O-104 The Impact of application of CRISPR /dCas9 systems for increasing the expression of FSH receptor in human granulosa cells

Abstract

Abstract Study question What are the molecular and physiological effects on human granulosa cells, when CRISPR/dCas9 epigenome edition technology is efficiently used to increase FSH receptor (FSHR) activity? Summary answer The significant increase in expression of FSHR maintained by dCAS9 systems has effects on several fundamental cellular events involving steroidogenic, life-sustaining,cell death related signaling sub-pathways. What is known already Manipulation of the expression of FSHR can result in biological events that include fundamental activities of FSH as regulation of folliculogenesis, production of steroidogenic hormones. FSHR can be activated in vitro by FSH analogues, and signaling networks activate ERK1/2 pathway through p38 mitogen-activated protein kinases. CRISPR/dCAS9 can be used to activate or inhibit gene action without silencing the gene. In spite of being used in many systems, interference of FSHR expression by epigenome edition is very limited as well as the use of in vitro tools to increase FSHR expression for investigating IVM, folliculogenesis, PCOS, FSH hyper/hyposensitivity, cancer biology. Study design, size, duration In-vitro cultured HGrC1 (human granulosa cell line) cells were treated by follitropin-alfa (Gonal-f). The experimental groups were designed in triplicates with cells i. without any genome edition and cells that have i. non-targeting gRNA, ii.dCAS9 activated FSHR, iii. dCAS9 activated FSHR and FSH treatment for 5 min, 15 min, or 1 hr; iv. only FSH treatment for 5 min, 15 min or 1 hr. FSHR related gene and protein expressions and estrogen production were evaluated. Participants/materials, setting, methods For human FSHR gene, two separate gRNAs were designed. Annealing, digestion, ligation, transformation, colony formation, plasmid isolation, Sanger sequencing analysis, MOI were accomplished for dCAS9/SAM system. HGrC1 (immortalized human granulosa cell line) cells were treated with 75 IU/ml Gonal-f in related groups. After treatment, in triplicates, gene/protein expression for MTOR, FSHR, AKT, MAPK8, MAPK1, TP 53, CASP 3, ESR1, CYP 19A1 were evaluated by qPCR, WB and IF. Estradiol was measured by chemiluminescence immunoassay. Main results and the role of chance FSHR has increased significantly in dCas9, dac9 + 5min Gonal-f, dCas9 + 15min Gonal-f, dCas9 + 1h Gonal-f; mostly in 5 min compared to groups without dCas9. The system plasmids did not show any non-specific effects.MAPK1, MAPK8, ESR1, MTOR, AKT, P53 and CASP3 gene expressions showed significant increase in dcas9 system groups compared to control and NT, whereas in all time-dependent treatments, a significant increase was seen in groups that have dcas9+Gonal-f compared to the ones treated with only Gonal-f. As time dependent manner has been investigated, expressions of MAPK1, MAPK8 and ESR1 were highest at min 5, expressions of CASP and AKT were highest at min 15 and the expression of MTOR was highest at hour 1, whereas FSHR and p53were strongly expressed at all time points (p < 0.05 for all mentioned results). Gene expression profiles were also confirmed for P-38 MAPK, AKT, FSHR by WB. IF stainings have revealed higher expression of AKT and MAPK in both dCas9 and dcas9+Gonal-f 5 min compared to the control group, however the intensity of the staining was not satisfactory enough. Interestingly, estrodiol(E2) levels were found to be higher in the 5 minutes only Gonal-f treatment and dcas9 with Gonal-f treatment, while the other groups were similar to control. Limitations, reasons for caution -This study has been conducted in cell line, rather than primary granulosa cells which are not easy targets for epigenome edition. -No testosterone derivatives were used as substrate for aromatase. This has been reflected in the results as significant increase in FSHR and related genes but non-significant increase in E2 production. Wider implications of the findings -dcas9/SAM system can be used efficiently increase other FSH-receptors, which is a potent way to reveal molecular sub-pathways in a temporal manner;eg. in male reproductive system or in other cells of female RS. -FSH related conditions as PCOS can be examined in vitro. -FHSR unresponsive conditions can be modeled in vitro. Trial registration number not applicable