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Objective:To study the effect and related mechanism of Fuyou granule on danazol-induced precocious puberty model in rats. Method:Totally 21 cages of SD female rats were randomly divided into normal group， model group， Leuprorelin（0.1 g·kg<sup>-1</sup>） and Fuyou mixture group（37.9 g·kg<sup>-1</sup>）， and high-dose， mid-dose and low dose Fuyou granule<italic> </italic>groups（17.0，8.5，4.3 g·kg<sup>-1</sup>）. Rats at 5 days of age were given a single subcutaneous injection of 300 μg danazol to establish the precocious puberty model. After 10 days of modeling， drug intervention was started. Vaginal opening was examined at the age of 20 days， and the gonadal development was observed by hematoxylin-eosin （HE） staining. The levels of serum luteinizing hormone （LH）， follicle-stimulating hormone （FSH） and estradiol （E<sub>2</sub>） were determined by radioimmunoassay. The mRNA expressions of hypothalamic gonadotropin releasing hormone （GnRH）， Kiss-1， G protein-coupled receptor 54 （GPR54） were detected by Real-time fluorescent quantitative polymerase chain reaction （Real-time PCR）， and the expression of GnRH cells in the hypothalamus was detected by immunohistochemistry. Result:Compared with the normal group， the vaginal opening of the model group was significantly earlier， and the uterus and ovarian coefficients were significantly increased （<italic>P</italic><0.05）， indicating that the danazol-induced precocious puberty model was successfully established. The expression levels of GnRH， Kiss-1， and GPR54 also increased significantly （<italic>P</italic><0.05）， indicating that the danazol model can activate the HPG axis in advance， thereby inducing precocious puberty<bold>. </bold>Compared with the model group， the mid-dose Fuyou granule group significantly delayed the time of vaginal opening （<italic>P</italic><0.01）， high-dose Fuyou granule group<italic> </italic>significantly reduced uterine wall thickness and uterine coefficient （<italic>P</italic><0.05，<italic>P</italic><0.01）， mid-dose group reduced ovarian coefficient and uterine wall thickness （<italic>P</italic><0.05）. All the three dosage groups of Fuyou granule significantly reduced the content of serum hormones E<sub>2</sub>， LH and FSH （<italic>P</italic><0.05，<italic>P</italic><0.01）， reduced the expression levels of hypothalamic GnRH， Kiss-1 and GPR54 mRNA （<italic>P</italic><0.05）， and decreased the expression of GnRH cells （<italic>P</italic><0.05）. Conclusion:Fuyou granule can achieve therapeutic precocity by regulating the Kiss-1/GPR54 system and down-regulating the expression of GnRH to inhibit the activation of the HPG axis.
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@#[Abstract] Objective: To investigate the value of pre-treatment kisspeptin (KISS-1) expression for diagnosis and prognosis prediction of pancreatic cancer. Methods: The clinical data of 90 patients with pancreatic cancer (pancreatic cancer group) in Cancer Hospital of Hubei Province from April 2015 to December 2018 were retrospectively analyzed; in addition, 40 patients with benign pancreatic lesions were selected as the benign pancreatic lesion group and 30 healthy people were chosen as control group. The serum levels of KISS-1 and CA19-9 in each group were detected by ELISA. The diagnostic efficacies of CA19-9 and KISS-1 in pancreatic cancer and their relationship with the prognosis of pancreatic cancer were analyzed. Results: The serum KISS-1 level in the pancreatic cancer group was significantly higher than that in the benign pancreatic lesion group and the control group (both P<0.01); the area under the curve (AUC) of serum KISS-1, CA19-9 and their combination for pancreatic cancer detection was 0.757 (95% CI: 0.684-0.831, P=0.000), 0.900 (95% CI: 0.854-0.946, P=0.000), and 0.906 (95% CI: 0.861-0.950, P=0.000), respectively. The AUC value of the combined detection was statistically different from that of KISS-1 (Z=3.124, P=0.024), and the AUC value of CA19-9 was also statistically differently from KISS-1 (Z=3.253, P=0.025). Correlation analysis showed that there was a significant negative correlation between KISS-1 and CA19-9 (r=-0.358, P=0.002). The results of survival curve analysis showed that the survival time of patients with serum KISS-1≥73.6 pg/ml was significantly better than that of patients with KISS-1<73.6 pg/ml (χ2=4.520, P=0.036); KISS-1<73.6 pg/ml was independently related to the patient's prognosis with an OR of 2.37 (1.08-4.75). Conclusion: Serum KISS-1 is helpful for the early diagnosis of pancreatic cancer, and the combined detection of KISS-1 and CAI9-9 can improve the sensitivity and specificity of pancreatic cancer diagnosis, and also is beneficial for prognosis evaluation.
