Effect of silencing PARG in human colon carcinoma LoVo cells on the ability of HUVEC migration and proliferation

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  • A Corrigendum to this article was published on 18 December 2013

Abstract

Our aim was to investigate the influence of silencing poly-(ADP-ribose)glycohydrolase (PARG) in human colon carcinoma LoVo cells on the ability of human umbilical vein endothelial cell (HUVEC) migration, proliferation and its possible mechanisms. PARG mRNA expression was detected by reverse transcriptase (RT) and real-time-PCR. PARG, poly-(ADP-ribose)polymerase (PARP), p38, p-p38, extracellular signal-regulated kinase (ERK), p-ERK, nuclear factor (NF)-κB, phosphorylated IκBα (p-IκBα), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (b-FGF), intercellular cell adhesion molecule (ICAM)-1 and matrix metalloproteinases (MMP)-9 expressions were detected by western blot. The influence of PARG-short hairpin (sh)RNA on the ability of HUVEC migration and proliferation were observed by transwell migration and Counting Kit-8 (CCK-8) assay. Both RT-PCR and western blot results showed that the expression of PARG in PARG-shRNA cells was decreased and expressions of PARP, p38, p-p38, ERK, p-ERK, NF-κB, p-IκBα, VEGF, b-FGF, ICAM-1 and MMP-9 in those cells were lower than that in the untransfected and control-shRNA groups (P<0.05). Migration assay showed that migratory inhibition rate for HUVEC was decreased (55.23%) in cocultured PARG-shRNA cells; moreover, CCK-8 assay showed that the proliferation of HUVECs cultured with the supernatant of PARG-shRNA cells was also comparatively lower. Hence, concluding that PARG silencing could inhibit the ability of HUVEC migration and proliferation by downregulating the activity of NF-κB in LoVo cells that in turn decreases angiogenic factors such as VEGF, b-FGF, ICAM-1, MMP-9, as well as phosphorylation of p38 and ERK.

Introduction

PARG (poly-(ADP-ribose)glycohydrolase) has many subtypes and different subcellular locations; there are at least three subtypes in the human PARG gene, including 99, 102 and 110 kD being the most important one; moreover, they exist both in the cytoplasm and nucleus.1 The main biological function of PARG is to regulate the function of poly-(ADP-ribose)polymerase (PARP), where reports have suggested that the activity of PARP was decreased in lung adenocarcinoma cells devoid of the PARG gene,2 and that the expression of PARP-1 was also decreased in breast carcinoma cells after being treated with GLTN (PARG inhibitor).3 The results of our previous study also4 revealed the correlation between PARG and PARP expression in colon carcinoma tissues whereby GLTN could inhibit the PARG expression in these cells.

Furthermore, p38 protein kinase and extracellular signal-regulated kinase (ERK) being mitogen-activated protein kinase (MAPK) family members and belonging to the serine/threonine kinases category; which upon phosphorylation generate p-p38 and pERK activities, usually translocate from the cytoplasm into the nucleus when being activated. At present, several studies have been correlated their important biological roles through activating their downstream gene targets, affecting the degradation of extracellular matrix of tumor cells,5 aggregation, adhesion and migration6 and angiogenesis of cancer cells.7 Hence, the belief is there that they have an important role in promoting invasion and metastasis of tumors. Moreover, a PARP inhibitor was found to inhibit p38 expression and cause the phosphorylation of retinal neurons in mice model of chronic hypoperfusion and chorionic cells in chicks.8, 9

It has also been reported that inhibiting PARP could adjust p3810 and that ERK phosphorylation of the MAPK pathway, could then downregulate nuclear factor (NF)-κB transcriptional11 activity and consequently decrease the expressions of NF-κB-dependent genes such as intercellular cell adhesion molecule (ICAM-1), which is important during inflammatory processes. As, our previous results also demonstrated that inhibiting PARP could inhibit NF-κB activity in mice colon cancer CT26 cells;12 vascular endothelial growth factor (VEGF), b-FGF (basic fibroblast growth factor), ICAM-1 and matrix metalloproteinases (MMP)-9 being well known NF-κB-dependent angiogenic-related factors, reflecting the relative migratory and proliferative ability of tumors were assessed in our experimental trial.13, 14, 15, 16, 17, 18

