The Mutations And Pathogenic Mechanism Of CARD14 Gene In Patients With Ovarian Endometriosis

Background and Objective:Endometriosis?EMs?is a common disease in women of childbearing age.The incidence has increased significantly in recent years.A considerable number of patients have the clinical manifestations of pelvic pain and infertility,which also seriously affected the health and life quality of middle-aged and young women.EMs is a complex gynecological refractory disease characterized by exhibiting with a wide range of lesions and various forms.The pathogenesis of EMs is complex and there are many doctrines,now it is generally believed that genetic and environmental factors can affect the incidence of EMs.Studies has found that EMs is closely related to the onset of ovarian cancer and may be a precancerous lesion of the tumor.Recently,more and more studies has confirmed this hypothesis and found that ectopic lesions in EMs patients contain many genetic somatic mutations,including KRAS,TP53,PTEN,BRCA2,ERBB3and other tumor-related genes,which suggested mutations in the EMs may play an important role in the pathogenesis of EMs.Otherwise,there are also many studies show that inflammation is closely related to the pathogenesis of EMs,for example,EMs patients with ectopic lesions and serum inflammatory factors concentrations increased significantly,which relatived to the progesterone levels reduced in EMs patients.The caspase recruitment domain family member 14?CARD14?gene acts as an upstream member which can activate the nuclear factor kappa B?NF-?B?pathway and promote the phosphorylation of transcription factor 65?p65?translocate into the nucleus by releasing the dissociation of the downstream inhibitor IKK alpha?IKK??,then activate the NF-?B signaling pathway,which plays an important role in the development of inflammation.Considering that EMs contains many somatic mutations,meanwhile inflammatory factors play an important role,we intend to detect the presence of CARD14 mutations in ectopic lesions of EMs patients,which in turn may activate inflammation-related signals and promote the pathogenesis of EMs.Methods:The patients diagnosed with ovarian endometriotic cysts by pathologist were collected.The ectopic endometrial lesions and paired peripheral blood were obtained from a toatl of 101 cases of EMs.The genomic DNA of tissues and blood samples was extracted and PCR amplification was performed.Followed by direct DNA sequencing,the sequences of tested samples were aligned to identify the potential mutations in the CARD14 gene.1)The pCMV6-hCARD14-DDK wild-type plasmid was obtained from Origene company,then the pCMV6-hCARD14-DDK mutant plasmid was constructed;then the corresponding lentivirus were constructed 2)HEC-1B cells were infected with these lentivirus,respectively.There were 3 groups:empty control,CARD14 wild-type and CARD14 mutant.Western blotting?WB?was used to detect the protein expression levels of CARD14;3)Cell functional assays were performed,namely,CCK-8 assay was for cell proliferation rate;Transwell assay was used to detect the cell invasion rate;cytometry was used to detect the cell apoptosis rate and cell cycle;scratch test was used to test the cell healing ability;clone forming assay was used to test the cell tumorigenicity;4)TMT technique was used to detect the differentially expressed proteins between CARD14 wild-type,mutant,and empty control.Results:1.The CARD14 p.V239A?c.716T>C?somatic mutation were found in2 out of 101?2.0%?cases of ovarian endometriotic lesion.The mutation caused valine mutated to alanine at the 239^th residue in CARD14?p.V239A?.Protein structural model results showed that this mutation could lead to changes in protein structure.No association was observed among the main clinical features and CARD14 mutations in our sample cohort.2.In the cell biological function test,clone forming assay detected CARD14mutant?p.V239A?can enhance the tumorigenicity of HEC-1B cells?P<0.01?.Cell scratch test indicated that the mutations could enhance the cell migration ability?P<0.01?.There was no statistically significant difference in cell proliferation,invasion,apoptosis and cycle?P>0.05?.3.The TMT technology detected and analyzed a total of 177 up-regulated proteins and 155 down-regulated proteins between HEC-1B cells infected with CARD14 wild-type and mutant lentivirus,respectively.The GO function and KEGG pathway analysis revealed that there are 38 immune associated proteins,These results implicated that CARD14 mutations might facilitate the pathogenesis of EMs via acting with these genes/signaling pathways.Conclusions:1.The CARD14 p.V239A?c.716T>C?somatic mutation?2.0%?was found in ovarian endometriosis for the first time,and this mutation can lead to changes in protein structure.2.CARD14 p.V239A mutation can promote cell healing ability and enhance the tumorigenicity of HEC-1B cells,there was no obvious effect on cell proliferation,invasion,apoptosis and cycle,suggested that CARD14 may play an active role in the development of ovarian endometriosis by influencing cell healing and tumorigenicity.3.The results of TMT proteomics showed that there were 38 differentially expressed proteins associated with immunity between HEC-1B cells infected with CARD14 wild-type and mutant.The analysis of GO function and KEGG indicated that CARD14 may participate in the pathogenesis of EMs through participation in immunity and inflammation-related signaling pathway.