LMTK3 knockdown retards cell growth and invasion and promotes apoptosis in thyroid cancer

Abstract

Lemur tyrosine kinase-3 (LMTK3) is a member of the serine/threonine tyrosine kinase family, which is thought to be involved in tumor progression and prognosis. The purpose of the present study was to determine the diagnostic significance and therapeutic targets in thyroid cancer. ELISA assay was used to detect the protein expression of serum LMTK3. Immunohistochemistry and reverse transcription‑quantitative polymerase chain reaction were employed to measure the expression of LMTK3. Flow cytometry was used to determine the cell cycle. Transwell assay was used to measure the invasion and migration of SW579 cells and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to detect cell apoptosis. The LMTK3 level was positively associated with disease stage and pathological type, whereas there was no correlation between LMTK3 level and gender, age, tumor size or lymph node metastasis. The serum LMTK3 level was significantly increased in 102 thyroid carcinoma patients compared with 52 benign thyroid tumor patients and 50 healthy volunteers (P=0.001). The protein and mRNA expression of LMTK3 was markedly higher in thyroid cancer patients compared with patients with benign thyroid tumors. Notably, LMTK3 knockdown retarded proliferation, invasion and migration in SW579 cells. In addition, downregulation of LMTK3 promoted apoptosis in SW579 cells. These findings indicated that LMTK3 knockdown retards the growth of thyroid cancer cells partly through inhibiting proliferation, invasion, migration and inducing apoptosis in SW579 cells. It may serve as a useful diagnostic biomarker and a novel therapeutic target for patients with thyroid cancer.

Figure 1.

Serum level of LMTK3 measured by ELISA in TCP, BTD and HV. Markedly elevated serum LMTK3 levels were detected in TCP compared with BTD and HV (n=6). Data are expressed as the mean ± standard error of the mean. ***P<0.001 vs. HV; ^###P<0.001 vs. BTD. LMTK3, lemur tyrosine kinase-3; TCP, thyroid cancer patients; BTD, benign tumor diseases; HV, healthy volunteers.

Figure 2.

LMTK3 level measured in TCP, BTD and HV tissue. (A) LMTK3 expression visualized by immunohistochemistry staining (original magnification, ×400; scale bar=100 µm). (B) The mRNA expression of LMTK3 was markedly elevated in TCP compared with BTD tissue samples. (C) Protein levels of LMTK3 were increased in TCP compared with BTD tissue samples. Data are expressed as the mean ± standard error of the mean (n=35). **P<0.01 vs. BTD group. LMTK3, lemur tyrosine kinase-3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TCP, thyroid cancer patients; BTD, benign tumor diseases; HV, healthy volunteers.

Figure 3.

LMTK3 knockdown retarded cell cycle and inhibited proliferation of SW579 cells. (A) The mRNA level of LMTK3 was reduced in the siRNA LMTK3 treated group compared with NC (n=6). (B) Protein expression of LMTK3 was significantly inhibited by treatment with LMTK3 siRNA compared with NC (n=3). (C) The effect of LMTK3 knockdown on cell cycle distribution in SW579 cells. Histograms illustrate SW579 cells treated with LMTK3 siRNA or exposed to NC, respectively (n=6). (D) Cell proliferation assessed by using an MTT assay. The result demonstrated that cell proliferation was decreased by treatment with LMTK3 siRNA, n=6. Data are expressed as the mean ± standard error of the mean. **P<0.01 vs. NC. LMTK3, lemur tyrosine kinase-3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; siRNA, small interfering RNA; NC, negative control.

Figure 4.

LMTK3 siRNA inhibits the migration and invasion of SW579 cells. (A) Migration/invasive cells were imaged following staining with hematoxylin (original magnification, ×200). (B) Histogram of the number of migrated cells. (C) Histogram of the number of invaded cells. Data are expressed as the mean ± standard error of the mean (n=6); **P<0.01 vs. NC. Ctrl, control group; NC, negative control; siRNA, small interfering RNA; LMTK3, lemur tyrosine kinase-3.

Figure 5.

LMTK3 knockdown promoted apoptosis in SW579 cells. (A) Apoptotic cells were determined by TUNEL staining and visualized (magnification, ×200). Green=apoptotic cells; blue=cell nucleus stained by DAPI. Representative images are presented. (B) Calculation of apoptotic cells. The results indicate that LMTK3 knockdown induced apoptosis in SW579 cells (n=6 independent experiments). Data are expressed as the mean ± standard error of the mean. **P<0.01 vs. NC. Ctrl, control group; NC, negative control; siRNA, small interfering RNA; LMTK3, lemur tyrosine kinase-3; DAPI, 4′,6-diamidino-2-phenylindole; TUNEL, terminal dUTP nick end labeling.