Till now, there has been no report that whether PARG could affect angiogenesis and the related expressions of p38 and ERK phosphorylation, p38, ERK, NF-κB, phosphorylated IκBα (p-IκBα), VEGF, b-FGF, ICAM-1 and MMP-9 in colon carcinoma cells. Therefore, we decided to unveiled this relationship in PARG-silenced LoVo cells, and simultaneously we checked the influence of silencing PARG in LoVo cells on the ability of human umbilical vein endothelial cell (HUVEC) migration and proliferation. However, we aimed preliminarily to observe the influence of PARG in colon carcinoma LoVo cells on the ability of promoting HUVEC proliferation, migration and its possible related mechanisms.

Materials and methods

In vitro

Cell culture

Human LoVo colon carcinoma cell line and human umbilical vein endothelial cells (HUVECs) were obtained from Professor Tang WX in Chongqing Medical University; purchased from the Institute of Biochemistry and Cell Biology (Shanghai, PR China), were grown and cultured in a 5% CO2 incubator at 37 °C in RPMI 1640 medium supplemented with 10% fetal bovine serum, and 100 U ml−1 of Penicillin and 100 μg ml−1 of Streptomycin (Sigma-Aldrich, Seelze, Germany).

Moreover, an ERK inhibitor U0126, a p38 inhibitor SB203580 and pyrrolidine dithiocarbamate (PDTC) a specific and selective inhibitor of NFκ-B were used to treat PARG-short hairpin (sh)RNA-transfected LoVo cells to show correlations between the latter, PARP and PARG.19, 20, 21

LoVo cell transfection

Transfection was done with lentiviral based shRNA vector (Sigma-Aldrich-TRCN00001265) with the PARG-shRNA interference sequence being: 5′-IndexTermCCGGGCGATCTTAGGAAACGGTATTCTCGAGAGTACCGTTTCCTAAGATCGCTTTTTG-3′ targeting PARG gene; while for the control, nontarget shRNA control transduction particles were used (Sigma-Aldrich). Transduction efficiency was optimized using pLKO.1 puro-Turbogreen fluorescein protein (GFP; Sigma-Aldrich) and steps were carried out according to the manufacturer’s instructions.

LoVo cells were plated in a 96-well plate at 1.6 × 104 in each well, and cells were incubated until they reached 70% confluence. After which, media was gently aspirated, 200 μl of fresh RPMI-1640 was added together with 8 μg ml−1 hexadimethrine bromide per well; then different concentrations of 2, 5, 10 and 15 μl of lentivirus particles with TurboGFP were added, and plate was left for incubation for 48 h and for each lentivirus construct triplicate wells were used. The latter was then subjected to fluorescence microscopy so as to assess the concentration of lentivirus that has resulted in optimal transduction efficiency. It was found that the 10 μl lentiviral particle concentration had displayed the maximum TurboGFP visible under microscopy (Figure 1).

Figure 1
figure1

(a) LoVo cells transfected with TurboGFP ( × 400). In all, 2, 5, 10 and 15 μl of lentiviral particles containing TurboGFP were transfected into LoVo cells. After 48 h of incubation at 37 °C, fluorescin was displayed in LoVo cells. (A) Represents 2 μl of lentiviral particles containing the TurboGFP group, (B) 5, (C) 10 and (D) 15 μl. (b) Total RNA extract result in untransfected, control-shRNA and PARG-shRNA cells. (cf) Effect of knockdown PARG by shRNA in LoVo cells. RT-PCR reveals complete inhibition of PARG expression in PARG-shRNA-transfected cells compared with untransfected and control-shRNA cells. β-Actin was used as an internal loading control. (g) Expression of PARG mRNA detected by real-time-PCR in different groups LoVo cells.

