| BackgroundBreast cancer(BC)is a malignant tumor with the highest incidence and mortality in women.The mortality of breast cancer is high in developed cities of China,just less than lung cancer.Breast cancer has a high degree of malignancy and is easy to metastasize.Currently,the breast cancer positive family genes,such as BRCA-1 and BRCA-2 has been shown associated with the pathogenesis of breast cancer.The pathological process of breast cancer is often accompanied by the differential expression of non-coding RNA.These noncoding RNAs may perform key functions.However,the pathogenesis of breast cancer is affected in many ways,and the exact mechanism is still unclear.Breast cancer is a blood-rich tumor.In the middle and advanced stage of breast cancer,multivessel artery participated in the blood supply and nutrition in tumors,leading to an increase in cancer stages.Mutiple therapy methods has been used in breast cancer,including radiotherapy,chemotherapy,endocrine therapy,molecular targeted therapy,etc.However,the limitation of the above methods is obvious in breast cancer patients with the middle and advanced stage.Transcatheter arterial chemoembolization(TACE)is a common method for the treatment of solid tumors atpresent,and the curative effect is certain.At present,regional arterial catheterization is a new and effective method for breast cancer treatment.This method can block and cut off the blood supply artery of primary tumor and reduce the blood supply of tumor.At the same time,the method can increase the concentration of local chemotherapeutic drugs,thus significant decrease the proliferation of primary tumor cells,increase apoptosis,and make the surrounding tissues ischemic,necrotic,and localize the lesion.The boundaries are clear,thus reducing tumor staging.However,due to the variety of breast cancer types and the complicated pathogenesis,it is difficult to choose the time of transcatheter arterial chemoembolization and post-chemotherapy surgery for breast cancer,so it is necessary to study the molecular mechanism of breast cancer,and to find ideal biomarkers for transcatheter arterial chemoembolization.At present,the main drugs that can be used in transcatheter arterial chemoembolization for breast cancer are anthracycline,vincristine,platinum,yew drugs,alkylators and antimetabolic drugs.The toxicity of chemotherapeutic agents to tumor cells is concentration-dependent.Although there is no uniform standard for the types and doses of drugs for transcatheter arterial chemoembolization for breast cancer,strict screening is required.Drug combinations with strong toxicity and weak adverse effects are the focus of clinical research currently.Pirarubicin is a new anthracycline drug which damage cells by blocking the replication of DNA and affects angiogenesis in breast,bladder and colon cancer.Mitomycin disaggregates DNA and obstructs the replication of DNA in many cancers,including breast cancer.Both pirarubicin and mitomycin are ideal for arterial embolization chemotherapy.Although arterial chemoembolization is stable in many cancers,however,experts and scholars are still divided on the use of non-operative transcatheter arterial chemoembolization for advanced breast cancer and the selection of surgical timing after transcatheter arterial chemoembolization.Therefore,it is necessary to further clarify the molecular mechanism of the tumorigenesis and development of breast cancer.To explore the appropriate therapeutic effect of transcatheter arterial chemoembolization,search for molecular markers that can reflect the curative effect of transcatheter arterial chemoembolization for breast cancer,and further promote the practice and theory of transcatheter arterial chemoembolization for breast cancer.Part one: Clinical observation of transcatheter arterial chemoembolization with pirarubicin and mitomycin in the treatment of breast cancer ObjectiveTo observe the clinical effects of transcatheter arterial chemoembolization of combined with pirarubicinthp and mitomycin in the treatment of unresectable advanced breast cancer.Methods1.Basic clinical dataTotal of 40 patients with locally advanced breast cancer admitted from June2013 to June 2016 in the affiliated Cancer Hospital of Zhengzhou University.All patients with complete follow-up data were confirmed by puncture biopsy or surgery and pathology.2.Transcatheter arterial chemoembolizationExperimental group(TACE): the patients were supine.The blood supply branches of breast tumor were perfused with chemotherapy drugs(THP 60 ~ 