The Mutations And Functional Analyses Of The MYH8 Gene In Samples With Ovarian Endometriosis

Background and purpose:Endometriosis(EMS)is a common gynecological disease in which endometrial glands and stroma survive outside the uterine cavity and infiltrate.The main symptoms include pelvic pain,sexual intercourse pain and infertility.Although EMS is a benign disease,it often shows the biological characteristics of malignant tumors,such as cell proliferation enhancement,spread and erosion to surrounding tissues.It is found that specific gene mutations play an important role in the occurrence and development of EMS.Myosin is a group of highly conserved proteins that are expressed in all eukaryotic cells.Its main function is to participate in the mechanical energy required by the transformation of cells,and play a role in the process of cell migration and muscle contraction.Myosin heavy chain 8(myh8)is an important member of myosin family,which can mediate biological processes such as cell migration and adhesion.It has been found that myh8 gene somatic mutations exist in a variety of tumors,including colorectal cancer,lung cancer,ovarian cancer and endometrial cancer;functional studies show that abnormal expression of myh8 can lead to the malignant tumors.Because EMs have tumor-like characteristics such as abnormal proliferation,apoptosis,invasion and migration,we hypothesized that patients with EMs may have mutations in the MYH8 gene and play an important role in the development of EMs,affecting downstream related signaling pathways and jointly promoting the pathogenesis of EMs.Method:Venous peripheral blood samples and the ectopic endometrial lesions from 152 ovaries EMs patients and 485 benign ovarian disease non-EMs control women were collected from Jiangxi Maternal and Child Health Hospital.After extraction of total DNA from blood and lesions,PCR amplification,purification of PCR products,direct sequencing and sequence comparison,the mutation and frequency of myh8 gene in patients with ovarian endometriosis were analyzed.The pcmv6-myh8-ddkoverexpression plasmid was purchased by origin company,and different myh8 mutant overexpression plasmids were obtained by site mutagenesis.The above plasmids were transfected into 293 T cells,and pcmv6-myh8-ddk wild-type and mutant lentiviruses were packaged respectively.After the above viruses infect Ishikawa cells,the biological effects of Ishikawa cells were detected: After the virus infected Ishikawa cells,the changes of cell biological effects were detected:Transwell chamber experiment was used to detect cell invasion and migration,flow cytometry was used to detect the apoptotic level and cell cycle of cells,CCK-8method was used to detect cell proliferation viability,cell scratch experiment was used to verify cell migration ability,plate cloning experiment was used to examine cell tumorigenic ability to verify Whether MYH8 gene mutation can really promote the pathogenesis of ovarian endometriosis.Then iTRAQ proteomics technology was used to detect the proteomic changes of pcDNA3.1 no-load control,MYH8 wild type,and two MYH8 mutants,and analyze the downstream genes and signal pathways that MYH8 mutations may affect.Result:There were 2 cases of MYH8 C.1441A> C(p.I481L)and C.4057G> A(p.E1353K)somatic mutations(1.3%)in 152 cases of ovarian endometriosis specimens.The results of protein structure simulation suggested that the mutation might cause structural changes of MYH8 protein.Subsequently,the correlation between the MYH8 mutation group of EMs patients and the clinical data of patients without EMs was analyzed,and no significant clinical indicators related to the mutation were found.These MYH8 gene mutations were not detected in 485 non-EMs control women.1.Transwell chamber experiment results showed that MYH8 mutant(p.I481 L,p.E1353K)could enhance the invasion and migration ability of Ishikawa cells(P <0.05)compared with MYH8 wild type;while there was no statistical difference in comparison of cell proliferation,cell plate tumor formation,apoptosis and cycle(P> 0.05).2.The results of iTRAQ technology showed that 256 differentially expressedproteins were up-regulated and 47 differentially expressed proteins were downregulated in 2 MYH8 mutant overexpressed Ishikawa cells compared with MYH8 wild type.Bioinformatics analysis revealed that there were differences in the expression of 6 cell motility-related proteins between MYH8 wild-type and mutant over-expressing Ishikawa cells,suggesting that MYH8 mutations may promote the pathogenesis of EMs through these differentially expressed genes.Conclusion:1.In this study,new mutations(1.3%)of MYH8 p.I481L(c.1441A>C)and p.E1353 K c.4057G>A were found in ovarian endometriosis for the first time,and this mutation can cause protein Structural change.2.These two new MYH8 mutations can enhance the invasion,migration and plate tumorigenesis ability of Ishikawa cells(P < 0.05),but have no significant effect on cell proliferation,plate tumorigenesis,apoptosis and cycle,suggesting that the new MYH8 mutations may promote the occurrence of ovarian endometriosis by affecting the metastatic ability of cells.3.The results of iTRAQ proteomics showed that there were differences in the expression of 6 cell motility related proteins between MYH8 wild type and mutant over-expressing Ishikawa cells,suggesting that MYH8 mutation may promote the pathogenesis of EMs through these genes.4.No significant correlation was found between MYH8 mutation and clinical data of patients.