Subsequently, 10 μl of lentivirus particles of either nontarget shRNA or PARG-shRNA were added to wells previously containing hexamethrine bromide and were incubated for 48 h. Following which, media was removed and 200 μl of fresh media together with Puromycin to a final concentration of 8 μg ml−1 (Sigma-Aldrich) was added to each well. Media was replaced with fresh media containing Puromycin every 2–3 days until resistant colonies could be identified.

Finally, the experimental group consisted of LoVo cells transfected with PARG-shRNA (PARG-shRNA) while the controls included both untransfected LoVo cells (control) and LoVo cells transfected with nontarget shRNA (control-shRNA). Reverse Transcriptase (RT)-PCR and western blot analysis were used to detect PARG knockout in human LoVo colon carcinoma cells.

Reverse transcriptase-PCR (RT-PCR)

Total RNAs was extracted from control, control-shRNA and PARG-shRNA LoVo cells with Trizol reagent (Takara, Dalian, China) and reversely transcribed into DNA, respectively. Genes were detected with a template of cDNA using the following oligonucleotide primers: PARG, 5′-IndexTermCCACCTCGTTTGTTTTCA-3′(sense) and 5′-IndexTermCCAACATCTGGCAAAGGA-3′(antisense); β-actin, 5′-IndexTermGTCAAGAAAGGGTGTAACGCAAC-3′(sense) and 5′-IndexTermTCCTGTGGCATCCACGAAACT-3′(antisense). In total, 35 PCR cycles were used for the amplification of RT products (94 °C for 30 s, 50–58 °C for 30 s, 72 °C for 1 min and then 5 min for the last extension). Then, PCR amplification products were separated on a 1.8% agarose gel. The experiment above was performed in triplicate.

Real-time-PCR

The Trizol reagent (Takara) was used to extract total RNA genes from control, control-shRNA and PARG-shRNA LoVo cells, and genes were detected with a template of cDNA using the following oligonucleotide primers: PARG, forward primer: 5′-IndexTermTTGGTCCCGGAGCCACGAAGAT-3′, reverse primer: 5′-IndexTermTGGCATCACCCCCAAAGGCA-3′; GAPDH primers, forward primer: 5′-IndexTermGGTGAACGCTGTGAACGG-3′, reverse primer: 5′-IndexTermTGTTAGTGGGGTCTCGCTCCT-3′. RT-PCR was carried out according to the manufacturer’s instructions. The absolute value of slope of each sample should be <0.1, and the amplification efficiency should be >99%. was used to analyze the data and calculate the amplification quantity. GAPDH acted as an internal loading control.

Western blot analysis

Cells were washed once with phosphate-buffered saline and collected by scraping into individual EP tubes. Cell protein extraction was done according to protein extraction protocols and protein concentrations were determined using the Coomassie Blue assay (Pierce Biotechnology, Woburn, MA, USA). Then, proteins (20 μg per lane) were loaded in 10% polyacrylamide gels (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), which were separated by electrophoresis; later transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) and unspecific reactions on the membranes were blocked with 5% non-fat dry milk for 2 h. Primary antibodies against PARG (Abcam, Cambridge, UK), PARP, NF-κB, p-p38, ERK, p-ERK, NF-κB, p-IκBα, VEGF, b-FGF, ICAM-1, MMP-9 and β-actin antibodies (the dilution concentration of antibodies were 1:1000, 1:1000, 1:1000, 1:2000, 1:1000, 1:2000, 1:1000, 1:1000, 1:1000, 1:1000, 1:1000, 1:1000 and 1:500, respectively; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were incubated overnight at 4 °C. Secondary antibodies (peroxidase-conjugated goat or anti-rabbit immunoglobulin G) were incubated for 1 h at 37 °C. Blots were washed three times, exposed to chemiluminescence reagents (Pierce Biotechnology), displayed to photographic film (Bio-Rad, Hercules, CA, USA) and evaluated by densitometric analysis using the Quantity One software.18 All respective experiments were performed in triplicates.

