| Breast cancer,the most common cancer among women,remains the leading cause of cancer-related deaths in the world.The five-year survival rate for breast cancer ranges from 40 percent in low-income countries to 80 percent in developed countries.Data from the American Cancer Association show that almost 1.3 million women worldwide are diagnosed with breast cancer every year,and more than 465,000 of them die of breast cancer.Currently,treatments for tumors mainly include radiation and chemotherapy.However,in the course of chemotherapy,patients with metastatic breast cancer often produce multidrug resistance(MDR),which ultimately leads to the failure of chemotherapy.New more effectively treatments are urgently needed to treat metastatic breast cancer.Therefore,it is necessary to make a deeper study of the molecular mechanism of drug resistance and explore drug resistance targets in breast cancer,so as to provide important research and theoretical basises for reversing the anti-drug treatment of breast cancer.ABC transporters are able to pump drugs out of the cell,so they could reduce the accumulation of drugs inside the cells and thus increase cell resistance.ABCC4,as a member of the ABC transporter family,is related to the resistance to various chemotherapy drugs in many cancers.The resistance of breast cancer cells to chemotherapy drugs is closely related to the high expression of ABC transporter.However,the expression,the biological roles and potential mechanisms of ABCC4 in breast cancer drug-resistant have not been reported yet.MicroRNAs(miRNAs)are highly conserved small non-coding small RNAs,and they could regulate the expression of target genes at post-transcriptional levels.Growing evidence has shown that miRNAs play important roles in multidrug resistance in cancer chemotherapy.Studies have disclosed that miR-124-3p has an inhibitory effect during tumor development.A study has reported that miR-124-3p expression was downregulaed in gastric cancer tissue,and it could target CD151 and restrain its expression,inhibiting gastric cancer cell proliferation and migration as well as promoting apoptosis,which plays an important role in the process of the occurrence and development of gastric cancer.In addition,miR-124-3p expression was down-regulated in the study of bladder cancer and nasopharyngeal carcinoma,which are involved in tumor growth and metastasis.However,the function and possible molecular mechanisms of miR-124-3p in the occurrence and development as well as multidrug resistance in breast cancer are still unclear.Therefore,this study intends to explore the influence of miR-124-3p,ABCC4and double-targeted miR-124-3p and ABCC4 on resistance of breast cancer and possible molecular mechanisms through molecular biology and cell biology experimental methods.First,the expression of miR-124-3p and ABCC4 was detected by immunohistochemical staining and qRT-PCR in breast cancer tumor tissue and normal tissue adjacent to carcinomas.I constructed multidrug-resistant breast cell line MCF-7-ADR by exposing increasing concentrations of Adriamycin.qRT-PCR was used to determine the levels of miR-124-3p and ABCC4 in normal breast cell MCF-7-10A and breast cancer cell MCF-7 cells and breast cancer resistant cell MCF-7-ADR.In vitro,we constructed the miR-124-3p overexpression,ABCC4knockdown and dual-targeting miR-124-3p and ABCC4 lentivirus vectors.Virus packaging was conducted to gain adenoviruses(LV-miR-124-3p,LV-sh-ABCC4 and LV-miR-124-3p+sh-ABCC4)and then MCF-7-ADR cells were infected with adenoviruses to gain Stable transfected cell lines(MCF-7-ADR-miR-124-3p,MCF-7-ADR-sh-ABCC4 and MCF-7-ADR-miR-124-3p+sh-ABCC4).Then CCK-8,flow cytometry,transwell and scratch assays were conducted to detect the effects of miR-124-3p overexpression,ABCC4 knockdown and dual-targeting miR-124-3p and ABCC4 on cell function and