Effect Of PTEN/SMAD4 On DNA Damage Repair Pathway Selection In Salivary Adenoid Cystic Carcinoma

Background:Salivary adenoid cystic carcinoma(SACC)is a kind of slow growing and highly malignant epithelial tumor in the head and neck.It is easy to spread along the nerves,and is prone to relapse with high hematogenous metastasis.Among the three types of SACC,solid type SACC had the highest degree of malignancy,worst prognosis and low long-term survival rate.The clinical treatment for SACC is limited,and the efficacy of postoperative radiotherapy and chemotherapy is unstable.To find the therapeutic target of SACC,optimize the therapeutic scheme and enhance the effect of radiotherapy and chemotherapy will help to improve the therapeutic effect of SACC and improve the quality of life of patients.The mechanism of DNA damage repair is closely related to the drug resistance of tumors.When cells produce DNA damage due to internal and external injuries,the cells will have DNA damage response(DDR),and stimulate different repair mechanisms.Among them,double strand breaks(DSB)are the most serious form of damage.The classical Homologous recombination(HR)led by BRCA1 as the core protein of DSB and C-non Homologous end joining(c-NHEJ)mediated by Ku70 are the most serious form of cell repair.Alt-non-homologous end joining(Alt-NHEJ)initiated by c-NHEJ and POLQ were the main pathways.There was a certain degree of competition among different repair pathways,and different tumors had different DNA repair pathways.Group in the early research has shown that three kinds of classification of salivary gland adenoid cystic carcinoma with different molecular phenotype,cribriform type and tube type SACC more expression of PTEN/SMAD4 high expression,solid type SACC much performance for the expression of PTEN/SMAD4 is missing,so that the expression of PTEN and SMAD4 missing is characteristic of solid type SACC gene phenotype,and through gene expression profile chip technology found in the loss of PTEN SACC POLQ expression increases can cause DNA damage repair,prompt should according to different molecular phenotype of SACC targeted therapy.Therefore,this study aims to explore which DNA damage repair pathway PTEN/ Smad4 double-deletion SACC uses to repair DSB,and to provide a new idea for further understanding the occurrence and development of SACC as well as clinical treatment.Objective and method:1.PTEN/ Smad4 double knockdown cell model was constructed in vitro to simulate solid SACC,and the effects of PTEN/ Smad4 double knockdown on DSB injury response were detected by PCR and Western Blot.2.The expression of BRCA1,KU70 and POLQ proteins in SACC was detected by PTEN/ Smad4 double knockdown;PTEN/ Smad4 overexpression cell model was constructed in vitro and protein expression of DSB repair pathway was detected.3.SACC was double knocked down by PTEN/ Smad4 cell culture,and POLQ was silenced with RNA interference to explore the effects of POLQ on DSB injury response and nuclear karytype at m RNA and protein levels.4.In the context of DNA damage,the response to DSB damage in SAC83 cells after further down-regulation of POLQ in PTEN/ Smad4 double knockdown SACC was investigated.Results:1.PCR and Western Blot results showed that compared with SACC83,the m RNA levels of DNA damage response genes H2 AFX,ATM and ATR in PTEN/ Smad4 double knockdown SACC83 were higher,and the expression level of γH2AX protein was higher.2.Western Blot results showed that the protein expressions of HR and c-NHEJ related genes BRCA1 and KU70 were decreased in PTEN/ Smad4 double knockdown SACC83,while the protein expressions of alt-NHEJ core proteins POLQ and PARP1 were increased.The expression of HR and c-NHEJ related genes BRCA1 and KU70 protein was increased and the expression of alt-NHEJ core protein POLQ was decreased in SACC83 with PTEN/ Smad4 overexpression.3.After successfully interfering with POLQ in PTEN/ Smad4 double-knockdown SACC83,compared with PTEN/ Smad4 double-knockdown SACC83,both PCR and Western Blot results showed that the expression of genes related to DNA damage response and γH2AX protein in PTEN/ Smad4 / Polq co-knockdown SACC83 was decreased at the m RNA level.Chromosomal radioactivity defects and chromosomal breakage were observed in PTEN/ Smad4 double knockdown SACC.PTEN/ Smad4 /Polq co-knockdown reduced the number of chromosome abnormality in SACC.4.POLQ was down-regulated in PTEN/ Smad4 double-deletion SACC,and the expression of DNA damage-responsive genes was increased.The results of the recovery experiment indicated that the double deletion of PTEN/ Smad4 affected DNA damage repair in SACC,which was dependent on POLQ.Conclusion:Alt-NHEJ was used as the main repair method in PTEN/ Smad4 double knockdown(solid type)SACC,while HR and c-NHEJ were used as the main repair route in PTEN/Smad4 overexpression SACC(sieve type and tubular type).The loss of PTEN/ Smad4 induces the DSB injury response by altering the expression of POLQ.