Transwell migration assay

Cell migration assay was done using 8.0-μm pore size Transwell inserts (Costar, Milpitas, CA, USA) as described formerly with some modification.22 The under surface of the membrane was coated with fibronectin (10 μg ml−1; Sigma, Ronkonkoma, NY, USA) in phosphate-buffered saline for 2 h at 37 °C. The lower chamber was filled with 500 μl of RPMI 1640 medium with 10% fetal bovine serum. LoVo cells were resuspended into the migration medium (serum-free RPMI1640) and 1 × 105 cells in 200 μl of medium were added to the upper chamber after being washed twice with phosphate-buffered saline. After 24 h of incubation at 37 °C, HUVECs were plated 105 per transwell and incubated for 8 h, the cells on the upper surface of the membrane were gently and carefully scraped out using cotton tips. The migrant cells that were adherent to the lower surface of the membrane were fixed in 10% formalin at room temperature for 10 min, and then stained with Hematoxylin and Eosin stain. The results of the cell migration assay were evaluated by counting the number of cells on the lower surface of the membrane under an inverted microscope in five different fields at a magnification of × 400.22

Counting kit-8 (CCK-8) assay

One hundred microliter of respective HUVEC cell suspensions (5 × 103 cells per well) were dispensed into triplicate in 96-well plates and incubated for 24 h; then LoVo cells supernatant to each well were added, respectively, according to the concentration of the supernatant 1:2, 1:4, 1:8, 1:16, 1:32 and 1:64. After 72 h, 10 μl of the Cell CCK-8 (Dojindo, Kumamoto, Japan) solution was added to the wells and plates were incubated for 1 h. Absorbance (optical density (OD)) was read using a universal microplate reader (Bio-Tek, Winooski, VT, USA) at 450 nm and a graph of A450 against concentration was plotted. Each mark represents the mean of collected readings and the procedure was repeated at least three times. The percentage of inhibition of proliferation was calculated by (mean OD for control-mean OD for PARG-shRNA)/mean OD for control × 100%.

Enzyme-linked immunosorbent assay (ELISA)

Tested supernatants were obtained on the sixteenth hour from untransfected, control-shRNA and PARG-shRNA-cultured LoVo cells. ELISA was performed on 100 μl aliquots of the cultures’ supernatants from both PARG-shRNA and control-shRNA cells; to estimate the concentration of VEGF and b-FGF. The ELISA kit was used according to manufacturer’s instructions and the OD450 was measured using a microplate reader model 3550 (Bio-Rad, Hercules, CA, USA).

In vivo

Intratumoral microvessel density (iMVD) for splenic allografted tumor in animal model

A total of 18 Balb/c female mice, 6–8-weeks old, weighing 18–21 g were supplied by the animal laboratories of the Chongqing Medical University, China, and the animal experiments were conducted and approved in accordance with our Institutional guidelines and Ethics Committee. CT26 (mouse-derived colonic carcinoma cell line) cells were cultured and successfully transfected similarly to LoVo cell transfection (Materials and methods), where the untransfected, control-shRNA and PARG-shRNA groups were grown according to similar conditions of cell culture, and the cell number was adjusted to a final concentration of 1 × 107 cells per ml. Mice were anaesthesised intraperitoneally with 2% chloral hydrate (15 ml kg−1), and cancer cells 5 × 105 in 0.05 ml phosphate-buffered saline was injected under splenic capsule of each mouse. All mice had free access to food and water throughout the experiment. On day 14, all mice were killed and the spleen was processed for immunohistochemical staining CD34 for iMVD, as it is a marker of vascular endothelial cells. Steps for immunohistochemical staining was carried out according to the SABC method (Wuhan, China) (BOSTRE company) while assessment for iMVD was graded as per Yang et al.,23 where three areas having uptaken the best stains were evaluated and a mean of them corresponded to the mean value of iMVD.

