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Abstract

BACKGROUND:

Gastric cancer is one of the most common malignancies worldwide. Recent studies reported that Piwil3 was overexpressed in various cancers, including gastric cancer (GC). This study was intended to investigate its function and mechanism in GC progress.

METHODS:

Quantitative real time PCR(RT-PCR) and western blotting assays were utilized to measure mRNA and protein expression levels, respectively. SiRNA transfection was performed to suppress the expression of Piwil3. CCK-8 assay, cell invasion and migration assays were used to determine the cell proliferative, cell invasive and migratory ability.

RESULTS:

The expression of Piwil3 was significantly increased in GC tissues compared with matched normal tissues. The specific siRNA significantly inhibited the protein and mRNA expressions of Piwil3, and effectively inhibited the proliferation and induced G0/G1 phase arrest in GC cells. Downregulation of Piwil3 significantly suppressed the migration and invasion of GC cells. Moreover, the downregulation of Piwil3 also significantly suppressed the tumor volumes in nude mice. Mechanism investigation showed that the downregulation of Piwil3 significantly decreased the mRNA and protein expressions of metastasis-related genes, including RhoC, MTA1, MMP2 and MMP9, and also modulated the phosphorylation levels of JAK2 and STAT3 but not their protein levels.

CONCLUSIONS:

These findings indicate that overexpression of Piwil3 promotes the proliferation, migration and invasion of GC cells partially through JAK2/STAT3 signal pathway.

Keywords

Piwil3 gastric cancer proliferation metastasis JAK2/STAT3

1. Introduction

Gastric cancer (GC) is the third leading cause of cancer-related death worldwide (account for 8.8% of total cancers), and is considered to lead the highest mortality rates in Eastern Asia and the lowest in Northern America [1]. Most patients with GC have develop advanced stages when diagnosed and their 5-year survival rates are less than 7% [2, 3].Tumor cell metastasis is major responsible for the high mortality rates but the molecular mechanism of GC metastasis remains unexplained.

PIWI belongs to Argonaute family which includes PIWIL1, PIWIL2, PIWIL3, and PIWIL4. It has been shown that PIWIs are aberrantly expressed in a variety of cancers and play important roles in tumorigenesis and development of cancers. For example, Rosenkranz et al. demonstrated dynamic expression of PIWIL1, PIWIL2 and PIWIL3 during mammalian oncogenesis [4]. The expression of PIWIL1–4 were also increased in the primary tumor and metastatic tissues in epithelial ovarian cancer [5]. PIWILs also regulate the progress of breast cancer [6]. However, the expression and bio-function of Piwil3 in GC development and its underlying mechanism remain largely unclear.

In the present study, we reported that the expression of Piwil3 was significantly increased in GC tissues. Downregulation of Piwil3 induced by specific siRNA significantly suppressed the proliferation and induced G0/G1 phase arrest in GC cells in vitro and in vivo. Moreover, the downregulation of Piwil3 also repressed the migration and invasion of GC cells in vitro. Furthermore, mechanism investigation revealed that Piwil3 regulated the expression of metastasis-related genes in JAK2/STAT3 signaling pathway.

2. Methods and materials

2.1 Patients and samples

Totally, 52 pairs of GC tissues and matched normal tissues were collected from the patients with GC, who were enrolled in Huaihe hospital according to the protocols approved by the Ethics Review Board.

2.2 Cell culture

Human GC cell lines (AGS, N87, SGC7901, MKN45 and BGC823) were purchased from Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM (St. Louis, MO, USA) containing 10% FBS and 1% penicillin/streptomycin at 37 ${}^{\circ}$ C in a humidified atmosphere of 5% CO ${}_{2}$ .

2.3 SiRNA transfection

MKN45 cells were selected to perform transfection because of its highly expressed Piwil3. The cells were seeded onto 12-well culture plates at a density of 6 $\times$ 10 ${}^{4}$ cells/well and transfected with the specific siRNAs for Piwil3 or controls (Genepharma, Shanghai, China) by using Lipofectamine ${}^{\rm TM}$ 2000 (Invitrogen, Shanghai, China) for 48 h according to the manufacturer’s protocol.

