| Research Background: Breast cancer is the most common malignancy globally.At present,there are many therapeutic methods for breast cancer,including surgery therapy,chemotherapy,endocrine therapy,molecular targeted therapy;and the mortality of breast cancer patients has decreased.However,some patients are still recurrence and metastasis within 5 years of treatment,which is the main cause of death in breast cancer patients.Breast cancer stem cells(BCSCs)also have stem cell characteristics,such as capability of self-renewal and multi-directional differentiation.BCSCs have higher tumorigenesis,abilities of invasion and metastasis,resistance to radiotherapy,chemotherapy,and hypoxia compared with non-stem cells in breast cancer.The most common markers of breast cancer stem cells are CD24,CD44,ALDH1,OCT4,and SOX2.Recent researches indicate that BCSCs play significant roles in the development,progression,recurrence and metastasis of breast cancer.Ubiquilin1(UBQLN1),which is a member of the highly conserved ubiquitin-like protein family,is widely expressing in tissues,and its coding gene is located on human chromosome 9(9q22),encoding a 589 amino acid sequence or a 561 amino acid sequence.UBQLN1,can provide polyubiquitination targets to specific proteins and promote proteasome degradation,especially in the process of endoplasmic reticulum-associated protein degradation(ERAD)as well as a transfer protein.Recent researches focused on that UBQLN1 plays crucial roles in neurological diseases caused by protein aggregation,such as amyotrophic lateral sclerosis,Alzheimer's disease,and Huntington's disease.However,there are few studies on the role of UBQLN1 in tumorigenesis and development;and its role in the development of breast cancer is still unclear.Epithelial-mesenchymal transformation(EMT)is a biological process in which epithelial cells lose polarity and are transformed into cells with interstitial phenotypes by specific procedures.The most significant molecular event is the decrease of E-cadherin,whereases N-cadherin and the mesenchymal marker Vimentin is increased in EMT.Some transcription factors for example,Snail1,Slug,Twist and ZEB1/2 directly or indirectly regulate the expression of EMT-related proteins,such as E-cadherin,to induce EMT process.EMT plays important roles in physiological and pathophysiological processes,such as embryonic development,tumor invasion and metastasis.Additionally,EMT is closely related to the biological functions of cancer stem cells.Objectives:(1)To investigate the effects of UBQLN1 on apoptosis,proliferation,migration and invasion of human breast cancer cells;(2)To explore the roles of UBQLN1 in EMT process in human breast cancer;(3)To explore the roles of UBQLN1 in BCSCs stemness.(4)To demonstrate the mechanism of UBQLN1 regulating the stemness and EMT through PI3K/Akt signaling pathway in human breast cancer cells.Methods: 1.To detecte the protein expression of UBQLN1 in normal breast cells and breast cancer cells: Western blot analysis was used to detect the expression of UBQLN1 in these three cell lines,including normal mammary epithelial cell line MCF-10 A,luminal breast cancer cell line MCF-7,and basal breast cancer cell line MDA-MB-231.2.To down-regulate the expression of UBQLN1 in two human breast cancer cell lines and verify the efficiency of down-regulation:(1)Breast cancer cells MCF-7 and MDA-MB-231 were transfected with siRNA specific for UBQLN1 and negative control(NC)by using Lipofectamine 2000;and real time qRT-PCR and western blot analyses were used to verify the transfection efficiency.(2)Lentiviral expression vectors of shRNA against UBQLN1(shUBQLN1)and shScramble infected breast cancer cells MCF-7 and MDA-MB-231 using puromycin to screen the infected cells.The infection efficiency was confirmed by real-time qRT-PCR and Western blot analyses.3.The roles of down-regulation of UBQLN1 on apoptosis,proliferation,migration and invasion of breast cancer cells:(1)Western blot ananlysis was used to detect the apoptosis-related protein caspase3(cleaved),Bcl2 and Bax and also the expression levels of invasive-associated proteins MMP2 and MMP9 in breast cancer cell lines;(2)Colony formation,scratch-wound and transwell assays