Abstract
Background17alpha-hydroxylase/17, 20-lyase encoded by CYP17 is the key enzyme in androgen biosynthesis pathway. Previous studies demonstrated the accentuation of the enzyme in patients with polycystic ovary syndrome (PCOS) was the most important mechanism of androgen excess. We chose CYP17 as the therapeutic target, trying to suppress the activity of 17alpha-hydroxylase/17, 20-lyase and inhibit androgen biosynthesis by silencing the expression of CYP17 in the rat ovary.MethodsThree CYP17-targeting and one negative control oligonucleotides were designed and used in the present study. The silence efficiency of lentivirus shRNA was assessed by qRT-PCR, Western blotting and hormone assay. After subcapsular injection of lentivirus shRNA in rat ovary, the delivery efficiency was evaluated by GFP fluorescence and qPCR. Total RNA was extracted from rat ovary for CYP17 mRNA determination and rat serum was collected for hormone measurement.ResultsIn total, three CYP17-targeting lentivirus shRNAs were synthesized. The results showed that all of them had a silencing effect on CYP17 mRNA and protein. Moreover, androstenedione secreted by rat theca interstitial cells (TIC) in the RNAi group declined significantly compared with that in the control group. Two weeks after rat ovarian subcapsular injection of chosen CYP17 shRNA, the GFP fluorescence of frozen ovarian sections could be seen clearly under fluorescence microscope. It also showed that the GFP DNA level increased significantly, and its relative expression level was 7.42 times higher than that in the control group. Simultaneously, shRNA treatment significantly decreased CYP17 mRNA and protein levels at 61% and 54%, respectively. Hormone assay showed that all the levels of androstenedione, 17-hydroxyprogesterone and testosterone declined to a certain degree, but progesterone levels declined significantly.ConclusionThe present study proves for the first time that ovarian androgen biosynthesis can be inhibited by silencing CYP17 expression. It may provide a novel strategy for therapy of hyperandrogenism diseases, and also set an example for the use of RNAi technology in endocrine diseases.
Highlights
Polycystic ovary syndrome (PCOS) is one of the most common gynecological endocrine diseases, which has an incidence rate of 5–10% in women of reproductive age
Cells were harvested and total RNA was extracted for CYP17 mRNA measurement
It showed that all three constructed lentivirus shRNAs had silencing effects on CYP17, and their silence efficiency was 65.5%, 73.7% and 77.4%, respectively (Fig 3B)
Summary
Polycystic ovary syndrome (PCOS) is one of the most common gynecological endocrine diseases, which has an incidence rate of 5–10% in women of reproductive age. Hyperandrogenism is closely associated with PCOS, and is regarded as the most important presentation of PCOS. The patient with PCOS often presents with anovulation which is associated with follicular dysfunction induced by hyperandrogenism [4]. Androgen excess can impair glucose tolerance, leading to insulin resistance, and causing a series of metabolic diseases [5,6]. Some anti-androgen drugs (such as cyproterone metformin), single or in combination, have been used to treat the patient with PCOS, but therapeutic effects are unsatisfied and side effects are still under concerns [7,8]
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