| Objective: Breast cancer is the most common cancer in women,There are approximately 1.67 million new cases of breast cancer worldwide each year.It is estimated that there will be about 22 million breast cancer patients worldwide by 2030.With the development of treatment methods,the prognosis of breast cancer patients has improved significantly,but there are still 30-40% of patients eventually relapse and metastasis until death.Triplenegative breast cancer(TNBC)has become a difficult problem in the field of breast cancer treatment because of its high malignancy,invasiveness,and inability to benefit from endocrine therapy and anti-HER-2 targeted therapy,limited means of treatment and poor prognosis.Therefore,it is urgent to find new and effective targets for the treatment of triple-negative breast cancer to improve the prognosis of triple-negative breast cancer patients.Cancer stem cells(CSCs)are a special part of tumor cells in tumor tissue.These cells constantly self-renew,divide and proliferate through some mechanisms,and promote the recurrence and metastasis of tumors.In 2003,Al-Hajj et al.isolated and identified breast cancer stem cells for the first time in breast cancer.Compared with other types of breast cancer,the proportion of cancer stem cells in triple negative breast cancer cell lines is higher.Therefore,targeted therapy for cancer stem cells may be one of the most effective methods for breast cancer in the future,especially for triple negative breast cancer.MicroRNAs(miRNAs)are a class of noncoding RNAs.They regulate cell proliferation,differentiation,and apoptosis via either perfect or imperfect base pairing with the 3’-untranslated region(3’-UTR)of their target mRNAs.As a tumor suppressor,miR-1 has been widely recognized by the academic community.Previous studies have found that mir-1 can significantly inhibit the proliferation of tri-negative breast cancer cells both in vivo and in vitro.Moreover,the expression of 13 kinds of miRs was found to be decreased in the study of breast cancer stem cells,among which miR-1 was one of them.Therefore,the study of miR-1 can be used as a starting point for the targeted treatment of breast cancer stem cells.The purpose of this study was to observe the expression of miR-1 in triple-negative breast cancer tissues and adjacent non-neoplastic tissues,to explore the effect of miR-1 on the biological behavior of breast cancer stem cells and its mechanism,and to provide a new theoretical basis for the treatment of triple-negative breast cancer.Method: 1.Total RNA was extracted from FFPE tissue samples of triple negative breast cancer,and the expression level of miR-1 was detected by real-time PCR.2.Breast cancer stem cells were isolated by flow cytometry.3.miR-1 agomir,NC agomir and EVI-1 overexpression vectors were transfected into breast cancer stem cells by Lipofectamine 2000.The self-renewal ability was measured by microsphere formation test.CCK-8 method was used to detect the proliferation ability.Western blotting was used to detect the expression of apoptotic pathway related factors and epithelial mesenchymal transition(EMT)pathway related factors.4.The target genes were identified by dual luciferase assay.5.A nude mouse xenograft model was established to evaluate the effect of miR-1 expression on the tumorigenic ability of breast cancer stem cells.6.Western blotting and immunohistochemistry were used to detect the changes of EVI-1 and calreticulin(CRT)in transplanted tumors.7.Expression vectors of different domains of CRT were constructed and transfected.8.Microsphere formation assay was used to determine the effect of calreticulin domains on the self-renewal ability of cancer stem cells.Flow cytometry was used to analyze the effect of calreticulin domains on apoptotic ability of cancer stem cells.Western blotting assay was used to detect the effects of calreticulin domains on the expression of EMT-related factors in cancer stem cells.Results: 1.By analyzing FFPE tissue samples of triple-negative breast cancer,it was found that miR-1 was low expressed in triple negative breast cancer.2.Breast cancer stem cells were successfully isolated by flow cytometry.3.MiR-1 can negatively regulate the expression of calreticulin.4.Overexpression of miR-1 can inhibit the formation and proliferation of breast cancer stem cell microspheres,promote apoptosis and inhibit the ability of EMT process.Meanwhile,over-expression of EVI-1 can antagonize the antitumor ability of miR-1.5.Dual luciferase assay confirmed that EVI-1 was the target gene of microRNA-1,and microRNA-1 could negatively regulate the expression of EVI-1.6.Bioinformatics method showed that calreticulin was the target gene of EVI-1,and EVI-1 could positively regulate the expression of calreticulin.7.Overexpression of miR-1 can inhibit the growth of transplanted tumors in nude mice,and expression of EVI-1 and calreticulin in transplanted tumors in nude mice is significantly inhibited by miR-1.8.Calreticulin N-domain promotes self-renewal of breast cancer stem cells significantly stronger than P-domain and C-domain.9.Calreticulin N-domain inhibits apoptosis of breast cancer stem cells significantly stronger than P-domain and C-domain.10.Calreticulin P-domain and C-domain may have the ability to inhibit EMT of cancer stem cells,which can be antagonized by N-domain.Conclusion: 1.miR-1 is low expressed in tri-negative breast cancer tissues.2.miR-1 can inhibit the proliferation of breast cancer stem cells and the ability of EMT,and promote its apoptosis,while EVI-1 can antagonize the anti-cancer ability of mir-1.3.miR-1 may indirectly inhibit CRT expression by directly binding to and down-regulating EVI-1,thus affecting the biological function of breast cancer stem cells.4.Overexpression of miR-1 inhibited the growth of transplanted tumor from breast cancer stem cells in nude mice.5.The mechanism of miR-1 inhibiting breast cancer stem cell transplantation may be through inhibiting the expression of EVI-1 and CRT.6.CRT N-domain promotes self-renewal of breast cancer stem cells significantly stronger than P-domain and C-domain.7.The Ndomain of CRT inhibits the apoptosis of breast cancer stem cells significantly more than the P-domain and C-domain,and the P-domain and N-domain may be antagonistic to each other.8.CRT P-domain and C-domain may have the ability to inhibit EMT of cancer stem cells,which can be antagonized by N-domain. |
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