| According to the World Health Organization,breast cancer is a malignant tumor that seriously puts women’s health at risk.On average,one out of every eight adult women suffers from breast cancer.The incidence and mortality of breast cancer are the highest among female malignant tumors.Although scholars from all over the world have carried out a lot of research on the pathogenesis of breast cancer in recent years,and have achieved rapid progress,such as precise treatment under the guidance of breast cancer molecular typing,research and development of various new targeted drugs,etc.,The progression-free survival and overall survival of breast cancer patients have improved significantly compared with the past,but there are still a considerable number of patients with advanced disease progression.Therefore,the recurrence and metastasis of breast cancer still poses a serious threat to the safety of the patient’s life.Therefore,there is a need to further improve the development and development of breast cancer and its molecular mechanisms in order to provide new guidance and ideas for accurate diagnosis and treatment of breast cancer.microRNAs(microRNAs,miRNAs,miRs)are a series of endogenously expressed single-stranded short-sequence small RNAs newly discovered in recent years,with a length of about 19-25 nucleotides,which are characterized by not coding for proteins and widely existing.It is evolutionarily conserved in eukaryotic cells.Many previous studies have shown that microRNAs are closely associated with a variety of diseases,including neoplasma.The discovery of micro RNA provides a great supplement for improving the molecular mechanism of breast cancer occurrence and development,and has broad prospects for clinical translation.miR-217-5p is a mature sequence and an important member of the miR-217 family.Recent studies have found that miR-217 has abnormal expression and functions in many malignant tumors,but the role of miR-217-5p on the occurrence and development of breast cancer has not been clearly studied.In this study,the expression of miR-217-5p in normal breast epithelial cell lines and breast cancer cell lines was detected,and the expression of miR-217-5p in breast cancer tissue and adjacent tissue was compared.The method of miR-217-5p was overexpressed and knocked down in breast cancer cell lines to verify its effects on cell proliferation,migration,invasion and epithelial-mesenchymal transition in breast cancer cells,and to further explore its function Mechanism;the possible target genes downstream of miR-217-5p were screened by bioinformatics methods,and further experiments confirmed that MTDH is one of the downstream target genes of miR-217-5p.The research results in this topic provide a better understanding of the development and molecular mechanisms of breast cancer development,provide new ideas for early diagnosis and accurate treatment of breast cancer,and provide new targets for the development of related new drugs.Part One Expression of miR-217-5p in breast cancer tissues and cell linesObjective: This part of the study aims to study the expression of miR-217-5p in breast cancer tissues and cell lines,and compare it with its corresponding normal tissues and normal cell lines,in order to further explore its role in the occurrence and development of breast cancer.provide the basis for research.Methods:1.The breast cancer tissues and their corresponding adjacent normal tissues of 35 breast cancer patients were collected,and RT-qPCR was used to detect the expression of miR-217-5p in cancer and adjacent normal tissues,respectively.2.Select normal breast epithelial cell line MCF10 A and breast cancer cell lines SK-BR3,BT549,MCF7 cell lines,and use RT-qPCR to detect the expression of miR-217-5p in each cell line respectively.Results:1.Compared with normal tissues adjacent to breast cancer,the expression of miR-217-5p in breast cancer tissues was significantly down-regulated,and the difference was statistically significant(P<0.05).2.Compared with normal mammary epithelial cell MCF10 A,the expression of miR-217-5p in cell lines MCF7,BT549,and SR-BR3 was significantly decreased by 29.24%,70.46%,and 43.52%,respectively,and the results were statistically significant.Significance(P<0.05).Summary:The expression of miR-217-5p in breast cancer tissues and cell lines was lower than its corresponding adjacent normal tissues and normal breast epithelial cell lines,which may play a role in inhibiting the occurrence and development of breast cancer.Part Two The effect and mechanism of miR-217-5p on breast cancer cell proliferation,migration,invasion and epithelial-mesen-chymal transitionObjective: Based on the research in the first part,the function