Expression Of MiR-200b In Plasma Exosomes Of Patients With Ovarian Cancer And Its Mechanism Of Promoting Tumor Metastasis

Background and Objective: Ovarian cancer is one of the three major malignant tumors of female reproductive system,and the mortality is the highest.Epithelial ovarian cancer(EOC)accounts for 85%-90% of ovarian cancer,which seriously threatens women’s health.Because the early symptoms of ovarian cancer are not obvious,the treatment rate is low,and effective early diagnosis methods are lacking,the diagnosis and treatment of ovarian cancer has always been a clinical problem.In the era of precision medicine,exploring new markers for early diagnosis of ovarian cancer is essential to improve the survival of patients.Exosomes contain DNA,RNA,protein,lipid and other active substances.They are important intercellular information and genetic material transmission carriers.They are widely distributed in body fluids such as blood and urine.They can promote the progress of tumors by regulating the interaction between tumor cells and immune cells in tumor microenvironment.Mi RNAs are a kind of endogenous non coding RNAs with a length of 19 ～ 25 nt.They can cause the degradation of target gene or inhibit its translation by complementary pairing with the specific base of target gene,and widely negatively regulate the expression of target gene,so as to participate in the occurrence and development of malignant tumor.Macrophages are an important type of immune cells in the tumor microenvironment,and their high infiltration is closely related to the occurrence and development of tumors.Macrophages mainly have two polarization states: classical activated M1 macrophages and alternative activated M2 macrophages.M1 type macrophages can kill and inhibit malignant tumor cells.On the contrary,M2 type macrophages can promote tumor growth,angiogenesis and cancer cell metastasis.Some scholars sequenced and analyzed the differentially expressed miRNAs in plasma exosomes of EOC patients,and found that miRNAs such as miR-200 b were significantly highly expressed in plasma exosomes of EOC patients.Previous studies have shown that tumor-derived exosomes miR-19b-3p can promote the polarization of M2 macrophages,thereby promoting the metastasis of lung adenocarcinoma.Therefore,can plasma exosome miR-200 b affect the progress of EOC by regulating the polarization of macrophages in the tumor microenvironment?We predicted and analyzed the target gene of miR-200 b and found that miR-200 b could bind to KLF6 3’UTR.KLF6 has been confirmed to be involved in the regulation of macrophage polarization.Therefore,we speculate that exosomal miR-200 b may regulate the polarization of macrophages by regulating the expression of KLF6,so as to promote the tumor progression of EOC.To verify the hypothesis:we isolated and identified plasma exosomes from EOC patients and normal women,and detected the expression of miR-200 b in plasma exosomes;secondly,we used M0 macrophages to transfect miR-200 b mimic/mimic NC to detect the effect of miR-200 b on the polarization direction of macrophages;At the same time,CCK-8,transwell,etc.observed the effects of macrophages with different polarization directions on the proliferation and invasion of ovarian cancer;At the same time,CCK-8,transwell and other methods were used to observe the effects of macrophages with different polarization directions on the proliferation and invasion of ovarian cancer;Finally,qRT-PCR,Western blot and luciferase activity experiments further confirmed the direct binding of miR-200 b with KLF6 3’UTR,and explored the molecular mechanism of miR-200 b regulating macrophage polarization.The development of this project provides a new direction for early diagnosis of EOC and a new strategy for miR-200b/KLF6 pathway as a new target for EOC treatment.Part Ⅰ: Study on the expression of miR-200 b in plasma exosomes of normal women and EOC patientsObjective:We extracted exosomes from the plasma of normal women and EOC patients,and analyze the expression level of miR-200 b in