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Androgen receptor (AR) plays a key regulatory role in the development of castration resistant prostate cancer (CRPC), and the level of constitutive active variants represented by androgen receptor variant 7 (AR-V7) is increasing during the progress of CRPC, which can be used as a molecular marker of disease progress and prognosis of patients with CRPC. It is an important target to overcome castration resistance and improve the quality of life and survival of patients. In this paper, the function of AR-V7 and its molecular regulation mechanism in CRPC are reviewed. The research shows that the generation of AR-V7 is related to the structural rearrangement of AR gene, gene amplification and the selective splicing of AR gene transcripts, and it is affected by the coordinated regulation of multiple signal pathway molecules such as TGF-β; AR-V7 changes the transport and nuclear localization mechanism of AR protein, and further affects the transcriptional expression of downstream target genes. AR-V7 antagonizes AR activity and blocks the differentiation process driven by AR and androgen, and inhibits the expression of tumor suppressor genes to stimulate the proliferation of tumor cells, thus promoting the progress of Pca. Related targeting studies have revealed AR-V7 targets and CRPC treatment strategies. Currently, they mainly focus on AR-V7 protein degradation, mRNA expression inhibition and N-terminal domain targeting intervention. With the development of in-depth research, the molecular mechanism of AR-V7 in the progress of Pca will be gradually clarified, which will certainly play a greater role in the prevention and treatment of CRPC.
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Biotinidase defciency (BD) is an autosomal recessive disorder caused by bi-allelic mutation in the BTD gene. Clinical manifestations in BD mainly depends on residual biotinidase enzyme activity, although there are some exceptions. Broadly BD disorders are classifed as profound BD and partial BD. Further profound BD can be early onset, late onset, and sometimes may be asymptomatic. Clinically late-onset profound BD can present with spectrum of manifestations ranging from single organ to multiple organ involvement, typically afecting function of brain, eye, ear, and skin. Here, a frst-born child to consanguineous parents with late-onset profound BD presenting with hyperventilation secondary to lactic acidosis, hypotonia, evolving spasticity, and abnormal neuroimaging fndings caused by novel homozygous variant, c.466-3T>G in the BTD gene is reported.
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Objective:To explore the efficacy and mechanism of Guben Qingyuan prescription combined with androgen deprivation therapy （ADT） in the treatment of castration-resistant prostate cancer （CRPC）. Method:A CRPC-bearing mouse model was established. When the tumor volume reached about 100 mm<sup>3</sup>， 50 CRPC-bearing BALB/c nude mice were randomly divided into the model group， ADT group， and ADT+low-， medium-， high-dose Guben Qingyuan prescription groups， with 10 mice in each group. After grouping， it was ensured that there was no statistically significant difference in tumor volume between groups. The mice in the model group was treated with the same amount of normal saline （10 mL·kg<sup>-1</sup>） by gavage， twice a day， while those in the other groups were provided with bicalutamide （5 mg·kg<sup>-1</sup>） for intragastric administration， once a day， and then with goserelin （0.36 mg·kg<sup>-1</sup>） for intraperitoneal injection on the 10th day. On the basis of ADT， the ones in the ADT+Guben Qingyuan prescription groups further received Guben Qingyuan prescription at the low （2.5 g·kg<sup>-1</sup>）， medium （25 g·kg<sup>-1</sup>）， and high doses （50 g·kg<sup>-1</sup>） by gavage， twice a day. After 25 days of continuous administration， the tumor tissue was harvested for recording the tumor growth and calculating the tumor inhibition rate. The mRNA and protein expression levels of androgen receptor （AR）， androgen receptor splice variant-7 （AR-V7）， and prostate-specific antigen （PSA） were detected by real-time polymerase chain reaction （Real-time PCR） and Western blot assay. Result:The tumor inhibition rates of the ADT+low-， medium-， and high-dose Guben Qingyuan prescription groups were 27.95%， 46.71%， and 44.46%， respectively， and the inhibition rates in the ADT+medium- and high-dose Guben Qingyuan prescription groups were significantly increased as compared with that in the ADT group （<italic>P</italic><0.05）. As revealed by comparison with the ADT group， Guben Qingyuan prescription at the medium and high doses significantly down-regulated the mRNA and protein expression levels of AR， AR-V7， and PSA （<italic>P</italic><0.05）. Conclusion:Guben Qingyuan prescription combined with ADT is efficient in controlling the tumor growth in CRPC-bearing mice， which is related to the regulation of AR/AR-V7 signaling pathway.
