Study On The Mechanism Of Baicalin Inhibiting Breast Cancer Through The Targeted Regulation Of MiR-338-3p On MORC4

Background and purpose:Breast cancer is a very common malignant tumor clinically,and it ranks first in the mortality rate of female malignant tumors worldwide.The causes of breast cancer are complex,the degree of malignancy is high,and it is easy to metastasize,which brings great challenges to the treatment of breast cancer.With the continuous research on breast cancer in recent years,the molecular mechanism of breast cancer has attracted more and more attention.Surgical treatment,radiotherapy,etc.are currently common breast cancer treatment methods.With the continuous advancement of these technical methods,the quality of life of breast cancer patients has also been significantly improved.Because many breast cancer patients have metastasized in the early stage,surgical resection cannot completely cure,and radiotherapy,chemotherapy and other treatment methods have side effects and high requirements for the patient's physical conditions,they are actively looking for effective treatment methods,and to prolong the breast cancer patients survival time is currently a problem.Micro RNA(miRNA)is a large family of noncoding RNA with a length of 15 to22 nucleotides.It has become a hot spot in the field of regulating gene transcription and expression.Many research evidences have proved that miRNA plays a key role in the expression of genes after transcription.About 50% of miRNAs in mammals are involved in the regulation of gene activity encoding protein expression.miRNA plays a role in almost all cells in human tissues,and miRNA expression changes are also closely related to the occurrence of many diseases.In addition,miRNA itself is also regulated by the expression and regulation of multiple genes and external factors in the body,mainly reflected by changes in miRNA expression levels and changes in metabolic levels.The miRNA regulates the expression of downstream genes related to the complementary binding site of the 3?UTR end of the target gene,and the same miRNA can have multiple binding sequences at the same time,so miRNA can regulate the expression of multiple genes,and the same gene can also be miRNA regulation,the complex regulatory network between miRNA and target genes together affect the body's normal life activities and pathological progress.miRNAs are closely related to tumors.The expression profiles of miRNAs in tumor tissues are different from those in normal tissues.These miRNAs have been shown to be involved in tumor metastasis and growth.miRNAs are a research hotspot for targeted gene therapy for tumors.mi R-338-3p is widely expressed in human tissues and participates in tumor progression.mi R-338-3p is lowly expressed in various tumors such as ovarian cancer and colorectal cancer,and mi R-338-3p has a tumor cell malignant phenotype.The role of mi R-338-3p in breast cancer progression is currently unclear.Microrchidia(MORC)is a highly conserved nuclear protein superfamily that participates in a variety of biological processes,including cell proliferation,cell senescence,DNA injury repair,and its dysregulation is associated with cancer.Early preliminary experiments found that mi R-338-3p and MORC4 3'UTR have complementary binding sites.Reviewing the literature found that MORC4 was overexpressed in tumor tissues,which can promote tumor progression.Scutellaria baicalensis is a traditional Chinese medicine in China,which has the functions of detoxification,clearing heat and hemostasis.Baicalin is an active ingredient extracted from Scutellaria baicalensis,which belongs to flavonoids.It has antibacterial,anti-inflammatory,and anti-oxidant effects,and has obvious improvement functions on cerebral ischemia-reperfusion injury and liver injury.Baicalin also has an anti-tumor effect,which can inhibit the proliferation and metastatic potential of various tumor cells such as osteosarcoma and lung cancer in vitro,and induce apoptosis.Baicalin is currently known to inhibit malignant progression of breast cancer,but its specific