Statistical analysis

Statistical analysis was performed by , one-way analysis of variance or q test was used to analyze the difference between groups, using the SPSS 13.0 software (SPSS Company, Chicago, IL, USA) package. A P-value <0.05 (P<0.05) was set as the criterion for statistical significance.

Results

Knockdown of PARG expression in LoVo cells by transfection with an shRNA lentivirus

To verify the efficacy of PARG inhibition; RT-PCR and western blot analysis were used; where total RNA (Figure 1b) and mRNA levels showed that after transfection PARG was almost completely inhibited in PARG-shRNA cells compared with both control groups (Figure 1a); where PARG mRNA expression in untransfected LoVo cells was 100.0±1.2%, in the control-shRNA group was 96.8%±2.4% and in PARG-shRNA was 6.6±1.8%. (Figures 1d and f). Although, in western blotting, PARG protein expression in untransfected LoVo cells was 100.0±1.5%, in the control-shRNA group was 101.7±1.9% and in PARG-shRNA cells was 8.3±1.5% (Figures 1c and e).

Expression of PARG mRNA detected by real-time-PCR in various groups of LoVo cells

The relative Ct number of PARG mRNA in the untransfected LoVo cells was 1.631, in the control-shRNA group was 1.653 and in the PARG-shRNA group was 1.787. Compared with controls group, the relative Ct number in the PARG-shRNA group was obviously increased (P<0.05). But there was no significant difference between the two control groups relative Ct number (P>0.05; Figure 1g).

The effect of silencing PARG on PARP, p38, ERK, p-p38 and p-ERK expressions in LoVo carcinoma cells

Our results showed that PARP expression was inhibited in PARG-shRNA cells, which confirmed our results compared with the two control groups. p38, p-p38, ERK and p-ERK expressions in PARG-shRNA cells were decreased compared with untransfected and control-shRNA cells (P<0.05; Figures 2a and b).

Figure 2
figure2

(a and b) PARG inhibition affects the expressions of PARP, p38, p-p38, ERK and p-ERK in various groups LoVo cells. 1-Untransfected LoVo cells, 2-control-shRNA; and 3-PARG-shRNA. (c and d) Western blot analysis highlights a decrease in expression of cytoplasmic p-IκBα together with a fall in the intranuclear expression of NFκ-B in PARG-shRNA cells compared with both untransfected and control-shRNA cells (*P<0.05). (e and f) PARG inhibition was found to inhibit expressions of VEGF, b-FGF, ICAM-1 and MMP-9 in LoVo cells. 1-Untransfected Lovo cells, 2-control-shRNA; and 3-PARG-shRNA.

Intranuclear expression of NF-κB in PARG-shRNA LoVo cells

Western blot analysis of cytoplamic extraction fraction showed that in PARG-shRNA cells, expression of p-IκBα was decreased as well as the relative intranuclear protein expression of NF-κB. Therefore, suggesting that in cells where PARG was silenced there was a decrease in intracytoplasmic expression of p-IκBα resulting in an intranuclear decrease of NF-κB (Figures 2c and d).

The influence of silencing PARG on angiogenesis-related factors

Compared with the control groups, the expressions VEGF, b-FGF, ICAM-1 and MMP-9 in the PARG-shRNA group were obviously decreased in comparison with the untransfected and control-shRNA cells (P>0.05) (Figures 2e and f).

Effect of silencing PARG on the ability of HUVEC migration in vitro

The number of migratory HUVECs through the microporous membrane in transwell migration assay was of 125±4 in the cocultured untransfected cells, control-shRNA was of 122±5 and in PARG-shRNA was of 56±2. Compared with the cocultured control groups, the number of migratory cells in the experimental group was obviously decreased (P<0.05) and the migratory inhibition rate was of 55.23% (Figures 3 a–c).

Figure 3
figure3

Influence on migration of HUVECs in different groups LoVo cells ( × 400). (a) HUVECs cocultured with untransfected LoVo cells; (b) HUVECs cocultured with control-shRNA transfected cells; and (c) HUVECs cocultured with PARG-shRNA-transfected cells.