driamycin(ADR)drug sensitivity of MCF-7-ADR cells.Meanwhile,qRT-PCR and Western blot were used to study the drug resistance molecular mechanisms in MCF-7-ADR cells.Finally,in vivo,we established MCF-7-ADR tumor-burdened animals and a further study of the effects of miR-124-3p overexpression,ABCC4 knockdown and dual-targeting miR-124-3p and ABCC4 on multidrug resistance and the possible molecular mechanisms in breast cancer.This research mainly focused on the effects of miR-124-3p,ABCC4 and dual-targeting miR-124-3p and ABCC4 on multidrug resistance and its molecular mechanisms in breast cancer,which will provide a new avenue for the treatments of the resistance to drug-resistant in breast cancer.This project is divided into three parts:Part ?:The study of expression of ABCC4 and miR-124-3p in breast cancer tissue and cells;Part ?:The study of dual-targeting miR-124-3p and ABCC4 on multidrug resistance and its mechanisms in breast cancer cells;Part ?:The study of dual-targeting miR-124-3p and ABCC4 on multidrug resistance in vivo.Part ?:The study of expression of ABCC4 and miR-124-3p in breast cancer tissue and cellsMethods1.Immunohistochemical staining was used to detect the expression of ABCC4protein in breast cancer tumor tissue and normal tissue adjacent to carcinomas.2.qRT-PCR was used to detect the expression of miR-124-3p and ABCC4 in breast cancer tumor tissue and normal tissue adjacent to carcinomas.3.Multidrug-resistant breast tumor cell line MCF-7-ADR was established by exposing increasing concentrations of adriamycin.4.CCK-8 assay was used to assess the toxicity of adriamycin to MCF-7 cells and MCF-7-ADR cells as well as their IC50 values.5.Cloning abilities of MCF-7 and MCF-7-ADR cells was detected by colony formation assay.6.qRT-PCR was used to determine the levels of miR-124-3p and ABCC4 in normal breast cell MCF-10A and breast cancer cell MCF-7 cells and breast cancer resistant cell MCF-7-ADR.Results1.Immunohistochemical staining results showed that ABCC4 expression was significantly increased in breast cancer tissue,compared to normal tissue adjacent to carcinomas.2.The results from qRT-PCR analysis showed that ABCC4 expression was significantly increased in breast cancer tissue,while miR-124-3p level was significantly decreased in breast cancer tissue,compared to normal tissue adjacent to carcinomas.3.We observed that MCF-7-ADR cells were normally grown in monolayer under microscope.CCK-8 assay revealed that the resistance index was 17.498.MCF-7-ADR cell clone formation ability was significantly increased,compared with MCF-7 cells.All the results implied that MCF-7-ADR cell was successfully constructed.4.The results from qRT-PCR analysis revealed that ABCC4 expression was significantly higher in MCF-7 cells and MCF-7-ADR cells than that in MCF-10A,while miR-124-3p level was significantly decreased in MCF-7 cells and MCF-7-ADR cells.Part ?:The study of dual-targeting miR-124-3p and ABCC4 on multidrug resistance and its mechanisms inbreast cancer cellsMethods1.The construction of miR-124-3p overexpression lentivirus vector:The pCDH-CMV-MCS-EF1-copGFP-T2A-Puro and miR-124-3p plasmids were both digested with EcoRI and NotI,and then they were ligated and miR-124-3p overexpression lentivirus vector was gained.pLenti-miR-124-3p plasmid was transformed.Colony PCR and gene sequence in identifying the recombinant vector were performed.2 The construction of ABCC4 interference lentiviral vector:The design and synthesis of ABCC4 interference sequence were performed.pLenti-hU6-GFP-Puro vector was digested with AgeI and EcoRI.ABCC4 interference sequence and pLenti-hU6-GFP-Puro lentiviral vector was ligated and ABCC4 interference lentiviral vector(pLenti-sh-ABCC4)was gained.Then pLenti-sh-ABCC4 plasmid was transformed.Colony PCR and gene sequence in identifying the recombinant vector was carried out.3.The