2.4 Cell proliferation assay

Cell proliferation was evaluated by using Cell Counting Kit 8 (CCK8, Beyotime, Shanghai, China) according to the manufacturer’s protocol. Briefly, MKN45 cells with transfection of the siRNA or control were seeded onto 96-well culture pates at a density of 4 $\times$ 10 ${}^{3}$ cells/well. The proliferation ability of the cells was analyzed after incubation for 0 h, 12 h, 24 h, 48 h and 72 h, respectively. The optical density (OD) values were read at 450 nm using a microplate reader (Thermo, USA).

2.5 Cell cycle

Flow cytometry was used to analyze cell cycle. After transfection for 48 h, the cells were harvested and fixed in 70% ice-cold ethanol (stored at $-$ 20 ${{}^{\circ}}$ C) overnight. The cells were washed with PBS and re-suspended in DNA staining solution (40 $\mu$ g/ml propidium iodide, 250 $\mu$ g/ml RNase and 2 mM EDTA) and incubation for 30 min at 37 ${{}^{\circ}}$ C. Cell cycle was analyzed using flow cytometer (FACSCalibur, BD Biosciences).

2.6 Cell invasion and migration assay

Invasion and migration activity of MKN45 cells were analyzed by using 24-well trans-well chambers coated with or without Matrigel (BD Biosciences) on the upper surface of the membrane with a pore size of 8 $\mu$ m (Sigma). In brief, MKN45 cells (1 $\times$ 10 ${}^{4}$ cells/well) with transfection with siRNA or control were suspended in culture media (100 $\mu$ L, serum free) and added into the upper trans-well chamber. The lower chamber was soaked swith 500 $\mu$ L DMEM medium containing 10% FBS. After incubation for 24 h, the chambers were washed three times and fixed with 4% paraformaldehyde for 10 min. Finally, the chambers were stained with crystal violet and the cells which passed through the membrane were counted visually under a microscope (OLYMPUS).

2.7 Quantitative reverse-transcriptase polymerase chain reaction

Total RNA was extracted from the cells with transfection of siRNA or control by using TRIzol (Invitrogen), respectively. Two microgramme of RNA was used to synthesize cDNA by using a ?rst strand cDNA kit (Sigma, Munich, Germany), according to the manufacturer’ protocol. PCR amplification was performed using a SYBR Green PCR kit (Thermo) and run on an ABI 7300 Thermocycler (Applied Biosystems, Foster City, CA, USA). The parameter of PCR reaction was 95 ${}^{\circ}$ C for 10 minutes, followed by 40 cycles at 95 ${}^{\circ}$ C for 15 s and at 60 ${}^{\circ}$ C for 45 s. The primers were showed in Table 1. Relative expression was calculated using the 2 ${}^{-\Delta\Delta\text{CT}}$ method.

Table 1
Primers used in FQ-RT-PCR analysis

Gene	Primer sequence	Size of PCR product	sSpecies
Piwil3	Forward: 5’-GTGTATCGGGATGGAGTGGGA -3’ Reverse: 5’-TGGCAAGGCGCTGGAACT-3’	146 bp	Human
RhoC	Forward: 5’-TGCCTCCTCATCGTCTTCA-3’ Reverse: 5’-GCCCTTAATGTCACGCACGATTTC-3’	310 bp	Human
MTA1	Forward: 5’-AACAAGCCAAATCCGAAC-3’ Reverse: 5’-GGGACCCCAAGAATACCA-3’	150 bp	Human
MMP2	Forward: 5’-GCTGGAGACAAATTCTGGAGATACA-3’ Reverse: 5’-GTATCGAAGGCAGTGGAGAGGA-3’	281 bp	Human
MMP9	Forward: 5’-GCTGGCAGAGGAATACCTGTAC-3’ Reverse: 5’-CAGGGACAGTTGCTTCTGGA-3’	112 bp	Human
GAPDH	Forward: 5’-CACCCACTCCTCCACCTTTG-3’ Reverse: 5’-CCACCACCCTGTTGCTGTAG-3’	110 bp	Human

