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TCEA3 Attenuates Gastric Cancer Growth by Apoptosis Induction

Background The aim of this study was to investigate and interpret the expression level and potential function of TCEA3 in gastric cancer. Material/Methods qRT-PCR was used to determine the expression level of TCEA3 in gastric cancer tissues. Pearson χ2 test was performed to clarify the correlation between TCEA3 expression and patients’ clinicopathologic characteristics. Biological function of TCEA3 was tested by proliferation assay and colony formation assay. Flow cytometry was used to study the potential function of TCEA3 in apoptosis induction. Results TCEA3 expression was significantly downregulated in gastric cancer tissues compared with paired normal tissues. Poor prognoses were observed in the low TCEA3 expression group of patients in contrast to the high TCEA3 expression group. Functionally, upregulation of TCEA3 inhibited gastric cancer cell proliferation and colony formation. We also found that TCEA3 may attenuate cell growth through apoptosis induction. Conclusions Our findings suggest that TCEA3 attenuates the proliferation and induces apoptosis of gastric cancer cells.


Background
Gastric cancer (GC) is one of the most common cancers and is the second leading cause of cancer deaths worldwide [1][2][3]. Despite great achievements in treatment of gastric cancer, the long-term survival rate for advanced gastric cancer patients is still quite low and the absolute number of gastric cancer patients and deaths is still a large burden [2,4,5]. Uncontrolled growth of cancer cells leads to most of the mortalities and play a critical role in the poor prognosis [6,7]. However, the underlying molecular mechanisms are still poorly understood. Therefore, a better understanding of the molecular mechanisms related to the carcinogenesis of gastric cancer is essential and urgent to develop novel avenues for gastric cancer therapy.
TCEA is one of most important transcription elongation factors; it can directly bind RNA polymerase II, allowing it to read through various transcription arrest sites [8][9][10]. TCEA has 3 distinct isoforms. TCEA1 is ubiquitously expressed, TCEA2 is a testis-specific isoform [11,12], and TCEA3 is another isoform of TCEA about which little is known. In previous studies, Tcea3 was reported to control cell fate of mESCs via regulating the Lefty1-Nodal-Smad2 pathway and that its appropriate level is essential for the balanced pluripotency of mESCs [13]. Moreover, TCEA3 was identified to bind with TGF-beta receptor I and induces Smad-independent, JNK-dependent apoptosis in ovarian cancer cells [8]. However, the potential role of TCEA3 in gastric cancer is still unclear.
The present study demonstrated that TCEA3 expression was significantly downregulated in gastric cancer tissues compared with paired normal tissues. Worse prognoses were observed in patients in the low TCEA3 expression group compared to those in the high TCEA3 expression group. Functionally, upregulation of TCEA3 inhibited gastric cancer cell proliferation and colony formation. We also found that TCEA3 can attenuate cell growth through apoptosis induction.

Tissue specimens and cell culture
We collected 34 tissue specimens from patients diagnosed with gastric cancer at the First Affiliated Hospital of Wenzhou Medical University. Tissues were harvested freshly after sample dissection and preserved at -80°C. Immortalized normal gastric mucosal epithelial cell line GES-1, gastric cancer cell lines MKN-45 and SGC-7901 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in RPMI 1640 medium containing 10% FBS (GIBCO, Carlsbad, CA, USA) and cultured at 37°C with 5% CO 2 .

Western blot
CDK6 and b-actin protein expression was performed as previously described [14]. The primary antibody for TCEA3 was purchased from ProteinTech (ProteinTech Group, Wuhan, China). GAPDH primary antibody was purchased from Abcam (Abcam, USA).

Plasmid and transfection
TCEA3 full-length cDNA was cloned from GES-1 cDNA and then inserted into the Lenti-virus vector PCDH. One day before transfection, MKN-45 and SGC-7901 cells were plated onto 6-well plates. After 24 h, when the cell confluence was about 70%, transfection was performed with Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions.