mechanism of action is not yet clear.Many studies have found that baicalin can play a role by acting on the expression of miRNA.This experiment explored the mechanism of baicalin combined with mi R-338-3p on the targeted regulation of microrchidia family CW-type zincfinger 4(MORC4)on breast cancer progression.The experiment is divided into two parts,the first part discusses the role of baicalin affect breast cancer cell apoptosis,invasion and migration through mi R-338-3p on MORC4 targeting regulation,the second part explores the effect of baicalin on breast cancer cell xenograft tumor growth in nude mice.Part One Baicalin affects breast cancer cell apoptosis,invasion and migration through mi R-338-3p's targeted regulation of MORC4Methods:1.Normal breast epithelial cells MCF-10 A and breast cancer cells MCF-7,MDA-MB-231 were treated with baicalin at 0,25,50,100,200 ?M respectively,and the proliferation of cells was detected by Cell Counting Kit-8(CCK-8)method for 24 h.2.Normal breast epithelial cells MCF-10 A and breast cancer cells MCF-7,MDA-MB-231 were treated with 100 ?M baicalin,and the proliferation activity of cells was detected by CCK-8 method at 12,24 and 48 h.3.Using quantitative real time polymerase chain reaction(q RT-PCR)to detect the expression level of mi R-338-3p in 65 breast cancer tissues and the corresponding adjacent tissues.4.Using q RT-PCR to detect the expression level of mi R-338-3p in normal breast epithelial cells MCF-10 A and breast cancer cells MCF-7,MDA-MB-231.5.Treated breast cancer cells MCF-7,MDA-MB-231 with baicalin at 0,25,50,100 and 200 ?M,and detect the expression of mi R-338-3p by q RT-PCR.6.Breast cancer cells MCF-7,MDA-MB-231 were transfected with mi R-338-3p mimics and mimics control,respectively,and then treated with 100 ?M baicalin for24 h.Transwell cells were used to detect cell invasion and migration.Flow Cytometry detection of apoptosis.7.Bioinformatics online software predicted mi R-338-3p target gene,luciferase reporter system and RNA Binding Protein Immunoprecipitation(RIP)experiment to verify the targeting relationship.8.Using q RT-PCR to detect the expression level of MORC4 m RNA in 65 breast cancer tissues and the corresponding adjacent tissues.9.Using q RT-PCR to detect the expression levels of MORC4 m RNA in normal breast epithelial cells MCF-10 A and breast cancer cells MCF-7,MDA-MB-231.10.Treated breast cancer cells MCF-7,MDA-MB-231 with baicalin at 0,25,50,100 and 200 ?M,and detected the expression of MORC4 m RNA by q RT-PCR.11.Transfected mi R-338-3p mimics,mimics control,mi R-338-3p inhibitor,inhibitor control in breast cancer cells MCF-7,MDA-MB-231,and detected the expression of MORC4 protein in the cells by Western blot.12.Co-transfection of mi R-338-3p mimics and pc DNA,co-transfection of mi R-338-3p mimics and pc DNA-MORC4 in breast cancer cells MCF-7,MDA-MB-231,Transwell chamber to detect cell migration and invasion,Flow cytometry was used to detect apoptosis.Results:1.The proliferative activity of normal breast epithelial cells MCF-10 A after 25,50,100,200 ?M baicalin treatment did not change;breast cancer cells MCF-7 and MDA-MB-231 after 50,100,200 ?M baicalin treatment,the proliferative activity was significantly decreased;the proliferative activity of breast cancer cells MCF-7 and MDA-MB-231 after 25 ?M baicalin treatment did not change significantly.2.The proliferative activity of normal breast epithelial cells MCF-10 A after 100?M baicalin treatment for 12,24,and 48 h did not change;breast cancer cells MCF-7,MDA-MB-231 after 12,24,48 h treatment with 100 ?M baicalin proliferative activity decreased significantly.3.The expression level of mi R-338-3p in breast cancer tissues was significantly lower than that in adjacent tissues.4.The expression level of mi R-338-3p in breast cancer cells MCF-7 and MDA-MB-231 was lower than that of normal breast epithelial cells MCF-10 A.5.The expression level of mi R-338-3p in breast cancer cells treated with baicalin at 50,100,200 ?M was significantly increased,and mi R-338 in breast cancer cells MCF-7 and MDA-MB-231 treated with 25 ? M baicalin