2.8 Western blot assay

The concentrations of total proteins in GC cells were determined by using a BCA Protein Assay Kit (Thermo Scientific). An equal amount of proteins was subjected to SDS polyacrylamide gel electrophoresis, followed by electro-transferring onto PVDF membranes. Five percent skimmed-milk powder in PBS with 0.1% tween-20 was used to block the PVDF membranes for 1 h. The membranes were incubated with diluent antibodies of ICAM, RhoC, MTA1, MMP-2 and MMP-9 for overnight at 4 ${}^{\circ}$ C, and then incubated with goat anti-rabbit secondary antibodies (Beyotime, Shanghai, China) for 1 h at room temperature. The bands were visualized using enhanced chemiluminescence detection kit (Santa Cruz Biotechnology, California).

2.9 Cell growth in vivo

MKN45 cells with transfection of siRNA or control were re-suspended in PBS. 2 $\times$ 10 ${}^{6}$ of the cells were injected into the subcutaneous tissues of nude mice. The sizes of formative tumors were measured every four days [7]. After injection for 48 days, the mice were sacrificed, and the formative tumors were photographed weighted on a digital balance.

2.10 Statistical analysis

The correlation of Piwil3 expression with the patient’s clinicopathological variables were analyzed by the chi-square test. All data were presented as the mean $\pm$ SD of three independent experiments and processed with SPSS 18.0 software. Multiple comparisons were performed by one-way ANOVA and Dunnett’s test. $P<$ 0.05 was considered statistically significant.

3. Results

3.1 Piwil3 is frequently increased in gastric cancer tissues and cell lines

The expression of Piwil3 is showed to be increased in various cancers, but in GC its expression is unclear. We initially detected the expression of Piwil3 in GC and matched normal tissues, and the results showed that the mRNA expression of Piwil3 was significantly higher in the GC tissues than that in the matched normal tissues ( $P<$ 0.01) (Fig. 1A). The human gastric cancer tissues were histopathologically classified into intestinal ( $n=$ 25) and diffuse types ( $n=$ 27). The human gastric cancer tissues were further classified according to the Piwil13 expression level into the High-group (Piwil13/GAPDH $>$ 1) and Low-group (Piwil13/GAPDH $<$ 1). High Piwil13 expression was detected in 18 of 25 cases of intestinal-type (72%) and 20 of 27 cases of diffuse-type (74%) of gastric carcinoma (Fig. 1B and C). In the intestinal type of gastric cancer, no significant correlation was found between Piwil13 expression and the clinicopathological findings. In the diffuse-type, Piwil13 expression was correlated with the depth of invasion ( $P<$ 0.05), the lymph node metastasis ( $P<$ 0.05) and the clinical stage ( $P<$ 0.05) (Table 2).

Table 2
Correlation between Piwil13 expression and clinicopathological parameters in two types of gastric carcinoma

Clinicopathological features	Intestinal-type gastric carcinoma ( $n=$ 25)			Diffuse-type gastric carcinoma ( $n=$ 27)
	Piwil13 low	Piwil13 high	P value	Piwil13 low	Piwil13 high	P value
Age	66.37 $\pm$ 10.65	68.41 $\pm$ 8.15	0.43	71.05 $\pm$ 9.38	70.32 $\pm$ 7.65	0.35
Gender			0.74			0.69
Male	2	6		4	12
Female	5	12		3	8
Depth of invasion			0.52			0.021 ${}^{*}$
Early (T1)	4	10		5	4
Advanced (T2/3/4)	3	8		2	16
Differentiation			0.78			0.81
High grade	4	9		4	9
Low grade	3	9		3	11
LN metastasis			0.25			0.032 ${}^{*}$
Negative	3	8		5	5
Positive	4	10		2	15
Clinical stage			0.50			0.043 ${}^{*}$
I/II	2	9		5	7
III/IV	5	9		2	13

Figure 1.