to detect the effects of UBQLN1 on the proliferation,migration and invasion of breast cancer cells.4.To detect the expression of EMT related proteins E-cadherin,N-cadherin,Vimentin,Snail1 and Twist in MCF-7 and MDA-MB-231 cell lines by immunofluorescence assay and Western blot analysis.5.The relationship between UBQLN1 and stemness of human breast cancer cells:(1)Western blot analysis was used to detect the breast cancer stem cells markers,such as ALDH1,OCT4 and SOX2,after downregulation of UBQLN1;(2)Immunomagnetic beads were used to isolate the CD44+/CD24low-breast cancer stem cells from luminal breast cancer cell line MCF-7,the qRT-PCR analysis was used to detect the expression of UBQLN1;(3)Mammosphere formation assay was used to detect the self-renewal ability of breast cancer stem cells.6.Western blot analysis was used to detect the expression levels of PTEN,Akt,p-Akt after downregulation of UBQLN1.Results: 1.UBQLN1 was significantly higher in breast cancer cell lines MCF-10 A,MCF-7 and MDA-MB-231 than that in normal breast epithelial cell line(P<0.01).2.Lentiviral expression vectors of shRNA against UBQLN1(shUBQLN1)and shScramble infected breast cancer cells MCF-7 and MDA-MB-231 using puromycin to screen the infected cells.The infection efficiency was confirmed by real-time qRT-PCR and Western blot analyses,successfully established a stable cell line that down-regulates UBQLN1(P<0.05).3.The effects of UBQLN1 on apoptosis,proliferation,migration and invasion in human breast cancer cells:(1)Cell scratch-wound assay showed that the cell migration ability was significantly decreased in shUBQLN1 group compared with the control group(P<0.01);(2)Colony formation assay showed the number of clonies and clone diameter were decreased in shUBQLN1 group than the control group(P<0.05);(3)Transwell assay showed that the number of perforated membrane was significantly decreased in shUBQLN1 group than that in the control group after 18 hours cultured(P<0.05);(4)Western blot analysis showed that the expression levels of MMP2 and MMP9 were dramatically decreased in shUBQLN1 group compared with the control group(P<0.01).In additional,caspase3(cleaved)and Bax was markedly increased,while Bcl2 was significantly decreased(P<0.05).4.The relationship between UBQLN1 and epithelial-mesenchymal transition in human breast cancer cells:(1)The protein expression level of E-cadherin was markedly increased,while the protein level of N-cadherin,Snail1,Vimentin and Twist were dramatically decreased in the shUBQLN1 group compared with the control group,(P<0.05).(2)The EMT related protein was significantly increased,while the fluorescence intensity of N-cadherin was decreased in shUBQLN1 group compared to control group.5.The relationship between UBQLN1 and human breast cancer stem cells:(1)ALDH1,OCT4 and SOX2 were significantly decreased in shUBQLN1 group compared with the control group(P<0.05).(2)The results of extraction of stem cells from MCF-7 cells by immunomagnetic beads showed that ALDH1 was highly expressed in the selected cell population,indicating that breast cancer stem cells were successfully selected.UBQLN1 was significantly higher in CD44+/CD24low-stem cells compared with CD24+ and CD44-/CD24-cells(P < 0.01).(3)The numbers and sizes of mammosphere were significantly decreased in shUBQLN1 group compared with the control group(P<0.05).6.The mechanism of UBQLN1 regulating the stemness and EMT of through PI3K/AKT signaling pathway in breast cancer cells.Western blot analysis shown that the expression of PTEN was significantly increased,whereases the expression of P-Akt(ser473)was decreased in shUBQLN1 than that in the negative control(P<0.05).Conclusions:1.UBQLN1 can promote the cells proliferation,migration and invasion and inhibit the apoptosis in human breast cancer cells.2.UBQLN1 can promote the process of EMT in human breast cancer cells.3.The expression level of UBQLN1 was positively correlated with the stemness in human breast cancer cells.4.UBQLN1 can activate PI3K/Akt signaling pathway in human breast cancer cells,and UBQLN1 may effect on stemness and epithelial-mesenchymal transition of human breast cancer cells through PI3K/Akt signaling pathway... |
PDF Full Text Request