Expression of heparanase regulates cell proliferation, apoptosis, migration and invasion in vitro in papillary thyroid carcinoma cells with multiple signaling pathways involved

Background Heparanase (HPSE) is an endo-β-D-glucuronidase, which is found overexpressed in various human cancers. The purpose of our work was to investigate the possible role of HPSE and the involved signaling molecules in the development of papillary thyroid carcinoma. Methods In this study, the expression plasmid of HPSE and RNA interference with shRNA specic for HPSE were used to elucidate the effects of HPSE on proliferation, apoptosis, migration and invasion in papillary thyroid carcinoma cells of B-CPAP. The probable downstream signaling molecules of HPSE were also explored. Results The results showed that upregulation of HPSE signicantly promoted cell proliferation, migration and invasion of B-CPAP cells, and interfered with cell apoptosis. On the contrary, knockdown of HPSE exhibited the opposite effects. Compared with the parental cells, HPSE silencing cells showed attenuated capabilities of proliferation, migration and invasion, yet the apoptotic rate of transfected cells was increased. The activations of various signaling molecules correlated with cell biological behavior were found to be regulated by HPSE upregulation or knockdown. Conclusions Our results suggested that HPSE probably contributed to the progression and metastasis of papillary thyroid carcinoma, which were associated with multiple signaling pathways. HPSE could be a potent molecular target for the therapeutic strategy of papillary thyroid carcinoma. same red/blue. of HPSE mRNA in HPSE transfected cells was upregulated, that shRNA transfected cells less control or HPSE or shRNA transfected cells. HPSE protein in HPSE transfected cells shRNA transfected cells lower or


Background
Heparanase (HPSE) is an endo-beta-glucuronidase and regarded to promote the invasion and metastasis of tumor cells. It has been con rmed that HPSE facilitates the proliferation and metastasis of ovarian cancer [1], and downregulation of HPSE resulted in the inhibition of adhesion and aggressiveness of hepatocellular carcinoma cells [2]. HPSE was also an important regulator in tumor microenvironment, such as tumor angiogenesis. The mRNA levels of HPSE were negatively associated with the prognosis factors of PDAC patients. HPSE in uenced the expression of VEGF-C and promoted invasion in BxPC-3 cells [3]. It was found that HPSE are key proteins in uencing tumor angiogenesis and cell proliferation and invasion in cervical cancer, possibly via the NF-κB signaling pathway [4].
Mechanism(s) underlying the progression of different tumors by HPSE regulation could be multifaceted.
It was found that HCCLM3 cells with high HPSE expression showed higher trans-endothelial migration (TEM) rate. Downregulation of HPSE or inhibition its activity resulted in suppression of TEM in HCC cells [5]. A role for HPSE in modulating autophagy was established in normal and malignant cells, thereby conferring growth advantages as well as resistance to chemotherapy [6]. MiR-558 upregulated the expression of HPSE in gastric cancer cell lines. MiR-558 facilitates the progression of gastric cancer through directly targeting the HPSE promoter to attenuate Smad4-mediated repression of HPSE expression [7].
A study of thyroid carcinoma found that the expression of HPSE2 was increased and tumors showed a typical combination of positive labeling for neoplastic cells and negative immunostaining in colloid [8].
However, the function of HPSE and the underlying mechanisms in human papillary thyroid carcinoma have not been su ciently disclosed and resolved. In this study, HPSE was upregulated or knocked down by HPSE plasmid or shRNA transfection to investigate the roles of HPSE in proliferation, apoptosis, migration and invasion of papillary thyroid carcinoma cells. Furthermore, the activity of downstream signaling(s) along with HPSE upregulation or knockdown was also explored.