had no significant change in mi R-338-3p expression level.6.The migration and invasion ability of breast cancer cells MDA-MB-231 treated with baicalin were significantly reduced,and the level of apoptosis increased;compared with the transfection of inhibitor control,the breast transfected with mi R-338-3p inhibitor After the cancer cells MDA-MB-231 were treated with baicalin,the cell migration and invasion capabilities were significantly increased,and the level of cell apoptosis decreased.7.mi R-338-3p and MORC4 were in a targeted relationship with each other.8.The expression level of MORC4 m RNA in breast cancer tissue was significantly higher than that in adjacent tissues.9.The expression level of MORC4 m RNA in breast cancer cells MCF-7 and MDA-MB-231 was significantly higher than that of normal breast epithelial cells MCF-10 A.10.Baicalin at 50,100 and 200 ?M could inhibit the expression of MORC4 m RNA in breast cancer cells MCF-7 and MDA-MB-231,and baicalin at 25 ?M could not inhibit the MORC4 m RNA in breast cancer cells MCF-7 and MDA-MB-231.11.The expression level of MORC4 protein in breast cancer cells MCF-7 and MDA-MB-231 after transfection of mi R-338-3p mimics was lower than that of cells transfected with mimics control;the expression levels of MORC4 protein in breast cancer cells MCF-7 and MDA-MB-231 after transfection with mi R-338-3p inhibitor were higher than those of transfected with inhibitor control cells..12.Compared with the cells co-transfected with mi R-338-3p mimics and pc DNA,the breast cancer cells MCF-7 and MDA-MB-231 cells after co-transfected with mi R-338-3p mimics and pc DNA-MORC4 had both migration and invasion capabilities significantly increased and decreased apoptosis.Part Two Effects of baicalin on the growth of breast cancer cell xenograft tumor in nude miceMethods:1.Breast cancer cells MCF-7 were inoculated subcutaneously into transplanted tumors in nude mice,and mi R-338-3pinhibitor lentiviral expression vector and negative control lentiviral expression vector were subcutaneously injected.,and the baicalin aqueous solution(100mg / kg)was intragastrically administered daily for 7days from the 7th day of cell inoculation.Measure the tumor volume,35 days after inoculation,take the transplanted tumor tissue and weigh the weight of the transplanted tumor.2.Take the transplanted tumor tissue,q RT-PCR to detect the expression of mi R-338-3p,and Western blot to detect the expression of MORC4,matrix metalloprotease 2(MMP-2),matrix metalloprotease 9(MMP-9),B cell lymphoma/lewkmia-2(Bcl-2),Bcl-2 Associated X Protein(Bax)protein.Results:1.The growth volume and weight of transplanted tumors in nude mice treated by intragastric administration of baicalin decreased significantly;compared with subcutaneous injection of negative control lentiviral expression vector,nude mice injected subcutaneously with mi R-338-3pinhibitor lentiviral expression vector after intragastric administration of baicalin,the growth volume and weight of the transplanted tumor in nude mice increased significantly.2.The expression level of mi R-338-3p in the transplanted tumor tissue of nude mice treated by intragastric administration of baicalin increased,the expression level of MORC4,MMP-2,MMP-9,Bcl-2 protein decreased,and the expression level of Bax protein increased;and Compared with subcutaneous injection of negative control lentiviral expression vector,nude mice injected subcutaneously of mi R-338-3p inhibitor lentiviral expression vector after baicalin gavage,mi R-338-3p expression in nude mice transplanted tumor tissue The level decreased,MORC4,MMP-2,MMP-9,Bcl-2 protein expression level increased,and Bax protein expression level decreased.Conclusions1.Baicalin inhibited the proliferation of breast cancer cells,increased the expression level of mi R-338-3p in breast cancer cells.2.mi R-338-3p targeted and negatively regulateed MORC4 expression in breast cancer cells.3.Baicalin inhibited the expression of MORC4 through mi R-338-3p.4.Baicalin inhibited the growth of breast cancer cell transplanted tumors in nude mice through mi R-338-3p.