Study On The Correlation Between CD44 Variant(CD44v16) And Breast Cancer Cell Stemness

OBJECTIVE:This study aimed to study the relationship between a new CD44variant(CD44v16)and the stemness of human breast cancer cell line MCF-7 in vitro and in vivo.Furthermore,we explored the mechanism of CD44v16 increasing the stemness of breast cancer MCF-7 cells,which provided a new theoretical basis and treatment for reducing breast cancer recurrence and metastasis and overcoming multidrug resistance in breast cancer cells.METHODS:The Lenti-EF1a-CD44-EGFP-p GK-puro was constructed according to the CD44v16 sequence of the target gene(Note:When constructing the vector,EGFP is non-fused to the target gene,and the target gene is added with 3x Flag at the C-terminus),and the lentivirus was prepared and infected with the target cell MCF-7to construct the MCF-7-CD44v16 stable expression cell lines.Constructionof knockdown vector Lenti-U6-sh RNA-GFP-puro,preparation of lentivirus and infection of target cell MCF-7/ADR,construction of MCF-7/ADR-CD44v16-sh RNA stable cell lines.Real-time quantitative PCR was used to detect the relativeexpression of CD44v16 m RNA in each group cells.Flow cytometry was used to detect the expression of CD44+/CD24-cells in each group before and after transfection.Western blotting analysis showed the the changes of CD44v16 proteinexpression in each group of cell lines before and after transfection weredetected.The different protein level protein expression differences of four stemness transcription factors OCT4,SOX2,c-MYC and KLF4 in each group of cell lines before and after transfection were detected.Breast cancer stem cells were effectively enriched from breast cancer cells before and after transfection by serum-free suspension culture,then we observe the formation of stem cell microspheres.The subcutaneous xenograft models of human breast cancer cell lines(MCF-7,MCF-7-CD44v16,MCF-7/ADR,MCF-7/ADR-CD44v16-sh RNA)were constructedto measure the volume of the tumor every two days and dynamicallymonitor the body weight changes of nude mice in each group.After tumor peeling,the protein expression of OCT4,SOX2,c-MYC and KLF4 were determinedby immunohistochemistry.RESULTS: MCF-7-CD44v16 and MCF-7/ADR-CD44v16-sh RNA stable cell lines are successfully constructed.q RT-PCR results display that the expression level of CD44v16 m RNA in MCF-7-CD44v16 group was significantly higher than that in control group MCF-7(P<0.01),while the expression level of CD44v16 m RNA in MCF-7/ADR-CD44v16-sh RNA group was significantly lower than that in control group MCF-7/ADR(P<0.01).FCM results show that the proportion of CD44+/CD24-cells in MCF-7-CD44v16 group was(50.73�2.12)%,which was significantly higher than that of control group MCF-7(4.49�0.28)%).However,the proportion of CD44+/CD24-cells in breast cancer in MCF-7/ADR-CD44v16-sh RNA group was(7.45�0.60)%,which was significantly lower than that in MCF-7/ADR((55.73�2.23)%).The differences were statistically significant(P<0.001).Western blot results showed that compared with the control group MCF-7,the expression levels of CD44v16,OCT4,SOX2 and c-MYC proteins in MCF-7-CD44v16 group were significantly up-regulated,while the expression level of KLF4 protein was down-regulated(P<0.05).In contrast,compared with the control group MCF-7/ADR,the expression levels of CD44v16,OCT4,SOX2,and c-MYC proteins in the MCF-7/ADR-CD44v16-sh RNA group were significantly down-regulated,while the expression level of KLF4 protein was up-regulated(P<0.05).Four groups of cells can be suspended in the form of microspheres in serum-free medium.The volume of stem cell microspheres in the CD44v16 overexpression group was significantly increased,while the volume of microspheres in the CD44v16 knockdown group was significantly reduced.The nude mouse xenograft model was successfully constructed.The growth rate of transplanted tumors in MCF-7-CD44v16 group was significantly higher than that in control group MCF-7(P<0.05).The growth rate of transplanted tumors in MCF-7/ADR-CD44v16-sh RNA group was significantly lower than that in control group MCF-7/ADR(P<0.05).Histochemical staining of transplanted tumor immunohistochemistry showed that the expression of stemness transcription factors OCT4,SOX2 and c-MYC in MCF-7-CD44v16 group was significantly increased(P<0.05),but the expression of KLF4 protein was increased(P<0.05).In contrast,the expression of CD44,stemness transcription factors OCT4,SOX2,and c-MYC protein in the MCF-7/ADR-CD44v16-sh RNA group was significantly(P<0.05),but the protein expression of KLF4 was increased(P<0.05).The difference between the groups was statistically significant(P<0.05).CONCLUSION: CD44v16 can significantly increase the stemness of breast cancer cells and increase the growth rate of subcutaneous xenografts in breast cancer cells,which may be achieved by regulating several tumor stemness transcription factors OCT4,SOX2,c-MYC and KLF4.This suggests that CD44v16 may become a new breast cancer stem cell-specific surface marker and as a new target for the treatment of breast cancer.