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Objective • To investigate the changes and distribution of thyroid transcription factor-1 (TTF1) expression around the puberty and to explore the position relationship among gonadotropin-releasing hormone (GnRH), KiSS1 and TTF1 expression in the hypothalamus of female SD rats. Methods • Female SD rats were divided into three groups: juvenile (JUV), early puberty (EP), and adult (AD). Tissue immunofluorescence staining was used to detect the expression of TTF1, KiSS1 and GnRH immunoreactive cells in the hypothalamus and the relative position among them. Real-time PCR was used to measure the expression of KiSS1, GnRH, TTF1 on mRNA levels in the hypothalamus, anteroventral periventricular nucleus (AVPV), and arcuate nucleus (ARC) respectively. Western blotting was performed to detect the changes in protein level of KiSS1 and TTF1. Results • TTF1 was densely expressed in hypothalamus nucleus AVPV, ARC and median eminence (ME) of female rats. GnRH,KiSS1 and TTF1 were adjacently expressed in ARC and ME. The mRNA level of TTF1 in the hypothalamus showed an upward trend after a slight decrease, while in AVPV and ARC tended to be consistent and showed an upward trend. The GnRH mRNA expression levels were significantly increased and reached the peak at AD. The mRNA expression levels of KiSS1 showed a sharp rise which was prior to the peak expression of GnRH mRNA at EP and then sustained the high level until AD. The protein expression level of TTF1 reached the peak at AD and the KiSS1 expression showed a sustained growth. Both of them showed an upward trend and basically consistent with the mRNA expression trend. Conclusion • Neuronal nuclei protein TTF1 mainly expressed in the nuclei AVPV, ARC, and ME of female rat hypothalamus. It was prominent in cells of ARC and ME which were localized GnRH, KiSS1, TTF1 positive neural cells. During the development of puberty onset, KiSS1 mRNA preceded GnRH mRNA to reach the peak at EP. The expression of TTF1 mRNA increased and reached a peak at AD, which was consistent with the overall increase of KiSS1 and GnRH expression. Protein expression of KiSS1 showed a corresponding upward trend together with their mRNA expression. TTF1 protein expression increased and peaked in AD.
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Objective·To investigate the changes and distribution of thyroid transcription factor-1 (TTF1) expression around the puberty and to explore the position relationship among gonadotropin-releasing hormone (GnRH), KiSS1 and TTF1 expression in the hypothalamus of female SD rats. Methods?·?Female SD rats were divided into three groups: juvenile (JUV), early puberty (EP), and adult (AD). Tissue immunofluorescence staining was used to detect the expression of TTF1, KiSS1 and GnRH immunoreactive cells in the hypothalamus and the relative position among them. Real-time PCR was used to measure the expression of KiSS1, GnRH, TTF1 on mRNA levels in the hypothalamus, anteroventral periventricular nucleus (AVPV), and arcuate nucleus (ARC) respectively. Western blotting was performed to detect the changes in protein level of KiSS1 and TTF1. Results?·?TTF1 was densely expressed in hypothalamus nucleus AVPV, ARC and median eminence (ME) of female rats. GnRH,KiSS1 and TTF1 were adjacently expressed in ARC and ME. The mRNA level of TTF1 in the hypothalamus showed an upward trend after a slight decrease, while in AVPV and ARC tended to be consistent and showed an upward trend. The GnRH mRNA expression levels were significantly increased and reached the peak at AD. The mRNA expression levels of KiSS1 showed a sharp rise which was prior to the peak expression of GnRH mRNA at EP and then sustained the high level until AD. The protein expression level of TTF1 reached the peak at AD and the KiSS1 expression showed a sustained growth. Both of them showed an upward trend and basically consistent with the mRNA expression trend. Conclusion?·?Neuronal nuclei protein TTF1 mainly expressed in the nuclei AVPV, ARC, and ME of female rat hypothalamus. It was prominent in cells of ARC and ME which were localized GnRH, KiSS1, TTF1 positive neural cells. During the development of puberty onset, KiSS1 mRNA preceded GnRH mRNA to reach the peak at EP. The expression of TTF1 mRNA increased and reached a peak at AD, which was consistent with the overall increase of KiSS1 and GnRH expression. Protein expression of KiSS1 showed a corresponding upward trend together with their mRNA expression. TTF1 protein expression increased and peaked in AD.
