| Breast cancer is one of the leading causes of cancer death in women worldwide.Although surgical treatments,radiotherapy,chemotherapy,and biotherapeutic treatments have achieved good results,approximately 25% of patients eventually relapse.One of the reasons is the presence of breast cancer stem cells(BCSCs),which are characterized by self-renewal,multidirectional differentiation,and distant metastasis.Therefore,targeted removal of BCSC or reduction of the number of BCSC subset may be a key strategy for effective treatment of breast cancer.Our previous study found that overexpression of mi R-7 reduced BCSC subpopulation and partially reversed epithelial-methylcamal transition(EMT)in human breast cancer MDAMB-231 cell line,but molecular mechanisms that remain unclear.BCSC subset is often associated with chemoresistance,progression,metastasis,and recurrence of breast cancer.Therefore,studying the mechanisms that mi R-7 reduces BCSC subset may help to find new strategies for treatment of breast cancer.ALDH1(Aldehyde dehydrogenase 1)is an intracellular aldehyde oxidative dehydrogenase that is essential for tumor cell growth and differentiation.Studies have shown that ALDH1 is high expressed in stem cells,protects stem cells from endogenous toxicants and exogenous drugs,plays an important role in maintaining stem cell characteristics,and can be a biomarker for CSC.This study uses ALDH1A3(member of the ALDH family)as a target gene to investigate whether mi R-7 would down-regulate ALDH1A3 gene expression,reduce BCSC stem cell-like properties and cell proliferation activity,and to attempt to demonstrate that ALDH1A3 can not only be used as a biomarker for BCSC,but also regulate the biological function of BCSC.Lnc RNA XIST(Long non-coding RNA X-inactive specific transcript,XIST)plays an important role in the X chromosome inactivation process.In addition,XIST is closely related to the formation and development of tumors.Decreasing the expression of XIST can inhibit the proliferation,invasion and migration of tumor cells,promote apoptosis,and play a role in suppressing tumor growth.However,the relationship between XIST and the cancer inhibitor mi R-7 has not been reported.Therefore,we have another focus in this study,which is whether there is mutual regulation between mi R-7-XIST and regulation of the downstream transcription factor slug that in turn affects the expression of epithelial specific antigen(ESA),a surface marker,on BCSC,to analyze the mechanisms that mi R-7 reduces BCSC subset.OBJECTIVE:To explore the regulation mechanisms of mi R-7-ALDH1A3 and mi R-7-XIST-Slug-ESA axis that reduces BCSC subset,and to provide new theoretical and experimental basis for treatment of breast cancer by targeting BCSC.METHODS:1.Flow cytometry(FCM)was used to detect the number of ALDHbr cells in human breast cancer cell lines BT549 and MDA-MB-231.CD44+CD24-and ESA+CD44+CD24-BCSC were respectively sorted from the MDA-MB-231 cells by magnetic activated cell sorting(MACS);Simultaneously,the number of ALDHbr cells was detected in MDA-MB-231 cells by FCM.The MDA-MB-231 cells were transfected with either the mi R-7 mimic or mi R-7 inhibitor or with the si ALDH1A3 by using LipofectamineTM 2000 reagent.Meanwhile,the number of ALDHbr cells was measured in MDA-MB-231 after transfection.Based on algorithm prediction(http://www.microrna.org),we wanted to know whether there are binding sites between mi R-7 and ALDH1A3 3'UTR gene,and then we constructed the recombinants of psi CHECK2-ALDH1A3-3'UTR,psi CHECK2-ALDH1A3-3'UTR-Mut,respectively to verify the regulatory effect of mi R-7 mimic on the ALDH1A3 gene expression in MDA-MB-231 and SK-BR-3 cells by the dual luciferase assay,respectively.The lentiviral vector expressing mi R-7 and control vector were constructed,and was transfected into the MDA-MB-231 cells respectively by using LipofectamineTM 2000 reagent.After screening,Lenti-mi R-7 and Lentivector monoclonal cells were selected,and RT-q PCR and Western Blot were used to detect the molecular expression of mi R-7 and ALDH1A3 in monoclonal cells.The si ALDH1A3 was transfected into MDA-MB-231 and BCSC,and cell surface molecule expression was detected by FCM.ESA+CD44+CD24-Lenti-mi R-7(BCSC-Lenti-mi R-7)and Lentivector(BCSC-Lentivector)were sorted by MACS,and cell cycle proteins Cyclin D1 and Ki67 were detected to analyze the mi R-7 effects on the proliferation of BCSC.The tumor-bearing mice model were established by injection of BCSCLenti-mi R-7,BCSC-Lentivector in nonobese diabetic/ severe combined immunodeficient(NOD/SCID)mice,respectively.RT-q PCR,Western Blot and immunohistochemistry experiments were used to comprehensively analyze the expression of ALDH1A3 and other molecules in tumor tissues.In addition,the influence of subcutaneous injection of si ALDH1A3 into tumor site on tumor growth in NOD/SCID mice was also evaluated.2.Based on algorithm prediction(http://www.microrna.org),we found that there are a 3 binding sites between mi R-7 and XIST,and that there is a binding site between mi R-7 and the transcription factor Slug 3' UTR.The si XIST was transfected into BCSC originated from the MDA-MB-231 cells.The effect of silencing XIST on the expression of mi R-7 was detected by RT-q PCR and the number of BCSC was detected by FCM.The recombinants of psi CHECK2-XIST1-3 and psi CHECK2-XIST1-3 Mut,psi CHECK2-Slug-3'UTR and psi CHECK2-Slug-3'UTR Mut dual luciferase plasmids were constructed and then were respectively transfected