The Study Of DNA Damage Repair Factor PBK/TOPK Affecting Radiosensitivity Of Colon Adenocarcinoma Through Glycolytic Pathway

Objective The study is to demonstrate that the radiosensitivity of colon adenocarcinoma can be affected by knocking down DNA damage repair factor PBK/TOPK and combining with radiotherapy,which have an impact on aerobic glycolysis.Methods Colonic adenocarcinoma cell line LS174 T was selected as the research object.The expression of PBK/TOPK was knocked down by lentiviral transfection.The transfection was confirmed by Real time-PCR and Western blot.The clone formation experiment was performed.The cell with koncking down PBK/TOPK was test group,which be irradiated by the doses of 0Gy,4Gy and 8Gy.So it could be choosed to make another test by using 0Gy and 8Gy to irradiating.After irradiation,total RNA and total protein were extracted at 24 h,48h and 72 h.The RNA and protein levels of PBK/TOPK,glycolytic key enzymes GLUT1 and HK2 were detected by Real time-PCR and Western blot.It could make clear that the radiosensitivity of colon adenocarcinoma with knocking down PBK/TOPK can be affected by the key enzymes GLUT1 and HK2 in the glycolytic pathway.The obtained measurement data were expressed by means of mean ± standard deviation.The comparison between the mean of the two samples obeying the normal distribution and the variance was analyzed by t test,and the comparison between the mean of the two groups was analyzed by variance.Results The results of cell clone formation experiments were compared.1)After knocking down PBK/TOPK in normal LS174 T cells,the number of cell clone formation was(0.6167±0.01528)and the negative control group(0.7767±0.01528)and blank.The group(0.78±0.01)was statistically significant(P<0.05),and the negative control group was not significantly different from the blank group(P>0.05).2)After 4Gy irradiation,the cell clone formation number was knocked down.The difference between the group(0.4113±0.0102)and the negative control group(0.6234±0.003)and the blank group(0.6278±0.0020)wasstatistically significant(P<0.05).3)After the 8Gy irradiation,the group was knocked down(0.11±0.01)compared with the negative control group(0.2258±0.0083)and the blank group(0.2384±0.0027),the difference was statistically significant(P<0.05).At the same time,the colony of the knockdown group was smaller.Therefore,we chose a significant difference between 0Gy and 8Gy to see if PBK/TOPK is associated with key glycolytic enzymes.By Real time-PCR and Western blot,we found that: 1)Compared with the normal group,the relative expression of PBK/TOPK,GLUT1 and HK2 decreased in LS174 T cells after 8Gy irradiation alone;the relative expression of PBK/TOPK was decreased gradually in LS174 T cells over time.GLUT1 and HK2 also changed.The relative expression of GLUT1 decreased with time,and reached the lowest at 48 h,then showed an increasing trend.The relative expression of HK2 showed a completely opposite change,and the highest expression at48 h.2)In the LS174 T cells,the relative expression levels of GLUT1 and HK2 were also changed in the PBK/TOPK simple knockdown group.The relative expression of GLUT1 decreased with time,and reached the lowest at 48 h,then showed an increasing trend.The relative expression of HK2 showed a completely opposite change,and the highest expression at 48 h.3)In the knockdown of PBK/TOPK combined with 8Gy irradiation group,the relative expression of PBK/TOPK decreased firstly and then increased.The relative expression of GLUT1 showed an increasing trend within 48 h,and reached the peak at 48 h,followed by a decreasing trend.The change in the relative expression of HK2 is gradually decreasing with time.Conclusions We initially explored the knockdown of PBK/TOPK combined with8 Gy irradiation,showing reduced colony formation,indicating decreased cell proliferation and increased death,indicating that knockdown of PBK/TOPKcombined with 8Gy irradiation can enhance the radiosensitivity of colon adenocarcinoma.Further experiments have shown that knockdown of PBK/TOPK combined with 8Gy irradiation can change the expression levels of key enzymes GLUT1 and HK2 in the glycolytic pathway.The relative expression of GLUT1 increased in 48 h,reached the peak at 48 h,and theshowed an decreased trend.And the relative expression of HK2 gradually decrease withtime.In summary,knockdown of PBK/TOPK in combination with 8Gy irradiation incolon adenocarcinoma can increase the sensitivity of colon adenocarcinoma by affecting the key enzymes GLUT1 and HK2 in the glycolytic pathway.