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The timing of puberty onset varies greatly among individuals, and much of this variation is modulated by genetic factors. This study aimed to identify the kisspeptin receptor (KISS1R) gene variations and to investigate the associations between these variations and central precocious puberty (CPP). Korean girls with CPP (n = 194) and their healthy controls (n = 99) were included in this study. The entire coding region and the exon-intron boundaries (exon 1 through 5) of the KISS1R gene were directly sequenced. Seven polymorphisms were identified in the KISS1R gene. A missense change c.1091T>A, and an intron variant c.738+64G>T showed significantly higher allele frequencies in CPP patients than in controls (c.1091T>A: 30.7% vs. 22.2%, P = 0.031; c.738+64G>T: 45.6% vs. 35.9%, P = 0.023). The missense variant (c.1091T>A) was a nonsynonymous polymorphism that induces amino acid substitution of p.Leu364His. The haplotype CAGTGTC was detected more frequently in the CPP group (P = 0.042). The sequence variants of the KISS1R gene can be inducible factors in the development of CPP. The association between sequence variants and CPP should be validated by further evidence obtained from larger samples of children with CPP.
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Adolescent , Child , Female , Humans , Amino Acid Substitution , Clinical Coding , Gene Frequency , Genetic Variation , Haplotypes , Introns , Puberty , Puberty, PrecociousABSTRACT
Objective:To explore the effects of KISS1 gene transfected by lentivirus on the proliferation,invasion and migration abilities of the colorectal cancer HCT116 cells,and to clarify their mechanisms.Methods:The human colorectal cancer cells with the lowest expression level of KISS1 gene were selected.The lentiviral vectors were builted and transfected the KISS1 gene,and the cells were divided into control group (treated with PBS),empty vector group (treated with empty vector) and over-expression group(treated with KISS1 gene vector).The multiplicity of infection (MOI) of the cells was detected by fluorescence microscope.Real-time PCR and Western blotting methods were used to detect the expression levels of KISS1 mRNA and protein(metastin);CCK-8 method was used to detect the proliferation ability of the cells;Transwell chambers method was used to detect the invasion and migration abilities of the cells.Results:Among LoVo,SW620,SW480,HCT-116,and HT29 cells,the expression levels of KISS1 mRNA and protein were lowest in HCT116 cells,so they were chosen as the research carrier.After transfected with lentiviral vectors,the HCT116 cells could stably express the enhanced green fluorescent protein(EGFP) gene,and the MOI was over 80%.Compared with control group and empty vector group,the expression levels of KISS1 mRNA and protein in the cells in over-expression group were significantly increased (P0.05).Conclusion:The KISS1 gene transfected by lentivirus vector can over-express KISS1 protein and inhibit the proliferation,invasion and migration abilities of the colorectal cancer cells,and the mechanism may be related to the expression of KISS1 protein.
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Kiss1 gene encodes Kisspeptins,an intercellular signal peptide whose corresponding receptor is Kiss1R. The initial study found that the Kiss1/Kiss1R system has the effect on inhibiting tumor metastasis. More and more evidence suggests that it can act on the hypothalamic-pituitary-gonadal(HPG) axis and is the key to puberty initiation and progression. It plays an important role in the neuroendocrine regulation of reproduction. In this paper, we reviewed the studies on Kiss1/Kiss1R system,its intracellular signal transduction pathway,role on HPG axis,and clinical application.
