Study On Expression,Function And Molecular Mechanisms Of FAM96B In Hepatocellular Carcinoma

Part Ⅰ FAM96 B expression in hepatocellular carcinoma and its clinical significance:an analysis based on data miningObjective To investigate the expression of family with sequence similarity 96 member B(FAM96B)in hepatocellular carcinoma(HCC)and its association with clinicopathological features and prognosis based on data mining.And to predict the interactions protein network and the possible signal pathways of FAM96 B in HCC.Methods The Oncomine database was used to analyze the expression difference of FAM96 B in HCC tissues and normal liver tissues.HCC dataset was collected from the Cancer Genome Atlas(TCGA)database.The correlation between expression level of FAM96 B and clinicopathological features of HCC was analyzed by SPSS21.0.The STRING database was used to analyze the network of proteins interacting with FAM96 B.Gene set enrichment analysis(GSEA)was used to predict the possible signal pathways of FAM96 B in HCC.Results The Oncomine database results showed that FAM96 B expression in HCC tissue was lower than in normal liver tissue.The Linked Omics database analysis results showed that FAM96 B expression was associated with years to birth,pathology N stage.The mRNA expression level of FAM96 B gene and the clinicopathological characteristics of 361 patients with HCC were obtained from the TCGA database.According to SPSS21.0 statistical analysis,it can be concluded that FAM96 B expression was associated with gender and AFP.STRING database showed that there was an interaction between FAM96 B and CIAO1(score=0.999),MMS19(score=0.999),NARFL(score=0.986)and so on.GSEA research results show that the FAM96 B mRNA high expression samples were enriched into P53 signaling pathway,apoptosis,cell cycle and so on.Moreover,the mRNA expression level was positively correlated with the gene expression in the above pathway,that is,when the expression of FAM96 B gene was up-regulated,the above pathway was activated.Conclusions The analysis results by multi database showed that FAM96 B was low expression in HCC tissues.Moreover,it played an important role in HCC by regulating interacting proteins such as CIAO1 and activating P53 signaling pathways.Part Ⅱ Expression,clinical significance and cell function of FAM96 B in HCCObjective FAM96 B has been found to be aberrantly expressed in some tumors,but the expression level of FAM96 B in HCC and its relationship with tumor progression remain unclear,and the possible molecular signaling pathways involved in FAM96 B and its possible upstream regulators need to be further explored.Therefore,the aim of this study is to investigate the expression levels of FAM96 B in HCC cells and clinical tissues and its effect on cell proliferation and invasion.The affection of FAM96 B on tumor progression in vivo conditions is also included in this study.Finally,we further analyze the expression of FAM96 B in HCC and the relationship between its expression and clinicopathological features and the relationship with the prognosis of HCC patients.Methods The mRNA and protein expression levels of FAM96 B in normal liver cell line L02 and other HCC cell lines,HCC and their paired adjacent noncancerous tissues were mainly determined by qRT-PCR and Western blot(WB).Immunohistochemical experiment(IHC)was used to detect the expression and distribution of FAM96 B in HCC and their paired adjacent noncancerous tissues.The correlation between clinical HCC pathological parameters and FAM96 B was further analyzed by T test,the risk factors affecting the prognosis of HCC patients were analyzed by KM survival analysis,and the independent risk factors affecting the prognosis of HCC patients were further analyzed by multivariate survival analysis(forward LR method).Overexpression and interference of FAM96 B were achieved by Lipofectamine3000 transfection plasmid or sh RNA method,then Transwell assay was used to explore the cell invasion ability,CCK-8 and Ed U assay were used to detect the proliferation level of the cells,respectively.HCC cell lines stably overexpressing FAM96 B were constructed by lentiviral transfection,subcutaneous tumorigenesis and hepatocarcinoma orthotopic tumorigenesis models were constructed to study the effect of FAM96 B on tumor progression under in vivo conditions.Results FAM96 B was