Effect Of BRIT1 On Proliferation And Metastasis Of Hepatocellular Carcinoma And Its Mechanism

ObjectiveHepatocellular carcinoma(HCC)is one of the most common clinical malignancies and the leading cause of cancer-related death in our country and worldwide.Its high degree of malignancy,low survival rate,and postoperative metastasis are still difficult problems to overcome in clinic.The BRIT1 gene plays an important role in the DNA damage response and DNA repair signaling pathway.Its deletion or low expression may have an inseparable link with cancer initiation and progression,but so far the function of BRIT1 in HCC(especially in its metastasis)has not been reported.We speculate that the BRIT1 gene may be a very important new tumor suppressor gene.Therefore,studying the expression and mechanism of BRIT1 in HCC has important theoretical significance for understanding the occurrence,development and metastasis of HCC,in order to provide scientific basis for the development new anti-HCC drugs and a novel predictive biomarker of HCC metastasis.MethodsUsing Western blot technique to detect the protein levels of 13 common hepatocell ular carcinoma cell lines,and to screen for low and high expression of HCC line as the experimental object.CCK-8 method was used to observe the effect of BRIT1 overexpression on the proliferation of HCC.Secondly,the effect of BRIT1 overexpression on the migration of HCC was examined by cell scratch technique and Transwell technique.Again,Matrigel-coated transwell chambers were used to simulate vascular and lymphatic basement membranes to observe the effect of BRIT1 overexpression on the invasive ability of HCC.Finally,using Western blot technique to observe the relationship between BRIT1 protein and the migration-related protein SERPNA5 in HCC.The RNA oligo of the target gene BRIT1 was designed and synthesized,RNAi negative control was used as a control.The first part of the screened HCC line with the highest level of BRIT1 expression was transiently transfected and the BRIT1 silencing effect was optimized to recede BRIT1.The HCC model was silenced and CCK-8 were used to perform cell growth curves.Cell scratch,Transwell and Matrigel methods were used to perform cell migration and invasion assays.The resulting BRIT1 silencing HCC model was propagated and migrated accordingly.Then,using Western Blot to explore the relationship of BRIT1 and SERPINA5 in HCC metastasis.The hematopoietic metastasis models of NOD/SCID mice were constructed by intravenous injection Hep3 B and Hep3 B.BRIT1 HCC cells.After observing the body weight,calculating the organ coefficient and performing histopathological analysis to investigate the effect of BRIT1 overexpression on the hematogenous metastatic potential of NOD/SCID mouse.ResultsTaking Hep3 B cell line as control,the growth curve measured using the CCK-8 method showed that the Hep3 B.BRIT1 cell proliferation rate was significantly reduced(P<0.01).Migration experiments using the scratch method and Transwell method showed that the migration ability of Hep3 B.BRIT1 unilateral cells was significantly weakened and the number of migration was significantly reduced(P<0.01).Invasion experiments using the Matrigel-coated transwell chambers method showed that the number of invasive Hep3 B.BRIT1 cells was significantly reduced(P<0.05).Western Blot results showed that the expression level of BRIT1 protein was significantly increased in Hep3 B.BRIT1 cells,and the expression level of SERPINA5 was up-regulated(P<0.01).The RNAi condition with the best silencing effect was detected by the protein level: siRNA: LipoFiter = 30 pmol: 1 μl transfected for 72 h,BRIT1 protein expression was down-regulated by 69% in siSNU449 hepatoma cells(P<0.01).The growth curve measured by CCK-8 method showed that the proliferation rate of siSNU449 cells was significantly increased(P<0.01).Using the scratch method and the Transwell methods showed that the unilateral cell migration ability of siSNU449 was significantly increased and the number of migration was significantly increased(P<0.01).Invasion experiments using the Matrigel-coated transwell chambers method showed that the number of cells invaded by siSNU449 cells was significantly increased(P<0.05).Western Blot results showed that after the expression of BRIT1 was down-regulated by more than 60% in siSNU449 cells,the SERPNA5 protein also showed a down-regulated trend in siSNU449 cells,and the difference was significant(P<0.05).By observing the weight of the constructed NOD/SCID mice HCC models by intravenous injection,it was found that 6 mice in Hep3 B group and 2 mice in Hep3 B.BRIT1 group lost their weight during the experimental period.When dissected each group of mice models,it was found that 4 mice in Hep3 B and 2 mice in Hep3 B.BRIT1 groups had abnormal visceral abnormalities such as gastrointestinal tract.After analyzing the organ coefficient,the hepatic coefficient of Hep3 B group and Hep3 B.BRIT1 group was lower than that of PBS control group(P<0.01),and the lung organ coefficient of Hep3 B group was slightly higher than that of PBS control group(P<0.05).The hepatic and lung organ coefficients in the Hep3 B.BRIT1 group were closer to normal than those in the Hep3 B group.Pathological observation and analysis showed that 70% of the Hep3 B group and 30% of the Hep3 B.BRIT1 group had lung pathological changes.Conclusions1.BRIT1 gene has a certain correlation with the proliferation,migration and invasion of HCC cells,and BRIT1 plays an inhibitory role when it is over-expressed,and BRIT1 gene plays a promoting role after being silenced.2.BRIT1 gene may also play a role in regulating the metastasis ability of HCC cells by regulating the expression of SERPINA5.The up-regulation of BRIT1 expression can increase the expression of SERPINA5.The down-regulation of BRIT1 expression can also cause the down-regulation of SERPINA5 expression.3.After injecting HCC cells into NOD/SCID mice by intravenous injection,it was observed that overexpression of BRIT1 gene would reduce the changes of pathological tissues in NOD/SCID mice.