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ABSTRACT Prolactin is best known for its effects of stimulating mammary gland development and lactogenesis. However, prolactin is a pleiotropic hormone that is able to affect several physiological functions, including fertility. Prolactin receptors (PRLRs) are widely expressed in several tissues, including several brain regions and reproductive tract organs. Upon activation, PRLRs may exert prolactin’s functions through several signaling pathways, although the recruitment of the signal transducer and activator of transcription 5 causes most of the known effects of prolactin. Pathological hyperprolactinemia is mainly due to the presence of a prolactinoma or pharmacological effects induced by drugs that interact with the dopamine system. Notably, hyperprolactinemia is a frequent cause of reproductive dysfunction and may lead to infertility in males and females. Recently, several studies have indicated that prolactin may modulate the reproductive axis by acting on specific populations of hypothalamic neurons that express the Kiss1 gene. The Kiss1 gene encodes neuropeptides known as kisspeptins, which are powerful activators of gonadotropin-releasing hormone neurons. In the present review, we will summarize the current knowledge about prolactin’s actions on reproduction. Among other aspects, we will discuss whether the interaction between prolactin and the Kiss1-expressing neurons can affect reproduction and how kisspeptins may become a novel therapeutic approach to treat prolactin-induced infertility.
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Humans , Male , Female , Prolactin/metabolism , Reproduction/physiology , Kisspeptins/metabolism , Prolactin/pharmacology , Receptors, Prolactin/metabolism , Hyperprolactinemia/complications , Signal Transduction , Sex Factors , Hypothalamus/metabolism , Infertility/etiologyABSTRACT
Kisspeptin signaling at the gonadotropin-releasing hormone (GnRH) neuron is now relatively well characterized and established as being critical for the neural control of fertility. However, kisspeptin fibers and the kisspeptin receptor (KISS1R) are detected throughout the brain suggesting that kisspeptin is involved in regulating the activity of multiple neuronal circuits. We provide here a review of kisspeptin actions on neuronal populations throughout the brain including the magnocellular oxytocin and vasopressin neurons, and cells within the arcuate nucleus, hippocampus, and amygdala. The actions of kisspeptin in these brain regions are compared to its effects upon GnRH neurons. Two major themes arise from this analysis. First, it is apparent that kisspeptin signaling through KISS1R at the GnRH neuron is a unique, extremely potent form or neurotransmission whereas kisspeptin actions through KISS1R in other brain regions exhibit neuromodulatory actions typical of other neuropeptides. Second, it is becoming increasingly likely that kisspeptin acts as a neuromodulator not only through KISS1R but also through other RFamide receptors such as the neuropeptide FF receptors (NPFFRs). We suggest likely locations of kisspeptin signaling through NPFFRs but note that only limited tools are presently available for examining kisspeptin cross-signaling within the RFamide family of neuropeptides.
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Humans , Amygdala , Arcuate Nucleus of Hypothalamus , Brain , Central Nervous System , Dopamine , Fertility , Gonadotropin-Releasing Hormone , Hippocampus , Neurons , Neuropeptides , Neurotransmitter Agents , Oxytocin , Synaptic Transmission , VasopressinsABSTRACT
Objective To investigate the expressions and correlations of Kiss-1 and matrix metalloproteinases-9 (MMP9) proteins in the colorectal cancer (CRC) tissues of patients with synchronous colorectal liver metastasis (SCRLM),and the association with the clinicopathologic factors and prognosis of patients.Methods The retrospective case-control study was adopted.The clinicopathological data of 96 patients with SCRLM and 69 patients with CRC and no metastasis who were admitted to the Eastern Hepatobiliary Surgery Hospital of the Second Military Medical University from January 2000 to May 2013 were collected.The 96 CRC tissues and 50 adjacent normal tissues (distance from resection margin ≥ 5 cm) were collected from 96 patients with SCRLM,and 69 CRC tissues were collected from 69 patients with CRC and no metastasis.Expressions of Kiss-1 and MMP9 protein were detected by immunohistochemistry (IHC).The follow-up of outpatient examination and telephone