| Background and ObjectivesBreast cancer (BC) still remains the most common malignant tumor in women, with 1.35 million cases diagnosed worldwide per year and causing 400,000 deaths.Recently, the therapeutic methods of breast cancer have been constantly developed. According to the molecular classification of breast cancer, the comprehensive treatment which includes surgery, endocrinetherapy, chemotherapy, radiotherapy, biologically targeted therapy and traditional Chinese medicine treatment has become the main model of breast cancer therapy.The molecular classification of breast cancer includes five types,consisting of luminal A,luminal B,HER2-positive,basal-like and nomal breast-like.The curative effect has improved continually and breast cancer has become one of the solid tumors with good outcome. However, global incidence of breast cancer has increased year by year. In our country,the morbidity of breast cancer has become the number one of women malignancy and has been trending younger lately. As we know, the development of breast cancer involves many changes in tumor-related markers, including dysfunctions and abnormal expression. To analyze the genome and proteome of breast cancer has showed that focusing on molecular heterogeneity maybe an available way to identify and develop novel therapeutics.In recent years, the aberrant expression of microRNAs(miRNAs) has been proved to be linked to the molecular pathogenesis of breast cancer.MiRNAs are small,endogenous,non-coding RNAs of 18 to 24 nucleotides in lengths.At post-transcriptional level,miRNAs negatively regulate gene expression by binding to the 3’untranslated region(3’UTR) of corresponding target messenger RNAs(mRNAs).Increasing evidence has shown that lots of miRNAs play an important role in various processes of the development of human cancers,including the proliferation,apoptosis,differentiation,migration and angiogenesis. Abnormal expression of miRNAs is observed in breast cancer and has been linked to the molecular phathogenesis of of breast cancer by means of their ability to impact the expression of crucial target mRNAs.MiRNAs can function as oncogenes or tumor suppressor genes depending on the target genes.For instance, miR-206,miR-34a,miR-200c,let-7,miR-335,miR-126,miR-101,miR-125a/b and miR-145 are downregulated and regulate multiple genes expression during breast cancer carcinogenesis and progression.In contrast, other miRNAs,such as miR-21,miR-155,miR-10b,miR-27a and miR-221/222 have been found to act as oncogenes in breast cancer. Exploration of cancer-specific miRNAs and their target genes is crucial for realizing their role in tumorigenesis and might be critical for defining novel therapeutic targets.The miR-29 family was firstly found in Hela cell in 2001,consisting of miR-29a,miR-29b and miR-29c.Researchers found that miR-29 family function as tumor suppressor genes in many human cancers.MiR-29b,the most important member of miR-29 family, can be divided to two molecules with same sequence:miR-29b-l and miR-29b-2. It has been pointed out that the expression of miR-29b relates to the molecular classifation of breast cancer.Compared to other subtypes of breast cancer,the expression level was much lower in HER2-overexpression subtype. HER2/neu (c-erb-B2) gene,which is closely correlated to tumor cell growth, invasion and metastasis,is the main pathogenic gene of breast cancer. We can detect amplifaction of HER2 gene and/or overexpression of HER2 product in 20-30% patients with breast cancer.As we know,HER2 overexpression is associated with poor prognosis. Therefore, to explore the function and mechanism of miR-29b will provide new ideas for the treatment especially in patients with HER2-overexpressing breast cancer.After prediction and screening,we discovered that the signal transducer and transaction factor 3 (STAT3) is a potential target gene of miR-29b. STAT3 is an important transcription factor of JAK/STAT pathway. STAT3 plays a key role in normal physiological function in cells after activation.STAT3 is the cross point