Piwil3 expression in gastric cancer tissues and cell lines. (A) The mRNA expression of Piwil3 is detected in 38 pairs of GC tissues and matched normal tissues by using qRT-PCR. (B) The mRNA expression of Piwil3 is detected in GC cell lines, including AGS, N87, SGC7901, MKN45 and BGC823 by using qRT-PCR. (C and D) Western blot assays reveal the protein level of Piwil3 in GC cell lines. “**” denotes $p<$ 0.01. Data are expressed as the mean $\pm$ SD, $n=$ 38.

Furthermore, we also detected the expression of Piwil3 in GC cell lines. As shown in Fig. 1D–F, the mRNA and protein expressions of Piwil3 were markedly increased in MKN45 cells compared with the other GC cell lines, including AGS, N87, SGC7901 and BGC823. These results suggested that Piwil3 is overexpressed in GC tissues.

Figure 2.

Down-regulation of Piwil3 suppresses the proliferation of MKN45 cells. (A) mRNA expression of Piwil3 is detected in MKN45 cells when transfected with the specific siRNA of Piwil3 for 48 h by using qRT-PCR. (B and C) The protein expression of Piwil3 is detected in MKN45 cells when transfected with the specific siRNA of Piwil3 for 48 h by using western blot. (D) The proliferation of MKN45 cells was analyzed after transfection with the specific siRNA for Piwil3 for 12, 24, 48 and 72 h by using CCK8, respectively. “**” denotes $p<$ 0.01, compared with the control cells; “ ${}^{\#\#}$ ” denotes $p<$ 0.01, compared with the mock cells. Data are expressed as the mean $\pm$ SD, $n=$ 6.

3.2 Specific siRNA for Piwil3 suppresses the proliferation of GC cells and induces GO/G1 arrest.

To investigate the bio-function of Piwil3 in GC cells, we selected MKN45 to perform the functional experiments because of its highly expressed Piwil3. The results showed that the specific siRNA significantly inhibited the mRNA and protein expressions of Piwil3 in MKN45 cells ( $P<$ 0.01) (Fig. 2A–C), suggesting that the specific siRNA is effective. We next detected the effect of Piwil3 on the proliferation of GC cells. The results showed that the specific siRNA for Piwil3 significantly suppressed the proliferation of MKN45 cells when transfection for 48 h ( $P<$ 0.05) or 72 h ( $P<$ 0.01) (Fig. 2D), suggesting that Piwil3 can promote the proliferative ability of GC cells. We further determined whether Piwil3 affected the cell cycle of GC cells. As shown in Fig. 3, the downregulation of Piwil3 significantly induced G0/G1 phase arrest when MKN45 cells transfected with the specific siRNA for 48 h ( $P<$ 0.01).

Figure 3.

Down-regulation of Piwil3 induces G0/G1 phase arrest in MKN45 cells. After MKN45 cells were transfected with the specific siRNA for Piwil3 for 48 h, the cell cycle distribution was identified by using flow cytometry. “**” denotes $p<$ 0.01, compared with the control cells; “ ${}^{\#\#}$ ” denotes $p<$ 0.01, compared with the mock cells. Data are expressed as the mean $\pm$ SD, $n=$ 6.

3.3 Downregulation of Piwil3 represses the migration and invasion of GC cells

Cell motility is an important factor in regulating cancer metastasis. We investigated whether Piwil3 could affect the abilities of migration and invasion of GC cells. The results showed that the specific siRNA significantly repressed the migration and invasion of MKN45 cells when transfection for 48 h ( $P<$ 0.01) (Fig. 4A and B). These suggested that Piwil3 is involved in GC metastasis.

Figure 4.

Down-regulation of Piwil3 suppresses the migration and invasion of MKN45 cells. After MKN45 cells were transfected with the specific siRNA for Piwil3 for 48 h, cell migration (A) and invasion (B) were analyzed by using trans-well assays. “**” denotes $p<$ 0.01, compared with the control cells; “ ${}^{\#\#}$ ” denotes $P<$ 0.01, compared with the mock cells. Data are expressed as the mean $\pm$ SD, $n=$ 6.