interview was performed to detect survival of patients till August 2014.Comparison of count data and correlation between expressions of Kiss-1 or MMP9 protein and clinicopathological factors were analyzed by the chi-square test and Fisher exact probability.Survival curve was drawn using the Kaplan-Meier method,and survival analysis was done using the Log-rank test.Correlation analysis was done by the Pearson correlation.Results Expression of Kiss-1 protein was located in the cytoplasm of tissue cells.The positive expression rates of Kiss-1 protein in CRC tissues of patients with SCRLM and with CRC and no metastasis and in adjacent normal tissues were 24.0％ (23/96),43.5％ (30/69) and 52.0％ (26/50),respectively,with a significant difference among the 3 tissues (x2 =14.307,P ＜ 0.05) and no significant difference between CRC tissues of patients with CRC and no metastasis and patients with SCRLM (x2 =0.845,P ＞ 0.05).The positive expression rate of Kiss-1 protein in CRC tissues of patients with SCRLM was significantly different from that in CRC tissues of patients with CRC and no metastasis and in adjacent normal tissues (x2 =0.702,11.594,P ＜ 0.05).Expression of MMP9 protein was located in the cytoplasm of tissue cells.The positive expression rates of MMP9 protein in CRC tissues of patients with SCRLM and with CRC and no metastasis and in adjacent normal tissues were 67.7 ％ (65/96),62.3 ％ (43/69) and 36.0％ (18/50),respectively,with a significant difference among the 3 tissues (x2=14.203,P ＜0.05) and no significant difference between CRC tissues of patients with CRC and no metastasis and patients with SCRLM (x2=8.038,P ＞ 0.05).The positive expression rate of MMP9 protein in CRC tissues of patients with SCRLM was significantly different from that in CRC tissues of patients with CRC and no metastasis and in adjacent normal tissues (x2 =13.475,13.475,P ＜ 0.05).The positive expression rates of Kiss-1 protein in CRC tissues of patients with SCRLM were 66.7％,21.9％ and 17.6％ in the high-,mederate-and low-differentiated tumor,50.0％,28.6％ and 17.5％ in the muscular layer of tumor invasion,outside of serosa and serosal layer,44.0％ and 16.9％ in patients with and without lymph node metastasis,respectively,showing significant differences among the tumor differentiation degree,depth of tumor invasion and lymph node metastasis (x2=6.546,6.172,7.453,P ＜0.05).The positive expression rates of MMP9 protein in CRC tissues of patients with SCRLM were 25.0％,66.7％ and 76.2％ in the muscular layer of tumor invasion,outside of serosa and serosal layer,44.0％ and 76.1％ in patients with and without lymph node metastasis,respectively,showing significant differences between the depth of tumor invasion and lymph node metastasis (x2 =12.094,8.690,P ＜ 0.05).All the 96 patients with SCRLM were followed up for a median time of 68 months (range,12-176 months).The median overall survival time,median tumor-free survival time,5-year cumulative survival rate and 5-year tumor-free survival rate were 31 months,26 months,69.6％ and 26.1％ in patients with SCRLM and positive expression of Kiss-1 protein and 26 months,19 months,24.7％ and 12.3％ in patients with SCRLM and negative expression of Kiss-1 protein,respectively,showing significant differences between the overall survival and tumor-free survival (x2=16.578,14.436,P ＜ 0.05).The median overall survival time,median tumor-free survival time,5-year cumulative survival rate and 5-year tumor-free survival rate were 31 months,19 months,24.6％ and 12.3％ in patients with SCRLM and positive expression of MMP9 protein and 28 months,16 months,58.1％ and 22.6％ in patients with SCRLM and negative expression of MMP9 protein,respectively,showing significant differences between the overall survival and tumor-free survival (x2=14.073,8.532,P ＜0.05).Of 96 patients with SCRLM,there were 23 patients with positive expression of Kiss-1 protein (10 with positive expression of MMP9 protein and 13 with negative expression of MMP9 protein) and 73 with negative expression of Kiss-1 protein (55 with positive expression of MMP9 protein and 18 with negative expression of MMP9 protein),with a negative correlation between expressions of Kiss-1 protein and MMP9 protein (r =-0.291,P ＜ 0.05).Conclusions The reduced expression of Kiss-1 protein and elevated expression of MMP9 protein are closely associated with invasion and metastasis of CRC and prognosis of patients.A combination detection of Kiss-1 and MMP9 proteins is expected to become a marker for predicting the prognosis of patients with SCRLM based on the negative correlation between them.
