Abstract
Poly (ADP-ribose) polymerases 6 (PARP6) is a novel member of the PARP family. Previous studies focused mostly on the role of PARP6 in colorectal cancer; however, the role of PARP6 in gastric cancer is currently unclear. In the present study, we found a high-level expression of PARP6 in gastric cancer cells and PARP6 promoted cell proliferation, migration and invasion. Moreover, we found a positive correlation exists between PARP6 and Survivin, which contributes to tumor ongoing survival. In sum, our data suggest that PARP6 may contribute to gastric cancer progression by activating the Survivin pathway.
Keywords: PARP6, Survivin, gastric cancer
Introduction
Gastric cancer is widely diagnosed as one of the leading causes of cancer death [1]. The most common cause is infection by the bacterium Helicobacter pylori, which accounts for more than 60% of cases [2]. Smoking, dietary factors and obesity are other risk factors [3,4]. However, the pathogenesis has not been fully elucidated yet.
Poly (ADP-ribose) polymerases (PARPs) are multifunctional protein translation enzymes that exist in most eukaryotic cells [5]. PARPs have been reported to be activated by damaging DNA fragments and were thought to have played an important role in DNA damage repair and apoptosis [6-8]. The PARP family has multiple members, including PARP1-4, PARP5α, PARP5β and PARP6-16 [9]. PARP1 was thought to act as an oncogenic gene in breast cancer [10], prostate cancer [11] and pancreatic cancer [12]. However, PARP6, a new member of the PARP family, has not been elucidated in cancer progression. Tuncel et al. [13] reported that PARP6 was mainly distributed in well-differentiated adenocarcinoma cells, and high expression of PARP6 could inhibit the proliferation of HeLa cells in cervical cancer cells. Honda et al. [14] reported that PARP6 was a poor prognostic molecular marker in hepatoblastoma patients. Recent studies revealed that PARP6 could inhibit the proliferation of colorectal adenocarcinoma cells, and was negatively correlated with Survivin in the tissues of colorectal adenocarcinoma [13,15]. But other researchers showed ther opposite result that PARP6 was a carcinogenic factor and there was a positive correlation between PARP6 and Survivin [16]. Survivin is a new member of the inhibitor of apoptosis (IAP) protein family [17]. It has been proven to be closely related to the occurrence, development and prognosis of various tumors [18], including gastric cancer [19,20].
To date, the relationship between PARP6 and Survivin, and the role of PARP6 in gastric cancer is not yet known. That is the subject of the present study.
Materials and methods
Tissue samples
65 postoperative paraffin samples of gastric cancer were obtained from Guizhou Provincial People’s Hospital during September 2016 - September 2017. 10 samples of normal gastric mucosa were examined as controls. All samples were obtained after approval by the Ethics Committees of Guizhou Provincial People’s Hospital. All cases were confirmed by surgery and routine pathology, and had no preoperative chemotherapy or radiotherapy. Patient characteristics are summarized in Table 1.
Table 1.
Expression of PARP6 and Survivin and their correlation with Clinicopathological features in gastric cancer
| Clinicopathological features | PARP6 expression | Survivin expression | ||||
|---|---|---|---|---|---|---|
|
| ||||||
| Low | High | P-value | Low | High | P-value | |
| Normal gastric mucosa | 10 | 0 | P<0.01 | 10 | 0 | P<0.01 |
| Gastric cancer | 9 | 56 | 5 | 60 | ||
| Age (years) | ||||||
| ≥50 | 6 | 36 | P>0.05 | 3 | 39 | P>0.05 |
| <50 | 3 | 20 | 2 | 21 | ||
| Sex | ||||||
| Male | 5 | 40 | P>0.05 | 3 | 42 | P>0.05 |
| Female | 4 | 16 | 2 | 18 | ||
| Metastasis | ||||||
| Yes | 2 | 33 | P>0.05 | 1 | 34 | P>0.05 |
| No | 7 | 23 | 4 | 26 | ||
| TNM stage | ||||||
| I+II | 5 | 40 | P>0.05 | 3 | 42 | P>0.05 |
| III+IV | 4 | 16 | 2 | 18 | ||
| Differentiation | ||||||
| Well and mod | 7 | 24 | P>0.05 | 0 | 31 | P>0.05 |
| Poorly | 2 | 32 | 5 | 29 | ||
Cell lines
Human gastric cancer cells (BGC823 cells) and human normal gastric mucosal epithelial cells (GES-1 cells) were cultured in RPMI 1640 (Hyclone, USA) with 10% fetal bovine serum (FBS) and and cultured at 37°C in 5% CO2.
