European Review for Medical and Pharmacological Sciences 2020; 24: 12709-12716 PRKD3 promotes malignant progression of OSCC by downregulating KLF16 expression Z. CHEN1, Q. HUANG2, W. XU1, H. WANG1, J. YANG1, L.-J. ZHANG1 1Department of Oral and Maxillofacial Surgery, The First People’s Hospital of Lianyungang, Lianyungang, China 2Department of Oral and Maxillofacial Head and Neck Surgery, The Second People’s Hospital of Hefei, Hefei, China Abstract. – OBJECTIVE: We aimed to clari- Key Words: fy the molecular mechanism of how PRKD3 pro- PRKD3, KLF16, Oral squamous cell carcinoma, Ma- motes the malignant progression of oral squa- lignant progression. mous cell carcinoma (OSCC). PATIENTS AND METHODS: 62 cases of OS- CC tissues and normal adjacent ones which Introduction were further confirmed by a qualified pathol- ogist were collected from patients in the De- partment of Pathology and Stomatology of our Oral squamous cell carcinoma (OSCC), one hospital. PRKD3 expression in the above tis- of the major diseases endangering human health, sue samples was studied by quantitative re- refers to the squamous cell carcinoma of the oral al-time polymerase chain reaction (qRT-PCR) mucosa. It is the most common malignant tumor analysis, and its relationship with clinicopath- of the oral cavity, accounting for about 90% of the ological characteristics of these OSCC patients malignant tumors of the oral cavity1-3. Worldwide, was analyzed. Meanwhile, a PRKD3 knockdown there are about 405,000 new cases of OSCC and expression model was constructed in OSCC cell lines for cell functional experiments. The rela- about 211,000 deaths every year, and the 5-year 1,2 tionship between PRKD3 and KLF16 was eluci- survival rate is less than 60% . The location of dated through bioinformatics and Luciferase re- OSCC is often limited and are close to important porter gene experiments. organs, such as the brain, important blood ves- RESULTS: Our data showed that PRKD3 ex- sels in the neck, and the upper respiratory tract, pression in OSCC specimens was remarkably etc., which seriously restricts the surgical scope; higher than that in adjacent ones. PRKD3 ex- meanwhile, the large maxillofacial defects and pression showed positive association with the functional defects caused by surgery directly incidence of distant metastasis, but not with 4,5 other clinical indicators such as gender, age, tu- affect the quality of life of patients . At present, mor stage or lymph node metastasis incidence. the clinical treatment of OSCC is still not very Patients with high PRKD3 expression had low- satisfactory, the main reasons are as follows. er overall survival compared to those with low First, there is no specific and effective treatment expression. In addition, OSCC cells migration for oral squamous cell carcinoma. Second, cur- ability and invasiveness were remarkably at- rent pathological diagnostic techniques cannot tenuated after PRKD3 was knocked down. Bio- accurately determine the biological character- informatics and Luciferase assay revealed that PRKD3 could directly bind to KLF16 and West- istics of OSCC, resulting in different prognosis ern blot suggested that KLF16 was upregulat- of patients with the same clinical stage for the ed after PRKD3 was knocked down. In addition, same treatment6-8. Therefore, it is of great sig- knocking down KLF16 reversed the inhibito- nificance for the early diagnosis, treatment and ry effect of PRKD3 knockdown on invasiveness prognosis judgment of OSCC to deeply explore and metastasis of OSCC cells. the molecular mechanism of the occurrence and CONCLUSIONS: The highly-expressed PRKD3, development of OSCC and find the markers of remarkably associated with metastasis incidence 9,10 and poor prognosis of OSCC patients, may accel- gene diagnosis and therapeutic targets . erate the malignant progression of OSCC through PRKD is a serine/threonine protein kinase, modulating KLF16 expression. belonging to the calcium/calmodulo kinase Corresponding Author: Laijian Zhang, BM; e-mail: [email protected] 12709 Z. Chen, Q. Huang, W. Xu, H. Wang, J. Yang, L.