Cell proliferation assay
Cell proliferation rates were calculated at 0, 24, 48, and 72 h after seeding in 96-well plates. Cells were cultured in 10% CCK-8 (DOJINDO) diluted in RPMI 1640 medium at 37°C until visual color conversion appeared. Quantification was carried out with a microtiter plate reader according to the manufacturer's instructions.

Colony formation
Cells were suspended in RPMI-1640 and seeded in 6-well culture plates at a density of 1×10 3 cells per dish. The plates were incubated for 2 weeks and the number of colonies was counted after staining with 0.1% crystal violet solution. Colonies of more than 50 cells were manually counted. The experiments were independently triplicated.

Flow cytometric analysis for apoptosis
Cells transfected with TCEA3 or negative control (NC) were plated in 6-well plates. After 48-h incubation, the cells were harvested and flow cytometry was performed with the AnnexinV/PI double-staining kit (BD Biosciences, USA) according to the manufacturer's instructions. All experiments were repeated 3 times.

MOLECULAR BIOLOGY
Statistics IBM SPSS 19.0 statistical software was used to analyze the data, which are expressed as mean ± standard deviation (SD).
The differences between groups were analyzed using the t test or one-way ANOVA, as appropriate. Differences were considered statistically significant at P<0.05.

Results
TCEA3 was downregulated and was associated with poor prognosis in gastric cancer In 34 gastric cancer patients, TCEA3 expression was investigated by qPCR. We found the expression level of TCEA3 in cancer tissues was significantly lower than in paired normal tissues (P<0.05) (Figure 1). To investigate the correlation between TCEA3 expression and the clinicopathologic characteristics of gastric cancer, we analyzed the expression of TCEA3 combined with clinicopathologic data. As shown in Table 1, while the expression of TCEA3 was not related to sex (P=0.6), we found significant association of age (P=0.66), tumor location (P=0.56), lymph node metastasis (P=0.19), or differentiation degree (P=0.23), TCEA3 with TNM stage (P=0.009) and depth of invasion (P=0.03).

TCEA3 inhibited the growth of gastric cancer cells
In gastric cancer cell lines MKN-45 and SGC-7901, TCEA3 also showed relatively lower expression than in immortalized normal gastric mucosal epithelial cell line (GES-1) (Figure 2A, 2B). To explore the potential role of TCEA3 in gastric cancer, we transfected MKN-45 and SGC-7901 cells with exogenous TCEA3 or negative control (NC). Then, we used proliferation and colony formation assays to evaluate the effect of TCEA3 on cell proliferative ability. As indicated in Figure 2C and 2D, the TCEA3 had a significantly slower function in proliferation for both MKN-45 and SGC-7901 cells. Consistent with the results of the proliferation assay, TCEA3 also remarkably inhibited colony formation ( Figure 3A, 2B). Our results show that TCEA3 can suppress the proliferation of gastric cancer cells.

TCEA3 induced gastric cancer cell apoptosis
To determine if the attenuation effect in cell growth by TCEA3 was due to the induction of apoptosis, flow cytometry was performed to evaluate the apoptotic cell rate. As shown in Figure 4A and 4B, the number of apoptotic cells significantly increased in MKN-45 and SGC-7901 transfected with TCEA3 compared to the NC group.

Discussion
TCEA3 was reported to bind with TGF-beta receptor I and induces Smad-independent, JNK-dependent apoptosis in ovarian cancer cells [8]. In the present study, we found that TCEA3 expression was significantly downregulated in gastric cancer tissues compared with paired normal tissues. Poor prognoses, especially the advanced TNM stage, were observed in the low TCEA3 expression group of patients in contrast to the high TCEA3 expression group, which indicates the potential correlation between TCEA3 and gastric cancer growth. Functionally, up-regulation of TCEA3 inhibited gastric cancer cell proliferation and colony formation. Interestingly, we also found that TCEA3 may attenuate cell growth through apoptosis induction.
Numerous suppressive genes have been demonstrated to be downregulated in gastric cancer and associated with the prognosis, like KIAA1324, MiR-361-5p, and EGR3 [15][16][17]. Apoptosis induction is a critical function by which suppressive genes