of numerous oncogenic tyrosine kinase pathway such as Src,EGFR and IL2/6-JAK. Emerging researches suggested that STAT3 could be an oncogene in a variety of tumors including breast cancer.Its excessive activation will promote abnormal overexpression of key genes, which are closely correlated with cell growth, apoptosis and differentiation. Activation of STAT3 can also induce the expression ofc-myc,cyclin D1/D2, MMP (matrix metalloproteinase) and VEGF (vascular endothelial growth factor).Additionally,STAT3 is correlated with biological behaviors of HER2-overexpressing breast cancer, such as lymph node metastasis, tumor invasion and drug resistance,etc.It was observed that STAT3 bound to the HER2 promotor and stimulated the expression of HER2 mRNA and protein.Then HER2 induced secretions of IL-6,which could stimulate STAT3 to form a positive loop of HER2-IL-6-STAT3.In our study,we focused on the function and mechnism of miR-29b in HER2-overexpressing breast cancer.Methods一. The expression of miR-29b in breast cancer and its clinical significance1. Breast cancer tissue samples,matched non-tumor adjacent tissue samples and clinicopathological characteristics collectionSixty-seven pairs of human breast cancer and their matched non-tumor adjacent tissue samples were collected from patients who underwent surgical resection at Nanfang Hospital between 2012 and 2013, and were diagnosed with primary BC based on histopathological evaluation.All sample tissues were fixed in 10% formalin and embedded in paraffin.These patients had no local or systemic treatments before the operation.2. Detection of the relative expression levels of miR-29b in tissue samplesTotal RNA was extracted with the miRNeasy FFPE Kit from QIAGEN.We used the taqman stem-loop qRT-PCR assay to detect the mature miRNA.cDNA was amplified using the SYBR Premix Ex taq.3. Judgement of the relative expression levels of miR-29b in tissue samplesThe mean cycle threshold(Ct) was determined by triplicate PCR runs,and the relative expression level was normalized to that of the internal control U6 snRNA and calculated using 2-△△Ct method.Relativechanges in the expression level of BC were found to be compared to the non-tumorous tissue adjacent to cancers.Therefore,the value of the relative ratio(lgC/N)< 0 was considered to indicate downregulation in cancer relative to non-tumorous control,and the ratio(lgC/N)≥0 was taken as normal/upregulation.4. Exploration of the correlation of miR--29b and patients’clinicophathological features5. Analyses of the relative expression level of miR-29b of different subtypes in 67 BC patients.二. Effect of miR-29b overexpression on the proliferation and invasion behaviors of HER2-overexpressing breast cancer1. Condition of cell culture and detection of the relative expression of miR-29b in cellsHuman BC cell lines SK-BR-3 and MDA-MB-435 were propagated in McCoy’5a supplement and DEME supplement,respectively.Total RNA was extracted from tissue samples using Trizol reagent.We used the taqman stem-loop qRT-PCR assay to detect the mature miRNA.cDNA was amplified using the SYBR Premix Ex taq.2. Transfection of miR-29b mimics in SK-BR-3 and MDA-MB-453 breast cancer cellsTransfection was performed with Lipofectamine 2000 reagent according to the manufacturer instructions.3. Effects of miR-29b overexpression on the proliferation and invasive behaviors of HER2-positive breast cancer cellsAfter transfection,the cell proliferation in vitro was performed by using a cell counting KIT-8(CCK-8 assay),cell cycle analisis and the apoptosis assay byFlow Cytometry(FCM).We used the transwell matrigel invasion assay to determine the invasive ability after transfection in two cell lines. Then we builded the animal model of BC to observe the tumor growing in breast cancer.H. To determine the target gene of miR-29b in HER2-overexpressing cellsand to test the regulation of miR-29b for the expression of STAT31. Construction of wild-type STAT33’UTR (STAT3_WT) mutant-type STAT3 3’UTR (STAT3_MUT) plasmid2. Dual-luciferase reporter assay:A mixture of 