Mechanism Of PFKFB3 Promotes Proliferation And Invasion In Hepatocellular Carcinoma And Its Effects On Lenvatinib Drug Resistance

Part 1 High Expression of PFKFB3 Could Lead to Poor Survival in Hepatocellular CarcinomaBackground:Hepatocellular carcinoma(HCC)has been a worldwide public health problem for several decades.It is difficult to identify the disease in the early stage,and most of the existing treatment methods are unsatisfactory.PFKFB3 is up-regulated in some cancers,but its role in HCC is still not clear.Thus,we designed this research to explore PFKFB3 expression and its role in the prognosis of HCC patients.Methods:We used some bioinformatics tools to extract and analyze PFKFB3 mRNA data from public databases.We also assessed the expression of PFKFB3 though the immunohistochemistry of HCC tissues and peritumoral liver tissues from 89 HCC patients.Results:The level of PFKFB3 mRNA was lower in HCC than in other cancers.Female patients had higher PFKFB3 mRNA levels than male patients(P=0.03).There were no significant differences in the level of PFKFB3 mRNA with regards to age,weight,tumor grade,and nodal metastasis status.Immunohistochemistry results showed that higher serum AFP levels(P=0.004)and larger sizes of tumors(P=0.029)were associated with high PFKFB3 expression.The up-regulation of PFKFB3 at both the mRNA and protein expression levels led to worse OS and DFS(P<0.05).Conclusion:Our data suggest that PFKFB3 expression in HCC is an independent prognostic factor of OS and DFS.Part 2 Mechanism of PFKFB3 promotes proliferation and invasion in hepatocellular carcinomaBackground:The function of PFKFB3 in HCC is still not clear to us.In order to find the answer,we design the study to explore its impacts on HCC proliferation,invasion and migration.Methods:Firstly,the molecules and pathways related to PFKFB3 expression were screened by biological techniques.In vitro,the expression of PFKFB3 was regulated by plasmid transfection and the phenotypic changes of proliferation of HCC cells were detected by Edu staining.The invasion ability of HCC cells was detected by transwell invasion assay.The migration ability of HCC cells was detected by cell migration assay.Using lentivirus transfection technique,a HCC cell line with up-regulated expression of PFKFB3 was constructed and animal experiments were carried out to study the effects of PFKFB3 on the proliferation and metastasis of HCC in vivo.The relationships between the expression of PFKFB3 and the MMPs were detected by RT-PCR,Western Blot and IHC staining.Results:Bioinformatics data showed that the expression level of PFKFB3 was positively correlated with several tumor-promoting genes and closely related to the process of ECM.In clinical patients,the expression level of PFKFB3 was positively correlated with many molecules of MMP family.In vitro,upregulating the expression of PFKFB3 can enhance the proliferation,invasion and migration of HCC cells,while interfering with the expression of PFKFB3 will inhibit the proliferation,invasion and migration of HCC cells.In vivo,when the expression of PFKFB3 was increased,the proliferation and metastatic ability of tumor were significantly enhanced.The expression level of PFKFB3 can cause positive correlation changes of multiple expression levels in MMP family at both mRNA and protein levels.Conclusion:PFKFB3 plays a role in promoting tumor in HCC.The increased expression of PFKFB3 can enhance the ability of proliferation,invasion and migration of HCC.Multiple star molecules of MMP family are downstream targets of PFKFB3.PFKFB3 may regulate tumor malignant phenotype by regulating the expression of MMP family members.Part 3 The function and mechanism of PFKFB3 in Lenvatinib resistant HCC cellsBackground:The main purpose of this part is to explore the effects and mechanism of PFKFB3 on the biological behavior of HCC in vitro cellular and in vivo.In clinical practice,targeted drug resistance is the most important reason that limits the efficacy of targeted drugs.We found that the up-regulated expression of PFKFB3 may be the cause of drug resistance to lenvatinib,so we designed experiments to detect the relationship between the expression level of PFKFB3 and drug resistance in HCC.Methods:The lenvatinib resistant HCC cells were constructed by small gradient concentration increasing method.The drug resistance gene PFKFB3 was picked out by sequencing.Colony formation test,transwell invasion test and scratch test were used to detect the proliferation,invasion and migration of drug-resistant HCC cells.CCK8 assay was used to detect the change of sensitivity of HCC cells to lenvatinib after changing the expression level of PFKFB3.RT-PCR and Western Blot were used to detect the expression of PFKFB3 and its downstream MMPs in drug-resistant cells.Flow cytometry was used to detect the apoptosis level of lenvatinib HCC cells under different administration conditions,and RT-PCR and Western Blot,IHC were used to detect the expression of PFKFB3 and MMPs after different drug treatments.The experiment of subcutaneously transplanted tumor was conducted to study the inhibitory level of different treatment schemes on HCC cells resistant to lenvatinib under in vivo conditions.Results:PFKFB3 and many members of its downstream MMP family are up-regulated in lenvatinib drug-resistant HCC cells,and up-regulate the ability of proliferation,invasion and migration of drug-resistant cells.The increased expression of PFKFB3 can reduce the sensitivity of HCC to lenvatinib.Combined use of PFKFB3 inhibitors can inhibit the expression of PFKFB3 and downstream MMP family members at RNA and protein levels in vivo and in vitro,and inhibit the proliferation of lenvatinib HCC cells.Conclusion:The upregulation of PFKFB3 expression can cause HCC cells to be resistant to lenvatinib.Combined use of PFKFB3 inhibitors can inhibit the expression of PFKFB3 and MMPs and reverse the drug resistance of HCC cells to lenvatinib.