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

doi:10.19439/j.sjos.2023.02.002

Abstract

Abstract: PURPOSE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: POLQ knocking-down promotes the sensitivity of SACC-83 cell line to DNA damage.

Key words: Salivary adenoid cystic carcinoma, POLQ, DNA Repair, Etoposide

CLC Number:

R739.8

BAI Han, LIU Han, ZHU Lei, LIU Chao, LI Nan, XIAO Jing. Study on enhanced sensitivity to DNA damage in POLQ knocking-down salivary of adenoid cystic carcinoma-83 cells[J]. Shanghai Journal of Stomatology, 2023, 32(2): 120-125.

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