-J. Zhang (CaMK) superfamily11,12, and there are three tigation complies with the Helsinki Declaration members of the family, PRKD1, PRKD2 and Clinical Practice Guidelines. Exclusion criteria: PRKD312. PRKDs possess numerous molecular patients complicated with other malignancies, targets and play a pivotal role in various cell those with mental disease, those complicated biological functions, such as cell migration, cell with myocardial infarction, heart failure. The proliferation, protein transport, epithelial inter- clinical TNM staging of tumors was based on stitiation, angiogenesis and gene transcriptional the International Union Against Cancer (UICC) regulation13,14. However, its dysregulation will standard. This study was approved by the Ethics affect the normal behavior of cells and even- Committee of The First People’s Hospital of tually produce carcinogenesis, which is why Lianyungang, and the signed written informed abnormal expression of PRKDs has been found consents were obtained from all participants in many tumors14,15. The function of PRKDs before the study. is tissue-specific, serving as a tumor suppres- sor or oncogene in different tissues15. PRKD1 Cell Lines and Reagents is absent in some aggressive types of OSCC, Human OSCC cells (Fadu, SCC-25, CAL-27, while PRKD3, which acts as an oncogene, is Tca8113) and one normal human oral epithelial upregulated, making PRKDs an effective target cell (Hs 680.Tg) purchased from American Type for the treatment of OSCC16,17. Additionally, Culture Collection (ATCC; Manassas, VA, USA) inhibition of PRKD3 or treatment with PRKDs were cultured with Dulbecco’s Modified Eagle’s broad-spectrum inhibitors can be used as an Medium (DMEM) medium (Gibco, Rockville, effective treatment for certain types of OSCC17. MD, USA) supplemented with 10% fetal bovine However, until now, little is known about the serum (FBS; Gibco, Rockville, MD, USA) at substrates regulated by PRKDs, and few studies 37°C in an incubator with 5% CO2. have17 been conducted on the signal transduc- tion network of PRKDs. Therefore, we explored Transfection the signal transduction network of PRKDs and Lentiviral transfection was performed with sh- the key nodes in the network in OSCC through PRKD3 (GenePharma, Shanghai, China) when multiple omics, so as to provide correspond- cell density reached to 35%-50% according to ing measures and potentially effective treatment the manufacturer’s instructions. These cells were methods for the treatment of OSCC. harvested 48 hours later for these experiments, We apply quantitative real-time polymerase including qRT-PCR, Western Blot, cell function chain reaction (qRT-PCR) technique to detect experiment and so on. PRKD3 mRNA expression in 62 cases of OSCC tissues and discusses its association with certain Cell Counting Kit-8 (CCK-8) Assay clinical pathological characteristics of these OS- Cells were seeded in a 96-well plate (2×103 CC patients including tumor TNM (tumor node cells/well) with 100 uL culture medium. CCK-8 metastasis) stage, metastasis incidence, patient’ test (Dojindo Molecular Technologies, Kuma- age, gender, so as to illustrate the role of PRKD3 moto, Japan) was performed based on the manu- in the progression of OSCC and thus provide a facturer’s protocol. The absorbance value at 450 new train of thought and theoretical basis for nm of each well was measured by a microplate early diagnosis, gene therapy and prognosis pre- reader. diction of this cancer. Transwell Cell Migration Assay Transwell chambers (Millipore, Billerica, MA, Patients and Methods USA) were inserted by using a 24-well plate ac- cording to the manufacturer’s instructions. 3×105 Patients and OSCC Samples cells suspended in 200 μL of serum-free medium A total of 62 cases of pathologically con- was applied in the upper layer of the chamber firmed OSCC tissue samples and correspond- with 600 μL of medium in the bottom. After 48- ing clinical and pathological data were col- h incubation, migratory cells in the bottom were lected from the Department of Pathology and reacted with 15-min methanol, 20-min crystal vi- Stomatology of our hospital. All patients did olet and captured using a microscope. Migratory not accept radiotherapy, chemotherapy or any cells were counted in 10 random selected fields biological treatment before surgery. This inves- per sample. 12710 PRKD3 promotes malignant progression of OSCC by downregulating KLF16 expression Cell Wound Healing Test Statistical Analysis After 48 h of transfection, cells were digested, Statistical Product and Service Solutions centrifuged and resuspended in medium without (SPSS) 22.0 program (IBM Corp., Armonk, NY, FBS to adjust the density to 5 x 105 cells/mL. USA) was used for processing. The data were The density of the plated cells was determined presented as X ± SD (standard deviation). p<0.05 according to the size of the cells (the majority of was considered statistically significant. Continu- the number of cells plated was set to 50000 cells/ ous variables were analyzed by t-test, categorical well), and the confluency of the cells reached variables were analyzed by χ2-test or Fisher’s 90% or more the next day. After the stroke, cells exact probability method. Kaplan-Meier method were rinsed gently with phosphate-buffered sa- was used to evaluate the prognosis survival time line (PBS) for 2-3 times and observed again after of patients, and the differences between different incubation in low-concentration