Background
Hepatocellular carcinoma (HCC) is a highly aggressive and heterogeneous disease. The latest national cancer statistics released by the National Cancer (China) showed that the morbidity of hepatocellular carcinoma is 36.5 per 10
4 people and that it ranks fourth in malignant tumors [
1,
2]. Therefore, we explored the mechanisms underlying the carcinogenesis and progression of HCC to benefit therapy.
ADAM17 was initially identified as an important member of the ADAM family by Black RA’s team in 1997. Because the enzyme is responsible for releasing soluble tumor necrosis factor-alpha (TNFα) from the plasmalemma, it is also known as TNFα converting enzyme (TACE/ADAM17) [
3]. It has been reported that the dysregulation of ADAM17 contributes to the pathology of various cancers. For instance, ADAM17 protein was highly expressed in esophageal squamous cell carcinoma (ESCC) and promoted the development, invasion and metastasis of ESCC [
4‐
7]. Similarly, ADAM17 silencing suppressed the invasion and proliferation of MCF7 cells in vitro [
8]. Furthermore, Wang H P’s study suggested that Nox1 promoted colorectal cancer metastasis by stabilizing ADAM17 [
9]. Although several studies have shown that ADAM17 promotes the occurrence and development of hepatocellular carcinoma [
10,
11], the potential regulatory mechanism has not been fully elucidated.
MMP21 is the last uncharacterized MMP which is different from other MMPs and is rarely produced in normal tissues without inflammatory or oncogenic stimuli, which may implicate a role for MMP21 in normal tissue homeostasis [
10]. The prodomain of MMP21 contains a peptide sequence similar to that of TNFα. It has also been reported that MMP21 is expressed in cancer cells located in the invasive front of tumors rather than dysplastic cells and enhances tumor metastasis in some solid tumors [
12‐
15]. Positive correlations between MMP21 and tumor diameter, depth of invasion, vessel invasion,lymph node distant metastases,and tumor-node-metastasis stage were observed in gastric cancer. The overall survival rate was significantly lower in MMP21- and MMP28- positive patients [
15]. However, the expression pattern of MMP21 in HCC and whether MMP21 could be activated by other proteinases remain unknown. Notably, in this study, we investigated whether ADAM17 may regulate the maturation of MMP21 and influence the progression of HCC.
Methods
Patients and specimens
All experimental procedures involving the use of human tissue included the relevant receipt of written informed consent and were approved by the institutional review board at The Xiangya Hospital of Centre South University. For formalin-fixed paraffin-embedded HCC samples, human specimens were collected from the XiangYa Hospital tissue biobank, and the protocol for staining was approved by the local ethics committee of The Xiangya Hospital.
Immunohistochemistry and evaluation of immunostaining intensity
The tumor tissue was subjected to immunohistochemistry and stained with primary antibodies against ADAM17 (Abcam, ab2051, 1:100) and MMP21 (Abcam, MAB2079Z, 1:200) followed by light microscopy examination. The immunostaining intensity for the two proteins was reviewed and independently scored by pathologists who were blinded to the clinical data and scored independently according to the staining intensity and the proportion of stained tumor cells. According to the staining intensity, samples were scored as follows: no staining = 0; light yellow (weak staining) = 1; yellow brown (moderate staining) = 2; and brown (strong staining) = 3. The scores were expressed in terms of the proportion of cell staining as follows: scores of 0, 1, 2, and 3 indicated 0, ≤ 30%, 30%–70% and ≥ 70% positive cells, respectively. Thus, the two combined scores (from the two independent pathologists) were taken as the final score, where 0 indicated negative (−); 1–2, weak positive (+); 3–4, strong positive (++); and 5–6, very strong positive (+++). In the statistical analyses, (++) and (+++) were classified as the positive group, while (−) and (+) were classified as the negative group.
Cell culture
Human HCC MHCC97H and Huh7 cells were cultured in DMEM supplemented with 10% fetal calf serum and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO2. Human HCC SMMC7721 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.
Tranfection assay
Specific small interfering RNA (siRNA) against ADAM17 (si-ADAM17) and MMP21 (siMMP21) and siRNA scrambled control (si-NC), were purchased from RIBBIO (Shanghai, China). Huh7 and SMMC7721 cells were transfected with plasmids or oligonucleotides using DharmaFECT Reagent (Invitrogen, Carlsbad, CA, USA). The sequences for siADAM17, siMMP21,siNC were as follows: siADAM17: 5′-GCTTGTTCATCGAGTGAAA-3′, 5′-GGATGGTCTAGCAGAATGT-3′; siMMP21: 5′-GATCCATAATGCAACCAAA-3′, 5′-ACTGGAAGGTAGTTAATGA-3′; siNC: TTCTCCGAACGTGTCACGTTT.
Cell invasion assay
For the transwell invasion assay, 24-well transwell units with an 8-μm pore size polycarbonate filter (Millipore) were used according to the manufacturer’s instructions. Briefly, filters were coated with Matrigel to form a continuous thin layer. Then, cells were seeded in DMEM in the upper chamber. The lower chamber was filled with DMEM with 10% FBS. Following 24 h of incubation at 37 °C, cells remaining in the upper compartment were removed using cotton swabs. The cells that invaded through the filter into the lower compartment were fixed with 4% paraformaldehyde and stained with crystal violet (0.5% in 20% methanol). To quantify invasive cells, three independent fields of invasive cells per well were photographed.