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Kisspeptins are peptide products of Kiss-1 gene and have been substantially associated with the initiation of puberty by virtue of their ability to cause pulsatile release of GnRH. Kisspeptin consists of 54 amino acids domain called metastin and its biological activity may be localized to the C-terminal (C-10, C-13, and C-14) segments. Kisspeptin binds to its cognate receptor GPR54 in the hypothalamic neurons and it is a G-coupled membrane receptor. This study is an attempt to understand the tentative conformation of the peptides in its native membrane mimicking environment. A 14 amino-acid derivative of kisspeptin (Asp-58-Val59-Ser60-Ala61-Tyr62-Asn63-Trp64-Asn65-Ser66-Phe67-Gly68-Leu69-Arg70-Tyr71NH2) was synthesized by f-moc (9-fluorenyl methoxy carbonyl) solid phase synthesis strategy. The synthetic peptide was cleaved and purified by Reverse phase-HPLC. CD spectroscopic analysis of secondary structure of the peptide revealed random coil disordered conformation in the aqueous environment. However, the peptide adopted more ordered β-sheet conformation in the solvents such as TFE and HFIP.
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Kisspeptin/G-protein couple receptor-54 (GPR54) system plays a key role in the activation of the gonadotropic axis at puberty. Central precocious puberty (CPP) is caused by the premature activation of hypothalamic gonadotropin-releasing hormone secretion. This study was aimed to identify KISS1 gene variations and to investigate the associations between KISS1 gene variations and CPP in Korean girls. All coding exons of KISS1 gene were sequenced in Korean girls with CPP (n = 143) and their healthy controls (n = 101). Nine polymorphisms were identified in KISS1 gene. A novel single-nucleotide polymorphism (SNP), 55648176 T/G, was identified for the first time. SNP 55648184 C/G and 55648186 -/T were detected more frequently in CPP group than in control group. SNP 55648176 T/G was detected less frequently in CPP group than in control group. Haplotype GGGC-ACCC was detected less frequently in CPP group. The genetic variations of KISS1 gene can be contributing factors of development of CPP. The association between the gene variations and CPP should be validated by further evidence obtained from large-scaled and functional studies.
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Child , Female , Humans , Base Sequence , Genetic Markers/genetics , Genetic Predisposition to Disease/epidemiology , Kisspeptins/genetics , Molecular Sequence Data , Point Mutation/genetics , Polymorphism, Single Nucleotide/genetics , Prevalence , Puberty, Precocious/epidemiology , Reproducibility of Results , Republic of Korea/epidemiology , Risk Assessment , Sensitivity and SpecificityABSTRACT
Kisspeptin/G-protein couple receptor-54 (GPR54) system plays a key role in the activation of the gonadotropic axis at puberty. Central precocious puberty (CPP) is caused by the premature activation of hypothalamic gonadotropin-releasing hormone secretion. This study was aimed to identify KISS1 gene variations and to investigate the associations between KISS1 gene variations and CPP in Korean girls. All coding exons of KISS1 gene were sequenced in Korean girls with CPP (n = 143) and their healthy controls (n = 101). Nine polymorphisms were identified in KISS1 gene. A novel single-nucleotide polymorphism (SNP), 55648176 T/G, was identified for the first time. SNP 55648184 C/G and 55648186 -/T were detected more frequently in CPP group than in control group. SNP 55648176 T/G was detected less frequently in CPP group than in control group. Haplotype GGGC-ACCC was detected less frequently in CPP group. The genetic variations of KISS1 gene can be contributing factors of development of CPP. The association between the gene variations and CPP should be validated by further evidence obtained from large-scaled and functional studies.
