Abstract: Background and purpose: As a protein kinase that regulates the cell cycle, polo-like kinase 4 (PLK4) is involved in mitosis initiation, centrosome maturation, cytokinesis, DNA damage detection, etc., which is highly expressed in a variety of tumors. Whether it is involved in the proliferation, invasion and migration of esophageal squamous cell carcinoma (ESCC), and the specific molecular mechanism still remain unclear. This study examined the expression of PLK4 in ESCC cell lines and clinical tissue specimens, and its effect on cancer cell proliferation, invasion and migration. Methods: Total cell RNA was extracted with TRIzol, and cDNA was synthesized with reverse transcription kit for real-time fluorescence quantitative polymerase chain reaction (RTFQ- PCR) to detect the mRNA expression level of PLK4 gene in normal esophageal epithelial cells Het-1A and ESCC cell lines TE-1, TE-8 and TE-13. After the cells were collected by centrifugation, total cell protein was extracted with RIPA lysate. Western blot was used to detect the expression level of PLK4 protein in normal esophageal epithelial cells Het-1A and ESCC cell lines TE-1, TE-8 and TE-13. A total of 93 cases of ESCC tissues and paired adjacent tissues (more than 5 cm from the edge of the primary tumor) confirmed by histopathology were collected at the Affiliated Hospital of Henan Medical College from January 2017 to December 2019. The clinical tissues were quick-frozen by liquid nitrogen followed by total tissue protein extraction by RIPA lysate. Western blot was used to detect the expression level of PLK4 protein in ESCC tissues. We constructed a receiver operating characteristic curve and analyzed the relationship between PLK4 expression and clinicopathological parameters. The expression of PLK4 in TE-13 cells was inhibited by siRNA interference technology. The siRNA interference fragments of PLK4 were designed and synthesized, followed by transfection with Lipofectamine ^TM 2000 to inhibit the expression of PLK4 in TE-13 cells. The effect of siRNA interference fragments on PLK expression was detected by RTFQ- PCR experiments and Western blot. After the expression of PLK4 was down-regulated in TE-13 cells, cell counting kit-8 (CCK-8) experiment and clone formation experiment were used to detect cell proliferation ability, and transwell chamber experiment and scratch healing experiment were conducted to detect cell invasion and migration abilities. The effects of down-regulation of PLK4 on the expressions of key proteins mTOR, p70S6K, p-mTOR ^Ser2448 and p-p70S6K ^{Thr421/Ser424} in the mTOR/p70S6K signaling pathway were detected by Western blot experiments. Results: The results of RTFQ-PCR and Western blot experiments showed that the mRNA and protein expression levels of PLK4 gene in ESCC cell lines were significantly higher than those in normal esophageal epithelial cells (P＜0.05). Compared with normal tissues adjacent to cancer, the protein expression level of PLK4 in ESCC tissue specimens was abnormally increased (P＜0.05). The receiver operating characteristic curve exhibited an area under curve (AUC) of 0.841, a 95% CI of 0.786-0.895 with 74.2% (69/93) sensitivity and 89.2% (83/93) specificity (P＜0.0001). There was no relationship between expression level of PLK4 protein and gender, age and tumor size (all P ＞ 0.05) in ESCC tissues. However, expression level of PLK4 protein was related to the degree of differentiation, clinical stage and lymph node metastasis (all P＜0.05). The lower degree of ESCC differentiation had higher expression rate of PLK4. The high expression rate of PLK4 in ESCC tissues of poorly differentiated degree was 86.7%, and the high expression rate of PLK4 protein in ESCC tissues of patients with stage Ⅲ-Ⅳ was 92.3%. The high expression of PLK4 was positively correlated with clinical stage (P＜0.05). The results of CCK- 8 and clone formation experiments showed that down-regulating the expression of PLK4 significantly inhibited the proliferation of TE-13 cells (P＜0.05). The results of the transwell chamber experiment and the scratch experiment showed that down-regulating the expression of PLK4 significantly inhibited the invasion and migration abilities of TE-13 cells (P＜0.05). Inhibiting the expression of PLK4 decreased the protein expressions of mTOR and p70S6K in TE-13 cells (P＜0.05), and the expressions of p-mTOR ^Ser2448 and p-p70S6K ^{Thr421/Ser424} decreased (P＜0.05). Conclusion: PLK4 is highly expressed in ESCC cells and tissues. Inhibition of PLK4 expression inhibited the proliferation, invasion and migration of ESCC cells. PLK4 may promote the malignant process of ESCC cells through the mTOR/p70S6K signaling pathway.

Key words: Polo-like kinase 4, Esophageal squamous cell carcinoma, Clinicopathological characteristics, Proliferation, Invasion and migration