Methods

Total RNA was extracted from tissues and cells according to the instructions of RNAiso Plus reagent (Takara, Dalian, China), and mRNA was reverse transcribed into cDNA using the PrimeScript ™ RT reagent Kit (Takara, Dalian, China). Besides, qRT-PCR was performed according to the instructions of the SYBR® Premix Ex TaqTM II (Takara, Dalian, China). Sequences of primers are shown in Table 1. The experiment was repeated three times. Fold change (relative expression) = 2^−ΔΔCt.

Sequences of primers

Proteins were extracted by lysis buffer (PMSF: RIPA = 1:100). Subsequently, proteins were electrophoresed by 5% concentrated gel and separated by 12% isolated gel, then transferred by membrane of PVDF. After blocking with 5% skim milk, the PVDF membranes were incubated with anti-TPPP3 antibody (GeneTex, USA, dilution 1: 1000) and anti-β-actin antibody (Sangon Biotech, China, dilution 1:800) overnight at 4°C, respectively. And then, these PVDF membranes should be incubated with fluorescent secondary antibody (CST, USA, dilution 1:10,000) for 60 minutes at room temperature. The Odyssey imaging system (LI-COR Biosciences, Lincoln, USA) was applied to analyze the gray value of corresponding protein [¹³].

The primary antibody used for immunostaining was the TPPP3 polyclonal antibody (GeneTex, USA). Immunohistochemical SP method was used to routinely dewax, gradual hydration of gradient alcohol, and repair antigen etc. The diluted TPPP3 primary antibody (dilution 1:100) was incubated at 4°C overnight, and immunohistochemical staining kit SP-9002 (zsbio, Beijing, China) were used. After DAB color reagent was added, hematoxylin was counterstained to the slice.

TPPP3 is mainly distributed in the cytoplasm. Light yellow, brownish yellow or tan fine particles appearing in the cytoplasm are positive staining. Strength of positive cells staining (contrast of staining with background) as follows: 0 (no positive staining), 1 (light yellow), 2 (brown), 3 (tan). Percentage of positive cells as follows: 1 (<10%), 2 (10% −50%), 3 (51% −75%), 4 (>75%). The products of staining intensities and the percentage of positive cell expressions was used as the immunohistochemical score. The immunohistochemical score ≥ 1 is considered positive, otherwise it is considered negative [¹⁴].

Lentivirus vector of TPPP3 overexpression (LV-TPPP3) and the control lentivirus vector (LV-CON307) were completed by Genechem Technology Co., Ltd. (Shanghai, China), and the lentivirus has puromycin resistance and green fluorescence protein (GFP) expression sequence. According to the operation manual of lentivirus, cells were inoculated at the density of 5 × 10⁴/ml, and transfected multiplicity of infection (MOI) was 30. The transfection efficiency of cells was observed under the inverted fluorescence microscope (Olympus, Japan) in 72 hours after transfection. When the fluorescence efficiency over 80%, the transfected cells were used for subsequent experiments. The successful transfection of 5–8 F and HONE1 was determined by qRT-PCR and western blot.

Cell proliferation was detected by CCK-8 (Dojindo Lab, Japan) and clone formation assays. For CCK-8 assay, NPC cells were seeded into 96-well plates at a density of 2500 cells/well and then incubated with CCK-8 solutions at different times (0, 24, 48, 72, and 96 h) for 2 h at 37°C. The optical density (OD) value of cells in each group was detected by Varioskan™ LUX Multimode Microplate Reader (Thermo Scientific, USA) at 450 nm.

For the colony formation assay, 500 cells were plated into each well of 6-well plates. Medium was refreshed every three days and cultured until clones were obviously visible. Colonies were fixed with 4% paraformaldehyde for 20 min, stained with 1% crystal violet for 20 min and counted by visual inspection. Clone formation rate = (number of clone formation/number of seeded cells) × 100% [¹³].

For wound healing assay, cells were seeded into 6-well plates (6 × 10⁵ cells/well) and cultured at 37°C, 5% CO₂ until cell grown to a confluent monolayer. Subsequently, a uniform scratch was made down the center of the well using a micropipette tip, and then the cells were cultured in serum-free medium. The migration of each group of cells at the time of scratching 0 h and 36 h later were observed under an inverted microscope. The relative distance of the cells to the scratched area was measured using Image J software, and relative cell migration rate (%) = relative distance of cell migration/original distance of scratched area × 100%.

Cell migration assays were performed using 24-well transwell units with 8 µm pore size polycarbonate inserts (Corning, USA). About 3 × 10⁴ cells were harvested in 200 µl serum-free medium and seeded into the upper chamber, and 500 µl medium containing 10% fetal bovine serum was added to the lower chamber. After 24 h of incubation, the cells on the top of the membrane were wiped off with a cotton swab. Cells on the lower surface were fixed with 4% polymethanol for 30 min, and stained with 1% crystal violet for 20 min. Five random fields of the lower surface were photographed under inverted microscope (Olympus, Japan).

For the invasion assay, the transwell chambers were coated with appropriate concentration of Matrigel (Corning, MA). About 4 × 10⁴ cells were harvested in 200 µl serum-free medium and seeded into the upper chamber. Invasion assay was performed similar to migration assay [¹³].

Based on {"type":"entrez-geo","attrs":{"text":"GSE12452","term_id":"12452"}}GSE12452, the expression profile data of 31 NPC tissues were grouped by the mean value of TPPP3 expression. The GSEA was performed to analyze the biological processes involved in the expression of TPPP3 gene in NPC and the possible related pathways with the molecular functions and biological processes of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). |NES| >1, NOM p-val < 0.05, FDR q-val < 0.25 were set as the threshold of significant enrichment.