Role And Mechanism Of PLG In The Resistance To Anoikis In Hepatocellular Carcinoma

Objective: To investigate whether Plasminogen(PLG)can affect the ability of proliferation,invasion and migration of hepatocellular carcinoma cells by inhibiting the anoikis phenomenon and enhancing the anoikis resistance,and to explore whether mitochondrial energy metabolism and oxidative phosphorylation pathways are the relevant mechanisms of PLG exerting the above effects.Methods:(1)Online bioinformatics analysis was used to predict the expression of PLG in liver cancer patients and its prognostic value;(2)The expression level of PLG in liver cancer tissues and adjacent tissues was detected by Western Blot(WB)and immunohistochemical staining,and the relationship between PLG expression level and clinicopathological characteristics of patients with HCC was analyzed by univariate chi-square analysis,and the factors affecting the prognosis of patients with HCC were also analyzed by univariate Cox regression method,as well as the prognostic value of PLG expression level in patients with HCC;(3)Flow cytometry was used to detect and analyze the apoptosis of HCC cells in adherent and suspended states;(4)Western Blot was used to detect the expression abundance of PLG in normal liver cells L-02 and other liver cancer cell lines such as Huh7,HCCLM3,MHCC97-H,SMMC-7721 and Hep3 B.Then,SMMC-7721 and HCCLM3 with high PLG expression were suspended for 24 h,and the changes of PLG expression were detected under the attachment and suspension conditions;(5)The expression of PLG in SMMC-7721 and HCCLM3 was endogenous down-regulated by specific PLG sh RNA lentivirus,then the changes and effects of apoptosis in SMMC-7721 and HCCLM3 in adherent and suspended state after down-regulated PLG were detected by flow cytometry.Finally,the ability of SMMC-7721 and HCCLM3 cells to proliferate,invade,and migrate was examined using plate cloning and proliferation formation assay,Transwell and Scratch assay;(6)Subcutaneous tumor formation was conducted in NOD/SCID mice,and the effect of PLG on down-regulation of tumor progression was verified by comparing tumor volume and weight changes;(7)WB technique was used to verify whether PLG inhibited anoikis in HCC cells and enhanced anoikis resistance by regulating pyruvate dehydrogenase kinases(PDKs).Results:(1)Online bioinformatics prediction results indicated that in poorly differentiated HCC,HBs Ag(+)HCC,and HCC without vascular invasion,the survival prognosis of HCC patients with high PLG expression was worse,and the OS and PFS were shorter than those with low PLG expression;(2)The IHC and WB results of liver cancer tissue samples showed that the expression of PLG in liver tissues was higher than that in adjacent tissues,and the activated PLG was mainly localized in the nucleus.Patients with high PLG expression had poor prognosis.AFP,tumor size and PLG expression were independent risk factors for poor prognosis in patients with HCC;(3)WB confirmed that PLG was highly expressed in liver cancer tissues and liver cancer cells(SMMC-7721 and HCCLM3),and the expression level of PLG after suspension for 24 h was higher than that under attached state;(4)There was no significant difference in the apoptosis rate of SMMC-7721,HCCLM3 and L-02 in attached state,but the apoptosis rate of SMMC-7721 and HCCLM3 was lower than that of normal liver cells L-02 after suspension for 24h;(5)Compared with L-02,PLG protein expression in SMMC-7721 and HCCLM3 was the highest,and after suspension for 24 h,PLG expression in SMMC-7721 and HCCLM3 was increased,but there was no significant change in L-02;(6)Compared with sh-Ctrl group,apoptosis rate of sh-PLG expression in SMMC-7721 and HCCLM3 cells showed no significant change in attached state,but in suspension state,the apoptosis rate of SMMC-7721 and HCCLM3 was higher than that of sh-Ctrl group after down-regulation of PLG expression,the proliferation,invasion and migration of SMMC-7721 and HCCLM3 were decreased;(7)Compared with sh-Ctrl group,the size and weight of subcutaneous neoplasms in sh-PLG group were reduced;(8)PLG may enhance anoikis resistance of HCC cells by regulating the PDK1/2/4 pathway.Conclusion:(1)PLG was highly expressed in HCC tissues and liver cancer cells and inhibit anoikis phenomenon in liver cancer cells,PDK1/2/4 may be one of the mechanisms that regulate PLG to inhibit anoikis or enhance anoikis resistance in liver cancer cells.