| Research Background and purpose: Breast Cancer(BC)is one of the most common malignant tumors in women,and its incidence ranks first among female malignant tumors,seriously threatening the health of women all over the world.In recent years,the incidence of breast cancer in China has been increasing year by year,bringing great social and economic burden.Although surgical methods,chemotherapy,endocrine therapy,targeted therapy and immunotherapy are constantly updated,the mortality rate of breast cancer patients is still high.About 20% to 30% of breast cancer patients will develop metastasis at diagnosis and after treatment of the primary tumor,and about 90% of cancer-related deaths are attributed to metastasis.The 5-year overall survival rate of breast cancer patients with distant metastasis is only 25%.Therefore,it is particularly important to actively explore the pathogenesis and metastasis mechanism of breast cancer.INHBA(statin subunit βA),A member of transforming growth factor β superfamily,not only plays an important role in the biological process of human early embryo development and tissue terminal differentiation,but also participates in the occurrence and development of malignant tumors and is closely related to clinical prognosis.However,the function and underlying mechanisms of INHBA in breast cancer remain unclear.The Wnt/β-catenin signal transduction pathway is a highly conserved pathway in biological evolution,which is strongly correlated with many aspects of cancer biology,including tumor cell proliferation,migration,maintenance of tumor stem cell characteristics,angiogenesis and tumor immunity.Wnt receptor expression was found to be elevated in breast cancer cells,especially in metaplastic and triple-negative breast cancers.Is there a correlation between INHBA and Wnt/β-catenin signaling pathway? Whether INHBA can participate in the proliferation,migration and invasion of tumor cells through the Wnt/β-catenin signaling pathway remains unclear.Therefore,it is of great scientific significance to explore the role and related mechanism of INHBA in breast cancer.Through this study,the function and related molecular mechanism of INHBA promoting the proliferation,migration and invasion of breast cancer are clarified,which is expected to provide a new theoretical basis for the occurrence and development mechanism of breast cancer,and also provide an effective potential target for the treatment and diagnosis of breast cancer.Part 1 :INHBA promotes breast cancer cell proliferation,invasion and epithelial-mesenchymal transformation(EMT)Objective: To investigate the biological function of INHBA in breast cancer progression and whether it promotes the occurrence of EMT in breast cancer cells.Methods : :(1)the expression levels of INHBAm RNA and protein in four breast cancer cell lines(MDA-MB-468,SUM149 PT,MCF-7,SKBR3)and human normal breast epithelial cell line MCF-10 A were compared by q RT-PCR and Western Blot.(2)The expression of INHBA and EMT-related proteins(E-cadherin and N-cadherin)in paraffin specimens of 141 cases of invasive breast cancer were detected by immunohistochemistry,and the relationship between the high expression of INHBA protein and clinicopathological features was analyzed.Meanwhile,the correlation among INHBA,E-cadherin and N-cadherin was analyzed.(3)The expression of INHBA in breast cancer cell line MCF-7 was interfered by lentiviral vector,and the overexpression and interference efficiency were determined by q RT-PCR and Western Blot.(4)The effects of INHBA knockdown on the proliferation and activity of breast cancer cells were detected by CCK-8 and Ed U experiments.(5)Transwell assay was used to detect the effects of INHBA on migration and invasion characteristics of MCF-7 cells.(6)Western Blot assay was used to detect the phenotypic changes of EMT(E-cadherin and N-cadherin expression)after interfering the expression of INHBA in MCF-7.Results:(1)The experimental results are consistent with those retrieved by GEPIA database: compared with normal human mammary epithelial cell line MCF-10 A,the levels of INHBA m RNA and protein in four breast cancer cell lines(MDA-MB-468,SUM149 PT,MCF-7 and SKBR3)were significantly increased,and the expression level of INHBA m RNA and protein in MCF-7 cell line was the highest.(2)Compared with benign hyperplasia breast tissue,the expression rate of INHBA protein in breast cancer tissue was higher,and the difference was statistically significant(χ2=12.283,P < 0.05).The high expression of INHBA was significantly correlated with histological grade(χ2=6.191,P < 0.05),lymph node metastasis(χ2=14.223,P < 0.05)and TNM stage(χ2= 6.038,P < 0.05).There was no significant correlation with age(χ2=0.191,P > 0.05),tumor diameter(χ2=1.049,P > 0.05)and molecular type(χ2=4.666,P > 0.05).