80 mg / ml,MMC10 mg).The drugs were injected slowly for 10 min.The tumor embolization process was: intubation to the feeding artery of breast tumor and embolization to complete occlusion of the supply artery of the tumor.Control group(CHEM): chemotherapy drugs were given systemic intravenous chemotherapy with THP 60~ 80 mg,MMC 10 mg.3.Result criterionResults according to the criteria for evaluating the curative effect of solid tumors,the response evaluation criteria in solid tumors were evaluated,and the clinical chemotherapeutic effects of the two groups were compared.Complete response(CR)the tumor disappeared completely and there were no new lesions.Partial partial response(partial response.PR)refers to the reduction of the tumor ? 50% and no new lesion;the stable state of the disease,no change refers to the contraction of the tumor DX < 50% and no new lesion;the progressive disease(PD)means that the tumor does not shrink or has a new lesion,in which CR and PR are effective.That is,the total validNC and PD are invalid.4.Follow-up of tumor progression and survivalthe effect was good and the operation was performed.The progression and survival rate of the tumor were followed up for 3 months,9 months,12 months,18 months.Results1.The clinical efficacy of transcatheter arterial chemoembolizationAfter transcatheter arterial chemoembolization,local mass and axillary lymph nodes of 20 patients began to shrink and soften at day 2~3.The degree of motion increased,the color of skin became lighter and the mobility of patients with adhesion between cancer and chest wall was obviously improved at day 2~3.There were 5cases of clinical complete remission of crus,12 cases of clinical partial remission,3cases of no change,0 cases of progressive disease.The total effective rate was 85%.The systemic intravenous chemotherapy group had 1 case of complete clinical remission.There were 10 cases of partial remission,9 cases of no change and 0 cases of progressive disease.The total effective rate was 59.4%.The difference between the two groups was statistically significant(P =0.031,? 2 = 7.87).2.Histological changesIn the CHEM group,the difference between the tumor mass and surrounding tissue was not obvious.Histological examination showed invasive carcinoma,obvious degeneration,obvious tumor embolus in vascular,and no clear cancer in left supraclavicular lymph node.Compared with the CHEM group,the anatomical specimens of the patients in the TACE group can be clearly observed at the interface between the tumor mass and the surrounding tissue,and the grayish white section and punctate necrosis could be observed,and the distribution of most blood vessels in the tumor mass was decreased.Microscopically,the left supraclavian lymph nodes after TACE showed no clear patchy necrosis of cancer tissue.Inflammatory infiltration and proliferation of fibrous tissue can be found around flake necrosis,and endothelial cell proliferation.3 Results of tumor progression and survival follow-upAll patients were followed up to June 31,2017.The follow-up period was 3 ~ 18 months,and 6 patients died.Among them,3 cases died of lung metastasis,2 cases died of liver metastasis,1 case died of brain metastasis.The survival rate of CHEMgroup at 3,9,12,18 months were 100.0%(20 / 20),90%(18 / 20),85%(17 / 20),and80%(16 / 20).The survival rate of TACE was 100.0%(20 / 20),90%(18 / 20),95%(18 / 20),and 90%(18 / 20)respectively.There was no difference between the two groups at 3 and 9 months.The 12-and 18-month survival rates were significantly different between the two groups(P =0.003,P =0.001).ConclusionTACE has potential application in the treatment of advanced unresectable BC.The clinical effect of TACE still needs further study.TACE can significantly inhibit the growth of BC,angiogenesis and metastasis,and has less adverse reactions.Part two: The effects of different concentrations of pirarubicin combined with mitomycin on breast cancer cells in vitro ObjectiveTo explore the effects of different concentrations of pirarubicin combined with mitomycin on the proliferation,tubule formation,migration and invasion of breast cancer cells,and to provide the experimental basis for exploring the molecular targets of transcatheter arterial chemoembolization.Materials and methods1.Routine culture of breast cancer cell line MCF-7(ER positive breast cancer cell line,low malignant degree)and MDA-MB-231(high metastatic breast cancer cell line).2.MTT kit was used to detect the absorbance of cell suspension at 570 nm and further study the effect