Plasmids, lentiviral production and infection
A whole-length PARP6 cDNA was subcloned into a vector (PLenti CMV Puro Empty) to get an overexpression vector (PLenti CMV-PARP6). A 21mer sequence (5-GGCGATGCCAACATTAATACT-3) was designed to generate the lentiviral vector of PARP6 short hairpin RNA (shRNA). Lentivirus production and transduction were done according to a previous method [21].
Immunohistochemistry
Biopsy specimens were fixed in 10% formalin, embedded in paraffin and stained with He-matoxylin-Eosin. Immunostaining was performed using the Leica Bond-Max automation system (Leica Biosystems, Bannockburn, IL). Primary antibody Survivin (1:100; ZA-0530, ZSGB-BIO, Beijing, China), or PARP6 (1:300, NBP1-8733, Novus Biologicals, LLC, Littleton, CO, USA) were used. The expression of PARP6 and Survivin was semi-quantitatively evaluated by using imunohistochemical scoring model: -, the majority of the cells were weak or negative; +, 1%-20% of the tumor cells showed moderate immunoreactivity; ++, over 20% of the tumor cells showed moderate or intense immunoreactivity. The high expression of PARP6 and Survivin was graded as ++, and low expression was graded as - or +. The assessment was made by a single senior pathologist (Y.Z).
Western blotting
Protein samples (Control, PARP6 knockdown, and PARP6 overexpression BGC823 cells) were separated on a 10% SDS gel. Proteins were transferred to PVDF membrane (Millipore, Billerica, Massachusetts). After being blocked for 2h at room temperature, the membranes were incubated with primary antibody PARP6 (1:1500, NBP1-52950, Novus Biologicals, LLC, Littleton, CO, USA), Survivin (1:1000; ZA-0530, ZSGB-BIO, Beijing, China), and GADPH (1:20000, 10491-1-AP, ProteintechTM) overnight at 4°C. The proteins were then incubated with the corresponding secondary antibody for 1 h at room temperature. After a thorough wash of the membrane the band was illuminated by using Clarity Western ECL-Substrate (Bio-Rad Laboratories, Inc.: Hercules, CA, USA,).
Wound healing assay
For wound healing scratch assay, control cells, overexpressed PARP6 BGC823 cells and knockdown PARP6 BGC823 cells were seeded in 6-well plates. Scratch wounds were made in confluent cell monolayers using a pipette tip. Monolayers were washed twice with PBS and the cells were cultured in serum-free media. The same area of the gap was imaged by using a microscope equipped with a digital camera at 0, 24 and 48 h after scratching was performed.
CCK-8 assay
Cells were seeded in 96-well plates at an initial concentration of 1×104 cells per well. The cells were incubated with 100 μl of complete medium and cultured for 24 h, 48 h and 72 h in a 5% CO2 incubator at 37°C. Then 100 μl serum free RPMI 1640 containing 10% CCK8 (CCK-8; Dojindo, Kumamoto, Japan) reagent was added in each well, and the cells were cultured for 2 h at 37°C. Finally, the absorbance (D value) of each well at 450 nm was detected by enzyme-linked immunosorbent assay to reflect the cell proliferation ability.
Cell migration and invasion
The top chambers (Corning Incorporated, Corning, NY, USA) were seeded with cells in serum-free media and 20% FBS was used as the chemoattractant in the bottom chamber. Cells were allowed to migrate for 24 h at 37°C. Then, migrated cells were fixed with 4% paraformaldehyde and stained using 0.1% Crystal violet solution. Invasion assay cells were seeded in 200 μl serum-free media into the upper wells which were previously coated with Matrigel basement extract, and 500 μl of media into the bottom wells. After 24 h of CO2 incubation at 37°C, the invasive cells on the bottom surface of the membrane were fixed and stained using 0.1% Crystal violet solution. For further quantification of the migration and invasion cells, five random regions of the filter were counted under microscope. Three filters were used and the experiments were carried out in triplicate.
Statistical analysis
Data were analyzed using the SPSS 13.0 softwares (SPSS Inc., Chicago, Ill., USA). One-way ANOVA and chi-square test were used to compare the data between the groups. Correlations between PARP6 and Survivin expression were determined by Spearman’s correlation coefficient. P<0.05 was considered significant.
