The Effect Of TRF2 Knockdown On Telomere Function And Biological Behavior Of Gastric Cancer Cells

Background and Objective:Gastric cancer is the most common malignant tumor of the digestive system,and the morbidity and mortality of gastric cancer ranks among the top of all tumors in China.Although surgical treatment is main treatment of gastric cancer,chemotherapy also plays a very important role in the treatment of gastric cancer.However,chemotherapy is easy to make gastric cancer resistant and it often produces toxic and side effects,resulting in unsatisfactory therapeutic effects and prognosis of gastric cancer.Therefore,it is of great significance to explore new treatment methods for gastric cancer.At present,the drugs used for chemotherapy mainly play a role by damaging the DNA of tumor cells and inducing their apoptosis.Studies have shown that the expression level of TRF2 is abnormally increased in gastric cancer cells,and the latest research found that TRF2 not only has telomere protection-related functions,but also participates in DNA damage repair response as a DNA damage repair factor to promote tumor cell survival.In order to further explore the role of TRF2 in gastric cancer cells,this study explored its effect on the development of gastric cancer cells by inhibiting the expression of TRF2,and further explored its molecular mechanism.Methods:Construct sh RNA lentiviral vector to infect human gastric cancer cell line BGC-823 to stably knock down TRF2 expression.Fluorescence in situ hybridization(FISH),immunofluorescence combined with fluorescence in situ hybridization(IF-FISH)technology was used to detect whether knockdown of TRF2 gene expression could cause telomere DNA damage response and cause telomere dysfunction Foci(TIF)and whether it causes chromosomal aberrations(abnormal telomere signal).The effects of TRF2 knockdown on the proliferation,invasion,and viability of human gastric cancer cell line BGC-823 were tested by scratch test,clone formation test,CCK8 test,flow cytometry,and trypan blue rejection test.Western Blot and real-time quantitative PCR(q PCR)were used to detect changes in autophagy-related proteins(LC3I/II,P62,ATG5)and iron death-related proteins(SLC7A11,GPX4).Finally,TRF2 knockdown gastric cancer cell BGC-823 was treated with autophagy and iron death inhibitors to observe the effect of inhibitors on gastric cancer cell migration,proliferation,cell viability and survival ability.Results:The sh RNA lentiviral expression vector was successfully constructed and knocked down the expression of human gastric cancer cell line BGC-823 TRF2.FISH and IF-FISH showed that TRF2 knockdown can cause significant telomere DNA damage and abnormal telomere signal.Scratch experiments,clone formation experiments,CCK8 experiments,flow cytometry,trypan blue rejection experiments show that TRF2 knockdown causes proliferation,cell viability,invasion,and survival of human gastric cancer cell line BGC-823 significantly decreased.Western Blot and real-time quantitative PCR(q PCR)results showed that TRF2 knockdown can enhance the autophagy level of human gastric cancer cell line BGC-823 and reduce the expression of ferroptosis-related proteins SLC7A11 and GPX4.By comparison,it was found that the TRF2 knockdown BGC-823 cell lines treated with autophagy and ferroptosis inhibitors were significantly stronger than TRF2 knockdown gastric cancer cells BGC-823 in terms of invasion,proliferation and survival.Conclusion:Knockdown of human gastric cancer cell line BGC-823 TRF2 results in telomere dysfunction,and reduces gastric cancer cell proliferation,cell viability,invasion,and viability.Further,we analyzed that the effect of TRF2 knockdown on gastric cancer cells depends on cell autophagy and ferroptosis.The results showed that TRF2 knockdown resulted in autophagic cell death and ferroptosis in gastric cancer cells.Therefore,we found that high expression of TRF2 in gastric cancer cells may be a protective mechanism to maintain the survival of gastric cancer cells.Knocking down the gastric cancer cell TRF2 can not only promote gastric cancer cell apoptosis through the traditional ATM-dependent DNA damage response,but also cause gastric cancer cell autophagic cell death and iron death.It provides new ideas for the research and treatment of the molecular mechanism of gastric cancer.