Effect Of BSP Gene Editing Using CRISPR-Cas9 On Biological Characteristics Of Breast Cancer Cells

CRISPR-Cas9 system,which is comprised of a nonspecific Cas9 nuclease and a cr RNA that identifies DNA sequences is the adaptable immune mechanism used by many prokaryotes to protect themselves from foreign nucleic acids,such as viruses or plasmids.CRISPR-Cas9 was already applied to gene editing in many species since it was first used in DNA cleavage in vitro in 2012.As a new gene editing technology,the CRISPR-Cas9 has the advantage of fast,simpleness,high efficiency and can be applied to nearly all species.CRISPR now has already successfully applied in gene editing in many species,building g RNA library,building animal disease models,especially in the research field of gene related diseases and molecular mechanism of cancers.Bone sialoprotein(BSP)is a highly phosphorylation and glycosylation secreted protein,which contributes to breast cancer cell adhesion,proliferation,migration,invasion and host immune defense,angiogenesis,which all related to breast cancer cells metastasis.Since Bellahcene's studies first proved that the expression of BSP is related to the bone metastasis of human breast cancer,there are plenty of studies on it.Many methods were applied to inhibiting the expression of BSP,such as antisense,antibody and RNAi technology.But all these strategies were based on the level of post-transcriptional or post-translational processing.This study is mainly to deplete BSP levels in the human bone-seeking breast cancer cell line MDA-MB-231BO(231BO)using CRISPR-Cas9,and explore the biological characteristic changes by cell proliferation,wound healing and colony formation assays for further understanding the molecular mechanism of BSP in bone metastasis of breast cancer cells.The main findings of this research were stated as follows:1.The stable Cas9-expressing clones of bone-seeking breast cancer cell(231BO-Cas9)were established successfully.The Cas9 protein was detected in 231BO-Cas9 cells by flow cytometry,western blot,and immunocytochemistry,which can be located on cell nucleus through the nuclear localization signal(NLS)'s guidance.2.The proliferation and migration of 231BO-Cas9 cells were not changed in real time cellular analysis and wound-healing.The structures of cell membrane,organelles and nucleus in 231BO-Cas9 and 231 BO cells were consistent under the transmission electron microscope,which imply that Cas9 expression has no effect on the ultrastructure of breast cancer cells.3.The surveyor assay and DNA sequencing showed that BSP-g RNA can guide Cas9 to cleave the BSP genes by the way of insertion-deletion(indel)mutation,which demonstrate that Cas9 can be located on cell nucleus and has the endonuclease activity in 231BO-Cas9 cells.4.Two BSP-silenced breast cancer cell lines(231BO-Cas9-g RNA)were established by CRISPR-Cas9,which showed 34.5% and 36.7% decrease in BSP,respectively,when compared with the control cells.CCK-8 assay showed that the cell proliferation was decreased in 231BO-Cas9-g RNA cells.Wound-healing showed a decrease of 28% and 32.9%,respectively,in 231BO-Cas9-g RNA cells.Colony formation showed a 32.3% or a 37.2% decrease in two 231BO-Cas9-g RNA cell lines respectively.These date suggest that CRISPR-Cas9 mediated BSP gene editing could decrease the levels of BSP,in turn inhibiting the proliferation,migration and colony formation of bone-seeking breast cancer cells.