The Mechanisms Of Biomechanically Induced Tumor Repopulating Cells

The mechanical microenvironment varies with tumor growth and development,and impacts tumor progress.The mechanical properties of extracellular matrix can not only affect the malignant phenotype of tumor,but also promote the progress of tumor and affect the invasion and metastasis of tumor.Our previous research has developed a mechanical method to select tumor repopulating cells(TRCs)by culturing them in soft three-dimensional(3D)fibrin gels in vitro.These TRCs were selected as highly self-renewing tumorigenic subpopulation.TRCs have been reported in the screening of anticarcinogen and researchs in tumor immunotherapy.However,there is not a comprehensive study on the origin of TRCs.In this study,we used HeLa cells,the earliest human cancer-derived cell line,and sequencing the time series samples of TRCs with next generation sequencing and whole-genome DNA methylation sequencing to build a transcription factor/miRNAs/target gene regulation network.Combining the GO and KEGG pathway enrichment analysis of differentially expressed genes,we look forward the key transcription factors,miRNA and pathways in the selecting of TRCs,systematically investigating the possiblily mechanism of the generation of TRCs combined with experimental verification.Main research contents and results are as follows:(1)TRCs cultured in 90 Pa soft 3D fibrin gels perform stronger invasion and metastasis ability than those cultured in ordinary plate culture,and the expression levels of stemness genes such as SOX2,NANOG and OCT4 were significantly increased.(2)Through transcriptome and epigenetic analysis,the results of our study comprehensively discussed the mechanism of bioinduction of tumor regeneration cells,which provided a new target for the follow-up research of anticancer drugs.In this paper,we evaluated the transcriptome alteration of TRCs,and found out that a variety of molecules related with integrin/membrane and stemness were continuously altered by mechanical environment.Some key regulators such as MYC,STAT3 and hsa-miR-199a-5p,they regulated membrane genes and the downstream mechanotransduction pathways such as Hippo/WNT/TGF-?/PI3K-AKT pathways,thus further affecting the expression of downstream cancer-related genes.(3)By integrating networks for membrane proteins,WNT pathway and cancer-related genes,we identified key molecules in the selection of TRCs,such as ATF4,SLC3A2,CCT3 and hsa-miR-199a-5p.Silencing ATF4 or CCT3 inhibited the selection and growth of TRCs whereas reduction of SLC3A2 or hsa-miR-199a-5p promoted TRCs growth.(4)Further studies showed that CCT3 promoted cell proliferation and stemness in vitro,while its suppression inhibited TRCs-induced tumor formation.We also contemplated CCT3 as a stemness-related gene.(5)We performed difference methylation gene analysis during TRCs selection,and found out non-CG methylation level was higher in TRCs.Non-CG methylation disappeared in differentiated cells and had high level in induced pluripotent stem cells and human embryonic stem cells.We speculated this methylation pattern of non-CG methylation may be a feature of DNA methylation during TRCs screening.Our findings provide insights in the mechanism of TRCs selection through transcriptome analysis and epigenetic analysis,and may provide a research target for the development of anticarcinogen.