Endometriosis and Cell Migration

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A research article from China Medical University investigated how a long non-coding RNA (lncRNA) known as HOTAIR contributes to the development of endometriosis.

Endometriosis is a disease affecting the female reproductive system by altering the uterine lining or endometrium. This lining should be present within the uterus, however, in this disease it is present outside of the uterus. Common symptoms are often prevailed by pelvic pain and infertility.

The cell migration and proliferation patterns of endometriosis are similar to that of cancer. Endometriosis can even affect other organs like the urinary bladder through deeply infiltrating endometriosis (DIE). Identifying potential preventions to this disease is crucial towards finding a cure.

LncRNAs can form during disease development and are able to influence transcriptional processes. HOTAIR is a type of lncRNA that acts as a molecular scaffold and has been overexpressed in various cancers. Through epigenome manipulation, it can increase cancer cell invasiveness and metastasis. Evaluating its expression in ovarian cancer could provide a promising method of treatment.

Both endometriosis and healthy patients were included in the research study. RNA samples were extracted from peripheral blood leukocytes. Taqman genotyping was used to detect single nucleotide polymorphisms (SNPs) where disease related functional SNPs were selected based on their ability to alter RNA structure. SNPs present in miRNA can influence endometriosis susceptibility. PCR tests were used to measure HOTAIR levels in test subjects and conduct site-directed mutagenesis.

Gene transfected cells were identified with green fluorescein protein (GFP) presence. DNA synthesis was monitored through immuno-fluorescent staining. All cells were seeded into a 96-well cell migration assay with Oris stoppers provided by Platypus Technologies. The stoppers create a cell free zone within the cell wells. An Oris detection mask was also utilized to quantify migrated cells.

Haplotype analysis which is used to identify linked alleles on the same chromosome was preformed to identify pro-disease alleles in test subjects. These alleles were compared to allelic distribution data. Results indicated that those with the G-G haplotype of rs1838169-rs17720428 had the most association with developing endometriosis. Genetic substitutions at these haplotype locations can cause changes in thermo-stability in the HOTAIR structure. It was found that HOTAIR levels in blood cells containing the two G-G haplotypes are 1.8 times higher than those with wild type (WT) genetic background. Furthermore, the HOTAIR structure also had more stability at the two functional SNPs. See the figure below for the results pertaining to WT and GG HOTAIR levels in blood cells.

HOTAIR associated epigenetic regulators were found to also be increased in endometriosis subjects. Anti-HOTAIR miRNAs were exposed to ovarian clear cancer cells to identify a possible endometriosis treatment. Results showed that anti-HOTAIR contributed to suppressing cell growth and mobility.

The genetic variations at specific alleles in HOTAIR were able to affect local RNA structures and indicate the degree of susceptibility to endometriosis. An increased risk of endometriosis was identified by identifying the exon regions that stabilize the HOTAIR structure (rs1838169 and rs17720428).

Resources:

Genetic impacts on thermostability of onco-lncRNA HOTAIR during the development and progression of endometriosis