The Mechanism Of LSD1 Regulating MiRNA Sorting Into Exosomes To Remodel Pre-Metastatic Niche

Breast cancer has been considered as the highest incidence rate of female cancer globally,which seriously threatens women’s health.Although the treatment options for breast cancer have been continuously improved in recent years,the overall survival rate of patients with metastatic breast cancer is still low.Breast cancer tends to metastasize to different organs,including bone,lung,liver,and brain.Bone metastasis accounts for about 75% of the cases of distant metastasis of breast cancer.Furthermore,Luminal subtype of breast cancer is most prone to bone metastasis.It is important to explore the molecular mechanism of bone metastasis of Luminal breast cancer,which will lay the foundation for early diagnosis and treatment of breast cancer bone metastasis.Exosomes are extracellular vesicles secreted by almost all cells and play an essential role in intercellular communication.RNA,proteins,lipids and other substances in tumor cells can be carried into the recipient cells by exosomes to regulate the function of the recipient cells.Therefore,exosomes released by tumor cells are extensively involved in remodeling the tumor microenvironment and forming the premetastatic niche.As an essential epigenetic regulator,LSD1 plays a vital role in breast cancer.Knockdown and functional mutation of LSD1 promote invasion and metastasis of Luminal breast cancer cells.However,the mechanism of how LSD1 regulates bone metastasis of breast cancer cells remains unclear.Bone metastasis of a tumor begins with the remodeling of pre-metastatic niche in the bone,so we explore the molecular mechanism by which LSD1 regulates the bone metastasis of breast cancer by remodeling the pre-metastatic niche.To investigate the role of LSD1-regulated exosomes in bone metastasis of breast cancer cells,breast cancer cells were transiently transfected with Control si RNA or LSD1 rescue construct,separately.Western blot assays confirmed the expression of exosomal markers CD63 and TSG101.Furthermore,transmission electron microscopy and particle size analysis showed that the morphology and size of exosomes met the standard.Subsequently,the enriched exosomes were stained with PKH67 and coincubated with target cells(human bone marrow mesenchymal stem cell h BMSC and mononuclear macrophage RAW264.7).The results showed that the exosomes of breast cancer cells regulated by LSD1 could enter the target cells.It is proved that the exosomes that have been successfully isolated and identified can be taken up by target cells.Subsequently,we conducted animal experiments to explore the effects of LSD1-regulated exosomes of breast cancer cells in the pre-metastatic niche.The Control,LSD1 KD,and Rescue exosomes of breast cancer cells were extracted and injected into mice through caudal vein injection.Then the tibia was taken and stained.It was found that contrasting to the control group,the number of bone trabeculae in the tibia of mice with LSD1 KD exosomes was significantly reduced,and the bone trabecular structure disappeared in the tibia.The distance between bone trabeculae increased,osteoclasts increased,and osteoblasts decreased significantly in LSD1 KD group.These results suggest that LSD1 KD exosomes of breast cancer cells can promote the formation of an osteolytic pre-metastatic niche.Subsequently,the effects of LSD1-related exosomes of breast cancer cells on remodeling pre-metastatic niche were explored through animal experiments.The results showed that the number of bone trabeculae in the tibia of mice in the LSD1 KD group was significantly reduced and the spacing between bone trabeculae was increased.We also further explored the influence of LSD1-related exosomes in remodeling pre-metastatic niche on breast cancer bone metastasis.A tumor bone metastasis model was constructed by caudal artery injection.The results showed that the exosomes of LSD1 KD breast cancer cells significantly promoted the metastasis and colonization of MCF7-Fluc cells in the tibia.Meanwhile,the number and volume of trabecular bone in the tibia were significantly reduced,and the spacing between trabecular bone was increased.The vivo experimental results showed that LSD1 KD exosomes of breast cancer cells could create a microenvironment conducive to breast cancer metastasis,promote bone