Subject(s)
Child , Female , Humans , Base Sequence , Genetic Markers/genetics , Genetic Predisposition to Disease/epidemiology , Kisspeptins/genetics , Molecular Sequence Data , Point Mutation/genetics , Polymorphism, Single Nucleotide/genetics , Prevalence , Puberty, Precocious/epidemiology , Reproducibility of Results , Republic of Korea/epidemiology , Risk Assessment , Sensitivity and SpecificityABSTRACT
Objective: To investigate the effects of methylation of KISS1 gene promoter and KISS1 expression on biological characteristics of colorectal cancer HCT116 cells induced by methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-Aza-dC). Methods: The promoter methylation status of KISS1 gene and its mRNA and protein expressions in HCT116 cells were detected by methylation-specific PCR, real-time fluorogenic quantitative-PCR and Western blotting, respectively; after treatment with 5-Aza-dC in vitro. The abilities of invasion and migration of HCT116 cells were detected by Transwell assay, and the cell proliferation was detected by Cell Counting Kit-8 (CCK-8). Results: The demethylation of KISS1 promoter was induced after treatment with 5-Aza-dC. The expressions of KISS1 mRNA and protein in HCT116 cells were significantly increased in a dose-dependent manner after treatment with different doses of 5-Aza-dC for 5 days as compared with those without treatment with 5-Aza-dC (P 0.05). The abilities of invasion and migration of HCT116 cells after 5-Aza-dC treatment were also reduced (P < 0.05). Conclusion: The DNA methyltransferase inhibitor 5-Aza-dC can reverse the methylation of KISS1 promoter, and reduce the abilities of invasion and migration of HCT116 cells by up-regulating the expression of KISS1. Copyright© 2014 by TUMOR.
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Objective To research the effect of the Kiss-1 gene promoter methylation on the Kiss-1 gene expression in colorectal carcinoma tissue,and to analyze the relationship between the Kiss-1 gene methylation and the clinical pathological features of colorectal carcinoma and its clinical significance.Methods The Kiss-1 gene promotor region methylation,Kiss-1 gene mRNA and protein expressions were detected respectively by methylation-specific PCR, Real-time PCR and Western blotting method in 126 cases of colorectal carcinoma tissue and para-cacinoma normal colorectal tissue.Results The positive rate of Kiss-1 gene methylation in colorectal carcinoma tissue (83.33%)was significantly higher than that in normal tissue (30.16%)(P0.05 ). Conclusion The Kiss-1 gene promoter methylation in colorectal carcinoma tissue is associated with the Kiss-1 gene expression level and the malignant characteristics of colorectal carcinoma;Kiss-1 gene promoter methylation may be used as a reference indicator for predicting the risk of metastasis of colorectal carcinoma.
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Puberty onset is triggered by re-emergence of the hypothalamic-pituitary-gonadal axis (HPGA),which is characterized by the significantly increasing amplitude and frequency of gonadotropin-releasing hormone (GnRH) secretion in human being.A series of studies found that many genes control puberty onset,including KISS1 and GPR54 gene,estrogen receptor (ESR) gene,energy balance-related genes,LIN28B gene,MKRN3 gene and so on.Studies have been confirmed that the mutation and single nucleotide polymorphisms (SNP) of the genes above are associated with early puberty.In this paper,the relationship between genetic alterations of these genes and early puberty are summarized as follows.-
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Objective To investigate the function of Kiss1 gene and estrogen receptor α gene (ERα gene) in puberty of rats,by detecting the expressions of Kiss1 mRNA and ERα mRNA in the hypothalamus and the serum luteinizing hormone (LH) and estradiol (E2) level of female Sprague-Dawley (SD) rats at various stage of development with Real-time PCR and enzyme-linked immunosorbent assay(ELISA).Methods Thirty-five female SD rats of 3 days were weaned on postnatal(PND)22 and then the vaginal opening condition was observed daily.The rats were sacrificed at PND 15(juvenile group,n =19) and PND 35 (pubertal group,n =16).The hypothalamus were segregated and the serum were extracted from heart blood.All of the samples were stored at-80 ℃ prepared.Then the mRNA were extracted from the hypothalamus and the cDNA obtained by reverse transcription were tested with real-time PCR.The relative mRNA expression level of Kiss1 gene and ERα gene were calculated.Results 1.Entire level:it was found that the pubertal group vaginal opening time was (32.1 ± 1.0) days,while the juvenile group was not found with vaginal opening until sacrificed.2.Real-time PCR:the expressions of Kiss1 and ERα gene were significantly increased in pubertal group (Kissl gene:5.39-± 2.52,ERα gene:1.57 ±1.87) compared with juvenile group(Kiss1 gene:1.06 ± 1.09,ERα gene:0.59-± 0.68),and the differences were statistically significant (all P ＜ 0.001).3.ELISA:the serum LH and E2 in pubertal group [LH (11.61 ± 0.95) IU/L,E2 (167.53 ± 31.09) ng/L] were significantly higher than LH [(5.46-± 1.89)IU/L] and E2 [(58.59 ± 29.96) ng/L] in juvenile group,and the differences were statistically significant (all P ＜0.001).Conclusion Kiss1 gene and ERα gene are involved in the start of the sexual development of female SD rat.