3.4 Downregulation of Piwil3 decreases the expressions of cancer metastasis-related genes

To elucidate the potential mechanism involved in Piwil3-induced the invasion and migration of GC cells, we detected the expressions of cancer metastasis-related genes, including RhoC, MTA1, MMP-2 and MMP-9. The results showed that the specific siRNA for Piwil3 significantly decreased the mRNA and protein expressions of RhoC, MTA1, MMP-2 and MMP-9 in MKN45 cells ( $P<$ 0.01) (Fig. 5). These suggested that Piwil3-induced the migration and invasion of GC cells might be associated with the expressions of RhoC, MTA1, MMP-2 and MMP-9.

Figure 5.

Piwil3 regulates the expressions of multiple cancer metastasis-related genes. After MKN45 cells were transfected with the specific siRNA of Piwil3 for 48 h, the mRNA (A) and protein (B and C) expressions of RhoC, MTA1, MMP2 and MMP9 were analyzed by by qRT-PCR and Western blotting, respectively. GAPDH serves as internal reference. “**” denotes $P<$ 0.01, compared with the control cells; “ ${}^{\#\#}$ ” denotes $P<$ 0.01, compared with the mock cells. Data are expressed as the mean $\pm$ SD, $n=$ 6.

3.5 Downregulation of Piwil3 blocks the activity of JAK2/STAT3 signaling pathway

Given the downregulation of Piwil3 significantly decreased the expressions of cancer metastasis-related genes, we investigated whether could its downregulation regulate the activity of JAK2/STAT3 signaling pathway. The results showed that the specific siRNA significantly decreased the phosphorylation levels of STAT3 and JAK2 ( $P<$ 0.01), whereas their protein levels were not significantly altered in MKN45 cells when transfection for 6 h (Fig. 6). These suggested that Piwil3 participated in the activity of JAK2/STAT3 signaling pathway.

Figure 6.

Piwil3 regulates JAK2/STAT3 signaling pathway. After MKN45 were transfected with the specific siRNA of Piwil3 for 48 h, the phosphorylation and protein levels of JAK2 and STAT3 were analyzed by using western blot. “**” denotes $p<$ 0.01, compared with the control cells; “ ${}^{\#\#}$ ” denotes $P<$ 0.01, compared with the mock cells. Data are expressed as the mean $\pm$ SD, $n=$ 6.

3.6 Downregulation of Piwil3 suppresses the growth of GC cells in vivo

Although the downregulation of Piwil3 could inhibit the proliferation of GC cells in vitro, its function in vivo also need to be identified. We constructed a lentivirus vector containing a specific shRNA for Piwil3. After MKN45 cells were infected with this lentivirus for 48 h, the mRNA and protein expressions of Piwil3 were significantly decreased ( $P<$ 0.01) (Fig. 7A and B). These cells were injected into the subcutaneous tissues of nude mice for 48 days, and in the period the sizes of formative tumors were measured several times. The results showed that the downregulation of Piwil3 significantly decreased the sizes of formative tumors in nude mice ( $P<$ 0.01), and reduced the weight of formative tumors ( $P<$ 0.01) (Fig. 7C). These suggested that Piwil3 can promote GC growth.

Figure 7.

Downregulation of Piwil3 suppresses cell growth in vivo. MKN45 cells infected with lentivirus containing the specific shRNA for Piwil or negative control were subcutaneously injected in nude mice. The mRNA (A) and protein (B) expressions of Piwil3 were analyzed by qRT-PCR and Western blotting, respectively. GAPDH serves as internal reference. Tumor volumes (C) were evaluated after injection for 6, 12, 18, 24, 30, 36, 42 and 48 days, respectively. The formative tumors (D) were taken out after injection for 48 days and their weight (E) were measured. “**” denotes $P<$ 0.01, compared with the control cells. Data are expressed as the mean $\pm$ SD, $n=$ 5.

4. Discussion

In the present study, we found that the expression of Piwil3 was increased in GC tissues compared with matched normal tissues. Downregulation of Piwil3 significantly inhibited the proliferation and induced G0/G1 phase arrest in GC cells in vitro and in vivo, and decreased the expressions of metastasis-related genes and also regulated JAK2/STAT3 signaling pathway.