of different concentrations of drug combination on the proliferation of breast cancer cells.In order to determine the time concentration of drug use,the optimal range of high dose and low dose is selected.3.The cell cycle was analyzed by DNA assay.The cells were stained with pyridine iodide.FACS Calibur Flow Cytometer was used to detect the content of DNA in G0/G1,S,and G2/M phase cells.4.Caspase3 activity assay was used to determine cell apoptosis.After adding detection buffer and Ac-LEHD-pNA,the results were detected.5.Tubule formation experiment.The matrigel matrix was coated on 96 well plate to inoculate breast cancer cells.After 8 hours of continuous culture,the cells were placed under 200 mesh microscopes.6.Cell migration ability was measured by scratch test after 24 hours of drug treatment.7.cell invasion ability was detected by Transwell after 24 hours of drug treatment.The invasive ability of breast cancer cells was detected.Results1.The optimal time for the combination of imirubicin and mitomycin was 24 h by MTT test.2.5 ?g/ml of pirarubicin and 1 ?g/ml of mitomycin were used as low concentration treatments,and 10 ?g/ml of pirarubicin and 5 ?g/ml of mitomycin were used as high concentration.2.Compared with control,the number of cells in G1 phase increased in low dose group and high dose group(P=0.022),the number of cells in S phase were decreased(P=0.012).The cell number in G1 phase in high dose group was significantly higher than that in G1 phase in low dose group,number of high dose cells in S phase were lower than that in low dose group(P=0.018).3.Compared with control,the caspase3 activity of low dose group and high dose group was significantly higher than control group(P=0.017),and the effect of high dose group was significantly higher than that of low dose group(P=0.031).4.Control,low dose group and high dose group formed tubules significantly inhibited(P=0.016),and the effect of high dose group was significantly higher than that of low dose group(P=0.033).5.Compared with control,the invasion and migration ability of low dose group and high dose group were significantly inhibited(P=0.017),and the effect of high dose group was significantly higher than that of low dose group(P=0.031).ConclusionThe effects of high concentration of pirarubicin combined with mitomycin on the proliferation,apoptosis,angiogenesis,migration and invasion of breast cancer cells are greater than those of low concentration drugs.Part three: Effects of pirarubicin and mitomycin on angiogenesis-related genes and non-coding RNAs in breast cancer cells ObjectiveTo investigate the effects of different concentrations of pirarubicin and mitomycin on angiogenesis related genes and non-coding RNAs in breast cancer cells.Materials and methods1.Tissue sources.Breast cancer tissue samples derived from the foregoing(part I)40patients with breast cancer after chemotherapy biopsy cases of paraffin wax.2.Routine culture of breast cancer cell line MCF-7(ER positive breast cancer cell line,low malignant degree)and MDA-MB-231(high metastatic breast cancer cell line).3.The expression of VEGF,EGFR,cyclin D1,and MMP2 in breast cancer were decteded by quantitative real-time PCR(qRT-PCR).The expression of breast cancer associated non-coding RNAs and microRNAs(ANRIL?BC016831?HIF1A-AS2?IGKV?LINC00052?MEG3?RP11-434D9.1?UCA1?let-7a?miR-21?miR-23?miR-29a?miR-124?miR-148a?miR-152?miR-206?miR-449a?miR-1266)by using qRT-PCR.Changes of the above genes and non-coding RNAs and microRNAs were detected in cancer cells.4.Western blot was used to detect the expression of BRCA-1,BRCA-2,VEGF cyclin D1 and MMP2 in breast cancer tissues and cells.Result1.The results of qRT-PCR detection of genes in tissues.Compared with before chemotherapy,20 cases in conventional chemotherapy group,VEGF was down-regulated in 8 cases,EGFR 7 cases,cyclin D1 3 cases,MMP2 5cases.But compared with before transcatheter arterial chemoembolization,20 cases in transcatheter arterial chemoembolization group,VEGF was down-regulated in 16 cases,EGFR 14 cases,cyclin D1 14 cases,MMP2 15 cases.Compared with systemic intravenous chemotherapy,lncRNA MEG3 was significantly up-regulated in transcatheter arterial chemoembolization group(P=0.001)and miR-1266 was significantly down-regulated(P=0.001).2.The results of qRT-PCR in breast cancer cellsCompared with control group,the levels of VEGF,EGFR,cyclin D1,MMP2,miR-1266 in low and high groups were decreased significantly(P < 0.05),and in high groups the levels of VEGF,EGFR,cyclin D1,MMP2,miR-1266 were significantly