FAM83D Associates With High Stemness And Metastasis Involving Up-Regulated CD44 Expression In Hepatocellular Carcinoma

Background:Hepatocellular carcinoma(HCC)is one of the most common malignancies with high mortality,and a serious public health problem in china.Benefiting from the great advance in the field of screening,diagnosis,surgery for HCC,the survival time and life quality of HCC patients have been significantly improved,but the high rate of HCC recurrence and metastasis after surgery or liver transplantation(LT)still limit the overall survival rate.How to perform more effectively prevention and therapy for HCC recurrence has become the key problem to further improve the prognosis of HCC patients.Genomic mutation and recombination drives HCC cells to acquire heterogeneous fetures and other malignant characteristics such as sternness and highly resistance to chemotherapy,which play a crucial role in promoting HCC cells survival.Deeply comprehensive interpretation of genomic mutation,abnormal structure,chromosome remodeling and copy number variation could provide important clues for revealing the underlying mechanism of HCC recurrence and metastasis and indenditifing accurate molecular markers for early diagnosis and therapy.Objectives:The study was designed to find the key oncogenes closely related to the initiation and development of HCC using the high throughput RNA sequencing technology.The other 150 pairs of HCC and adjacent non-HCC tissues were used to reveal the pattern and clinical significance of the candidate oncogene-FAM83D expression.The role of FAM83D in maintaining HCC cells sternness is also investigated.Furthermore,the potential molecular mechanism of FAM83D regulating HCC developmeng is elucidated by gene chip assay and protein mass spectrometry.Material and Methods:1.The differentially expressed genes between HCC and corresponding adjacent tissues were screened by high-throughput RNA sequencing and bioinformatics analysis.2.A total of 150 pairs of HCC tissues,matched paracancerous tissues and clinical pathological information was collected.Expression pattern of FAM83D in HCC was analyzed using immunohistochemistry and real-time PCR.The HCC patients were divieded into two groups according to FAM83D expression level,and the correlation between FAM83D and clinicopathological parameters,survival prognosis was analyzed.3.FAM83D-siRNA lentivirus was transfected into HCC cell lines to knockdown FAM83D expression in vitro.The spheoid formation assay,real-time PCR,Western blotting,flow cytometry analysis and tumor xenograt experiment were performed to detect the effect of FAM83D on sternness,cancer stem cells(CSCs)marker expression,the expansion of CSCs,tumorigenesis and metastasis in HCC.4.Transcriptome microarray experiment was performed to screen the gene with different expresson following FAM83D knockdown.GO and KEGG analysis was used to reveal the molecular mechanisms underlying FAM83D regulating sternness of HCC cells.5.The small molecule inhibitors of signaling pathway and CD44 knockdown assay were used to reveal the relationship between CD44 and MAPK,TGF-β and Hippo signaling pathway.CO-IP experiment and mass spectrometric analysis were performed to found the key upstream regulator of FAM83D regulating MAPK,TGF-β and Hippo signaling pathway.Results:1.We profiled mRNA expression in liver samples from 10 HCC and corresponding non-HCC tissues(no pre-LT therapy)with RNA sequence and found that 321 up-regulated genes and 223 down-regulated genes(>2 folds,P<0.01).Among the 544 genes,FAM83D was increased in 9/10 HCC tissues compared with adjacent liver tissues.2.Additionally,we expansively detected the FAM83D expression in 150 pair specimens with real-time PCR and immunohistochemistry,and verified FAM83D was apparently increased in HCC tissues.Moreove,overexpression of FAM83D in HCC was signicantly correlated with poor tumor characteristics,i.e.poor differentiation,portal vein tumor thrombus,tumor number,and greatest tumor diameter.Furthermore,patients with higher FAM83D developed more frequent HCC recurrence and presented poorer tumor free survival outcomes after LT.3.The FAM83D knockdown significantly reduced the expression of CD44s and CD44v,and also inhibited the number and volume of tumor spheroid in HCC.In vivo,the HCC cell lines with decreased FAM83D expression presented inhibitory ability of tumorigenicity,metastasis and lung clone.4.Gene expression microarray assay revealed that SMMC-7721 cells transfected with siRNA-FAM83D resulted in a total of 1565 differentially expressed genes including 686 up-regulated genes and 879 down-regulated genes,mainly located in MAPK,TGF-β and Hippo signaling pathways.Following the FAM83D knockdown,the level of ERK1/2-p,Smad2-p and YAP expression was apparently inhibited,but the YAP-p level was increased.5.The suppression of MAPK,TGF-β and Hippo signaling pathways signicantly inhibited the CD44 expression.Moreover,CD44 knockdown signicantly reduced the level of ERK1/2-p,Smad2-p and YAP expression,but increased YAP phosphorylation.These results suggested that CD44 and above three signaling pathways presented mutual regulation.6.CO-IP assay and mass spectrometry analysis defined that RAP1 is the key upstream regulator of FAM83D regulating MAPK,TGF-β and Hippo signaling pathways.Conclusion:1.FAM83D expression is significantly higher in HCC tissues than that in adjacent non-HCC tissues,and correlate with the malignant tumor characteristics and poor prognosis.2.FAM83D knockdown could significantly inhibit the CD44 expression and the sternness,tumorigenesis and metastasis of HCC cell lines.FAM83D provides a novel therapeutic approach against HCC recurrence after LT.3.The MAPK,TGF-β and Hippo signaling pathway are the potential molecular mechanisms involved in FAM83D regulating CD44 expression and HCC development.