To explore the possible mechanism of KiSS1 in control GnRH secretion participate insexual development onset and normal reproduction regulation by investigate the changesof expression of KiSS1, GnRH in hypothalamus and LH, FSH, E2 in serum,by using RNAinterference Mediated with Lentivirus-based Vectors, after Interfering expression ofKiSS1.
Constructed the eukaryotic expression plasmid of rat KiSS1 gene and four plasmidexpressing KiSS1 microRNA, then respectively co-transfected them into 293T cells.Real-time PCR detected the expression of KiSS1 mRMA in order to filter the mosteffective microRNA plasmid.Constructed recombinant entivirus and determined the titer,then they were intracerebroventricularly infused into the brain of Sprague-Dawley rats(21-day-old) . The three groups included interference virus group, Lentivirus-control,NS-control, Ten rats in each group animals were sacrificed at30-day-old, 35-day-old, 45-day-old. Then the expression of KiSS1 and GnRH mRNA wereconducted in the rat hypothalamus with Real-time PCR, the LH, FSH, E2 in serum wereexamined with Chemiluminescence method. HE staining was used to observe histomorphologyofovarian.
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Successfully filtered the most effective microRNA plasmid and constructed recombinantlentivirus. The titer of recombinant lentivirus was 8×108TU/ml. The levelof KiSS1 mRNA in interference virus group was significantly reduced after infectingrecombinant lentivirus compared with control group at 30d NS-controlgroup0.2±0.02, Lentivirus-control group 0.188±0.023, interference virus group0.106±0.018; at35d NS-control group 0.433±0.046; Lentivirus-control group0.41±0.034; interference virus group 0.218±0.025; at 45d NS-control group0.315±0.048; Lentivirus-control group 0.282±0.052; interference virus group0.215±0.033, at 30d F=112.40 P<0.01; at 35d, F=209.5 P<0.01; at 45d, F=5.2,P<0.05. The level of GnRH mRNA in interference virus group was significantly reducedafter infecting recombinant lentivirus compared with control group (at 30d NS-controlgroup 0.387±0.044; Lentivirus-control group 0.373±0.05; interference virusgroup 0.23±0.03; at35d NS-control group 0.517±0.048; Lentivirus-control group0.53±0.052; interference virus group 0.407±0.03; at 45d NS-control group0.468±0.03, Lentivirus-control group 0.479±0.038; interference virus group0.455±0.054,at 30d,F=24.6, P<0.01at35d,F=20.90 P<0.01). The difference was statistical significance. The level of LH in interferencevirus group is lower than other two groups at 35d (NS-control group 0.111±0.008mU/ml ; Lentivirus-control group 0.106±0.006mU/ml; interference virus group0.101±0.004mU/m F=5.98 P<0.05). The level of FSH in interference virus group islower than other two groups at 35d (NS-control group 0.219±0.015mU/ml;Lentivirus-control group 0.215±0.014mU/ml; interference virus group0.205±0.014mU/ml F=9.72 P<0.05). The level of E2 in interference virus group islower than other two groups(at 35d, NS-control group 56.04±4pg/ml;Lentivirus-control group 52.9±3.26pg/ml; interference virus group46.8±3.01pg/ml; at45d NS-control group 57.4±5.5pg/ml; Lentivirus-control group58.1±3.02pg/ml; interference virus group 52.4±3.57pg/ml; at 35d,F=25.25P<0.01; at 45d,F=7.63 P<0.05). Meanwhile, the time of vaginal orifice opening of theinterference virus group was significantly later than control virus group and normalsaline group (the interference virus group37.4±1.57d, control virus group35.2±1.19d, and normal saline group 34.9±0.99d, F=18.1 P<0.01). Thehistology of ovary showed similar results.
Our result show that the lentiviral vector of KiSS1-microRNA can inhibited theexpression of KiSS1 stabily, efficiently and specificly. Lentiviral with KiSS1-microRNAcan affect the expression of GnRH and The levels of sex hormone. Intralateroventricularmicroinjection of KiSS1-microRNA Lentiviral can delay sexual development of SD femalerat.
Lentivirus-mediated RNAi can effectively suppress the expression of KiSS1 stabily,as aresult the expression of GnRH gene can be suppressed, then affecting sexualdevelopment. It may provide a potential tool for the study of targeting control of sexualdevelopment in vivo and treating precocious puberty and other diseases.
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