Results
Expression of PARP6 and Survivin
PARP6 and Survivin expression was not detectable in normal gastric mucosa (0/10), while most gastric cancer samples had high-level protein expression of PARP6 (56/65) and Survivin (60/65) (Figure 1, Table 1). Furthermore, we detected the expression of PARP6 and Survivin in normal gastric mucosa cell lines (GES-1) and gastric cancer cell lines (BGC823). Results showed that the expression of PARP6 and Survivin were significantly elevated in BGC823 cells compared with GES-1 cells (Figure 2).
Figure 1.
PARP6 and Survivin expression in normal gastric mucosa and gastric cancer tissues. Cases were stained with hematoxylin and eosin (H&E) and immunohistochemistry of PARP6 and Survivin. (Magnification, 200×).
Figure 2.
The expression of PARP6 and Survivin in normal gastric mucosal GES-1cells, gastric cancer BGC823 cells, shRNA-PARP6 and overexpression-PARP6 BGC823 cells. The experiment was repeated independently three times.
Correlation between PARP6 and Survivin in gastric cancer
The correlation between PARP6 and Survivin expression in gastric cancer was examined by immunohistochemistry and western blot (Table 2, Figure 2). The Spearman correlation analysis (γ=0.720, P<0.05) showed that there was a positive correlation between PARP6 and Survivin in gastric cancer (Table 2). In addition, we found that upregulation of Survivin in PARP6 overexpressing gastric cancer cells, and downregulation of Survivin in PARP6 knockdown gastric cancer cells (Figure 2).
Table 2.
Correlation between PARP6 and Survivin expression in gastric cancer
| PARP6 expression | Total | |||
|---|---|---|---|---|
|
| ||||
| Low | High | |||
| Survivin expression | ||||
| Low | 5 | 0 | 5 | P<0.001 |
| High | 4 | 56 | 60 | γ=0.720* |
| Total | 9 | 56 | ||
Spearman correlation analysis.
Wound healing of BGC823 cell in vitro
We examined whether PARP6 could regulate cell motility using a wound healing assay. Results showed that the migration rate of knockdown of PARP6 cells was significantly lower than control cells at 24 and 48 hours. Meanwhile, overexpression of PARP6 cells was significantly higher than control cells (Figure 3).
Figure 3.
Wound healing of control, shRNA-PARP6 and overexpression-PARP6 cancer cells in vitro. (Magnification, 100×). The experiment was repeated independently three times.
PARP6 affects BGC823 cell Proliferation
Cell proliferation was measured by recording changes in absorbance (optical density, OD) at 450 nm wavelength. Results showed that knockdown of PARP6 significantly inhibited BGC823 cell proliferation at 24, 48 and 72 hours (Figure 4). While overexpression of PARP6 in BGC823 cells resulted in a greater percentage of cell proliferation (Figure 4).
Figure 4.
Proliferation of control, shRNA-PARP6 and overexpression-PARP6 cancer cells in vitro. Cell numbers were counted at the following time points: 24, 48, and 72 hours. Cell proliferation was measured using CCK8 assay. The experiment was repeated independently three times. *P<0.05, **P<0.01.
Effects of PARP6 on BGC823 cell migration and invasion
Transwell assay showed that PARP6 knockdown decreased cell migration and invasion of BGC823 cells, while overexpression of PARP6 increased cell migration and invasion of BGC823cells (Figures 5, 6).
Figure 5.
Migration of control, shRNA-PARP6 and overexpression-PARP6 cancer cells in vitro. (Magnification, 200×). The experiment was repeated independently three times. *P<0.05, **P<0.01.
Figure 6.
Invasion of control, shRNA-PARP6 and overexpression-PARP6 cancer cells in vitro. (Magnification, 200×). The experiment was repeated independently three times. *P<0.05, **P<0.01.
Discussion
PARP6, a new member of the PARPs family, is rarely to be studied. Previous studies showed conflicting results regarding the role of PARP6 and the correlation with Survivin in colorectal cancer [13,15,16]. In this study, we explored them in gastric cancer. Our results showed high level expression of PARP6 and Survivin in gastric cancer samples, and there was not expression of them in normal gastric mucosa (Figure 1). Moreover, we detected the expression of PARP6 and Survivin in cell lines. Results showed high level expression of PARP6 and Survivin in BGC823 cancer cells compared with GES-1 normal gastric mucosa cells (Figure 2). These results suggested that PARP6 maybe act as oncogene and had a very deep relationship to Survivin. So, we firstly analyzed the correlation of them. Immunohistochemical results showed a positive correlation between PARP6 and Survivin in gastric cancer (γ=0.720, P<0.05) (Table 2). Furthermore, we found that the expression of Survivin was synchronized with the expression of PARP6 in knockdown or overexpression PARP6 cancer cells (Figure 2). Thus, we concluded that PARP6 and Survivin have a perfect rank correlation, and PARP6 maybe the upstream regulatory factor.
