and survival in patients with gastric cancer [6]. In recent years, scientific attention has turned to the mechanistic and functional of PDCD4 in the tumorigenesis of early gastric cancer, much of which remains unknown [7].
Epithelial-mesenchymal transition (EMT) describes a developmental switch from a polarized epithelial phenotype to a highly motile mesenchymal phenotype, is associated with the cell invasion and motility in gastric cancer [8][9]. It plays an important role in gastric cancer initiation and progression [10].
The present study examined the expression of PDCD4 and EMT-associated proteins in gastric tissues of multiple pathologic conditions, and to explore the possible biological function of PDCD4 in gastric cancer cells. These findings shed important light on the molecular mechanisms of PDCD4 in regulation of EMT during PLGC and early gastric cancer and find novel diagnostic and therapeutic targets for early gastric cancer.

Methods
Patients and tissue specimens Gastric tissues were collected from patients from the First Affiliated Hospital of Anhui Medical University from July 2017 to June 2018. A total of 150 subjects were enrolled in this study, including 30 healthy subjects who underwent routine endoscopic screening for gastric cancer, 90 patients with atrophic gastritis associated with various degrees of dysplasia (mild, moderate and severe, n = 30 in each group) and 30 patients with early gastric cancer. All specimens were histopathologically confirmed. This study was approved by the Human Research Ethics Committee of Anhui Medical University. Written informed consents were obtained from all participants.

Immunohistochemistry
Paraffin sections were baked overnight and routinely processed. Endogenous peroxidase activity was quenched by 3% hydrogen peroxide for 10 minutes. Then, the antigen were then blocked for 20 minutes with Bovine serum albumin (BSA, 5%) and the slides were

Western blot
After the protein was processed, the protein liquid was separated by SDS-PAGE and transferred into nitrocellulose membranes (Millipore, Bedford, MA). The blot was blocked in blocking buffer (5% not-fat dry milk and 1% Tween-20 in PBS) for 2 hours at room temperature, and then incubated with appropriate primary antibodies in blocking buffer overnight at 4 o C. The membranes were washed and incubated with secondary antibodies and were analyzed with an enhanced chemiluminescent reagent. The bands from western blotting were quantified by Quantity One analysis software (Bio-Rad).

Immunofluorescence
The cells were fixed with 4% paraformaldehyde for 30 minutes, permeabilized with 0.04% Triton X-100 in PBS, blocked in DMEM with 10% FBS. Primary antibodies were incubated at Biopsies were divided into normal tissue, mild dysplasia, moderate dysplasia, severe dysplasia, early gastric cancer by hematoxylin and eosin staining (Fig. 1). The expression of PDCD4 and E-cadherin appeared to gradually decrease as the gastric lesions evolved from very mild forms to more severe forms, while the expression of N-cadherin and Vimentin were opposite to that of change ( Fig. 1 and Table1). As well, decreased PDCD4 expression was associated with changed EMT-associated proteins (Table 2). Therefore, PDCD4 may play an important role in PLGC and early gastric cancer via EMT pathway.

Transfection with PDCD4 into SGC-7901 cells
To investigate the function of PDCD4 in tumorigenesis and progression of gastric cancer, SGC-7901 cells were stably transfected with pcDNA3.1/PDCD4. Western blot was used to analyze the transfection results ( Fig. 2A). Immunofluorescence and RT-PCR also revealed the protein and mRNA expression of PDCD4 in stable PDCD4 high-expressed SGC-7901 cells (Fig. 2B-C). These results suggested that a stable PDCD4 high-expressed SGC-7901 cell line was successfully established, and can be used for the further biological study.
Effect of PDCD4 on EMT-associated proteins in transfected SGC-7901 cells In order to further study the effects of PDCD4 on EMT-associated proteins in transfected SGC-7901 cells, the expression of E-cadherin, N-cadherin, Vimentin, with both RT-PCR and immunofluorescence assay were detected. The results showed that transfection of SGC-7901 cells with the PDCD4 expression vector resulted in increased levels of E-cadherin expression while it reduced Vimentin and N-cadherin expression (Fig. 3). This data confirm that high expression of PDCD4 has a certain inhibition effect on EMT process, thus providing a basis for the gene target of gastric cancer.

Effect of PDCD4 on tumor growth of gastric cancer in xenograft model
Nude mice were selected for normal group, control group, pcDNA3.1 and pcDNA3.1/PDCD4-expressing xenograft for the assessment of the role of PDCD4 on tumor growth. After thirty days treatment, the results showed that the tumor appearance in the PDCD4 high-expressed group was visibly smaller than control group. Following removal and measurement of all tumors, it was clear that tumor weights were significantly lower in the PDCD4 high-expressed group than those in control group (Fig. 4). These findings indicate that over-expression of PDCD4 could inhibit the gastric cancer cell line in nude mice tumorigenic ability.