HPSE overexpression and RNA interference
Human papillary thyroid carcinoma of B-CPAP cells were purchased from Chinese Academy of Sciences and cultured in RPMI 1640 medium (Gibco, USA), supplemented with 10% FBS, under the condition of 37℃ and 5% CO2. The overexpression plasmid for HPSE and the empty vector were purchased from GeneChem (China). The shRNAs speci c for HPSE and the scrambled non-targeted shRNA were also purchased from GeneChem (China). All the plasmid were transfected into B-CPAP cells using Lipofectamine 3000 (Invitrogen, USA). The experiments for cells were repeated at least three times.
Quantitative real-time polymerase chain reaction (qPCR) Total RNA was extracted using RNApure kit (Aidlab, China) and reverse transcribed into cDNA using GoScript Reverse Transcription System (Promega, USA). qPCR was performed using the GoTaq qPCR The length of PCR products were 193bp and 138bp, respectively. The qPCR was performed for 40 cycles with an initial denaturation at 95℃ for 2min, ampli cation at 95℃ for 15s, and annealing at 60℃ for 1min. GAPDH was used as reference gene, and the results were analyzed using 2 -ΔΔCt method.

Western blot
Cells were lysed in RIPA containing protease inhibitor (Promega, USA) and phosphatase inhibitor (APPLYGEN, China) cocktail, and centrifuged for the supernatant extraction. 50μg total protein was used for electrophoresis and transmembrane routinely. with membranes at 4℃ overnight. Then the goat anti-rabbit (1:4000) or anti-mouse IgG (1:2000, both from ZSGB-BIO, China) was used, and membranes were developed using ECL (Millipore, USA). The software of Image J was used to evaluate the integrated optical density (IOD) of protein bands. The ratio of IOD (HPSE) and IOD (β-actin) of the same specimen was calculated as the relative expression level of HPSE protein.

EdU incorporation assay
The kFlour555 Click-iT EdU imaging kit (KeyGEN BioTECH, China) was applied to explore cell proliferation. The staining was performed brie y as follows: cells were cultured with diluted EdU of 20μmol/L for 2h. Cells were xed with 4% paraformaldehyde, and neutralized by glycine solution of 2mg/mL. After washed with PBS, cells were permeated with 0.5% Triton X-100, and then incubated with prepared Click-iT reaction cocktail in the dark. Hoechst33342 was used to counterstain nucleus. Under an inverted uorescent microscopy, 5 random elds of 200× were focused and cells with red or blue uorescence were counted in the same elds. Cell proliferative rates were represented as red/blue.
Cell apoptosis assay.
The cell apoptosis was examined by ow cytometry using an Annexin V-PE/7-AAD apoptosis detection kit (KeyGEN BioTECH, China). Cells were digested and collected using trypsin without EDTA. Cells ( 1×10 5 ) were washed twice in PBS and incubated in mixture of 5μL 7-AAD in 50μL binding buffer for 10min in the dark. After gently mixed with 450μl binding buffer, cells were added with 1μl Annexin V-PE and incubated for 10min away from light. Results are representative of three individual experiments.

Wound healing assay
Cell were passaged 24h before scari cation and seeded in 6 well plate (1×10 6 ). 200μL pipettes were used to draw a straight line, which was vertical to the bottom of plate, using ruler as a guide. The detached cells were removed by PBS. Cells in plate were kept on being cultured in medium without FBS and cell migration was monitored at 12h and 24h. 5 random elds of 100× were selected at 24h, and images were captured. The blank area (A) in the scratch was measured using Image J soft, and the wound healing rate was calculated as (A0h-A24h)/A0h×100%.
Cell invasion assay.
The cell invasion was performed in a transwell chamber (Costar, USA) of 24-well with an 8μm pore size insert, which was precoated with Matrigel (BD, USA). Cells (1×10 4 ) were seeded in the upper chamber, and cultured in medium with 1% FBS for 24h. Cells were allowed to migrate towards medium containing 10% FBS in the bottom chamber. Cells on the upper membrane surface were erased with a cotton swab, and the migratory cells attached to the lower membrane surface were xed with 4% paraformaldehyde and stained with crystal violet. The number of invasive cells was counted in 5 random elds of 200× under a microscope. Data shown are representative of three individual wells.