In view of that PARP6 was upregulated in gastric cancer cells, and a significant correlation exists between PARP6 and Survivin, we suspected that PARP6 may be carcinogenic in gastric cancer. To prove this idea, we further assessed the motility, proliferation, migration and invasion of PARP6 overexpression and knockdown gastric cancer cells (Figures 3, 4, 5 and 6). Our results supported the hypothesis that PARP6 plays a carcinogenic role in gastric cancer.
All of these results indicate that PARP6 plays a carcinogenic role in gastric cancer and there was a significant correlation between PARP6 and Survivin. Since Survivin is universally recognized as an oncogene [18-20,22,23], and PARP6 may be an upstream regulatory factor of Survivin, we provide new insight that PARP6 may play a carcinogenic role through the Survivin pathway in gastric cancer. Additional studies are needed to clarify this point.
Taken together, our results provide new insights into the regulation of PARP6 and Survivin in gastric cancer. They are promising targets for the development of new strategies for the diagnosis and treatment of gastric cancer.
Acknowledgements
This study was supported by the Guizhou Province Science and Technology Agency-Guizhou Provincial People’s Hospital Joint Fund, under Grant LH[2016]7136 and LH[2015]7108; National Natural Science Foundation of China (Grant No. 81560088); Science and Technology Foundation of Guizhou Province, under Grant J[2015]2086; and Doctoral Fund of Guizhou Provincial People’s Hospital, under Grant GZSYBS [2017]03. The research work was completed in the Clinical Medical Research Center of Affiliated Hospital of Guizhou Medical University.
Disclosure of conflict of interest
None.
References
- 1.Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ, He J. Cancer statistics in China, 2015. CA Cancer J Clin. 2016;66:115–132. doi: 10.3322/caac.21338. [DOI] [PubMed] [Google Scholar]
- 2.Chang AH, Parsonnet J. Role of bacteria in oncogenesis. Clin Microbiol Rev. 2010;23:837–857. doi: 10.1128/CMR.00012-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Sjödahl K, Lu Y, Nilsen TI, Ye W, Hveem K, Vatten L, Lagergren J. Smoking and alcohol drinking in relation to risk of gastric cancer: a population-based, prospective cohort study. Inter J Cancer. 2007;120:128–132. doi: 10.1002/ijc.22157. [DOI] [PubMed] [Google Scholar]
- 4.Yang P, Zhou Y, Chen B, Wan HW, Jia GQ, Bai HL, Wu XT. Overweight, obesity and gastric cancer risk: results from a meta-analysis of cohort studies. Eur J Cancer. 2009;45:2867–2873. doi: 10.1016/j.ejca.2009.04.019. [DOI] [PubMed] [Google Scholar]
- 5.Perina D, Mikoč A, Ahel J, Ćetković H, Žaja R, Ahel I. Distribution of protein poly(ADP-ribosyl)ation systems across all domains of life. Dna Repair. 2014;23:4–16. doi: 10.1016/j.dnarep.2014.05.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Kim BK, Im JY, Han G, Lee WJ, Won KJ, Chung KS, Lee K, Ban HS, Song K, Won M. p300 cooperates with c-Jun and PARP-1 at the p300 binding site to activate RhoB transcription in NSC126188-mediated apoptosis. Biochim Biophys Acta. 2014;1839:364–373. doi: 10.1016/j.bbagrm.2014.03.004. [DOI] [PubMed] [Google Scholar]
- 7.Feng X, Koh DW. Roles of poly(ADP-ribose) glycohydrolase in DNA damage and apoptosis. Int Rev Cell Mol Biol. 2013;304:227–281. doi: 10.1016/B978-0-12-407696-9.00005-1. [DOI] [PubMed] [Google Scholar]
- 8.D’Amours D, Desnoyers S, D’Silva I, Poirier GG. Poly (ADP-ribosyl) ation reactions in the regulation of nuclear functions. Biochem J. 1999;342:249–268. [PMC free article] [PubMed] [Google Scholar]