Scratch wounding assay
Transfected cells were plated in six-well plates and incubated at 37 °C until a confluent monolayer was formed (> 90%). With a 100-µl sterile pipette tip, a scratch was created. The cells were washed three times with PBS (pH 7.2) to remove cell fragments, and low serum DMEM was added. Micrographs were taken immediately after wounding and after 24 h, 48 h, 72 h, and 110 h. The closure percentage was calculated using the following equation: closure percentage = [1–(Tx/T0)] × 100%, where T0 is the wounded area at 0 h and Tx is the wounded area after x h.
Western blot
Cells were then harvested in lysis buffer. A BCA protein assay kit was used to determine the concentration of protein. Samples were separated on a 10% SDS–PAGE gel, followed by transfer to polyvinylidene difluoride (PVDF) membranes in an electrophoretic manner. The primary antibodies were used at a 1:1000 dilution, the loading control anti-tubulin was used at a 1:5000 dilution, and the secondary antibody was used at a 1:1000 dilution. The targeted proteins in the membrane were detected with an electrochemiluminescence detection system followed by exposure to X-ray film.
Elisa
To evaluate the production of MMP21 substrates, the transfected cells were prepared. ELISA kits (CUSA-BIO, Catalog Number. CSB-EL014668HU) were used to analyze the collected medium specimens for the proteins of interest. Supernatants were collected in triplicate for each cell line.
Immunoprecipitation
Cells underwent cytolysis in 1 ml of RIPA buffer, followed by a 10-min incubation on ice. Total cell lysates were centrifuged for 10 min at 20,000g at 4 °C. Ten microliters of primary antibody was used to incubate the supernatants for 60 min. Then, 20 µl of protein A/G PLUS-agarose was added to the lysate and incubated at 4 °C overnight. The beads were washed with ice-cold RIPA buffer four times. The samples were then suspended and denatured in SDS sample buffer (which contained 100 mM dithiothreitol, 10% glycerol, 50 mM Tris, pH 6.8, 2% SDS, and 0.01% bromophenol blue).
HPLC method
Recombinant human TACE/ADAM17 (rhTACE) (Catalog # 930-ADB) was purchased from R&D Systems. The amino acid polypeptide sequence (ALAQAVRRFQ) was ordered from Sangon Biotech. The assay buffer used in this study was 25 mM Tris, 2.5 µM ZnCl2, 0.005% Brij-35 (w/v), pH 9.0. First, rhTACE was diluted to 0.2 ng/µl in assay buffer. Second, the substrate was diluted to 20 µM in assay buffer. Third, 50 µl of 0.2 ng/µL rhTACE was added, and the reaction was initiated by adding 50 µl of 20 µM substrate. A substrate blank was included that contained 50 μl of assay buffer and 50 µl of substrate. The HPLC reaction conditions in our study were as follows: column: 250*4.6 mm, Sinchrom ODS-BP-5; solvent A: 0.1% TFA in 100% water; solvent B: 0.1% TFA in 100% acetonitrile; flow rate: 1.0 ml/min; wavelength (nm): 220; and volume: 10 µl.
Statistical analysis
SPSS 21.0 software was used for statistical analysis. Chi-square test or Fisher Exact test are used to compare the expressions of ADAM17 and MMP21 in cancer tissues and adjacent tissues. The relationship between the two indexes and the clinical data of hepatocellular carcinoma patients was analyzed by chi-square test. Spearman rank correlation analysis was used to analyze the expression correlation between ADAM17 and MMP21. P < 0.05 was considered statistically significant. Each experiment was repeated three times.
Discussion
HCC is one of the most prevalent human cancers. Tumor invasion is the main cause of mortality in patients with HCC [
16‐
19]. Recent studies have determined that ADAM17 dysfunction may play an important role in tumor invasion [
20]. In 2016, CD133-expressing CSCs were shown to be responsible for cell invasion and migration after radiation, and their radiation-induced metastatic potential could be prevented by suppression of ADAM17 [
21]. A recent study proved that ADAM17 promotes cell invasion and migration through the integrinβ1 pathway in HCC [
22]. In our study, we first used a database to confirm that ADAM17 was correlated with the OS and PFS of patients with HCC (Additional file
2: Figure S3). Later, we further identified that ADAM17 was markedly increased in HCC tissue samples. Moreover, we found that downregulation of ADAM17 could significantly suppress the invasion of MHCC97H and SMCC7721 cells. Therefore, ADAM17 may represent a novel target in the progression of HCC.
ADAM17 was originally identified as an enzyme responsible for processing TNFα from a precursor to a soluble circulating form [
3,
23]. As a protein cleaved by ADAM17, TNFα seems to lack a definable consensus cleavage motif [
19]. However, statistics in the MEROPS database showed that there is orderliness. It is more selective for alanine at the P1 position and revealed a preference for valine at the P1′ position among 60 cleavage sites registered in this database. Recently, some studies demonstrated that cleavage site specificities were in excellent agreement with the information derived from the MEROPS database [
24,
25]. Interestingly, compared with the cleavage peptide (LAQAVRSS), we found that the prodomain of MMP21 has a similar peptide sequence (LAQAVR) through the Swiss Prot database. Furthermore, MMP21 was recently shown to play an important role in tumor processing [
14‐
16,
26]. In our study, MMP21 was found to be upregulated in hepatocellular carcinoma and associated with microvascular invasion. In addition, the high expression of MMP21 in MHCC97H cells is often accompanied by high metastatic potential. Importantly, downregulation of ADAM17 could decrease the expression of MMP21, while overexpression of ADAM17 can markedly increase the expression of MMP21. Furthermore, ADAM17 can positively regulate the secretion of MMP21. Next, we confirmed that ADAM17 coimmunoprecipitated with MMP21 in the Huh7 cell line. On the other hand, it was further indicated by high-performance liquid chromatography that the precursor of MMP21 may be cleaved by ADAM17 protease.
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