Statistical analysis
The statistical software of SPSS 13.0 was applied to perform data analysis. One-way ANOVA was used to evaluate the differences between cells with various plasmid transfection. Multiple comparisons were made using LSD-t test within groups. All data were shown as mean±SD and data were regarded statistically signi cant when the p

Results
The overexpression or knockdown of HPSE in mRNA and protein level were demonstrated by qPCR and western blot Cells were transiently transfected with HPSE plasmid or shRNA, and the mRNA and protein level of HPSE were evaluated by qPCR and western blot. Our results showed that the expression of mRNA (Fig.1A) and protein (Fig.1B) in HPSE transfected cells were signi cantly upregulated, compared with contrl and empty vector groups. Moreover, the relative mRNA (Fig.1A) and protein (Fig.1B)  transfection, compared with control or empty vector group. It was implied that cell proliferation was inhibited when HPSE was downregulated by shRNA transfection (Fig.2).
The effects of HPSE plasmid or shRNA on cell apoptosis Cells were transiently transfected with HPSE plasmid or shRNA, and cell apoptosis was examined by Annexin V-PE/7-AAD double staining using ow cytometer. The apoptotic rate of cells with HPSE upregulation was 5.72±1.22%, and the control and empty vector transfected cells were found a higher apoptotic rates of 9.28±1.04% and 8.40±1.27% (p=0.024). In cells with HPSE knockdown, we observed the apoptotic rate of 17.01±1.43%, which was higher that other groups (p=0.0002). These results shown in Fig.3 indicated that HPSE protected cells survival avoid of apoptosis.
The effects of HPSE plasmid or shRNA on cell migration Cells were transiently transfected with HPSE plasmid or shRNA, and cell migration was observed by wound healing assay. The results showed that the wound healing rates were 17.7±1.5%, 16.0±1.6%, 28.8±3.8% and 12.6±2.5% in control, empty vector, HPSE plasmid and shRNA transfection groups, respectively (p=0.00017, Fig.4). These data con rmed that HPSE promoted cell migration, yet HPSE knockdown interfered with cell migration.  (Fig.6).