metastasis of breast cancer,and lead to bone destruction and dissolution.In vitro osteoblast differentiation model and osteoclast differentiation model were constructed by h BMSC cells and RAW264.7 cells.The results of in vitro experiments showed that LSD1 KD exosomes of breast cancer cells promote the formation of microenvironment before osteolytic metastasis by promoting osteoclast differentiation and inhibiting osteoblast differentiation.As an essential component of exosomes,miRNAs participate in the regulation of target cells.To explore which exosomal miRNA is regulated by LSD1 knockdown to remodel the pre-metastatic niche,we performed miRNA sequencing analysis of exosomes from LSD1 knockdown and Rescue breast cancer cells.We screened out miR-6881-3p,whose expression was down-regulated in LSD1 KD exosomes and recovered in Rescue group.Proved by experiment,miR-6881-3p could promote osteoblast differentiation and inhibit osteoclast differentiation.We predicted the target genes of miR-6881-3p and carried out functional enrichment analysis of the predicted target genes.Not surprisingly we found the target genes PBX1 and ASXL2 that played critical regulatory roles in osteoblast and osteoclast differentiation,which could be inhibited by miR-6881-3p.By supplementing miR-6881-3p and enriching exosomes of LSD1 KD Luminal breast cancer cells,LSD1 KD exosomes inhibited osteoblast differentiation and promoted osteoclast differentiation by down-regulating miR-6881-3p in exosomes.We performed combined analysis of miRNA sequencing results from Control,LSD1 KD,Rescue MCF7 exosomes,and cells.The results showed that more than 80% of miRNAs were down-regulated in LSD1 KD exosomes.However,there was no significant difference in the expression of these exosomal miRNAs in cells,indicating that LSD1 does not affect the intracellular expression of these exosomal miRNAs but affects their sorting to exosomes.We performed sequence analysis of these down-regulated miRNAs in LSD1-knockdown exosomes,and the consensus sequence was UAGGGC.This sequence is highly overlapped with the consensus sequence of miRNA that is modified by exosome sorting regulated by LSD1,suggesting that LSD1 may mediate exosomal miRNA sorting in breast cancer cells through hn RNPA2B1.We further confirmed the presence of hn RNPA2B1 in exosomes of breast cancer cells and the combination of hn RNPA2B1 and LSD1-related miRNA.At the same time,when LSD1 was knocked down in cells,the expression of hn RNPA2B1 was down-regulated.This indicates that LSD1 regulates the expression of hn RNPA2B1 protein.As a histone demethylase,LSD1 can regulate gene expression by binding to the promoter region of genes.Therefore,we designed specific primers in the promoter and 3’UTR region of hn RNPA2B1 and conducted Ch IP experiments.The results showed that LSD1 could specifically mediate the demethylation of H3K9 in the promoter region of hn RNPA2B1 and regulate the transcription of hn RNPA2B1.The above experimental results showed that LSD1 knockdown inhibited the demethylation of H3K9 in the promoter region of hn RNPA2B1,resulting in the down-regulation of the expression of hn RNPA2B1 and the reduction of the separation of miRNAs such as miR-6881-3p into exosomes.Finally,we conducted experiments by knocking down hn RNPA2B1 in breast cancer cells and supplementing miR-6881-3p in exosomes.The results showed that hn RNPA2B1 regulated osteoblast differentiation and osteoclast differentiation by regulating miR-6881-3p.This study showed that LSD1 could specifically bind to the hn RNPA2B1 promoter region,mediating H3K9 demethylation and activation of hn RNPA2B1 transcription.hn RNPA2B1 can bind to miR-6881-3p and sort it into exosomes.miR-6881-3p then proceeds to target cells through exosomes and inhibits gene expression by binding to the 3’UTR region of target genes PBX1 and ASXL2,thus affecting osteoblast differentiation and osteoclast differentiation.The pre-metastatic niche of breast cancer was reconstructed to regulate the occurrence of breast cancer bone metastasis.The LSD1-regulated miRNAs found in this study are expected to develop into markers for the early diagnosis of breast cancer bone metastases in the future,and the mimics of miR-6881-3p or monoclonal antibodies of PBX1 and ASXL2 may play a potential role in inhibiting bone resorption and alleviating bone-related events.