[Study on enhanced sensitivity to DNA damage in POLQ knocking-down salivary of adenoid cystic carcinoma-83 cells].

To investigate the effects of POLQ inhibition on proliferation, colony formation, cell cycle, DNA damage and repair in salivary adenoid cystic carcinoma-83 (SACC-83) cell line. POLQ knocking-down SACC-83 cells were constructed using short hairpin RNA (shRNA) transient transfection, and the inhibition efficiency was detected by qRT-PCR and Western blot. DNA damage in SACC-83 cells was induced by different concentration of DNA damage agent etoposide (VP-16-213), and the levels of γH2AX expression were detected by Western blot to evaluate DNA double-strain breaks. Under different concentration of etoposide-induced DNA damage condition, CCK-8 assay was used to evaluate the effect of POLQ inhibition on cell proliferation in SACC-83 cell line. Under etoposide-induced DNA damage condition, plate colony assay was performed to detect the effect of POLQ inhibition on cell clone formation ability in SACC-83 cell line, and flow cytometry was used to detect the effect of POLQ inhibition on cell cycle in SACC-83 cell line. Furthermore, under etoposide-induced DNA damage condition, Western blot was used to analyze POLQ, γH2AX, RAD51 and PARP1 protein expression. SPSS 20.0 software package was used for statistical analysis. The mRNA and protein expression of POLQ was inhibited by shRNA transient transfection. Increased γH2AX in SACC-83 was closely coupled with increased concentrations of etoposide. The results of CCK-8 assay showed that POLQ knocking-down suppressed cell proliferation ability in SACC-83 cell line, and the inhibitory effect was mitigated with increased concentration of etoposide(P＜0.001). The result of plate colony assay demonstrated that under etoposide-induced DNA damage condition, compared with the control group, POLQ knocking-down suppressed cell colony ability in SACC-83 cell line(P＜0.001). Moreover, the results of flow cytometry demonstrated that under etoposide-induced DNA damage conditions, compared with the control group, POLQ knocking-down induced S phase arrest(P＜0.01). Mechanistically, the results of Western blot showed that POLQ regulated DNA damage and repair by promoting expression of γH2AX(P＜0.05) and homologous recombination (HR) pathway-related protein RAD51 (P＜0.05), respectively, and down-regulating the alternative non-homologous end joining (alt-NHEJ) pathway-related protein PARP1(P＜0.01). POLQ knocking-down promotes the sensitivity of SACC-83 cell line to DNA damage.