50nm miR-29b mimics and 0.5μg of reporter plasmid was cotransfected into SK-BR-3 and MDA-MB-435 cells using Lipofectamine 2000.After 48h,luciferase activity were measured using a system of dual luciferase reporter assay.3. Detection of the relative transcriptional levels of STAT3 in SK-BR-3 and MDA-MB-453 cells was performed using qRT-PCR4. Detection of the relative levels of STAT3 protein was performed using western-blot四. MiR-29b-mediated downregulation of STAT3 is involved in tumor suppression of HER2-positive breast cancer cells1. Transfection of si-STAT3/control in SK-BR-3 and MDA-MB-435 breast cancer cellsTransfection was performed with Lipofectamine 2000 reagent according to the manufacturer instructions.2. Effects of knocking down STAT3 expression on the proliferation and invasive behaviors of HER2-overpressing breast cancer cellsAfter transfection,the cell proliferation in vitro was performed by using a cell counting KIT-8(CCK-8 assay),cell cycle analisis and the apoptosis assay by Flow Cytometry(FCM).We used the transwell matrigel invasion assay to determine the invasive ability after transfection in two cell lines.3. Rescue experimentCotransfection of pcDNA-STAT3/pcDNA 3.1 and miR-29b mimic/scramble was performed by using Lipofectamine 2000.After transfection,we used CCK-8 assay to analyze cell proliferation and transwell assay to observe the invasive ability in SK-BR-3 cell line.5. Effects of miR-29b overexpression on relative protein levels of STAT3 and its downstream targets1. Transfection of miR-29b mimic/scramble or si-STAT3/control in SK-BR-3 and MDA-MB-435 breast cancer cells3. Detection of the relative protein levels of STAT3 and its downstream targets were performed using western-blot4. Detection of the expression of HER2 and STAT3 in miR-29b mimic and scramble transfected groups in animal model was performed using IHC analysisResults一. The expression of miR-29b in breast cancer and its clinical significance1. The mean of 2-△△Ct of miR-29b in breast cancer tissues is 1.338±0.776, and mean of 2-△△Ct of miR-29b in matched non-tumor adjacent tissues is 3.528±1.980.The expression level of miR-29b in breast cancer tissues is significantly reduced(t=-8.060, P< 0.001).2. Among the 67 patients with primary breast cancer,50(74.63%) showed a reduction in the expression level of miR-29b.3. Univariate analyses showed that:①The patients with bigger tumor size(≥5cm) and HER2-positive status tended to have levels of miR-29b expression(P< 0.01; P< 0.01);②No significant correlation was found between miR-29b expression and age, histology, status of ER, PR and nodal metastasis (P>0.05)4. Multivariate analysis identified that the expression of miR-29b was closely related with tumor size(≥5cm) and HER2 positive status(P< 0.01; P< 0.01),the risk of miR-29b reduction in breast cancers patients with tumor size(≥5cm) and HER2 positive status was increased to 27.617 and 11.284,respectively.5. Compared to another three subtypes of breast cancer,HER2-overexpressing breast cancer patients was significantly decreased(VS.luminal A/B P< 0.01; VS. BasalP<0.05).二. Effect of miR-29b overexpression on the proliferation and invasive ability of HER2-overexpressing breast cancer cells1. The expression level of miR-29b was significantly decreased in two HER2-overexpressing breast cancer cell lines SK-BR-3 and MDA-MB-435relative to normal breast cell Hs578Bst,MDA-MB-231(Basal),BT474(luminal B) and MCF-7(luminal A).All of the P value was< 0.05.2. QRT-PCR detection found that miR-29b expression level to be markedly increased in SK-BR-3 and MDA-MB-435 cells with mimic transfection(P< 0.001).3. Using CCK-8 assay to test the effect of miR-29b overexpression on cell proliferation ability in SK-BR-3 and MDA-MB-435 cells:in 48h and 72h after transfection,the restoration of miR-29b expression by miR-29b mimics to suppress cell proliferation.4. The cell cycle distribution of SK-BR-3 and MDA-MB-435 cells showed that the number of cells in the G1 phase was greater in cells transfected with miR-29b mimics than in scramble.The number