MiR-205/RUNX2 Axis Regulates Breast Cancer Cell Stemness And Malignancy

Research Background:Breast cancer is the highest incidence of malignant tumor in women in the world,and it is the second largest cancer that causes death in women.With the in-depth study of the mechanism of the occurrence and development of breast cancer,breast cancer stem cell(BCSC)has been paid more and more attention.BCSC is a group of cells with self-renewal and multi-directional differentiation potential.Its self-renewal ability makes tumor stem cells proliferate and differentiate,which plays an important role in the occurrence,development,and recurrence of breast cancer.The surface markers of breast cancer stem cells include CD44,CD24,Ep CAM,ALDH1,and CD44~+/CD24~-is considered to be the most typical biomarker of breast cancer stem cells.Many mi RNAs are involved in regulating the self-renewal of tumor stem cells,including mi RNAs that promote or inhibit tumor stem cell proliferation,and are associated with development and prognosis.mi RNAs are abnormally expressed in breast cancer,gastric cancer and many other cancers.mi RNAs are involved in the growth,proliferation,and differentiation of breast cancer stem cells.Our research group found that mi R-205 is a tumor suppressor in the development of breast cancer in the early stage of the experiment.mi R-205 can inhibit the proliferation,invasion,EMT and stemness of breast cancer cells.RUNX2 is a member of the RUNX family and is essential for osteoblast differentiation and chondrocyte maturation.Our previous studies have shown that RUNX2 is up-regulated in breast cancer cells and overexpression of RUNX2 promotes the occurrence,development and invasion of breast tumors.With the emergence of epigenetics and related regulatory mechanisms,mi RNA is considered to be one of the most effective regulators at the post-transcriptional level of gene expression.Among them,micro RNA-205 acts as a tumor suppressor by targeting RUNX2 in pancreatic cancer and osteosarcoma.However,the relationship between micro RNA-205 and RUNX2 in breast cancer has not been reported.In this study,the effects of simultaneously up-regulating the expression of micro RNA-205 and RUNX2 on the proliferation,invasion,migration,and stemness of breast cancer cells were detected in vitro.In vivo nude mice tumor experiments were performed to observe the effects of micro RNA-205 and RUNX2 on tumor formation in mice.To explore the relationship between micro RNA-205 and RUNX2,it provides new strategy for the treatment of breast cancer patients.Research Objective:Based on the previous research group,we further verified the regulatory relationship between micro RNA-205 and RUNX2,and whether micro RNA-205 can inhibit the stemness and malignant biological behavior of breast cancer cells by targeting RUNX2.Research content:1. In human breast cancer MCF-7 cells,lentivirus infection was used to establishstable overexpressing micro-NC,micro RNA-205-5p,RUNX2,co-transfected with m icro RNA-205-5p and RUNX2 cell lines.2. The GFP fluorescence of all transfected cell lines was observed under fluorescence microscope,and the transfection efficiency was detected by q RT-PCR.3. Western blot and RT-PCR were used to detect the expression of RUNX2.Westernblot was used to detect the expression of breast cancer stem cell markers CD44 and CD24.Flow cytometry was used to screen the proportion of CD44~+/CD24~-stem cells.Mammosphere experiments were used to observe the ability of mammosphere formation in each group.4.In vitro experiment:cell proliferation,invasion,migration and unanchored growth ability were detected by MTT,plate cloning,Transwell,soft agar colony experiment,respectively.5.In nude mice tumor formation experiment:cells were injected into nude mice to observe their tumor formation ability.Research result:1.The cell lines stably overexpressing micro RNA-205-5p and RUNX2 was successfully constructed.2.Western blot and RT-PCR experiments showed that increased expression of micro RNA-205-5p led to a decrease in protein and m RNA levels of RUNX2,and overexpression of micro RNA-205-5p and RUNX2 will reverse the results.3. Western blot and RT-PCR experiments showed that increasing the expression ofmicro RNA-205-5p will cause the expression level of RUNX2 protein and m RNA to decrease,while overexpressing micro RNA-205-5p and RUNX2 will reverse the results.4. MTT,Transwell,soft agar colony assay showed that micro RNA-205-5p could inhibit the invasion and metastasis of breast cancer cells by targeting RUNX2 in MCF-7 cells.5. Compared with overexpressing micro RNA-205-5p and RUNX2 alone,the growth rate of tumor was slower and the volume of tumor was smaller.Conclusions:In breast cancer MCF-7 cells,micro RNA-205-5p can regulate the expression of RUNX2.Micro RNA-205-5p inhibits the malignant behavior and stenness of breast cancer cells by targeting RUNX2.