Effects Of POLD1 On Proliferation And Invasion Of Oral Squamous Cell Carcinoma And Its Regulatory Mechanism

PURPOSE:Oral squamous cell carcinoma(OSCC)is one of the most common tumors,with the highest incidence in head and neck squamous cell carcinoma,accounting for about 90%of oral cancers,and the incidence is increasing every year.Although surgery,radiotherapy and chemotherapy are widely used in the clinical treatment of OSCC,the 5-year survival rate of OSCC still hovers around 65%,and the survival rate of advanced patients is less than 30%.DNA polymerase delta subunit 1(POLD1)is associated with multiple tumors.However,the correlation between POLD1 and OSCC remains unknown.The aim of the study was to investigate the effects of POLD1 expression on proliferation,migration,invasion,and OSCC cell lines and its related mechanisms.We also attempted to find out the pathogenesis of OSCC at the molecular level,and to find potential biomarker for OSCC.METHODS:Online bioinformatics were used to analyze POLD1 expression in head and neck squamous cell carcinoma(HNSCC)samples and normal tissues.POLD1 expression was evaluated in forty OSCC and normal tissues using immunohistochemistry.OSCC cell lines(SCC9 and CAL27)were infected with lentivirus with lentiviral vector containing shRNA(POLD1)or empty vector,and stable cell lines were established by screening.Western blot assay was used to detect the silencing efficiency of POLD1 in stable cell lines and the expression levels in the nucleus,cytoplasm and cell membrane,respectively.Stable cell lines infected with lentiviral vector containing shRNA(POLD1)were called the experimental group,while stable cell lines infected with empty lentiviral vector were called the control group.CCK-8 assay and EDU assay were used to detect cell proliferation of SCC9 and CAL27 cells.Would healing assay and transwell assay were used to detect cell migration and invasion.The effect of POLD1 on cell cycle of SCC9 and CAL27 cells was determined by flow cytometry and the related pathway was studied via western blot.Peripheral blood samples of five OSCC patients before and six months after surgery were collected.High-throughput transcriptome sequencing was performed to detect the expression change of POLD1(mRNA)before and after surgery,in order to further explore the potential role of POLD1 as a biomarker for OSCC.T-test was used for comparison between groups,and GraphPad Prism and ImageJ software were used for data analysis and plotting.P<0.05 was considered statistically significant.RESULTS:Online database show that POLD1 was abnormally upregulated in head and neck squamous cell carcinoma.Immunohistochemical results showed that the expression of POLD1 in 40 pairs of OSCC tissues was higher than that in normal tissues,Western blot showed that POLD1 expressed both in the nucleus and cytoplasm and was silenced in experimental grouP.CCK-8 assay and EDU assay show that depletion of POLD1 in OSCC cells inhibited proliferation ability.Would healing assay and transwell assay showed that depletion of POLD1 in OSCC cells inhibited cell migration and invasion.Flow cytometry showed that the cell cycle of the experimental group was arrested in G2 phase and shortened in G1 phase.Western blots showed increased phospho-Cdc2(Tyr15)and phospho-Rb(Ser807)levels in the experimental group compared to the control group,but no significant changes were observed in phospho-Chkl(Ser345)and phospho-Chk2(Thr68)levels.Blood sequencing results showed that the expression of POLD1 was significantly decreased after six months surgery(P<0.001).CONCLUSIONS:Silencing POLD1 inhibited the proliferation,migration and invasion of SCC9 and CAL27 cells.Silencing POLD1 shortened the G1 phase and blocked SCC9 and CAL27 cells in G2 phase.The mechanism may be related to phospho-Cdc2/phospho-Rb pathway.Expression of POLD1(mRNA)was significantly decreased in patients with OSCC 6 months after surgery,further indicating the potential role of POLD1 as a biomarker and molecular therapeutic target for OSCC.