Silenced PARG on the ability of HUVEC proliferation

From our CCK-8 assay results, the percentage of HUVEC proliferation in the group treated with supernatant of PARG-shRNA-cultured cells was obviously decreased (P<0.05; Figures 4a and b) compared with both supernatants controls; hence, showing poor chances of proliferation in PARG-silenced cells.

Figure 4
figure4

(a and b) The effect on growth of HUVECs in vitro dependent on the concentration of LoVo cell supernatant. Concentrations were of 1:2; 1:4; 1:8; 1:16; 1:32; and 1:64.

The effect of silenced PARG on the concentrations of LoVo-secreted VEGF and b-FGF

We evaluated VEGF and b-FGF protein secretions in PARG-silenced LoVo cells conditioned medium using ELISA; control-shRNA cells served as control (Table 1). PARG inhibition markedly reduced concentrations of the secreted VEGF and b-FGF in LoVo cells through the ELISA method. Compared with the control group, concentrations of the VEGF and b-FGF in PARG-shRNA LoVo cells supernatants (P<0.05) were significantly lower. Only minimal expression was seen in PARG-shRNA cell lines. Concordance between VEGF and b-FGF mRNA and protein levels in PARG-shRNA cells was observed clearly.

Table 1 The effect of silenced PARG on the concentrations of LoVo-secreted VEGF and b-FGF detected by ELISA (*P<0.05)

The influence of ERK inhibitor U0126, p38 inhibitor SB203580 and NF-κB inhibitor PDTC on various factors in LoVo carcinoma cells

To further investigate the relationship between p38, ERK, and NF-κB signal pathways in human colon carcinoma cells, we used ERK inhibitor U0126 and p38 inhibitor SB203580 to treat LoVo cells, respectively. Then, NF-κB and p-IκBα expressions were determined by western blot analysis (Figure 5); compared with the untransfected group, the expressions of each protein in PARG-shRNA cells were obviously decreased (P<0.05) (Figure 5). NF-κB inhibitor PDTC was used to treat LoVo cells, then the changes of the PARP, phosphorylations of ERK and p38 expressions were noted. Compared with the untransfected group, the expression of PARP in PARG-shRNA cells was obviously decreased, but there were no obviously changes in the expressions of phosphorylated p38 and ERK (Figures 6a and c).

Figure 5
figure5

(a and b) The effect of inhibitor of ERK and p38 on expressions of p-IκBα and NF-κB. 1-Untransfected cells; 2-LoVo cells treated with U0126; and 3-LoVo cells treated with SB203580.

Figure 6
figure6

(a and b) The effect of inhibitor of NF-κB (PDTC) on expressions of PARP, p38, p-p38, ERK and p-ERK. 1-Untransfected cells; 2-LoVo cells treated with PDTC.

iMVD for splenic allografted tumor in animal model observation

On day 14 following injection of CT26 cells, the mice were killed, abdominal laparotomy was performed and the spleen iMVD was assessed by immunohistochemistry staining for CD34. Both controls had a P>0.05 (Figures 7a–c, Table 2) compared with the PARG-shRNA group. In PARG-shRNA compared with the untransfected and control-shRNA groups, iMVD was much lesser (Figures 7a–c, Table 2; P<0.05). Thus, PARG suppression does appear to have a critical role in the prevention of angiogenesis in vivo.

Figure 7
figure7

iMVD of intrasplenic allograft tumors in mouse modelvar (SABC, × 400) using immunohistochemistry for CD34. (a) iMVD of intrasplenic allograft tumor in the untransfected group, (b) iMVD of intrasplenic allograft tumor in the control-shRNA group and (c) iMVD of intrasplenic allograft tumor in the PARG-shRNA group.

