Characterisation of microRNAs in Human Stem Cells

In collaboration with David Baulcombe and Attila Molnar we have generated microRNA libraries for human embryonic stem cells (hESCs) before and after differentiation along the neuronal lineage and also from human mesenchymal stem cells (hMSCs). Both cell types are of medical importance and understanding how their proliferation and differentiation is regulated by microRNAs is also of scientific interest. The hMSC library was sequenced by 454 technology and the two subsequent hESC libraries by Solexa sequencing. Approximately a quarter of all currently known microRNAs were identified between the libraries, in addition to 3 novel microRNAs and 25 annotated piRNAs. For the hESC libraries, we verified the presence of embryonic specific microRNAs (miR-302 family) and neuronal specific microRNAs (miR-9/miR-9*), and demonstrated that expression of these miRNAs is regulated at the transcriptional level. Additionally, promoter assessments of miR-9 transcription revealed that multiple upstream regions may be important in neuronal specific expression. Almost half of all known human microRNAs are located within the introns of host genes. We used microarrays to analyse host gene expression and found that there was little correlation with microRNA expression, indicating that many microRNAs are not regulated at the transcriptional level by their host promoter. Furthermore, the expression of microRNAs from the same cluster, and also from the same hairpin precursor, did not always correlate when compared between the stem cell libraries. Taken together, this data indicates that microRNAs are regulated at a variety of levels both pre- and post-transcriptionally. Many microRNA isomers were also detected that differed in expression between human cell types, and upon differentiation of the hMSCs through the osteoblastic lineage. Interestingly, microRNAs and some of their isomers showed different affinities for Argonaute proteins in pulldown assays. We also profiled mRNAs that were immunoprecipitated with Argonaute in order to identify miRNA target

A polymorphism in a let-7 microRNA binding site of KRAS in women with endometriosis.

Endometriosis is found in 5-15% of women of reproductive age and is more frequent in relatives of women with the disease. Activation of KRAS results in de novo endometriosis in mice, however, activating KRAS mutations have not been identified in women. We screened 150 women with endometriosis for a polymorphism in a let-7 microRNA (miRNA) binding site in the 3'-UTR of KRAS and detected a KRAS variant allele in 31% of women with endometriosis as opposed to 5% of a large diverse control population. KRAS mRNA and protein expression were increased in cultured endometrial stromal cells of women with the KRAS variant. Increased KRAS protein was due to altered miRNA binding as demonstrated in reporter assays. Endometrial stromal cells from women with the KRAS variant showed increased proliferation and invasion. In a murine model, endometrial xenografts containing the KRAS variant demonstrated increased proliferation and decreased progesterone receptor levels. These findings suggest that an inherited polymorphism of a let-7 miRNA binding site in KRAS leads to abnormal endometrial growth and endometriosis. The LCS6 polymorphism is the first described genetic marker of endometriosis risk

Characterisation of microRNAs in human stem cells

In collaboration with David Baulcombe and Attila Molnar we have generated microRNA libraries for human embryonic stem cells (hESCs) before and after differentiation along the neuronal lineage and also from human mesenchymal stem cells (hMSCs). Both cell types are of medical importance and understanding how their proliferation and differentiation is regulated by microRNAs is also of scientific interest. The hMSC library was sequenced by 454 technology and the two subsequent hESC libraries by Solexa sequencing. Approximately a quarter of all currently known microRNAs were identified between the libraries, in addition to 3 novel microRNAs and 25 annotated piRNAs. For the hESC libraries, we verified the presence of embryonic specific microRNAs (miR-302 family) and neuronal specific microRNAs (miR-9/miR-9*), and demonstrated that expression of these miRNAs is regulated at the transcriptional level. Additionally, promoter assessments of miR-9 transcription revealed that multiple upstream regions may be important in neuronal specific expression. Almost half of all known human microRNAs are located within the introns of host genes. We used microarrays to analyse host gene expression and found that there was little correlation with microRNA expression, indicating that many microRNAs are not regulated at the transcriptional level by their host promoter. Furthermore, the expression of microRNAs from the same cluster, and also from the same hairpin precursor, did not always correlate when compared between the stem cell libraries. Taken together, this data indicates that microRNAs are regulated at a variety of levels both pre- and post-transcriptionally. Many microRNA isomers were also detected that differed in expression between human cell types, and upon differentiation of the hMSCs through the osteoblastic lineage. Interestingly, microRNAs and some of their isomers showed different affinities for Argonaute proteins in pulldown assays. We also profiled mRNAs that were immunoprecipitated with Argonaute in order to identify miRNA targetsEThOS - Electronic Theses Online ServiceBBSRC, Institute of Obstetrics and Gynaecology Trust.GBUnited Kingdo

