Protein Kinase N1 Promotes Proliferation and Invasion of Liver Cancer
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EXPERIMENTAL AND THERAPEUTIC MEDICINE 21: 651, 2021 Protein kinase N1 promotes proliferation and invasion of liver cancer XIA WANG1, YANSONG GE2, MINGQI SHI2, HANHAN DAI2, WEI LIU2 and PEIYUAN WANG2 1Department of Pathology, Binzhou Medical University, Yantai, Shandong 264003; 2Department of Radiology, Yantai Affiliated Hospital of Binzhou Medical University, Yantai, Shandong 264100, P.R. China Received March 11, 2020; Accepted March 22, 2021 DOI: 10.3892/etm.2021.10083 Abstract. Protein kinase (PK) N1, also called PKC‑related accounts for ~55%, with 110,000 deaths every year (1‑3). protein 1, participates in the proliferation, invasion and Currently, there are a variety of treatment options for liver metastasis of various malignant tumors. However, the role of cancer at different stages, including routine options (such PKN1 in liver cancer remains to be elucidated. The present as hepatectomy and transcatheter arterial chemoemboliza‑ study investigated the expression of PKN1 using immuno‑ tion), molecular targeted therapy (VEGF/VEGF monoclonal histochemistry in surgical specimens from 36 patients and antibody, EGF receptor inhibitors) and immunotherapy analyzed the correlation with VEGF, microvascular density (programmed death‑1/programmed death‑ligand 1), which can (MVD), cell proliferation index (Ki67) and clinicopatholog‑ prolong the survival time and prognosis of some patients (4‑6). ical parameters. PKN1 was highly expressed in hepatocellular However, due to the selection of indications, sensitivity to carcinoma (HCC) and was positively correlated with histo‑ treatment and other reasons, most patients have not received logical grading of HCC, Ki67 expression and MVD. PKN1 effective treatment. Therefore, it is important to explore the expression in moderately and poorly differentiated HCC was mechanism of proliferation, invasion and metastasis of liver significantly higher compared with highly differentiated HCC. cancer. In recent years, research on PKN1 in tumors has Expression of PKN1 was positively correlated with Ki67 attracted much attention and PKN1 has been reported as a and MVD, and Ki67 expression was positively correlated promising therapeutic target for prostate cancer (7,8). However, with MVD. The effects of PKN1 on proliferation, invasion the role of PKN1 in liver cancer remains to be elucidated. and apoptosis of liver cancer cells were detected in vitro. PKN1, also known as PKC‑related protein 1, is a Cell viability, migration and invasion were reduced and the serine/threonine protein kinase belonging to the PKC apoptosis rate was significantly improved when PKN1 expres‑ superfamily (9). It is reported that PKN1 may participate sion was silenced in liver cancer cells. Thus, PKN1 serves an in cytoskeletal reconstruction, cell adhesion, apoptosis, important role in the development and progression of liver tumor cells and other life processes (10‑12). Overexpression cancer. Inhibition of PKN1 activity may provide a promising of PKN1 serves an important role in the development of therapeutic target for liver cancer. neurodegenerative diseases, prostate cancer, ovarian cancer, endometrial cancer and other tumors (9,13‑16). PKN1 contains Introduction a unique regulatory domain in the amino terminus, which presents a loop of serine/threonine protein kinase that serves Liver cancer is one of the commonest malignant tumors world‑ a key role in activation of PKN1. PKN1 can act upstream of wide. In 2018, the estimated global incidence was ~841,000, mitogen‑activated PKs, C‑Jun N‑terminal kinase and p38, with ~782,000 related deaths, while the incidence in China or downstream of EGF signaling and TGF‑β (12,16,17). Activated PKN1 regulates the invasion of prostate cancer cells and phosphorylation of p38, which further regulates a signaling cascade of invasion‑related genes PXN, NEDD9 and NT5E/CD73 (8). Yang et al (18) demonstrated that PKN1, as Correspondence to: Professor Xia Wang, Department of Pathology, Binzhou Medical University, 346 Guanhai Road, Yantai, an important member of PI3K/AKT/mTOR signaling pathway, Shandong 264003, P.R. China affects the differentiation of prostate adenocarcinoma cells E‑mail: [email protected] and is closely associated with Gleason score. Inhibition of PKN1 blocks transcriptional activation in androgen‑dependent Professor Peiyuan Wang, Department of Radiology, Yantai Affiliated Hospital of Binzhou Medical University, 717 Jinbu Road, Yantai, cancer cells (7). In endometrial cancer cells, PKN1 modulates Shandong 264100, P.R. China TGF‑β and EGF‑dependent regulation of cell proliferation, E‑mails: [email protected] migration and invasiveness and therefore is a component of the network signaling downstream of TGF‑β and EGF (15). Key words: protein kinase N1, liver cancer, microvascular density, James et al (19) demonstrated that inhibition of PKN1 expres‑ proliferation sion stimulates apoptosis in malignant melanoma cells by regulating the WNT/β‑catenin pathway. 