Table 2 Microvessel density in intrasplenic allograft tumor in various groups (n=10, )

Discussion

In the rapid development of our modern era, biological research techniques for investigating the molecular mechanisms of diseases, lentivirus vectors come into role. Previously, many non-lentivirus vectors have had many inadequacies, where some could not efficiently transduct nondividing cells, while others could not target genes and some even brought in strong immune responses after being used on a long-term basis. These have had serious influences on vector systems being applied in research, but lentivirus vectors have remedied the above situations;24 where in this study, we have silenced PARG gene in human colon carcinoma LoVo cells by using a shRNA. Our results demonstrated that PARG-shRNA lentivirus vector was successfully transfected into LoVo cells, and that the lentivirus vector itself had no obvious influence on the expressions of PARG mRNA and PARG protein.

PARG is the main enzyme executing degradation of PAR and PARP; these two are the main enzymes that control metabolism of PAR and have an important role in maintaining the balance of PAR in body in normal physiological states.25 Until now, only one PARG gene has been discovered in the mammalian body,26 it has three subtypes produced by three cDNA coding: 110, 102 and 99 kDa,27 where 110 kDa has a major role in the nucleus, as well as in the cytoplasm,28 coexisting with cytochrome C.29, 30 Previously, a study2 reported that a large number of PAR protein gathered together in lung adenocarcinoma cells, which were transfected with PARG-shRNA inhibited the activity of PARP. Moreover, Frizzell et al.31 demonstrated that the activity of PARP is dependent on PARG, and that PARP together with PARG could unanimously affect the expressions of downstream genes. Moreover, our previous studies4, 32 also demonstrated that the correlation of expressions between PARG and PARP, PARP and VEGF, b-FGF were related in human colon carcinoma tissues; where GLTN (PARG inhibitor) could reduce the expressions of PARP, NF-κB, VEGF, b-FGF and MMP-9 by inhibiting PARG in these cells. Furthermore, the inhibition of PARP in colon carcinoma CT26 cells could also reduce the activity of NF-κB and the expressions of its dependent genes that are associated with colorectal metastasis (such as ICAM-1, p-selectin). As, previously the relationship of PARG with angiogenesis involving factors VEGF and b-FGF were not explored. Hence, on the basis of our preliminary studies, we transfected human colon carcinoma LoVo cells with PARG-shRNA lentivirus vector to observe the influence of silencing PARG gene in these cells on the ability of HUVEC migration and proliferation; where we collected supernatants of each group of LoVo cells to culture HUVECs separately and CCK-8 assays results showed that silencing of PARG could inhibit the ability of promoting proliferation of HUVECs. Meanwhile, HUVECs cocultured with PARG-shRNA cells in transwell migration assay whereby a decrease in migration of HUVECs was appreciated.

In vivo studies were conducted with the objective of enhancing and supporting our in vitro experiments as in our previous studies,12, 21 where CT26 cells were transfected under similar conditions as LoVo cells and our results demonstrated that transfection of PARG-shRNA CT26 cells had decreased iMVD in mouse spleen (*P<0.05) compared with both the untransfected and control-shRNA groups. This indicates that deficiency of PARG could inhibit the angiogenic behavior of colon carcinoma in our model.

It is well known that many factors involved in the invasion and metastasis of tumors where the MAPK signal pathway remains one of the most important, which affect cell growth, proliferation, differentiation, migration and apoptosis through the regulation of its downstream gene expressions.33 Jukka et al.34 highlighted that ERK and p38 affected MMP-9 expression where MMP-9 was found to be decreased on inhibiting ERK and p38 phosphorylation.34, 35, 36, 37, 38 Moreover, Bancroft et al.39 and Ckajima et al.40 pointed out that blocking the MAPK pathway was lead to the inhibition of VEGF secretion and that ERK-mediated VEGF increased while p38-mediated VEGF decreased. Another report7 stipulated that ERK and p38 signal pathways was also mediated by b-FGF, where the use of p38 inhibitor could completely block FGF-inducing tumor angiogenesis. Hence, in our study we decided to check the expressions and phosphorylations of p38 and ERK in LoVo cells after silencing PARG, which revealed that the expressions and phosphorylations of p38 and ERK in PARG-shRNA cells were obviously lower than in the control groups. These therefore, illustrate that silencing PARG could reduce p38 and ERK expressions and inhibit p38 and ERK phosphorylations, unveiling that PARG inhibition could reduce the MAPK pathway phosphorylations and limit its activities.