construction of dual-targeting miR-124-3p and sh-ABCC4 lentivirus vectors:pLenti-miR-124-3p vector was digested by NotI,and sh-ABCC4 fragment was inserted into pLenti-miR-124-3p vector.The pLenti-miR-124-3p+sh-ABCC4plasmid was transformed.Colony PCR and gene sequence in identifying the recombinant were performed.4.Recombinant lentivirus packaging and titration:the recombinant lentiviruses(pLenti-miR-124-3p,pLenti-sh-ABCC4orpLenti-miR-124-3p+sh-ABCC4),pCMV-?8.2 and pCMV-VSV-G plasmids were co-transfected into HEK293T cells.At 72 h post-transfection,GFP fluorescence expression was observed under the fluorescence microscope.The titration of virus was performed after concentration.5.The infection efficiency of three lentivirus particles(LV-miR-124-3p,LV-sh-ABCC4 and LV-miR-124-3p+sh-ABCC4)in MCF-7-ADR cells was observed under the fluorescence microscope.6.CCK-8,flow cytometry,Transwell and scratch assays were conducted to detect cell proliferation,cell cycle,apoptosis invasion and migration of MCF-7-ADR,MCF-7-ADR-miR-124-3p,MCF-7-ADR-sh-ABCC4,and MCF-7-ADR-miR-124-3p+sh-ABCC4 cells.7.The sensitivity of cells to adriamycin in all groups was detected by CCK-8assay.8.qRT-PCR was used to detect the expression of miR-124-3p and ABCC4,and the expression of ABCC4,p53,and P-gp protein was detected by Western blot in all groups.Results1.The results of colony PCR,sequencing and comparison of pLenti-miR-124-3p,pLenti-sh-ABCC4,and pLenti-miR-124-3p+sh-ABCC4 showed that the molecular sizes and sequences were consistent with data in the NCBI database.The results showed that vectors were successfully constructed.2.The fluorescence observation showed that the efficiency of recombinant vectors,pCMV-?8.2 and pCMV-VSV-G co-transfecting into HEK293T cells was more than 90%,which could be used for the next experiment.3.The results of titration of virus showed that the titration value of LV-miR-124-3p,LV-sh-ABCC4,and LV-miR-124-3p+sh-ABCC4 was determined to be 2×10~8 TU/mL.4.Biological function detection showed that both miR-124-3p overexpression and ABCC4 knockdown can promote cell proliferation,invasion and migration,and hinder the normal cell cycle process,as well as promote apoptosis.Moreover,dual-targeting miR-124-3p and ABCC4 has more obvious effects on cell function.5.CCK-8 assay exhibited that both miR-124-3p overexpression and ABCC4knockdown significantly promoted the sensitivity to adriamycin of MCF-7-ADR,and combination of miR-124-3p overexpression and ABCC4 knockdown has promotive effect on drug sensitivity of MCF-7-ADR cells,compared to miR-124-3p overexpression and ABCC4 knockdown MCF-7-ADR cells.6.Results from qRT-PCR and Western blot analyses showed that the expression of miR-124-3p was significantly upregulated in MCF-7-ADR-miR-124-3p cells and ABCC4 expression was significantly downregulated in MCF-7-ADR-sh-ABCC4cells through lentivirus-mediated gene recombination techniques.Moreover,miR-124-3p overexpression or ABCC4 knockdown significantly inhibited the p53protein expression in MCF-7-ADR cells,and the inhibition effect of dual-targeting miR-124-3p and ABCC4 is more apparent.miR-124-3p overexpression significantly inhibited protein expression of P-gp in MCF-7-ADR cells.Part ?:The study of dual-targeting miR-124-3p and ABCC4 on multidrug resistance in vivo.Methods1.The mice models bearing MCF-7-ADR solid tumors were constructed by injecting MCF-7-ADR cells subcutaneously in the armpit of right forelimb and the length and width of tumor were determined by the vernier caliper every week.Two weeks after injection of MCF-7-ADR cells,each mouse received an intraperitoneal(IP)injection of adriamycin(2 mg/kg)twice a week.After 35 days,the tumor-bearing mice were sacrificed,and the weights of tumors were determined.2.HE staining was used to detect the cell morphological and histological characteristics in tumor tissues