2 WANG et al: PROTEIN KINASE N1 IN LIVER CANCER In the present study, the expression of PKN1 in surgical The necrotic area and severe inflammatory area were specimens was investigated by immunohistochemistry and the avoided in the selection of liver cancer specimens and correlation with VEGF, MVD, Ki67 index and clinicopatho‑ significant areas of expression were selected for analysis. logical parameters was analyzed. At the same time, the effects The immunoreactivity of PKN1 and VEGF was evaluated of PKN1 on the proliferation, invasion and apoptosis of liver according to integral optical density (IOD) by Image‑Pro cancer cells were detected in vitro and the role of PKN1 in Plus 6.0 (Media Cybernetics, Inc.). Ki67 proliferation index liver cancer progression was further explored. was calculated with Image Pro Plus 6.0 as follows: Ki67 (%)=positive tumor cells/all tumor cells x100%. MVD was Materials and methods marked by CD34 (a classic endothelium marker). The mean number of stained microvessels was recorded from five undu‑ Patients and specimens. A total of 36 patients with hepa‑ plicated high‑power fields (magnification, x400) per specimen. tocellular carcinoma (HCC) who were treated at Binzhou Medical University Hospital were enrolled. Patients who had Cells culture and reagents. The human liver cancer cell lines received preoperative radiotherapy or chemotherapy or had (HepG2 and Hep3B) were purchased from the cell banks tumors of other sites were excluded. Complete clinical and of the Chinese Academy of Science and were cultured in pathological data were recorded and hepatectomy specimens Dulbecco's modified Eagle medium (DMEM) with high were collected by the Pathology Department of the Hospital. glucose (HyClone; Cytiva) and 10% fetal bovine serum (FBS; There were 27 males and 9 female patients, with a mean age of Gibco; Thermo Fisher Scientific, Inc.) at 37˚C in a humidified 58.8±9.6 years (range 36‑77 years). The study was approved by atmosphere with 5% CO2. Then, three siRNAs against PKN1 the Ethics Committee of Binzhou Medical University (Yantai, were designed (19) and synthesized by Shanghai GenePharma China; approval no. 2018‑012) as required by the Declaration Co., Ltd.: siPKN1‑1: 5'‑CCU CGA AGA UUU CAA GUU C‑3'; of Helsinki. Prior to sample collection, written consent to use siPKN1‑2: 5'‑GAA CAU GAU CCA GAC CUA CAG CAA U‑3'; their tissues was obtained from all patients. siPKN1‑3: 5'‑ACA GUA AGA CCA AGA UUG A‑3'; and Negative The present study is a retrospective study and all 36 patients control (NC) group 5'‑UUC UCC GAA CGU GUC ACG UTT‑3'. had single lesions, including 13 in the left hepatic lobe and 23 HepG2 and Hep3B cells were seeded in six‑well plates and in the right hepatic lobe, and adjacent normal tissue was also transfected with 100 pmol siRNA using Lipofectamine® 2000 obtained from the patients. The longest diameter ranged from (Thermo Fisher Scientific, Inc.). PKN1 expression was detected 2 cm to 13 cm, with an average of 6.6±3.4 cm (<6.6 cm in using western blotting to confirm transfection efficiency. Of 23 cases and ≥6.6 cm in 13 cases). Histological grading of HCC the three siRNAs, the two more efficient silencing sequences was divided into well‑differentiated (grade I; n=7), moderately were selected for the following assays. differentiated (grade II and III; n=21) and poorly differenti‑ ated (grade Ⅳ; n=8) according to Edmondson‑Steiner grading Reverse transcription‑quantitative (RT‑q) PCR. Total RNA (24 h system (20,21). after transfection) of liver cancer cells was isolated with RNAiso plus reagent (Takara Biotechnology Co., Ltd.) and cDNA was Immunohistochemistry. All samples of pathological speci‑ generated with a PrimeScript RT Reagent kit with gDNA Eraser mens were fixed in 4% paraformaldehyde for 24 h at room (Takara Biotechnology Co., Ltd.). Quantitative gene expression temperature, followed by gradient dehydration and paraffin was performed for PKN1 and GAPDH (internal control) by embedding; 4‑µm sections were prepared for immunohisto‑ LightCycler 480 SYBR‑Green I Master Mix Reagent kit and chemical staining. The sections were incubated with 0.01 mol/l the LightCycler 480 real‑time System (Roche Diagnostics). citrate buffer (pH 6.0) in a microwave oven at 98˚C, three Nucleotide sequences of specific primer for genes were as times for 5 min, for antigen retrieval. Endogenous peroxidase follows: PKN1 forward, 5'‑AAA GCA GAA GCC GAG AAC AC‑3' was blocked by treatment with 3% H2O2 for 20 min at room and reverse, 5'‑ACA CAG CCA ACT CCA GTT CC‑3'; GAPDH temperature. After pretreatment with normal goat serum forward, 5'‑GAA GGT GAA GGT CGG AGT C‑3' and reverse, (OriGene Technologies, Inc.) for 30 min at room temperature 5 ' ‑ G A A GAT GGT GAT GGG ATT TC‑3'. PCR amplification was to block nonspecific binding, the sections were incubated performed under the following conditions: Initial denaturation with rabbit anti‑PKN1 polyclonal antibody (cat. no. bs‑7478R; for 10 min at 96˚C, followed by 40 cycles at 95˚C for 15 sec BIOSS; 1:500), rabbit anti‑VEGF polyclonal antibody (cat. and 60˚C for 60 sec.