exosomes,providing a new direction for finding markers for early diagnosis of EOC..Methods:1.For exosome isolation,peripheral blood samples were taken from three healthy women,and six untreated patients with epithelial ovarian cancer in the second affiliated hospital of Nanchang University.Then,Exo Quick? Exosome Precipitation Solution was utilized to extract exosomes from the plasma in accordance with the manufacture’s introduction.Transmission electron microscopy(TEM)was utilized to analyze the morphology of exosomes,and the size of exosomes was ensured by nanoparticle tracking analysis(NTA)on a Nano Sight NS300.Besides,the expression of the markers of exosomes,including lysosome-associated membrane proteins 1(LAMP-1)and tumor susceptibility gene 101(TSG101),were measured by using western blot.2.Using qRT-PCR to detect the differential expression level of miR-200 b in plasma exosomes of normal women and EOC patients.Results:1.The typical circle and saucer-like structures characteristic of exosomes were observed using a TEM。2.Using NTA technology to analyze the size of exosomes,the peak diameter of exosomes is about 100 nm.3.Western blot was used to detect the exosomal marker protein,and the results showed that the expression of TSG101 and LAMP-1 can be seen in the exosomes of normal female exosomes and EOC patients.4.Using qRT-PCR to detect the expression level of miR-200 b in the plasma exosomes of the two groups,the results showed that miR-200 b was significantly highly expressed in the plasma exosomes of patients with ovarian cancer(P<0.05).Conclusions:1.Successfully isolated and identified plasma exosomes from normal women and EOC patients.2.The expression level of plasma exosomal miR-200 b in EOC group was significantly higher than that in normal female group,which provided a certain theoretical basis for exosomal miR-200 b as a marker for early diagnosis of ovarian cancer.Part Ⅱ: Effects of miR-200 b regulating macrophage polarization on the proliferation and invasion of EOC cellsObjective:To investigate the effect of miR-200 b on the proliferation and invasion of EOC cells by regulating the polarization direction of macrophages.Methods:1.Culture the human monocyte line THP-1 cells in RPMI-1640 medium containing10% fetal bovine serum,1% streptomycin/penicillin,and 0.05 n Mβ-mercaptoethanol.In a culture environment of 5% CO2 and 37? C,PMA was used to induce THP-1 cells to differentiate into macrophages.After 48 hours of incubation with PMA,THP-1 cells were collected and named M0 macrophages.2.We used the transfection reagent Lipofectamine 2000 to transfect miR-200 b mimic,mimic NC,etc.into M0 macrophages.Cells were collected 24 hours later,and the overexpression efficiency of miR-200 b was detected by qRT-PCR.3.The M0 macrophages transfected with miR-200 b mimic and mimic NC were labeled with Anti-CD86 and Anti-CD206 immunofluorescence antibodies,respectively.Observed CD86-positive and CD206-positive cells under the microscope.4.At room temperature,incubated the cells with PE-labeled CD206 antibody and FITC-labeled CD86 antibody for 30 minutes.Subsequently,the BD Accuri? C6 flow cytometer was used to detect the ratio of M1(CD86+)and M2(CD206+)macrophages.5.The expression levels of M1 marker i NOS m RNA and M2 marker Arg-1 m RNA in M0 macrophages transfected with miR-200 b mimic and mimic NC were detected by qRT-PCR.6.The concentrations of IL-1β and CCL-17 in cell supernatant of macrophages were measured by ELISA assay.7.M0 macrophages culture medium and M0 macrophages culture medium transfected with miR-200 b mimic and mimic NC were collected and co incubated with EOC cell line OVCAR-3(PBS as control).CCK-8 was used to detect the effect on cell proliferation ability.Transwell was used to detect cell invasion ability.Results:1.Transfection of miR-200 b mimic into M0 macrophages with transfection reagent Lipofectamine 2000 significantly promoted the expression of miR-200b(P <0.05).2.Immunofluorescence results showed that after miR-200 b mimic treatment,the number of CD86 positive cells(M1 