Discussion
HPSE is overexpressed in many types of human cancer and more expression of HPSE is associated with more tumor metastasis and invasion. Previous research has indicated that HPSE upregulation is correlated with poor prognosis and contributes to the lymphovascular invasion of breast cancer [9]. A systematic study showed that either the expression of HPSE in cancer and/or the serum concentration of HPSE may be a useful biomarker for the evaluation of surgery effects and prognosis prediction in ovarian cancer [10].
The mechanism(s) underlying tumor progression by HPSE have been shown variable. HPSE is associated with the aggressiveness of neuroblastoma, and Smad4 inhibits the tumorigenesis and progression of NB through suppressing the HPSE expression, through directly binding to its promoter and repressing the LEF1-facilitated transcription of HPSE. [11]. Downregulation of HPSE repressed glioma cell proliferation, whereas exogenous HPSE stimulated growth and activated ERK and AKT signaling. The anti-HPSE treatment of malignant glioma provides a novel strategy for the use of HPSE as a therapeutic target [12]. MiR-429 decreases the invasive capability by downregulating the expression of HSPE in gastric cancer cells [13]. Roneparstat, a non-anticoagulant heparin with potent anti-HPSE activity, inhibited the growth and brain metastatic breast cancer abilities of lapatinib-resistant cell clones. The inhibition of HPSEmediated signaling provides new insights to validate the use of Roneparstat for novel BMBC therapeutic strategies [14].
In this study, human papillary thyroid carcinoma of B-CPAP cells were transiently transfected with HPSE plasmid or shRNA to explore its effects on cell proliferation, apoptosis, migration and invasion. When HPSE was upregulated, cell proliferation was enhanced and apoptosis was inhibited. Cell migration and invasion were promoted. However, the downregulation of HPSE by speci c shRNA transfection exerted the opposite effects. Our results are in agreement with previous ndings in other human cancers, which indicated that HPSE facilitated the progression of papillary thyroid carcinoma.
To further investigate the probable cell signaling(s) downstream of HPSE, multiple antibodies of different signaling pathways were applied. Several studies have reported that ERK and Akt signaling were downstream of HPSE to facilitate cell proliferation. Differentiating Hpse-transgenic embryonic stem cells showed a higher growth rate, and overexpression of HPSE enhanced Erk and Akt phosphorylation in neural stem and progenitor cells [15]. The level of phosphorylated ERK and Akt was also found increased with HPSE transfection and decreased after HPSE knockdown in this study, which suggested that ERK and Akt were a part of HPSE signaling to mediate cell proliferation. It was reported that silencing HPSE expression activated apoptosis and autophagy of HCC cells [16]. Signi cantly increased apoptotic cells upon HPSE silencing was also found in melanoma cells, mediated by the activation of caspase 3/PARP1 pathway [17]. We showed here a negative correlation between the activities of cleaved caspase-3/PARP and the level of HPSE, which indicated that the caspase-3/PARP was probably involved in HPSE regulated cell apoptosis.
FAK were active signaling mediator in cell migration or invasion of various human cancers. FAK has previously been shown to be a downstream target of HPSE. Tumor cell invasiveness was reduced after knockdown of HPSE in myeloma and melanoma cells and lowering HPSE levels attenuates phosphorylation of FAK, a critical factor for the invasion of cancer cells. A study revealed the involvement of the HPSE-FAK axis in invasion of brain cancer cells [18]. 3-phosphoinositide-dependent protein kinase (PDK1) is participated in various cell signaling pathway, including Akt and PKC. Myristoylated alaninerich C-kinase substrate (MARCKS) is a substrate of PKC, activated in cell adhesion and motility. Molecular analysis suggested that PDK1 facilitated the progression of PIK3CA-driven head and neck squamous cell carcinoma (HNSCC). Both PIK3CA and PDK1 protein levels were correlated with tumor invasion and metastasis in human HNSCC clinical samples [19]. Knockdown of MARCKS suppressed cell migration and invasion of prostate cancer and facilitated cell-cell adhesion in cell colonies [20]. Our results showed that the activations of PDK1, PKC and MARCKS were induced after HPSE overexpression, and downregulated by HPSE knockdown. The phosphorylation of FAK was also altered by differential expression of HPSE. These ndings revealed that multiple cell signaling pathways were affected by HPSE expression, and its complex effects on cell proliferation, apoptosis, migration and invasion caused by upor down-regulation would be further investigated.

Conclusions
Our data disclosed in cells of papillary thyroid carcinoma that multiple signaling molecules were involved in HPSE affected cell proliferation, apoptosis, migration and invasion. Therefore, our results revealed an initial insights for the activation of cell signaling induced by HPSE, which would provide possible therapeutic target to neutralize the effects of HPSE on papillary thyroid carcinoma. The speci c inhibition for respective signaling pathway would be performed to further improve our conclusions, and the molecular mechanisms will be our main research direction. Declarations CY and ZL initiated and designed the study; CY, XC and YH performed the experiments and wrote the manuscript; SZ contributed to the paper translation and gave experimental instructions; DC analyzed the data; ZL gave pertinent suggestions for the manuscript revision. All authors read and approved the nal manuscript.

Funding
The study was supported by grants from the National Natural Science Foundation of China (No.81672644), given to the corresponding author Zhen Liu, who designed the study, interpreted the data and revised the manuscript.

Availability of data and materials
The datasets supporting the conclusions of the current study are available from the corresponding author on reasonable request. Please contact corresponding author, if you want to request the dataset.
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.   The wound healing rate was increased in HPSE transfected cells. The cell migration of B-CPAP was inhibited by HPSE knockdown (Magni cation 100×).