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Objective To explore the expression and significance of Kiss-1, Ki-67 and VEGF-C in papillary thyroid carcinoma(PTC) and thyroid follicular adenoma (FA).Methods Forty-four cases of PTC and twelve cases of FA paraffin-embedded tissues were used .Immunohistochemical staining and microscopic image analysis technique were used to analyze the expression of Kiss-1, Ki-67 and VEGF-C.Results The integrated optical density (IOD) of Kiss-1, and VEGF-C in the PTC groups was 475.56 ±126.02 and 805.29 ±226.05,respectively.The proliferation index of Ki-67 protein was (3.36 ±1.11) %and the difference between the PTC and FA groups was statistically significant (P<0.05).The IOD of the above two indices was 408.12 ±124.05 and 912.63 ±108.12 in the PTC with lymph node metastasis group , respectively, while the proliferation index of Ki -67 protein was (3.93 ±0.92) % and the difference vs the group without lymph node metastasis was significant ( P <0.05 ) .In the PTC with capsular infiltration group the IOD of above two was 425.58 ±87.38 and 891.37 ±149.36, the proliferation index of Ki -67 protein was (3.79 ±1.09) %and the difference with PTC group without capsular infiltrtion was statistically significant (P<0.05).Linear correlation analysis showed that Ki-67 and VEGF-C were with positively correlated in PTC and FA tissues (P<0.05),while Kiss-1 and Ki-67, VEGF-C were with negatively correlated in PTC and FA tissues (P<0.05).Conclusion Kiss-1, Ki-67 and VEGF-C can facilitate the differential diagnosis of PTC and FA , serving as prognostic indicators in patients with PTC .
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The effect of AMP-activated protein kinase (AMPK) on KiSS-1 mRNA levels was detected by realtime PCR in the hypothalamic GT1-7 neurons. The promoter activity of KiSS-1 gene was detected by DualLuciferase Reporter Assay System.The effects of AMPK on the protein expression and subcellular distribution of SP1 were determined by Western blot.The results showed that AMPK reduced the mRNA expression and promoter activity of KiSS-1 gene while SP1 increased the promoter activity of KiSS-1 gene. Besides,AMPK alse decreased the translocation of SP1.These results suggest that AMPK may inhibit the expression of KiSS-1 gene by decreasing the translocation of SP1 from cytoplasm to nucleus in the hypothalamus GT1-7 neurons.
ABSTRACT
PURPOSE: Leptin has been considered a link between metabolic state and reproductive activity. Defective reproductive function can occur in leptin-deficient and leptin-excessive conditions. The aim of this study was to examine the effects of centrally injected leptin on the hypothalamic KiSS-1 system in relation to gonadotropin-releasing hormone (GnRH) action in the initial stage of puberty. METHODS: Leptin (1 microg) was injected directly into the ventricle of pubertal female mice. The resultant gene expressions of hypothalamic GnRH and KiSS-1 and pituitary LH, 2 and 4 hours after injection, were compared with those of saline-injected control mice. The changes in the gene expressions after blocking the GnRH action were also analyzed. RESULTS: The basal expression levels of KiSS-1, GnRH, and LH were significantly higher in the pubertal mice than in the prepubertal mice. The 1-microg leptin dose significantly decreased the mRNA expression levels of KiSS-1, GnRH, and LH in the pubertal mice. A GnRH antagonist significantly increased the KiSS-1 and GnRH mRNA expression levels, and the additional leptin injection decreased the gene expression levels compared with those in the control group. CONCLUSION: The excess leptin might have suppressed the central reproductive axis in the pubertal mice by inhibiting the KiSS-1 expression, and this mechanism is independent of the GnRH-LH-estradiol feedback loop.