PIWI proteins, defined by highly conserved PiWi (P-element-induced wimpy testis) and PAZ (Piwi/Argonaute/Zwille) domains, have been reported to be involve in the development of several human cancers [8, 9]. Our data confirmed that the expression of Piwil3 was increased in GC tissues compared with matched normal tissues and the high level expression of Piwil3 was associated with the depth of invasion, the lymph node metastasis and the clinical stage in the diffuse type of gastric cancer. Wang et al. reported that the expression of PIWIL1–4 was significantly higher in GC tissues, and the expression of PIWIL1 might serve as an independent prognostic factor for GC [10], and these findings supported our results.

The MKN45 cell line is derived from a poorly differentiated adenocarcinoma with highly metastatic potential (PMID: 3962675). In our current study, it showed highly expressed Piwil3 level. This means that MKN45 cells will be a very nice model system to study the loss of function of Piwil3 in GC cells. When the expression of Piwil3 was down-regulated by using the specific siRNA in MKN45 cells, the cell proliferation, migration and invasion were significantly repressed, suggesting that Piwil3 can regulate the progress of GC, and to our knowledge this finding is first reported by us. We also performed the in vivo experiments revealing that the ability of tumor formation of MKN45 cells infected with a lentivirus containing specific shRNA for Piwil3 was significantly alleviated, and this result is consistent with the experiments in vitro. In addition, we tried to overexpress Piwil3 with lenti-Piwil3 in the moderately differentiated SGC-7901 gastric cancer cell line with minimum Piwil3 level. However, cells were killed 24 hr after transduction and this phenomenon repeated in another round of transduction. Transduction condition will be optimized in the future.

Tumor cell invasion is involved in breaking extracellular matrix, which can be degraded by type IV collagen-degrading enzymes, mainly MMP-2 and MMP-9 [11, 12]. MTA1 was reported to regulate the expressions of MMP-2 and MMP-9 in various cancers [13]. RhoC, one of the Rho GTPases, is most convincingly linked with cancer metastasis. Increasing evidences indicated that RhoC participated in the progress of various cancers, including breast cancer [14], non-small cell lung carcinoma [15], colon carcinoma [16], and hepatocellular carcinoma [17]. In order to preliminarily explain the reason for the regulation of migration and invasion by Piwil3 in gastric cancer, we examined the changes of metastasis-related genes mentioned above after the silencing of Piwil3. Our results revealed that Piwil3 regulated the expression of MTA1, MMP-2, MMP-9 and RhoC. These data suggested that Piwil3 promotes the proliferation, migration and invasion of GC cells through regulating the expressions of multiple cancer metastasis-related genes. However, how Piwil3 induced MTA1, MMP-2, MMP-9 and RhoC expression needs further research.

JAK2/STAT3 signaling pathway plays a crucial role in the development of cancer. STAT3 is an important transcription factor, and phosphorylated STAT3 (pSTAT3) directly induced the transcriptions of multiple cancer-related genes in nucleus. Increasing evidences indicated that the activation of pSTAT3 promoted neoplasm metastasis [18, 19]. Our results showed that the downregulation of Piwil3 significantly inhibited the phosphorylation levels of JAK2 and STAT3, suggesting that Piwil3 indeed participated in regulating the JAK2/STAT3 signaling pathway. Although Piwil3 could regulate the expressions of multiple cancer-related genes, the direct target of Piwil3 remains unclear. Therefore, in the future the exact mechanism by which Piwil3 regulates the JAK2/STAT3 signaling pathway needs to be explored.

In conclusion, our study demonstrated that the expression of Piwil3 was increased in GC tissues compared with matched normal tissues. Enhanced Piwil3 expression in the diffuse type of gastric cancer was associated with the depth of invasion, the lymph node metastasis and the clinical stage in the diffuse type of gastric cancer. Downregulation of Piwil3 suppressed the progress of GC through regulating the expressions of multiple cancer metastasis-related genes and JAK2/STAT3 signaling pathway, suggesting that Piwil3 may be a therapeutic target for GC.

Conflict of interest

The authors declare no conflict of interest.

Footnotes

Acknowledgments

This work is supported by Scientific Research Foundation of Henan (092102310090).

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