and possible related mechanisms of miR-217-5p on breast cancer cell proliferation,migration,invasion and epithelial-mesenchymal transition were further explored.Methods:1.Transient miR-217-5p mimic or inhibitor and its corresponding control into SK-BR3 or BT549 by liposome transient transfection,and overexpress or knock down miR-217-5p in cells;2.The changes of cell proliferation,migration and invasion after overexpression or knockdown of miR-217-5p were detected by CCK8 assay,plate clone formation assay,cell scratch assay,and Transwell assay,respectively;3.RT-qPCR and Western Blot experiments were used to detect the expression levels of E-cadherin,Vimentin and slug in cells after overexpression or knockdown of miR-217-5p,respectively;4.RT-qPCR and Western Blot experiments were used to detect p65 and its phosphorylation degree and the expression level of IκBα in cells after overexpression or knockdown of miR-217-5p,respectively.Results:1.After transiently transfecting miR-217-5p mimic or inhibitor into breast cancer cells with liposomes,the expression of miR-217-5p could be up-regulated 8168 times or down-regulated by 55%,and the differences were statistically significant(P< 0.001).2.It was found by CCK8 method and plate clone formation experiments that after overexpressing miR-217-5p in SK-BR3,the proliferation ability of cells could be significantly inhibited(P<0.05),while knockdown of miR-217-5p in BT549 After 217-5p,the proliferation ability of cells was significantly enhanced(P<0.05).Through cell scratch assay and plate clone formation assay,it was found that after overexpression of miR-217-5p in SK-BR3,the migration and invasion abilities of cells were significantly inhibited(P<0.05),while knockdown in BT549 After miR-217-5p,the migration and invasion abilities of cells were significantly enhanced(P<0.05);3.RT-qPCR and Western Blot experiments both indicated that after overexpression of miR-217-5p in SK-BR3 cells,the expression of E-cadherin was enhanced,while the expression levels of Vimentin and slug were decreased,and miR-217 was knocked down in BT549 cells After-5p,the expression of E-cadherin decreased,while the expression of Vimentin and slug increased;4.RT-qPCR experiments showed that after overexpression of miR-217-5p in SK-BR3 cells,the expression of p65 was decreased,while the expression of IκBα was increased.Western Blot experiments showed that the phosphorylation of p65 was also down-regulated.After down-regulation of miR-217-5p in BT549 cells,both p65 and its phosphorylation were enhanced,while the expression of IκBα was decreased;Summary: miR-217-5p can inhibit the proliferation,migration,invasion and epithelial-mesenchymal transition of breast cancer cells,and may exert its function by inhibiting the NF-κB pathway.Part Three Screening and validation of downstream target genes of miR-217-5pObjective: The possible downstream target genes of miR-217-5p were searched and verified.Methods:1.MTDH was predicted to be the downstream target gene of miR-217-5p through the Target Scan database,and was verified by the dual luciferase reporter assay;2.RT-qPCR was used to detect the expression of MTDH in breast cancer tissue and its corresponding adjacent normal tissue,and the correlation between it and miR-217-5p expression was analyzed by Pearson method;3.The miR-217-5p mimic or inhibitor and its corresponding control were transferred into SK-BR3 or BT549 cells by liposome method,and the expression of MTDH was detected by RT-qPCR and Western Blot experiments.Results:1.Target Scan database analysis showed that MTDH was the target gene of miR-217-5p,which was verified by dual luciferase reporter assay(P<0.05);2.In breast cancer tissues and breast cancer cell lines,the expression of MTDH is high in its corresponding adjacent normal tissues and normal breast cell lines.The expression of MTDH in breast cancer tissue was significantly negatively correlated with miR-217-5p(P<0.05);3.After overexpression or knockdown of miR-217-5p in breast cancer,the expression of MTDH in cells was down-regulated or up-regulated,suggesting that miR-217-5p negatively regulates MTDH.Summary:MTDH is a direct target gene of miR-217-5p.Conclusions:Our study can draw the following main conclusions: 1)miR-217-5p is significantly down-regulated in breast cancer tissues and cell lines compared with normal breast tissues and cell lines;2)miR-217-5p can significantly inhibit breast cancer cells Proliferation,migration,invasion and epithelial-mesenchymal transition,and may play a role through the NF-κB pathway;3)We confirmed that MTDH is a direct target gene of miR-217-5p by bioinformatics analysis and related experiments. |
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