highly expressed in the liver normal cell L02 and lowly expressed in the HCC cell lines.FAM96 B expression was detected in paired and adjacent tissues,confirming low expression in HCC tissues.FAM96 B was mainly expressed in the cell membrane and plasma in HCC tissues.Low expression of FAM96 B was significantly correlated with tumor diameter,tumor thrombus,AFP and tumor differentiation of HCC,and survival analysis found that tumor diameter,tumor thrombus,AFP,differentiation and abnormal expression of FAM96 B were all risk factors affecting the prognosis of HCC,while multivariate analysis identified tumor diameter,tumor thrombus and low expression of FAM96 B as independent risk factors affecting the prognosis of HCC.Overexpression of FAM96 B can significantly inhibit the proliferation and invasion of HCC cells,while interference with FAM96 B expression can significantly enhance the proliferation and invasion of HCC cells.In vivo,the subcutaneous tumorigenic growth of nude mice was significantly inhibited after overexpression of FAM96 B,the tumorigenic size and weight were significantly lower than those of the NC group.After FAM96 B overexpression,the orthotopic tumorigenic ability of the liver was significantly inhibited in nude mice.Conclusions FAM96 B expression is low in HCC and is associated with its proliferation and invasion;low expression of FAM96 B is an independent risk factor affecting the prognosis of HCC patients.FAM96 B inhibited the invasion and proliferation of HCC.Part Ⅲ FAM96 B regulated by the circ＿CPSF6 / miR-665 / FAM96 B axis,affecting the downstream AKT signaling pathwayObjective To deeply explore the downstream signaling pathways and upstream molecular biological regulatory mechanisms that FAM96 B may be involved in.Methods Used bioinformatics to predict miRNA molecules that can regulate FAM96 B,we further verified the site binding of FAM96 B to miR-665 by dual luciferase reporter assay and finally validated the regulation of FAM96 B by miR-665 by qRT-PCR and Western Blot.Bioinformatic prediction of circular RNAs that regulate miR-665 was used to further investigate the expression of each predicted circular RNA in HCC using qRT-PCR.Dual luciferase reporter assays were used to validate the bindingsof circ＿CPSF6 to miR-665.Finally,the upstream and downstream correlation of them were further verified by Western Blot interference and functional recovery experiments.The expression changes of proteins involved in downstream signaling pathway after overexpression of FAM96 B were studied by Western Blot.To roughly analyze the possible signaling pathways that FAM96 B may be involved in.Results The bioinformatics prediction suggested that FAM96 B expression might be regulated by miR-665,dual luciferase reporter experiments confirmed the binding site between FAM96 B and miR-665.Both mRNA and protein levels of FAM96 B were decreased when miR-665 was overexpressed in HCC.While interfering miR-665,both FAM96 B mRNA and protein levels were increased.The bioinformatics prediction suggested that circ＿CPSF6may regulate miR-665,and dual luciferase reporter experiments confirmed the presence of adsorption sites of circ＿CPSF6 with miR-665.When circ＿CPSF6 was overexpressed,miR-665 expression was decreased.When circ＿CPSF6 was interfered,miR-665 expression was increased.When circ＿CPSF6 was disturbed,FAM96 B expression was attenuated,and when simultaneously disturbed with circ＿CPSF6 and miR-665 expression,reducuated FAM96B expression was deregulated.When circ＿CPSF6 was overexpressed,FAM96 B expression was enhanced,when simultaneously overexpressed with circ＿CPSF6 and miR-665 expression,enhanced FAM96 B expression was deregulated.There were negative correlations between circ＿CPSF6 expression and miR-665 expression,or between FAM96 B expression and miR-665 expression;but expression of circ＿CPSF6 and FAM96 B were positively correlated.When disturbed circ＿CPSF6,cell invasion and proliferation of HCC were all enhanced.However,when simultaneously disturbed with circ＿CPSF6 and miR-665 expression,invasion and proliferation of HCC were relieved.Overexpression of FAM96 B,phosphorylated AKT was significantly inhibited,and cyclin 1 expression was diminished.Conclusions FAM96 B is regulated by the circ＿CPSF6 / miR-665 / FAM96 B axis in HCC,and its overexpression may further inhibit the downstream AKT signaling pathway.