- 9.Vyas S, Matic I, Uchima L, Rood J, Zaja R, Hay RT, Ahel I, Chang P. Family-wide analysis of poly(ADP-ribose) polymerase activity. Nat Commun. 2014;5:4426. doi: 10.1038/ncomms5426. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Gonçalves A, Sabatier R, Charafe-Jauffret E, Gilabert M, Provansal M, Tarpin C, Extra JM, Viens P, Bertucci F. Triple-negative breast cancer: histoclinical and molecular features, therapeutic management and perspective. B Cancer. 2013;100:453–464. doi: 10.1684/bdc.2013.1740. [DOI] [PubMed] [Google Scholar]
- 11.Salemi M, Galia A, Fraggetta F, Corte CL, Pepe P, Vignera SL, Improta G, Bosco P, Calogero AE. Poly (ADP-ribose) Polymerase 1 protein expression in normal and neoplastic prostatic tissue. Eur J Histochem. 2013;57:80–82. doi: 10.4081/ejh.2013.e13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Porcelli L, Quatrale AE, Mantuano P, Leo MG, Silvestris N, Rolland JF, Carioggia E, Lioce M, Paradiso A, Azzariti A. Optimize radiochemotherapy in pancreatic cancer: PARP inhibitors a new therapeutic opportunity. Mol Oncol. 2013;7:308–322. doi: 10.1016/j.molonc.2012.10.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Tuncel H, Tanaka S, Oka S, Nakai S, Fukutomi R, Okamoto M, Ota T, Kaneko H, Tatsuka M, Shimamoto F. PARP6, a mono(ADP-ribosyl) transferase and a negative regulator of cell proliferation, is involved in colorectal cancer development. Int J Oncol. 2012;41:2079–2086. doi: 10.3892/ijo.2012.1652. [DOI] [PubMed] [Google Scholar]
- 14.Honda S, Minato M, Suzuki H, Fujiyoshi M, Miyagi H, Haruta M, Kaneko Y, Hatanaka KC, Hiyama E, Kamijo T. Clinical prognostic value of DNA methylation in hepatoblastoma: four novel tumor suppressor candidates. Cancer Sci. 2016;107:812–819. doi: 10.1111/cas.12928. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Qi G, Kudo Y, Tang B, Liu T, Jin S, Liu J, Zuo X, Mi S, Shao W, Ma X. PARP6 acts as a tumor suppressor via downregulating Survivin expression in colorectal cancer. Oncotarget. 2016;7:18812–18824. doi: 10.18632/oncotarget.7712. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Wang H, Li S, Luo X, Song Z, Long X, Zhu X. Knockdown of PARP6 or survivin promotes cell apoptosis and inhibits cell invasion of colorectal adenocarcinoma cells. Oncol Rep. 2017;37:2245–2251. doi: 10.3892/or.2017.5441. [DOI] [PubMed] [Google Scholar]
- 17.Altieri DC, Marchisio PC, Marchisio C. Survivin apoptosis: an interloper between cell death and cell proliferation in cancer. Lab Invest. 1999;79:1327–1333. [PubMed] [Google Scholar]
- 18.Cheung CH, Huang CC, Tsai FY, Lee JY, Cheng SM, Chang YC, Huang YC, Chen SH, Chang JY. Survivin - biology and potential as a therapeutic target in oncology. Onco Targets Ther. 2013;6:1453–1462. doi: 10.2147/OTT.S33374. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Wang T, Zhang X, Guo X, Xian S, Yunfei Lu. 3-Bromopyruvate and sodium citrate target glycolysis, suppress survivin, and induce mitochondrial-mediated apoptosis in gastric cancer cells and inhibit gastric orthotopic transplantation tumor growth. Oncol Rep. 2016;35:1287–1296. doi: 10.3892/or.2015.4511. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Yang B, Huang J, Liu H, Guo W, Li G. miR-335 directly, while miR-34a indirectly modulate survivin expression and regulate growth, apoptosis, and invasion of gastric cancer cells. Tumor Biol. 2016;37:1771–1779. doi: 10.1007/s13277-015-3951-8. [DOI] [PubMed] [Google Scholar]
- 21.Chu M, Chang Y, Guo Y, Wang N, Cui J, Gao WQ. Regulation and methylation of tumor suppressor miR-124 by androgen receptor in prostate cancer cells. PLoS One. 2015;10:e116197. doi: 10.1371/journal.pone.0116197. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Denel-Bobrowska M, Marczak A. The role of survivin in the diagnosis and therapy of gynaecological cancers. Postep Hig Med Dosw. 2016;70:1182–1189. [PubMed] [Google Scholar]
- 23.Caldas H, Jiang YM, Fangusaro J, Mahotka C, Conway EM, Altura RA. Survivin splice variants regulate the balance between proliferation and cell death. Oncogene. 2005;24:1994–2007. doi: 10.1038/sj.onc.1208350. [DOI] [PubMed] [Google Scholar]