in tumor-bearing mice.3.qRT-PCR was used to detect the expression of miR-124-3p and ABCC4,and the protein expression of ABCC4,p53 and P-gp was detected by Western blot in tumor tissues in tumor-bearing mice.Results1.After treatment with adriamycin,compared with MCF-7-ADR group,tumor growth of mice with injecting MCF-7-ADR-sh-ABCC4 or MCF-7-ADR-miR-124-3p cells was significantly inhibited.And the growth inhibition efficiency of doxorubicin in the tumor of mice with injecting MCF-7-ADR-miR-124-3p+sh-ABCC4 cells was significantlyhigherthanthoseinMCF-7-ADR-sh-ABCC4and MCF-7-ADR-miR-124-3p groups.Compared with the MCF-7-ADR group,the weights of the tumor of mice with injecting MCF-7-ADR-sh-ABCC4 or MCF-7-ADR-miR-124-3p cells were significantly decreased,and the weights of the tumor of mice with injecting MCF-7-ADR-miR-124-3p+sh-ABCC4 cells were significantlylowerthanthoseinMCF-7-ADR-sh-ABCC4and MCF-7-ADR-miR-124-3p groups.2.HE staining results showed that there existed an obvious difference in the pathological morphology of tumor tissues of tumor-bearing mice,and the heterogeneity was the most obvious and a large number of tumor cells were observed in MCF-7-ADR group.Cancerous tissue necrosis,tumor cell number decrease,and cell shriveling were observed in tumor tissues of mice with injecting MCF-7-ADR-miR-124-3p,MCF-7-ADR-sh-ABCC4and MCF-7-ADR-miR-124-3p+sh-ABCC4 cells.That phenomenon was the most obvious in MCF-7-ADR-miR-124-3p+sh-ABCC4 group.3.Results from qRT-PCR and Western blot analyses showed that ABCC4mRNA and protein levels were significantly reduced in the tumor tissues of mice with injecting MCF-7-ADR-sh-ABCC4 or MCF-7-ADR-miR-124-3p cells,compared with MCF-7-ADR group,and the mRNA and protein levels of ABCC4 in tumor tissues of mice with injecting MCF-7-ADR-miR-124-3p+sh-ABCC4 cells were significantly reduced,compared with MCF-7-ADR-sh-ABCC4 and MCF-7-ADR-miR-124-3p groups.miR-124-3p level was significantly increased in tumor tissues of mice with injecting MCF-7-ADR-miR-124-3p or MCF-7-ADR-miR-124-3p+sh-ABCC4 cells,compared with MCF-7-ADR group.Moreover,miR-124-3p overexpression or ABCC4 knockdown significantly inhibited the p53 protein expression in the tumor tissues of mice,and the inhibition effect of dual-targeting miR-124-3p and ABCC4 is more apparent.miR-124-3p overexpression significantly inhibited protein expression of P-gp in the tumor tissues of mice.Conclusions1.ABCC4 expression was significantly increased in tumor tissues of patients with breast cancer,while miR-124-3p level was significantly decreased.Moreover,ABCC4 expression was also significantly increased in MCF-7 and MCF-7-ADR cells,while miR-124-3p level was significantly decreased.2.Overexpression of miR-124-3p and downregulation of ABCC4 inhibited the proliferation,invasion and migration of MCF-7-ADR cells.In addition,overexpression of miR-124-3p and downregulation of ABCC4 hindered cycle progression of MCF-7-ADR cells,promoted apoptosis,and enhanced the sensitivity to adriamycin of MCF-7-ADR cells,thereby inhibiting the breast cancer cell drug resistance.Moreover,the effects dual-targeting miR-124-3p overexpression and downregulation of ABCC4 is more obvious,and P-gp and mutant p53 proteins were involved in that process.3.In vivo,overexpression of miR-124-3p and downregulation of ABCC4significantly promoted sensitivity to adriamycin tumor of mice bearing MCF-7-ADR tumor,and the positive effect of dual-targeting miR-124-3p overexpression and downregulation of ABCC4 on sensitivity to adriamycin tumor of mice bearing MCF-7-ADR tumor is more obvious,and P-gp and mutant p53 proteins were involved in that process.Therefore,dual-targeting miR-124-3p overexpression and ABCC4 downregulation may serve as an ideal therapeutic target for reversing the drug resistance of breast cancer. |
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