Effect of PDCD4 on EMT process of gastric cancer in xenograft model
The results of immunohistochemical staining showed that PDCD4 positive signals was brown in colour and located in the cytoplasm and nucleus while E-cadherin was mainly located on the cytoplasm-facing side of the cell membrane and partially in the cytoplasm.
N-cadherin and Vimentin, appeared to be exclusively located in the cytoplasm.
Interestingly, PDCD4 high-expressed group observably diminished the expression of Ncadherin and Vimentin protein in the tumors, compared to control groups. In contrast, PDCD4 high-expressed group enhanced the expression of E-cadherin (Fig. 5). The present observations indicate that results from the in vivo study were in good agreement with the in vitro results.

Discussion
Gastric cancer is one of the most common types of cancer worldwide, particularly in East Asian populations [11]. Researchers have devoted much to study the mechanisms of carcinogenesis and tumor progression, but the exact information of tumor initiation is remained largely unknown [12].
PDCD4 is a novel tumor suppressor gene and a promising target for anti-cancer therapies [13]. PDCD4 is frequently down-regulated in various human cancers including gastric cancer [14]. However, the role of PDCD4 in patients with PLGC and early gastric cancer have not been fully elucidated. In the present study, we observed a much reduced level of PDCD4 expression in the majority of gastric tissues with severe dysplasia and early gastric cancer, as opposed to the normal gastric mucosa and tissues with mild dysplasia. Therefore, we speculate that PDCD4 might be a potentially valuable molecular target in diagnosis and therapy for early gastric cancer [15].
A key process in promoting tumorigenesis is the EMT, which is a process by which epithelial cells lose their epithelial attributes and acquire a mesenchymal cell phenotype [16]. At the molecular level, EMT is characterized by loss of epithelial cell markers, including the cell adhesion protein, E-cadherin, and by the acquisition of mesenchymal markers, such as N-cadherin and Vimentin [17]. In the past several years, EMT had emerged as one of the most interesting topics in the field of PLGC, and had caused widespread concern [18][19]. The current study indicates that N-cadherin/Vimentin expression in severe dysplasia and early gastric cancer tissues were higher than normal gastric mucosa tissues. However, E-cadherin and PDCD4 decreased in the process of PLGC and early gastric cancer. Interestingly, it exists on a negative correlation to the expression of N-cadherin/Vimentin. These findings indicate that PDCD4 participates in tumorigenesis through the regulation of EMT process [20].
To investigate the hypothesized regulatory role of PDCD4 on the EMT associated proteins, a stable PDCD4 high-expressed SGC-7901 cell line was established. Then, we detected the expression of EMT-associated proteins. The results show that expression of E-cadherin is increase,while expression of N-cadherin and Vimentin is decrease. Taken together, our data demonstrated that high expression of PDCD4 may inhibit gastric tumorigenesis through EMT process.
The in vitro data prompted us to test the anti-tumorigenic role of PDCD4 in vivo [21].Therefore, we established a successful animal model of PDCD4 high-expressed for the first time in a xenograft model in nude mice. The research shows that PDCD4 highexpressed suppressed tumor growth and pathological features, confirming the tumorigenic role of PDCD4. In addition, we also expand our current understanding of the regulatory role of PDCD4 on EMT in gastric cancer, positioning EMT pathway as downstream mediators of PDCD4 function.

Conclusion
The present study discovered that PDCD4 may be a potential therapeutic target and biomarker in gastric cancer patients in the forthcoming future. To our knowledge, our finding is significant because it might be beneficial in the tumorigenesis mechanism of early gastric cancer, and might provide insights in the development of gastric cancer

Consent for publish
Not applicable.

Availability of data and materials
The datasets supporting the conclusions of this article are included within the article. The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This research study was supported by the National Natural Science Foundation of China   Confirmation of stable PDCD4 high-expressed SGC-7901 cells. Plasmids of pcDNA3.1 and pcDNA3.1/PDCD4 were respectively transfected into SGC-7901 cells for 48 h, and then cells were harvested for isolating total cellular RNA or protein.
(a) The expression of PDCD4 was evaluated by western blot assay. GAPDH was served as an internal control of protein level. The relative density was normalized to GAPDH, which was determined by densitometric analysis. (b) The expression of PDCD4 was evaluated by Immunofluorescence assay. The positive cells in each each group were indicated by arrows. Images taken at ×400 magnification. (d) The mRNA expression of EMT associated proteins were measured using RT-PCR.
The data was analyzed using the 2-∆∆Ct method. Data are presented as means ± SD. *p < 0.05 and **p < 0.01 vs. control group. The tumor volume curve of mice xenograted with PDCD4 high-expressed group compared to controls over time. (c) Quantitative tumor weight assessment between the experimental and control group revealed significant differences.
Data are presented as means ± SD. *p < 0.05 and **p < 0.01 vs. control group.

Figure 5
Immunohistochemical evaluation of PDCD4 and EMT associated proteins in xenografted mice. Images taken at ×200 magnification.

Supplementary Files
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