Expression Of GSPT2 In Hepatocellular Carcinoma Patients And Its Molecular Epidemiology Study

Part 1 Expression and prognosis of GSPT2 in hepatocellular carcinomaObjective:To study the expression of GSPT2 gene in hepatocellular carcinoma?HCC?and its correlation with clinicopathological features and prognosis.Methods:Immunohistochemical?IHC?staining was performed to detected the expression of GSPT2 gene in 56 pairs of liver cancer tissues and corresponding adjacent tissues,and results was semi-quantitatively analyzed with H-score.Expression levels of mRNA and protein of GSPT2 gene in 3pairs of hepatocellular carcinoma and adjacent tissues were detected by qRT-PCR and Western blot.Chi-square test was utilized to analyze the relationship between GSPT2 gene expression and clinicopathological features.OS and DFS of different GSPT2 expression levels was compared using Kaplan-Meier and Log-rank test.The Cox proportional hazards model was performed to estimate HR with 95%C between different clinical factors and mortality from HCC.The TCGA database was mined to analyze the difference of GSPT2 gene expression in hepatocellular carcinoma patients and normal people and its relationship with prognosis.Results:1 Immunohistochemistry results showed that the median GSPT2 gene expression of liver cancer patients?11.36?was lower than the normal median?24.51??P=0.018?.According to the median expression?11.36?,the liver cancer patients can be divided into low expression group and high expression group,statistical analysis showed that the median of low expression group?4.47?was obviously lower than adjacent tissues?24.51??P<0.001?,the median of high expression group?25.14?had no statistical difference with adjacent tissues?P=0.179?.2 The relationship between the GSPT2 gene expression levels and clinical pathological characteristics analysis results showed that no significant correlation was found between GSPT2 expression level with patients'gender,age,tumor diameter,cirrhosis of the liver,presence of transfer,TNM staging and preoperative AFP levels,HBsAg infection,but related to the number of tumors?P=0.044?.3 Results of human liver cancer and adjacent tissues showed that the expression level of GSPT2 mRNA had a significant difference?P=0.041?between liver cancer tissues?1.00±0.34?and adjacent tissues?5.16±2.27?.Western blot results showed that the relative expression levels of GSPT2protein had a significant difference?P=0.009?between liver cancer?0.85±0.03?and adjacent tissues?1.00±0.04?.4 Survival analysis of liver cancer patients in the low expression group and the high expression group showed that the median OS had a significant difference(?²⁼4.021,P=0.045)between the low expression group?652.00?and high expression group?597.00?,and the median DFS also had a significant difference(?²⁼4.539,P=0.033)between the low expression group?434.00?and high expression group?327.00?.5 Cox regression model showed that cirrhosis?0.367?0.148-0.913??,TNM stage?4.268?1.376-13.234??and tumor number?2.389?1.091-5.231??wereindependentprognosticfactorsofDFS.TNMstaging?4.136?1.335-12.816??was an independent prognostic factor for OS.6 Bioinformatics analysis results showed that GSPT2 mRNA expression in asian patients with hepatocellular carcinoma?n=155?was significantly lower than that in normal people?n=10??Z=-2.961,P=0.003?.According to the the median of GSPT2 mRNA expression,patients with liver cancer was divided into high expression and low expression group,there had a significant difference?P<0.001?between low expression group and normal group,and no statistically significant difference between the normal group and the high expression group?P=0.545?.Kaplan-meier survival analysis was performed on the expression level of liver cancer patients,and the results showed that high expression of GSPT2 mRNA was correlated with poor prognosis of liver cancer?P=0.002?Conclusions:1 GSPT2 gene was expressed in the cytoplasm of cells,and the expression level of liver cancer patients was lower than that of normal people.2 The expression of GSPT2 gene in patients with liver cancer was not related to the clinicopathological characteristics such as age,gender,cirrhosis and