A polymorphism in a let‐7

Endometriosis is found in 5–15% of women of reproductive age and is more frequent in relatives of women with the disease. Activation of KRAS results in de novo endometriosis in mice, however, activating KRAS mutations have not been identified in women. We screened 150 women with endometriosis for a polymorphism in a let-7 microRNA (miRNA) binding site in the 3'-UTR of KRAS and detected a KRAS variant allele in 31% of women with endometriosis as opposed to 5% of a large diverse control population. KRAS mRNA and protein expression were increased in cultured endometrial stromal cells of women with the KRAS variant. Increased KRAS protein was due to altered miRNA binding as demonstrated in reporter assays. Endometrial stromal cells from women with the KRAS variant showed increased proliferation and invasion. In a murine model, endometrial xenografts containing the KRAS variant demonstrated increased proliferation and decreased progesterone receptor levels. These findings suggest that an inherited polymorphism of a let-7 miRNA binding site in KRAS leads to abnormal endometrial growth and endometriosis. The LCS6 polymorphism is the first described genetic marker of endometriosis risk

Estrogen withdrawal, increased breast cancer risk and the KRAS-variant.

The KRAS-variant is a biologically functional, microRNA binding site variant, which predicts increased cancer risk especially for women. Because external exposures, such as chemotherapy, differentially impact the effect of this mutation, we evaluated the association of estrogen exposures, breast cancer (BC) risk and tumor biology in women with the KRAS-variant. Women with BC (n = 1712), the subset with the KRAS-variant (n = 286) and KRAS-variant unaffected controls (n = 80) were evaluated, and hormonal exposures, KRAS-variant status, and pathology were compared. The impact of estrogen withdrawal on transformation of isogenic normal breast cell lines with or without the KRAS-variant was studied. Finally, the association and presentation characteristics of the KRAS-variant and multiple primary breast cancer (MPBC) were evaluated. KRAS-variant BC patients were more likely to have ovarian removal pre-BC diagnosis than non-variant BC patients (p = 0.033). In addition, KRAS-variant BC patients also appeared to have a lower estrogen state than KRAS-variant unaffected controls, with a lower BMI (P < 0.001). Finally, hormone replacement therapy (HRT) discontinuation in KRAS-variant patients was associated with a diagnosis of triple negative BC (P < 0.001). Biologically confirming our clinical findings, acute estrogen withdrawal led to oncogenic transformation in KRAS-variant positive isogenic cell lines. Finally, KRAS-variant BC patients had greater than an 11-fold increased risk of presenting with MPBC compared to non-variant patients (45.39% vs 6.78%, OR 11.44 [3.42-37.87], P < 0.001). Thus, estrogen withdrawal and a low estrogen state appear to increase BC risk and to predict aggressive tumor biology in women with the KRAS-variant, who are also significantly more likely to present with multiple primary breast cancer