of cells in the S phase dropped remarkably.5. The effects of miR-29b on apoptosis were also detected by FCM.Data showed that miR-29b could promote cell apoptosis in SK-BR-3 and MDA-MB-435 cells with miR-29bmimics transfection relative to scramble controls.6. We examined the effects of miR-29b overexpression on the invasive ability in SK-BR-3 and MDA-MB-435 cells by using transwell assay.Results showed a significant decrease in the cells per field with miR-29bmimics transfection compared to control groups.7. In BC animal model,enforced expression of miR-29b resulted in a decrease weight of tumor(P< 0.001);the volume of tumor is significantly different between two groups(P< 0.01).三. To determine the target gene of miR-29b in HER2-overexpressing BC and to test the regulation of miR-29b for the expression of STAT31. We conducted the wild or mutant type of STAT3 3’UTR luciferase reporter plasmid.2. The wild and mutant type of STAT3 3’UTR plasmids were cotransfected in SK-BR-3 and MDA-MB-435 cells with miR-29b mimic or scramble control.Relative luciferase activities were tested by the dual-luciferase reporter assay system and normalized by the firefly luciferase activity by that of Renilla luciferase.The cells cotransfected with wild type STAT3 3’UTR plasmid and miR-29b mimics showed a siginificant decrease in relative luciferase activity.No remarkable changes in relative luciferase activity were observed in cells cotransfected with mutant type STAT3 3’UTR plasmid and miR-29b mimic.The results showed that miR-29b could bind to human STAT3 mRNA 3’UTR.3. We used qRT-PCR to examine STAT3 mRNA levels in SK-BR-3 and MDA-MB-435 cells transfected with miR-29b mimic or scramble control.Fourty-eight hours after transfection,we observed a depression in STAT3 mRNA in miR-29b mimic transfected groups in both cell lines.4. Western-blot showed a significant reduction in STAT3 protein levels in both cell lines after transfection with miR-29b mimic relative to scramble control.四. MiR-29b-mediated downregulation of STAT3 is involved in tumor suppression of HER2-positive breast cancer1. We used STAT3-siRNA to knock down the expression of STAT3 in both HER2-overexpressing breast cell lines.2. CCK8 assay found a depression in cell proliferation in STAT3-siRNA group of SK-BR-3 and MDA-MB-435 cell lines.Inhibition of STAT3 significantly reduced the invasive cells per field.FCM detection showed a higher apoptosis ratio,a greater number of cells in the G1 phase but a remarkable reduction in the number of cells in the S phase in STAT3-siRNA groups.3. Rescue experiment indicated that after overexpression of miR-29b,restoration of STAT3 could partially reersed miR-29b inhibitory functions of HER2-overexpressing breast cancer.The results suggested that miR-29b directly targeted STAT3.五. Effects of miR-29b overexpression on relative protein levels of STAT3 and its downstream targetsWestern-blot found significant reductions in relative expression levels of STAT3 and its downstream proteins in si-STAT3 or miR-29b mimic group relative to their controls,respectively.Conclusions1. MiR-29b is downregulated in most cases of breast cancers and closely associated with bigger tumor size and positive status of HER2 expression in patients.Compared to another three subtypes of breast cancer,the expression level of miR-29b was significantly lower in tissues and cells fell to HER2-overexpressing subtype.lt indicated the tumor-suppressing of miR-29b in HER2-overexpressing BC.2. MiR-29b can suppress HER2-overexpressing breast cancer growth ability in vitro and in vivo.3. Further studies indentified STAT3 as a downstream target of miR-29b in HER2-overexpressing breast cancer,and miR-29b reduced both mRNA and protein levels of STAT3.4. STAT3 can promote HER2-overexpressing breast cancer cells proliferation,migration and cell cycle in vitro,and inhibit cell apoptosis.5. Western-blot also showed miR-29b depressed the protein levels of STAT3 and its downstream targets,including HER2,CCND2 and MMP2. |
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