Genetic Mutation Study In BPES And Role Of HotairM1 In Stemness Regulation Of Cancer Stem Cells

Objectives:Blepharophimosis-ptosis-epicanthus inversus syndrome(BPES)is a rare autosomal dominant disease caused by FOXL2 gene mutations,and it is clinically characterized by an eyelid malformation associated(type I)or not(type II)with premature ovarian failure(POF).Functional study of novel mutations is necessary for female patients to predict infertility and prompt therapy.In our study,we collected the blood samples of a Chinese BPES family to find out the type of BPES.Materials and methods:A clinical and molecular genetic investigation was performed in all members of a Chinese family with BPES.Blood samples were collected and genomic DNA was extracted.FOXL2 coding region was sequenced.Subcellular localization was detected by confocal microscopy.Expression levels of OSR2、SIRT1、St AR were detected by real-time PCR.Transactivation activities were performed by dual luciferase reporter assays and electrophoretic mobility shift assays.Results:A novel deletion mutation of FOXL2 was found in this Chinese family.This mutation(c.634＿641 del,CCCATGC)was located between the forkhead domain and the polyalanine domain,resulting in a frameshift mutation and a truncated protein.This mutant protein showed a strong cytoplasmic mislocalization,and an aberrant expression and transactivation activity of target gene St AR,implying a kind of type I mutation with a large possibility of infertility in female patients.Conclusions:This study identifies a novel mutation of FOXL2 and this mutant protein influenced the expression and transactivation ability of target genes in maintaining development and function of ovarian,indicating a type I BPES.Patients carrying this mutation,especially female patients should get prompt personalized therapy and follow-up.Objectives:Cancer stem cells are a small population of tumor cells that maintainstemness and have the ability to differentiate into normal tumor cells,which can be theimportant factors in the tumorigenesis,metastasis and drug resistance in tumors.Targetedtherapy to the cancer stem cells will be of benefit in solving the metastasis and drugresistance.Recently,the importance of epigenetics has been taken seriously.Many newepigenetic modifications such as long noncoding RNAs have been found,acting as potentialtargets in tumorigenesis and stemness.Hotair M1 plays an important role in the development.This study aims to use technologies and methods such as sphere formation,extreme limitingdilution assay,and chromatin immunoprecipitation assay to study the function andmechanism of lnc RNA in maintaining stemness.This study may provide new targets andtheoretical basis for the treatment of tumors.Materials and methods:The cancer stem cells were selected using flow cytometer todetect the expression of Hotair M1.Sphere formation assay,extreme limiting dilution assayand western blot were performed to detect the function of this lnc RNA in maintaining thestemness when Hotair M1 was knocked down or over-expressed.chromatin oligonucleotideprecipitation and chromatin immunoprecipitation assay were performed to detect theepigenetic mechanism of lnc RNA and its target gene.Results:The expression of Hotari M1 is low in cancer stem cells and the knock down ofHotair M1 will increase the proprotion of cancer stem cells,thus increasing the tumorproliferation,metastasis and tumor formation ability.The over-expression of Hotair M1 canreverse this phenomenon by decreasing the proprotion of cancer stem cells,and inhibitingthe proliferation,metastasis and tumor formation ability of tumors.HOXA1 is the target oflnc RNA Hotair M1.The inhibition of HOXA1 expression can significantly inhibit theproliferation and migration ability of tumors.Hotair M1 can silence HOXA1 expression byrecruiting the PRC2 complex,thus inducing trimethylation of H3K27 in HOXA1 promoter region,and inhibiting the expressiong of HOXA1.Conclusions:The expression of Hotari M1 is low in cancer stem cells and showing aninhibition of stemness.Hotair M1 knock down would expose the binding site of HOXA1promoter region and recruit the PRC2 complex,inducing trimethylation of H3K27,thusinhibiting the HOXA1 expression,and finally,increasing the proprotion of cancer stem cells,increasing the tumor proliferation,metastasis and tumor formation ability.