metastasis,but only related to the number of tumors.3 High expression of GSPT2 gene was associated with poor prognosis of liver cancer.Part 2 Effect of stable overexpression of GSPT2 gene on the biological function of HepG2 liver cancer cellsObjective:Construct HepG2 liver cancer cells with stable overexpression of GSPT2 gene to investigate the influence on the biological function of HepG2 liver cancer cells.Methods:The HepG2 liver cancer cells and 7402 liver cancer cells was transfected with overexpression plasmids.The expression of GSPT2 mRNA in liver cancer cells were detected by qRT-PCR.Flow cytometry was used to detect cell cycle changes and scratch assay test was used to detect the ability of cell migration.Lentiviral stable transfection was performed on HepG2 liver cancer cells to construct HepG2 liver cancer cells with stable overexpression of GSPT2 gene.The expression of GSPT2 gene mRNA and protein were detected by qRT-PCR and Western blot.CCK8 was used to detect cell viability.Flow cytometry was used to detect cell cycle and cell apoptosis.Scratch assay was used to detect the ability of cell migration.Transwell was used to detect the cell invasion ability.Results:1 qRT-PCR results of cells collected after transient transfection for 48h showed that GSPT2 gene was highly expressed in 7402 liver cancer cells and HepG2 liver cancer cells compared with the black control group?P<0.001?.Cell cycle detection by flow cytometry showed that there was no difference in the proportion of overexpression of GSPT2 in 7402 HCC cells in G1 phase compared with the control group,and there had a significant difference in the proportion in S phase?P=0.004?.Compared with the control group,the overexpression of GSPT2 in HepG2 liver cancer cells decreased from 57.99%to 54.56%in G1 phase?P=0.006?,and increased from 28.28%to 33.59%in S phase?P=0.001?.Cell scratch results showed that the cell scratch migration rate?47.67±8.26?after transient transfection of GSPT2 overexpressed plasmid was higher than that of the control group?28.8±7.44??P=0.042?.2 The expression of GSPT2 mRNA and protein in the lentivirus infected group were higher than the blank control group.The human HepG2 liver cancer cells line stably overexpression GSPT2 was successfully constructed?P<0.05?.3 The results of CCK8 experiment showed that the absorbance value of the overexpression group was higher than that of the control group after24hours?P<0.05?,indicating that the cell viability of the overexpression group was increased and the cell proliferation ability was enhanced compared with the control group?P=0.028?.4 Cell cycle was detected by flow cytometry and results showed that the GSPT2 gene overexpression group decreased from 51.52%to 39.53%compared with the control group in G1 phase,and the S phase increased from27.03%to 39.71%compared with the control group?P<0.05?.5 Cell apoptosis was detected by flow cytometry,and results showed the apoptosis rate?1.00±0.12?in the overexpression GSPT2 group was significantly lower than that in the control group?6.00±1.23?.6 Transwell invasion assay was used to detect the invasion ability of cells in each group.The results showed that the number of cells invaded into the lower compartment after the overexpression of GSPT2 was 428.75±24.53,while the number of cells in the control group was 296.75±12.69.The number of cell metastasis was increased and the cell invasion ability was enhanced in the overexpression group?P<0.001?.7 Scratch test was conducted to compare the influence of GSPT2 gene overexpression on the migration ability of HepG2 cells.After 48 hours,results showed that the cell scratch migration rate?25.00±6.00?after GSPT2overexpression was higher than that of the control group?9.70±6.87??P<0.001?.Conclusions:1 Transient overexpression of GSPT2 gene can promote the cell cycle and migration ability of HepG2 hepatocellular carcinoma.2 Stable transfection of GSPT2 overexpressed lentivirus can successfully construct GSPT2 overexpressed HepG2 liver cancer cells.3 Overexpression of GSPT2 in HepG2 liver cancer cells can promote cell proliferation and cycle,increase the migration and invasion ability of liver cancer cells,and inhibit apoptosis.