into the MDA-MB-231 and LD cells(established by our Lab from the human breast cancer post-surgery sample).The regulatory effects of mi R-7 mimic on the XIST and slug genes was verified.Based on algorithm prediction(http://www.microrna.org),it indicated that there is a binding site between slug and the upstream region of the ESA gene promoter.The si Slug was transfected into MDA-MB-231 cells,and FCM was used to detect ESA expression on the transfected cell surface.Chromatin immunoprecipitation(Ch IP-PCR)experiment was performed to verify the presence of a binding site between the slug transcription factor and the ESA gene promoter.The BCSC transplanted NOD/SCID tumor-bearing mice were established,and were randomly divided into three groups of equal number(four per group): 1)BCSC group,2)Drug group(doxorubicin + cyclophosphamide)and 3)mi R-7 Agomir group to observe the therapeutic effect on tumor-bearing mice.The expression of XIST,Slug,ESA,mi R-7 was detected by RT-q PCR.Immunohistochemistry and Western Blot experiments were performed to analyze the expression of Slug and ESA in tumor tissues.3.12 cases of breast cancer patient's cancer tissue and adjacent tissues were collected,and the molecular expression of ALDH1A3,XIST,Slug,ESA,and mi R-7 were analyzed by RT-q PCR and Western Blot assays.RESULTS:1.The sorted ESA+CD44+CD24-MDA-MB-231 cells by MACS may effectively enrich ALDHbr cells,and the expression of surface markers and biomarkers in BCSC indicated a parallel expression trend.Bioinformatics prediction and dual luciferase reporter system experiment confirmed that ALDH1A3-3'UTR is mi R-7 regulatory target.Overexpression of mi R-7 mimic inhibited the expression of ALDH1A3 and effectively reduced the number of ALDHbr cells and the BCSC surface markers in MDA-MB-231 cells.The lentiviral mi R-7 overexpression plasmid(designated Lenti-mi R-7)was successfully constructed,which was infected into BCSC.The monoclonal cell was selected by intermediated culture;FCM analysis of cell cycle in BCSCLenti-mi R-7 revealed that the G2-M phases(1.99% vs 8.12% or 7.60% compared with BCSCLentivector and BCSCs),and that the molecular expression of the cyclic proteins Cyclin D1,Ki-67 was significantly reduced,indicating that mi R-7 effectively reduced the proliferation of BCSC and the stemness of BCSC.In vivo NOD/SCID mouse experiment demonstrated that the expression of biomarker ALDH1A3 and cell surface marker ESA in BCSC-Lenti-mi R-7 group were significantly lower than BCSC-Lentivector group,and tumor growth was significantly inhibited.At the same time,NOD/SCID mice were challenged by 2×105 BCSC and tumor growth appeared on day 15,and the tumor growth in the tumor-bearing mice in the si ALDH1A3 injected group was significantly inhibited compared with the control groups.2.The results indicated that there was a negative regulation between XIST and mi R-7 in MDAMB-231 and LD cells confirmed by dual luciferase reporter system.Slug was one of the candidate genes and has one site in Slug-encoded m RNA containing a 3'-UTR element for mi R-7.The result of the mi R-7 mimic transfected cells showed that mi R-7 reduced the relative luciferase activity of the wild type vector and down-regulated slug gene expression.There is an each other inhibition effect between mi R-7 and XIST.The result of Ch IP-PCR confirmed that the slug acted as transcriptional regulator bound to the region of the ESA promoter,directly enhancing ESA gene expression.The above experiments indicated that mi R-7-XIST was involved in the regulation of slug expression,and slug served as a transcriptional regulatory molecule to regulate the expression of ESA,which is BCSC surface marker,forming a complete regulatory axis,thereby affecting the number of BCSC subset.In addition,si XIST can inhibit the cell cycle of BCSC(G2-M phase),and the data suggested that XIST was positively correlated with the ability of BCSC proliferation and differentiation.In vivo experiment,2×105 BCSC was injected subcutaneously with NOD/SCID mice,and mi R-7 Agomir was used as a targeted drug to treat tumor-bearing mice.The results showed that the expression of slug and ESA was significantly decreased in the tumor compared with the control groups,and that tumor growth was effectively inhibited,which was statistically significant(p(27)0.05).3.The detection of 12 breast cancer patients found that,compared with the adjacent tissue,the expression levels of ALDH1A3,XIST,Slug and ESA m RNA in cancer tissue were negatively correlated with the expression of mi R-7.The protein expression levels of ALDH1A3,Slug and ESA in cancer tissues were significantly increased.CONCLUSIONS:1.There are an inter-modulation between mi R-7 and XIST.mi R-7 has a targeted regulatory effects on ALDH1A3,slug and ESA,thus affecting the proportion of BCSC subset.2.In vivo and in vitro experiments demonstrated that the expression levels of ALDH1A3,XIST,slug and ESA are positively correlated with the number of BCSC subset,and the expression of mi R-7 is negatively correlated with the number of BCSC subset.3.mi R-7 inhibits ALDH1A3 and reduces BCSC biomarkers,and decreases slug and down regulates ESA expression,further reducing the BCSC surface marker and the proportion of BCSC subset.These findings from the study may provide a new idea for treatment of breast cancer by mi R-7 targeted ALDH1A3 and XIST and slug to down regulate ESA expression and BCSC subset. |
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