| Objective Liver cancer is one of the six most common malignancies in the world and is the third leading cause of cancer-related death.China is the country with the largest number of liver cancer patients in the world,which has brought a heavy burden on people’s health and social economy.Liver cancer has an insidious onset,and is prone to recurrence and metastasis,with limited treatment options and poor prognosis.Therefore,searching for biomarkers related to the prognosis of liver cancer is of great significance for improving the prognosis of liver cancer patients.Methods 1.The expression level and function of GOT2 in liver cancer cells:(1)The LIRI-JP data set was downloaded from the ICGC database to further verify the expression level of GOT2 in liver cancer and its relationship with prognosis.(2)The m RNA expression level of GOT2 in MHCC97 L,MHCC97H,HCCLM3,Huh-7,Hep3 B and Hep G2 were detected by real-time quantitative PCR.(3)The Hep G2 cell line was transfected with lentiviral vectors that knocked down or overexpressed of GOT2,and cell line with stable knockdown or overexpression of GOT2 were constructed by purinomycin screening.The transfection efficiency was detected by real-time quantitative PCR and western blot(WB)assay.(4)The effect of GOT2 expression level on proliferation activity of Hep G2 cells in vitro was detected by CCK-8 cell proliferation assay and plate clone formation assay.(5)The effect of GOT2 expression level on the migration and invasion ability of Hep G2 cells was detected in vitro by Transwell cell migration and invasion assay.2.The effect of GOT2 expression level on autophagy and its potential mechanism of liver cancer:(1)The protein expression levels of LC3 and P62 were detected by WB to evaluate the effect of GOT2 expression level on autophagy.(2)The autophagy inhibitor 3-methyladenine(3-MA,5m M)was used to interfere with transfected cells for 12 h to inhibit autophagy.The CCK-8cell proliferation experiment was used to detect the cell proliferation activity before and after intervention to explore the effect of autophagy on cell proliferation activity.(3)The protein expression levels of mTOR and p-mTOR were detected by WB to evaluate the effect of GOT2 expression level on mTOR pathway activity.(4)The mTOR pathway inhibitor rapamycin(100n M)was used to intervene the transfected cells for 12 h to inhibit the activity of mTOR pathway.Then,the protein levels of LC3 and P62 were detected by Western blotting to investigate the effect of mTOR pathway activity on autophagy.Results 1.The expression level and function of GOT2 in liver cancer cells:(1)The expression of GOT2 was downregulated in liver cancer tissues,and its low expression was associated with poor overall survival in liver cancer patients.(2)The relative expression level of GOT2 was low in MHCC97 L,MHCC97H,HCCLM3 cell lines with strong metastasis ability,while the relative expression level of GOT2 was high in Hu H-7,Hep3 B,Hep G2 cell lines with weak metastasis ability.The relative expression level of GOT2 was the highest in Hep G2 cell line.(3)The transfection results showed that compared with the sh-NC group,the m RNA expression level of GOT2 in the sh-GOT2 group decreased by 69%(t=22.600,P<0.001),the protein expression level decreased by 55%(t=14.410,P<0.001);Compared with the OE-NC group,the m RNA expression level of GOT2 in the OE-GOT2 group increased by about 2 times(t=7.757,P<0.001),and the protein expression increased by 1.4 times(t=2.880,P<0.05),indicating that the cell line with stable knockdown and overexpression of GOT2 was successfully constructed.(4)The results of CCK-8 cell proliferation experiments showed that the OD value of liver cancer cells in the sh-GOT2 group was significantly higher than that of the sh-NC group after 2days of culture,the OD value of liver cancer cells in the OE-GOT2 group was significantly lower than that of the OE-NC group after 4 days of culture,the difference was statistically significant(P<0.05).Moreover,the results of plate clone formation experiment showed that the number of cell clones in the sh-GOT2 group was more than that in the sh-NC group,the difference was statistically significant(t=8.819,P<0.001);the number of cell clones in the OE-GOT2 group was less than that in the OE-NC group,the difference was statistically significant(t=18.64,P<0.001).(5)The results of the Transwell migration experiment showed that the number of migrating cells in the sh-GOT2 group was significantly more than that in the sh-NC group,the difference was statistically significant(t=8.193,P<0.001);while the OE-GOT2 group induced fewer migrating cells than the migration cells induced by the OE-NC group,the difference was statistically significant(t=5.011,P=0.007).(6)The results of the transwell invasion experiment showed that the sh-GOT2 group induced more invasive cells than the sh-NC group,the difference was statistically significant(t=11.510,P<0.001);while the OE-GOT2 group induced fewer invasive cells than the OE-NC group,there was a statistically significant difference(t=15.24,P<0.001).2.The effect of GOT2 expression level on autophagy in liver cancer cells:(1)The results of western blot experiment showed that compared with the control group,the value of LC3-II/LC3 I was increased significantly(t=10.430,P<0.001),while the protein expression level of P62 was decreased(t=5.022,P<0.01)after the expression level of GOT2 was knocked down.On the contrary,after overexpression of GOT2,the value of LC3-II/LC3 I was decreased(t=6.591,P<0.01),and the protein expression level of P62 was increased(t=9.547,P<0.001).(2)After adding the autophagy inhibitor 3-MA,the value of LC3-II/LC3-I was decreased in the sh-NC group and the sh-GOT2 group,and the protein expression level of P62 was increased,the difference was statistically significant(P<0.05).The results of CCK-8 cell proliferation experiments showed that after adding 3-MA,the OD value of liver cancer cells in the sh-GOT2 group was decreased significantly(P<0.05).However,there was no significant difference in the OD value of liver cancer cells in the sh-NC group(P>0.05).(3)The results of western blot experiment showed that compared with the sh-NC group,the value of p-mTOR/mTOR in the sh-GOT2 group was significantly reduced(t=11.570,P<0.001).After adding rapamycin,the value of p-mTOR/mTOR in the sh-NC group was decreased significantly(t=5.167,P<0.01),but there was no significant difference in the expression levels of LC3-II/LC3-I and P62 protein(P>0.05).Moreover,the value of p-mTOR/mTOR in the sh-GOT2 group was significantly decreased after adding rapamycin(t=5.274,P<0.01),at the same time,the value of LC3-II/LC3-I was significantly increased(t=2.793,P<0.05),and the protein expression level of P62 was decreased significantly(t=3.219,P<0.05).Conclusions 1.The expression of GOT2 was down-regulated in liver cancer tissues,and its low expression was associated with poor overall survival of patients.2.GOT2 inhibits the proliferation,migration and invasion of liver cancer cells in vitro,which may be a potential tumor suppressor factor for liver cancer.3.Knockdown of GOT2 induces autophagy in liver cancer cells to promote cancer cell proliferation.4.Knockdown of GOT2 may induce abnormal autophagy in liver cancer cells by inhibiting the activity of mTOR pathway. |
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