(3)At the same time,the high expression of INHBA was correlated with E-cadherin and N-cadherin in breast cancer hyperplasia tissues,and the high expression of INHBA was negatively correlated with E-cadherin(r=-0.249,P < 0.05),that is,the expression of E-cadherin decreased in patients with high expression of INHBA.The high expression of INHBA was positively correlated with the expression of N-cadherin(r =0.442,P < 0.05),that is,the expression of N-cadherin was enhanced in the high expression of INHBA.E-cadherin expression was negatively correlated with N-cadherin expression(r=-0.536,P < 0.05),and N-cadherin positive expression rate increased in patients with low E-cadherin expression.(4)The experimental results of CCK-8 and Ed U showed that knocking down INHBA reduced the proliferation rate of MCF-7 cells.Conversely,overexpression of INHBA promoted the proliferation of MCF-7 cells(P<0.01).(5)Transwell results showed that interference with INHBA inhibited the migration and invasion of MCF-7 cells,while overexpression of INHBA promoted the migration and invasion of MCF-7 cells(P<0.05).(6)After INHBA was interfered with in MCF-7 cells,the expression level of E-cadherin increased,while that of N-cadherin decreased;On the contrary,the expression level of E-cadherin protein in the overexpressed INHBA group was lower than that in the control group,and the expression level of N-cadherin protein was higher than that in the control group(P<0.05).Conclusions :(1)The expression level of INHBA in breast cancer tissues and cells is significantly higher than that in benign hyperplasia breast tissues and normal breast cells,and the high expression of INHBA is closely related to the late TNM stage,lymph node metastasis and poor tumor differentiation in breast cancer patients.(2)INHBA promoted the proliferation,migration and invasion of breast cancer cells.(3)INHBA promotes the occurrence of EMT in breast cancer cells.Part 2 : INHBA promotes the proliferation,migration and invasion of breast cancer cells by activating Wnt/β-catenin signaling pathwayObjective: To investigate the effect of INHBA expression on m RNA and protein expression levels of core members of Wnt/β-catenin signaling pathway in breast cance,and to explore the potential mechanism of INHBA promoting the occurrence and development of breast cancer.Methods:(1)The expression of INHBA in breast cancer cell line MCF-7 was interfered by lentiviral vector,and the transfected cells were treated with XAV-939(Wnt/β-catenin signaling inhibitor).Blank control group,pc-INHBA group,blank + XAV-939 group,pc-INHBA + XAV-939 group.q RT-PCR and Western Blot were used to detect the effects of XAV-939 treatment on m RNA and protein levels of core members of Wnt/β-catenin pathway.(2)MTT assay was used to detect the effect of XAV-939 on the proliferation of breast cancer cells;Transwell assay was used to determine the effect of XAV-939 treatment on the migration and invasion of breast cancer cells.(3)Tumorigenesis experiment was designed in nude mice to detect the effect of INHBA gene knockdown on the proliferation ability of breast cancer cells;Tumor proteins were extracted and Western Blot was used to detect the effect of INHBA gene knockdown on the protein expression levels of core members of Wnt/β-catenin signaling pathway in breast cancer cells.Results:(1)The m RNA levels of β-catenin and GSK-3β,the core members of Wnt/β-catenin pathway,and the protein levels of β-catenin,p-GSK-3β and p-GSK-3β/GSK-3β were significantly increased in MCF-7 cells transfected with pc-INHBA plasmid.The signal inhibitor XAV-939 was used to treat MCF-7 cells transfected with pc-INHBA plasmid,and it was found that the effect of increased β-catenin and GSK-3β m RNA levels caused by INHBA overexpression was reversed.The protein expression levels of β-catenin and p-GSK-3β and p-GSK-3β/GSK-3β were significantly decreased(P < 0.01).(2)The proliferation,migration and invasion of MCF-7 cells were significantly enhanced after INHBA overexpression,and the effect of INHBA on the proliferation,migration and invasion of MCF-7 cells was reversed after the blocking of Wnt/β-catenin pathway by XAV-939 signaling inhibitor(P < 0.05).(3)Compared with NC group,tumor volume and weight of nude mice in sh RNA-INHBA group were significantly decreased,and β-catenin and p-GSK-3β protein expression levels were significantly decreased(P < 0.05).Conclusion :(1)INHBA can activate the Wnt/ β-catenin pathway,and increase the m RNA and protein expression levels of β-catenin and GSK-3β,the core members of the Wnt/β-catenin pathway,and the phosphorylation level of GSK-3β protein.(2)INHBA promotes the proliferation,migration and invasion of breast cancer cells by activating the Wnt/β-catenin pathway. |
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