macrophages)in M0 macrophage medium decreased significantly(P < 0.05),while the number of CD206 positive cells(M2macrophages)increased significantly(P < 0.05).3.Flow cytometry results showed that after miR-200 b mimic treatment,the percentage of CD86-positive cells was significantly reduced(P <0.05),but the percentage of CD206-positive cells was significantly increased(P <0.05).4.Compared with M0 macrophages transfected with mimic NC,the expression level of i NOS m RNA in M0 macrophages transfected with miR-200 b mimic was significantly reduced(P <0.05),while the expression level of Arg-1 m RNA was significantly increased(P <0.05).5.Compared with M0 macrophages transfected with mimic NC,the concentration of IL-1β in the culture supernatant of M0 macrophages transfected with miR-200 b mimic was significantly decreased(P <0.05),and the concentration of CCL-17 was significantly increased(P <0.05).6.Compared with the PBS group,M0 macrophage conditioned medium not only significantly inhibited the proliferation activity of EOC cells(P <0.05),but also significantly inhibited the invasion ability of EOC cells(P <0.05).7.Compared with mimic NC,miR-200 b mimic conditioned medium significantly enhanced the proliferative activity of EOC cells(P <0.05),but also significantly enhanced the invasive ability of EOC cells(P <0.05).Conclusions:1.miR-200 b promotes the polarization of macrophages to M2 and inhibits the polarization of macrophages to M1.2.miR-200 b promotes the proliferation and invasion of EOC cells by inducing macrophages to polarize to M2.Part Ⅲ: Study on the Molecular Mechanism of miR-200 b Regulating the Polarization of MacrophagesObjective:Explore the molecular mechanism of miR-200 b regulating the polarization direction of macrophages.Methods:1.The binding sites of miR-200b-3p and KLF6 3 ’-UTR were analyzed by star Base v2.0 database.By constructing KLF6 3’UTR wt and KLF6 3’UTR mut luciferase reporter vector and co-transfecting 293 T cells with miR-200 b mimic or mimic NC,luciferase activity experiment showed that miR-200 b directly bound to KLF63’UTR.2.Transfected miR-200 b mimic and mimic NC,or miR-200 b inhibitor and inhibitor NC into M0 macrophages respectively,used qRT-PCR and western blot to detect the expression level of KLF6.3.miR-200 b mimic and KLF6 overexpression vectors were co transfected in M0 macrophages.The proportion of M1 and M2 macrophages was detected by flow cytometry.The expression levels of i NOS m RNA and Arg-1 m RNA were detected by qRT-PCR.ELISA assay was used to measure the concentration of IL-1β and CCL-17 in the cell supernatant.Results:1.Bioinformatics prediction showed that KLF6 3’UTR had a targeted binding sequence of miR-200 b.Luciferase reporter gene experiment confirmed that miR-200 b can directly target and bind KLF6 3’UTR.2.The levels of KLF6 m RNA and protein in M0 macrophages transfected with miR-200 b mimic group were significantly lower than those transfected with mimic NC group(P < 0.05);In M0 macrophages transfected with miR-200 b inhibitor group,the expression levels of KLF6 m RNA and protein were significantly up-regulated(P < 0.05).3.M0 macrophages were infected with the lentivirus expressing KLF6 following miR-200 b mimic transfection.The percentage of M1 and M2 macrophages was measured by flow cytometry.Our data revealed that the inhibition of miR-200 b to macrophage M1 polarization,and the promotion of miR-200 b to macrophage M2 polarization were rescued by KLF6 overexpression.4.The overexpression of KLF6 reversed the inhibition of miR-200 b on the expression of i NOS m RNA and the promotion of Arg-1 m RNA.In addition,miR-200 b reduced the production of M1 macrophages-related cytokine IL-1β,and facilitated the production of M2 macrophages-related cytokine CCL-17 in M0 macrophages,while which were recused following over-expression of KLF6.Conclusion:There is a targeting interaction between KLF6 and miR-200 b.miR-200 b induces the polarization of macrophages to M2 and inhibits the polarization of macrophages to M1 by inhibiting the expression of KLF6.