| Breast cancer is the most common malignant tumor in women all over the world According to the International Agency for Research on Cancer(IARC),the mortality rate of breast cancer in China is on the rise.In 2030,the incidence of breast cancer in China will reach 234 thousand.So far,taxanes and anthracyclines are still effective chemotherapy drugs for breast cancer.However,clinical practice has proved that 30%-50%of breast cancer patients are not sensitive to these chemotherapy drugs,or although they are sensitive to these drugs,the side effects of these drugs on heart,liver,kidney and blood system seriously affect their physical and mental health.Traditional Chinese medicine(TCM)has the advantages of prolonging the survival time and improving the quality of life of breast cancer patients.According to the clinical manifestations and characteristics of breast cancer,it belongs to the category of "breast rock" and "breast stone carbuncle" in TCM.According to the TCM pathogenesis characteristics of breast cancer“liver depression and spleen deficiency,phlegm and blood stasis block each other",the research group proposed that the basic treatment of breast cancer is " soothing liver and strengthening spleen,Promoting blood circulation and resolving phlegm".Xiaoyao Powder has the function of soothing the liver and relieving the depression,strengthening the spleen and nourishing the blood,which is the classical ancient prescription of soothing the liver and strengthening the spleen Rhizoma Dioscoreae Nipponicae and Fritillaria thunbergii are the anti breast cancer chinese herbal medicines selected by the research group in many years of clinical practice and a large number of experiments.Longbei-Xiaoyao Powder(LXP)is composed of Rhizoma Dioscoreae Nipponicae and Fritillaria thunbergii on the basis of Xiaoyao Powder,and it has been used in the clinical treatment of breast cancer to achieve good results.Experimental studies have found that the anti-breast cancer mechanism of Xiaoyao Powder may be related to the regulation of apoptosis-related genes and target protein expression.The occurrence and development of breast cancer is a complex process of multiple genes,factors and pathways,including genetic changes and epigenetic changes.Comprehensive and in-depth exploration of its targets and mechanism of action from the perspective of genetics and epigenetics provides a scientific basis for LXP to treat breast cancer,and is of great significance for the promotion of comprehensive treatment programs for breast cancer with TCM.Objectives:1.On the premise of clarifying the effect of LXP on the proliferation and migration of human normal breast epithelial cells and breast cancer cells,as well as the effect and mechanism of miR-145 methylation and low expression on the proliferation and migration of breast cancer cells,the mechanism of LXP on breast cancer cells in vitro was clarified;2.To clarify the effect and mechanism of LXP on xenograft breast tumors in vivo.Methods:1.The effect and mechanism of longbeixiaoyao powder on inhibiting the proliferation and migration of breast cancer cells in vitro:?The inhibitory effect of LXP on human breast cancer cells:CCK-8 method was used to screen the inhibitory concentration of LXP on the proliferation of breast cancer cells;according to the drug concentration of IC50 value,cell scratch test was carried out to detect the inhibitory effect of LXP on the migration of breast cancer cells;?Lentivirus transfected miR-145 into breast cancer cell line MDA-MB-231,CCK8 method and scratch test were used to detect the effect of miR-145 on breast cancer cell proliferation and migration,and qPCR method was used to detect miR-145 on miR-145/c-Effects of Myc/TP53 signaling pathway and expression of apoptosis-related genes caspase3,Bcl-2 and Bax;? BSP and qPCR were used to detect the methylation of miR-145 promoter and the regulation of miR-145 expression,and the effect of LXP on miR-145/c-Myc/TP53 signaling pathway and caspase 3,Bcl-2 and Bax expression.2.Observe the effect and mechanism of LXP on breast cancer xenografts in vivo:In order to observe the effect of miR-145 and drug intervention on tumor volume,weight and survival status of nude mice,we constructed MDA-MB-231 breast cancer transplantation model and high expression of miR-145 MDA-MB-231 breast cancer transplantation model.The methylation of miR-145 promoter and the expression of miR-145 were detected by BSP and qPCR,and the expression of caspase 3,Bcl-2 and Bax were also detected.Results:1.Effect and mechanism of LXP on breast cancer cell proliferation and migration:?The IC50 of LXP on MDA-MB-231 cell line was 0.7613mg/ml at 48h and 0.7664mg/ml at 72h.The IC50 of LXP on ER positive breast cancer cell line MCF-7 was 1.261mg/ml at 48h and 1.095mg/ml at 72h.The IC50 of LXP on MCF-10A was 2.891mg/ml at 48h and 2.423mg/ml at 72h.The IC50 of MDA-MB-231 cells was the lowest and most sensitive to the chinese herbal compound,while the IC50 of normal mammary epithelial cells MCF-10A was the highest,indicating that the cells were most tolerant to LXP,and LXP had no significant inhibition or even proliferation promoting effect on MCF-10A within the IC50 of MCF-7 and MDA-MB-231.LXP can significantly inhibit the migration of breast cancer cells at the IC50 concentration of MDA-MB-231 