The Role Of ANLN Gene In DNA Damage Repair And Its Effect On Gastric Cancer Cells

Gastric cancer is a malignant tumor of digestive tract,which is still an important cause of cancer death at present.It is of great significance for early diagnosis and treatment of gastric cancer to search for effective therapeutic target molecules in gastric cancer.The actin binding protein(ANLN)gene is a gene that affects cell division.We investigated the effect of ANLN gene overexpression/silencing on proliferation,apoptosis and cycle of gastric cancer cells.To determine the effect of ANLN gene on DNA damage repair;Regulation of the ATR signaling pathway by ANLN gene in S phase sites;Finally,the influence of ANLN gene down-regulation on the development of gastric cancer was determined.First,we obtained survival data related to the expression level of ANLN in 231 cases of gastric adenocarcinoma and gastric tubular adenocarcinoma,407 cases of gastric cancer and normal gastric tissue,and 27 pairs of matched expression level of ANLN in gastric cancer tissues through the database.The protein expression of ANLN gene in 55 cases of gastric cancer was detected by immunohistochemistry.Secondly,the expression levels of ANLN gene in gastric cancer cell line SGC-7901 / MGC-803 and normal gastric mucosal cell line GES-1 were detected by Western blot and real-time quantitative PCR.The effect of silencing/overexpressing ANLN gene on the proliferation of gastric cancer cells SGC-7901 / MGC-803 was detected by CCK-8 assay.The effect of silencing/overexpressing ANLN gene on cycle and apoptosis of gastric cancer cells SGC-7901 / MGC-803 was detected by flow cytometry.The effect of the silencing/overexpressing ANLN gene on the DNA damage of gastric cancer cells SGC-7901 / MGC-803 was detected by comet assay.Changes in downstream ATR signaling pathway proteins after silencing/overexpression of the ANLN gene were detected by Western blot.Finally,the effect of CGK733(ATR signaling pathway inhibitor)on the proliferation of gastric cancer cells SGC-7901 / MGC-803 was detected by CCK-8 assay after the overexpression of ANLN gene.The effect of CGK733 on cycle and apoptosis of gastric cancer cells sgc-7901 / mgc-803 after overexpression of ANLN gene was detected by flow cytometry.Comet assay detected the effect of adding CGK733 after overexpression of ANLN gene on DNA damage of gastric cancer cells SGC-7901 / MGC-803.Changes in downstream ATR signaling pathway protein levels were detected by Western blot after the overexpression of ANLN gene and the addition of CGK733.The database data showed that the expression level of ANLN in normal gastric tissues was significantly lower than that in gastric cancer tissues,and the high expression of ANLN would significantly reduce the overall survival of patients with gastric cancer.Immunohistochemical experiments showed that the expression of ANLN gene was significantly correlated with the tumor size and stage of gastric cancer.Western blot and PCR results showed that the expression levels of ANLN in gastric cancer cell lines SGC-7901 and MGC-803 were significantly higher than that in normal gastric mucosal cell line GES-1.CCK-8 results showed that silencing the ANLN gene reduced cell viability(p<0.05);Overexpression of the ANLN gene increases cellular activity(p<0.05).Flow cytometry showed a decrease in the apoptosis rate of the overexpressed ANLN gene(p<0.05),ANLN gene silencing increased the apoptosis rate(p<0.05).At the same time,the number of G1 phase cells decreased and the number of S phase cells increased.The results of the comet experiment showed that the DNA damage was aggravated after the silencing of ANLN gene,and the cell damage was not significantly changed after the overexpression of ANLN gene.Western blot results of overexpression or silencing of ANLN indicated that the expression of p-ATR /p-Chk1/p-Cdk2 was significantly down-regulated and the expression of p-Cdc25a/cyclin E1 was significantly up-regulated after overexpression of ANLN gene,while the expression of ANLN gene was reversed after silencing of ANLN gene.In addition,overexpression of CGK733 decreased cell viability(p<0.05)and increased apoptosis rate(p<0.05).At the same time,the number of G1 phase cells decreased,the number of S phase cells increased and the DNA damage was aggravated.The expression of p-ATR /p-Chk1/p-Cdk2 was significantly up-regulated and the expression of p-Cdc25a/cyclin E1 was significantly down-regulated after the overexpression of ANLN and the addition of CGK733.In summary,the overexpression of ANLN gene can promote the proliferation and inhibit the apoptosis of gastric cancer cells.Downregulation of the ANLN gene had the opposite effect.Inhibition of ANLN expression leads to cell arrest in the S phase.ANLN gene regulates DNA damage repair through the checkpoint ATR signaling pathway during the S period,and it may become a new target gene for targeted therapy of gastric cancer.