Many studies41 have shown that NF-κB have a very important role in invasion and metastasis of colon carcinoma through upregulating of MMP-2, MMP-9 expressions. Beside, it was found that inhibiting PARP could also downregulate NF-κB activity, and further downregulate NF-κB-dependent gene expressions.42 Moreover, our experimental trials also demonstrated that inhibiting PARP in colon carcinoma could reduce the formation of PARP–NF-κB complex, and simultaneously reduce NF-κB activity and NF-κB-depending gene expressions such as MMP-2, MMP-9 and ICAM-1.43, 44 Furthermore, it is also well known that activating of NF-κB occurs by the phosphorylation of IκBα where NF-κB translocates to the nucleus. Hence, in this study our results demonstrated that p-IκBα and NF-κB expressions decreased after PARG inhibition, and that NF-κB-depending genes VEGF, b-FGF, ICAM-1 and MMP-9 expressions were also decreased. We also collected supernatants of each group of LoVo cells, where ELISA tests showed that silencing of PARG could inhibit the secretion of VEGF and b-FGF angiogenic factors. VEGF is known to have an important role in promoting invasion and angiogenesis in osteosarcoma cells, human neuroblastoma cells45, 46 in the secreted form and was present in the supernatant of human primary adipocytes in obesities, to promote inflammation in obesity-related type 2 diabetes.47 Although, b-FGF was also found in the secreted form48 where it was found to promote repair of damaged corneal cells, induce mesenchymal stem cells differentiation for vascularization in pig model for myocardial ischemia-reperfusion injury,49 and promote inflammatory reactions through the increasing of MMP-9 expression and secretion in monocytic cells during inflammation process;50 hence, our results suggest that the downregulation of the activity of NF-κB could decrease the expressions of NF-κB-dependent angiogenic factors such as VEGF, b-FGF, ICAM-1 and MMP-9 by inhibiting the activity of PARP and phosphorylations of p38 and ERK in LoVo cells could have some roles in inhibiting angiogenesis, which is the main source of tumor dissemination.

To further investigate the relationship between p38, ERK, NF-κB and PARG in human colon carcinoma cells; ERK inhibitor U0126, NF-κB inhibitor PDTC and p38 inhibitor SB203580 were used to treat LoVo cells, respectively. The results revealed that NF-κB activity decreased in LoVo cells that were treated by ERK inhibitor and p38 inhibitor; whereas the expressions of phosphorylated ERK and p38 did not change in LoVo cells that were treated by NF-κB inhibitor. Therefore, we stipulate that inhibiting phosphorylations of ERK and p38 pathways could downregulate NF-κB and in turn PARG activity in human colon carcinoma LoVo cells important in highlighting MAPK signaling pathway.

At the same time, we also noted that PARP expression was decreased when PARG silenced; we therefore, speculate that it may be related to the downregulation of NF-κB. Hence, we used NF-κB inhibitor PDTC to treat LoVo cells that showed that PARP expression in these cells was obviously decreased when compared with untransfected and control-shRNA cells. This hence, points out that NF-κB could have a feedback mechanism on inhibiting PARP expression; nevertheless, the detailed mechanisms need be further investigated. Hence, highlighting that PARG probably has a primordial role in promoting angiogenesis of human colon cancer, which could further promote invasion and metastasis.

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Acknowledgements

This work was supported by the National Nature Science Foundation of China (NSFC: 30870946).

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Correspondence to Y-l Wang.

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Keywords

  • colon cancer
  • HUVEC
  • migration
  • proliferation
  • PARG
  • ShRNA

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