and MCF-7(P<0.05),but has no significant inhibitory effect on the migration of human normal breast epithelial cells MCF-10A(P>0.05).?The results of meta-analysis showed that the expression of miR-145 in breast cancer tissue was significantly lower than that in adjacent and normal breast tissues,and the SMD was-2.90(95%CI=-3.11,-2.70).In addition,the expression of miR-145 in ER positive and HER-2 positive breast cancer tissues was significantly lower than that of ER and HER-2 negative breast cancer tissues.The expression of miR-145 in breast cancer patients with negative lymph node metastasis or less than 2 centimeters in diameter was significantly higher than that in breast cancer patients with positive lymph node metastasis or more than 2 centimeters in diameter(P<0.001).BSP detection showed that the miR-145 promoter of MCF-7 and MDA-MB-231 breast cancer cells was in a hypermethylated state,and qPCR detection showed that the expression of miR-145 in breast cancer cells was significantly lower than that of human normal breast epithelial cells MCF-10A.After miR-145 was transfected into MDA-MB-231 cells by lentivirus,it was found that miR-145 could inhibit the proliferation and migration of MDA-MB-231 breast cancer cells.Overexpression of miR-145 increased the expression of Bax and caspase-3 mRNA in MDA-MB-231 cells(P<0.01),decreased the expression of c-myc and bcl-2 mRNA(P<0.01;P<0.001),but had no significant effect on TP53(P>0.05).?LXP can partially reduce the methylation status of miR-145 promoter in MDA-MB-231 cells after intervention in MDA-MB-23 1 cells at a drug concentration of 0.8 mg per milliliter for 24 hours,and can also increase MDA-MB-231 cells miR-145,TP53,Bax and caspase3 mRNA expression levels(P<0.01),while down-regulating c-Myc and Bcl-2 mRNA expression levels(P<0.05;P<0.01).2.The effect and mechanism of LXP on breast cancer xenografts:?The nude mice in the miR-145 high-expression transplanted tumor group,the LXP group,the combination medication group and the normal control group were in good condition,sensitive,with normal activities,diet,water intake,and rosy skin,and nude mice in the model control group and the paclitaxel group were generally in poor condition.?The transplanted tumors in the armpits of nude mice in each group continued to grow,and the growth rate of the tumor mass in nude mice of the model control group was significantly faster than that of the miR-145 high-expression transplanted tumor group,paclitaxel group,and LXP group and combined medication group from the 4th day after administration.The tumor weight of tumor model group,high expression miR-145 transplanted tumor group,LXP group,PTX group and combination group were 1.01±0.20g,0.73± 0.13g,0.55± 0.10g,0.53± 0.10g and 0.42± 0.04g respectively.The tumor inhibition rates of miR-145 transplanted tumor group,LXP group,PTX group and combination group were 27.72%,45.54%,47.52%and 58.42%,respectively,and the combination group had the highest tumor inhibition rate of triple negative breast cancer.?Statistical analysis of the survival time using the Log-rank(Mantel-Cox)test(conservative)method showed that the survival time of the LXP group was better than that of the combined drug group,the highly expressed miR-145 transplanted tumor group,the PTX group and the tumor model group,but the difference was not statistically significant(P>0.05).?The study of the mechanism in vivo showed that the expression of miR-145 in the transplanted tumor tissue of the LXP and the combined drug group was significantly higher than that of the tumor model group(P<0.01).Compared with the tumor model group,the relative expression levels of TP53,caspase3 and BAX in the LXP group,PTX group and combination drug group increased to varying degrees,and the relative expression levels of c-Myc and BCL-2 showed different degrees of downregulation.Compared with the application of PTX alone,the combination of PTX with LXP had a synergistic effect on the relative expression of TP53,c-Myc and BCL-2 mRNA(P<0.05).Conclusions:1.Longbei Xiaoyao Powder can inhibit the proliferation and migration of breast cancer cells MCF-7 and MDA-MB-231,but it has no obvious effect on the proliferation of human normal breast epithelial cells MCF-10A in the effective dose of inhibiting breast cancer cells,showing a certain targeting,especially for the three negative breast cancer cells MDA-MB-231;2.Based on the literature meta-analysis and cell experiments,it was confirmed that the methylation and low expression of miR-145 are closely related to the occurrence and development of breast cancer.MiR-145 can inhibit the proliferation and migration of breast cancer cells by regulating the miR-145/c-Myc/TP53 signal pathway and the expression of apoptosis related genes;3.The mechanism by which LXP inhibits the proliferation and migration of breast cancer cells may be related to the regulation of miR-145/c-Myc/TP53 signaling pathway and the expression of apoptosis-related genes caspase3,Bcl-2 and Bax through the expression and demethylation of miR-145;4.LXP can regulate the miR-145/c-Myc/TP53 signal pathway and the expression of apoptosis-related genes caspase 3,Bcl-2 and Bax through the demethylation and expression of miR-145,so as to play the role of anti-tumor and prolong the survival period of mice. |
PDF Full Text Request