down-regulated than those in low concentration and high concentration groups(P < 0.05).The effect of high concentration on lncRNA MEG3 expression was stronger than that of low concentration(P < 0.05).3.Protein expression changesWestern blot was used to detect gene expression in breast cancer tissue.In 20 cases of conventional chemotherapy,VEGF was down-regulated in 6 cases,EGFR 7cases,cyclin D1 5 cases,MMP2 4 cases.But compared with before transcatheter arterial chemoembolization,in 20 cases in transcatheter arterial chemoembolization group,VEGF was down-regulated in 13 cases,EGFR 14 cases,cyclin D1 12 cases,MMP2 9 cases.Compared with control group,the levels of VEGF,EGFR,cyclin D1,MMP2 was significantly down-regulated in low concentration and high concentration groups in breast cancer cells(P < 0.05).The levels of VEGF,EGFR,cyclin D1,MMP2 in high groups was significantly lower than that in low groups(P < 0.05).4.Correlation study resultsThere was a negative correlation between BRCA-1/BRCA-2 gene and lncRNA MEG3 in transcatheter arterial chemoembolization.Negative correlation between BRCA-1/BRCA-2 gene and lncRNA MEG3 was also found in drug-treated cells.A positive correlation between BRCA-1/BRCA-2 and miR-1266 was fond in tissue and cells.ConclusionLncRNA MEG3 expression in breast cancer after transcatheter arterial chemoembolization was increased obviously,and the marker gene of breast cancer was negatively correlated with MEG3.miR-1266 in transcatheter arterial chemoembolization after breast cancer expression was significantly decreased and was positively correlated with breast cancer marker gene.It is indicated that MEG3 and miR-1266 were involved in the development of breast cancer,which can be used as transcatheter arterial chemoembolization therapy markers.Part four Mechanism research of pirarubicin&mitomycin-mediated anti-angiogenesis of breast cancer in vitro ObjectiveTo explore the mechanism of Pirarubicin and mitomycin in regulating angiogenesis in breast cancer cells.Materials and methods1 Conventional culture of breast cancer cell lines MCF-7 and MDA-MB-231.The cells were treated with low and high concentrations of pirarubicin and mitomycin chemotherapeutic drugs.2 Construction of MEG3 overexpression lentivirus vector and low expression lentivirus vector,and construction of miR-1266 overexpression vector.3 Detect the changes of MEG3 and miR-1266 levels by using qRT-PCR.4 Effects of MEG3 overexpression or suppression on angiogenesis of breast cancer cells by tubulogenesis experiment.5 Protein level changes of MMP2,N-cadherin,twist1 were tested by western blot.6 The targeting relationship between of lncRNA and miRNA was predicted by bioinformatics methods and online databases.7 Luciferase reporter gene experiment was used to explore the binding of MEG3 to miR-1266.8 Pull down experiment confirmed the combination of MEG3 and miR-1266.Results1.The successful construction of lentivirus vector and miR-1266 overexpression vector.The stable high expression MEG3 lentivirus vector and silencing MEG3 vector was successfully constructed,the miR-1266 overexpression vector were constructed.2.Overexpression of MEG3 significantly inhibited angiogenesis in breast cancer cells and the expression of VEGF(P=0.028).Suppression of MEG3 significantly abolished the inhibition effects of high concentration of chemotherapeutic drugs on angiogenesis of breast cancer cells(P=0.023).3.Overexpression of MEG3 significantly inhibited the expression of MMP2,N-cadherin,twist1 in breast cancer cells.Suppression of MEG3 significantly abolished the inhibition effects of high concentration of chemotherapeutic drugs on epithelial-mesenchymal transition markers(MMP2,N-cadherin,twist1).4.MEG3 overexpression significantly inhibited the level of miR-1266(P=0.001)5.Bioinformatics analysis and double luciferase reporter gene analysis showed that miR-1266 and MEG3 had direct binding relationship.6.Pull down study confirmed that lncRNA MEG3 directly regulated the level of miR-1266 in breast cancer cells.7.MiR-1266 overexpression significantly alleviated the inhibitory effect of high concentration of chemotherapeutic drugs on epithelial-mesenchymal transition and angiogenesis in breast cancer cells.Conclusion LncRNA MEG3 can be used as a molecular marker of transcatheter arterial chemoembolization for breast cancer.LncRNA MEG3/miR-1266 may be one of the important mechanisms of transcatheter arterial chemoembolization on the angiogenesis of breast cancer. |
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