Estrogen withdrawal, increased breast cancer risk and the KRAS-variant

<p>The <i>KRAS</i>-variant is a biologically functional, microRNA binding site variant, which predicts increased cancer risk especially for women. Because external exposures, such as chemotherapy, differentially impact the effect of this mutation, we evaluated the association of estrogen exposures, breast cancer (BC) risk and tumor biology in women with the <i>KRAS</i>-variant. Women with BC (<i>n</i> = 1712), the subset with the <i>KRAS</i>-variant (<i>n</i> = 286) and <i>KRAS</i>-variant unaffected controls (<i>n</i> = 80) were evaluated, and hormonal exposures, <i>KRAS</i>-variant status, and pathology were compared. The impact of estrogen withdrawal on transformation of isogenic normal breast cell lines with or without the <i>KRAS</i>-variant was studied. Finally, the association and presentation characteristics of the <i>KRAS</i>-variant and multiple primary breast cancer (MPBC) were evaluated. <i>KRAS</i>-variant BC patients were more likely to have ovarian removal pre-BC diagnosis than non-variant BC patients (p = 0.033). In addition, <i>KRAS</i>-variant BC patients also appeared to have a lower estrogen state than <i>KRAS</i>-variant unaffected controls, with a lower BMI (<i>P</i> < 0.001). Finally, hormone replacement therapy (HRT) discontinuation in <i>KRAS</i>-variant patients was associated with a diagnosis of triple negative BC (<i>P</i> < 0.001). Biologically confirming our clinical findings, acute estrogen withdrawal led to oncogenic transformation in <i>KRAS</i>-variant positive isogenic cell lines. Finally, <i>KRAS</i>-variant BC patients had greater than an 11-fold increased risk of presenting with MPBC compared to non-variant patients (45.39% vs 6.78%, OR 11.44 [3.42–37.87], <i>P</i> < 0.001). Thus, estrogen withdrawal and a low estrogen state appear to increase BC risk and to predict aggressive tumor biology in women with the <i>KRAS</i>-variant, who are also significantly more likely to present with multiple primary breast cancer.</p

MicroRNA signatures differentiate melanoma subtypes

Melanoma is an aggressive cancer that is highly resistance to therapies once metastasized. We studied microRNA (miRNA) expression in clinical melanoma subtypes and evaluated different miRNA signatures in the background of gain of function somatic and inherited mutations associated with melanoma. Total RNA from 42 patient derived primary melanoma cell lines and three independent normal primary melanocyte cell cultures was evaluated by miRNA array. MiRNA expression was then analyzed comparing subtypes and additional clinicopathologic criteria including somatic mutations. The prevalence and association of an inherited variant in a miRNA binding site in the 3′UTR of the KRAS oncogene, referred to as the KRAS-variant, was also evaluated. We show that seven miRNAs, miR-142-3p, miR-486, miR-214, miR-218, miR-362, miR-650 and miR-31, were significantly correlated with acral as compared to non-acral melanomas (p < 0.04). In addition, we discovered that the KRAS-variant was enriched in non-acral melanoma (25%), and that miR-137 under expression was significantly associated with melanomas with the KRAS-variant. Our findings indicate that miRNAs are differentially expressed in melanoma subtypes and that their misregulation can be impacted by inherited gene variants, supporting the hypothesis that miRNA misregulation reflects biological differences in melanoma

Cryptic splice sites and split genes

We describe a new program called cryptic splice finder (CSF) that can reliably identify cryptic splice sites (css), so providing a useful tool to help investigate splicing mutations in genetic disease. We report that many css are not entirely dormant and are often already active at low levels in normal genes prior to their enhancement in genetic disease. We also report a fascinating correlation between the positions of css and introns, whereby css within the exons of one species frequently match the exact position of introns in equivalent genes from another species. These results strongly indicate that many introns were inserted into css during evolution and they also imply that the splicing information that lies outside some introns can be independently recognized by the splicing machinery and was in place prior to intron insertion. This indicates that non-intronic splicing information had a key role in shaping the split structure of eukaryote genes. <br/