Study Of Mutation Of MED12 Gene In Uterine Leiomyomas

Background and Objective: Uterine leiomyomas(ULs) is a common benign tumor of gynecology. However, the most important clinical manifestation of ULs is abnormal uterine bleeding, pelvic pain and anemia, which has strongly effect on the women’s life. Many foreign studies have reported that hypermutation of Mediator complex subunit 12(MED12) gene in ULs. Among them, about 70% of patients with ULs lesions exist MED12 mutations, most of which(90%) are located in the 44 th glycine(MED12 p.G44). In order to determine the MED12 gene with ULs mutation spectrum in patients, we plan to identify the mutations in MED12 predilection region by large-scale sequencing and analysis clinical characteristics; Next, we construct MED12 wild type and mutant plasmids and transfect them into 293 T cells. Finally, we test proliferation, invasion, migration, apoptosis and cell cycle in the 293 T cells, on the cellular level to find whether the MED12 gene mutation can actually promote the tumorigenesis of ULs.Methods: Firstly, we extract total DNA of 362 uterine tissues in ULS from 322 patients. To find out the potential mutations, amplify the DNA by PCR and followed by sequencing and sequence alignment. Simultaneously, Clinical feature was analyzed to figure out the key mutation. And then the recombinant plasmid was successfully transfected into 293 T cells. Divided into four groups: empty plasmid, blank cells, the MED12 mutant and wild type, using Western blotting(WB) detection MED12 protein expression. CCK8 method was used to detect cell survival rate. Transwell was used to detect cell migration and invasion. Flow cytometry was used to detect cell apoptosis. PI staining was used to detect cell cycle.Results: By sequencing, we proved that a total of 158 cases of MED12 mutations(43.6%) in 362 cases of all ULs samples. The ratio of the 44 th glycine(G) mutated into aspartic acid(D) was the highest(18.5%). MED12 mutation had no correlation with the clinical characteristics. Nevertheless, our study did not find any mutation in MED12 L. Using CCK8 assay to test proliferation, we found MED12 mutations G44 D promoted survival of 293 T cells and had significant statistical difference(P < 0.01). However, apoptosis and cell cycle had no statistical difference. Mutant MED12 G44 D can through changes in cell proliferation, so as to promote the occurrence and development of uterine leiomyoma. Our experiments show that the mutant MED12(G44D) can change cell proliferation, thereby promoting the tumorigenesis of ULs.Conclusions:1. In this study, we found that there was a high frequency of MED12 gene mutation(43.6%, 158/362) in the ULs samples. In addition, the mutation of MED12 occurred less frequently in the adjacent layers of the uterus. Furthermore, we did not find a MED12 L mutation in patients with uterine fibroids. 2. MED12 mutations have different types of mutations indifferent tumor in multiple uterine fibroids, it shows different origins of different tumor of multiple uterine fibroids. 3. MED12 mutation increased the viability of 293 T cells, however, it had no significant impact on the process of apoptosis of 293 T cells. Therefore, MED12 may promote the tumorigenesis of ULs by affecting cell viability.