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The development and progression of metastatic castration-resistant prostate cancer is the major challenge in the treatment of advanced prostate cancer. The androgen receptor signaling pathway remains active in metastatic castration-resistant prostate cancer. Docetaxel and cabazitaxel are the first- and second-line chemotherapy, respectively, for patients with metastatic castration-resistant prostate cancer. These two taxanes, in general, function by (i) inhibiting mitosis and inducing apoptosis and (ii) preventing microtubule-dependent cargo trafficking. In prostate cancer, taxanes have been reported to inhibit the nuclear translocation and activity of the androgen receptor. However, whether this is attainable or not clinically remains controversial. In this review, we will provide a comprehensive view of the effects of taxanes on androgen receptor signaling in prostate cancer.
Subject(s)
Humans , Male , Antineoplastic Agents, Phytogenic/therapeutic use , Prostatic Neoplasms, Castration-Resistant/drug therapy , Receptors, Androgen/drug effects , Signal Transduction/drug effects , Taxoids/therapeutic useABSTRACT
Objective • To investigate the effects of ovarian granulosa cell androgen receptor splice variant insertion isoform (AR-ins) on the levels of sex hormones and insulin in patients with polycystic ovary syndrome (PCOS). Methods • Seventy-nine control patients (control group) of normal ovulation and one hundred and fifty-four PCOS patients (PCOS group) who received in vitro fertilization embryo transfer in the Assisted Reproductive Department of the International Peace Maternity & Child Health Hospital, Shanghai Jiao Tong University School of Medicine from Jun. 2017 to Jun. 2018 were recruited. The sociodemographic information, serum basic hormones and insulin data were collected according to medical history record, and the hormone levels were measured by chemiluminescence. The ovarian granulosa cells and follicular fluids of all the patients were collected. Nested PCR was used to identify the androgen receptor splice variant (AR SV) in the ovarian granulosa cells from PCOS patients, and then they were divided into two groups, i.e., PCOS Wt group (n=39) and PCOS AR-ins group (n=115). The levels of sex hormones and insulin in follicular fluids of the three groups were detected by chemiluminescence. The free androgen index (FAI) was calculated. The sociodemographic information, the levels of sex hormones and insulin in serum and follicular fluid of the three groups were compared by One-way ANOVA, Welch's ANOVA, Kruskal-Wallis H test and chi-square test. Results • Compared with the control group, the levels of serum sex hormone-binding globulin (SHBG) were significantly lower in the PCOS AR-ins group (P=0.000), and the levels of serum total testosterone (TT) (P=0.000), insulin (P=0.001) and FAI (P=0.000) were significantly higher in the PCOS AR-ins group. Compared with the control group and the PCOS Wt group, the differences of SHBG, TT, insulin levels and FAI in the follicular fluid in the PCOS AR-ins group were statistically significant (all P<0.05). Conclusion • PCOS patients with AR-ins have high androgen and insulin levels in serum, and this effect is more pronounced in the ovarian local microenvironment.
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The development and progression of metastatic castration-resistant prostate cancer is the major challenge in the treatment of advanced prostate cancer. The androgen receptor signaling pathway remains active in metastatic castration-resistant prostate cancer. Docetaxel and cabazitaxel are the first- and second-line chemotherapy, respectively, for patients with metastatic castration-resistant prostate cancer. These two taxanes, in general, function by (i) inhibiting mitosis and inducing apoptosis and (ii) preventing microtubule-dependent cargo trafficking. In prostate cancer, taxanes have been reported to inhibit the nuclear translocation and activity of the androgen receptor. However, whether this is attainable or not clinically remains controversial. In this review, we will provide a comprehensive view of the effects of taxanes on androgen receptor signaling in prostate cancer.
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As more and more patients with metastatic prostate cancer develop resistance to androgen-deprivation therapy (ADT) and consequently castration-resistant prostate cancer (CRPC), reasonable selection of therapies is becoming increasingly important for the prediction of the therapeutic results. Many studies show that androgen receptor splice variant 7 (AR-V7) is involved in the development and progression of CRPC and that the expression of AR-V7, absolutely higher in CRPC than in hormone-nave prostate cancer, plays a significant role in the mechanisms of resistance to abiraterone, enzalutamide and taxane chemotherapies. Further more, some clinical trials have revealed that the AR-V7 level may indicate the prognosis of different therapeutic options: AR-V7 negative in circulating tumor cells suggesting the effectiveness of a new hormonal therapy and taxane chemotherapy while AR-V7 positive indicating the poor result of a new hormonal therapy. These findings show that AR-V7 could be a biomarker for therapeutic options and the prognostic evaluation of CRPC.
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Objective To evaluate the prognostic value of the ratio of AR and AR-V7 expression in prostate cancer treated by castration therapy.Methods Immunohistochemical staining was performed in biopsy specimen of 136 prostate cancer patients received hormone therapy in Tongji Hospital of Huazhong University of Science and Technology from January 2010 to December 2015.Expression was determined using modified H score method.Patients aged from 53-96,the median age was 71,median tPSA value at diagnosed was 110.00 ng/ml(2.61-4 003.4 ng/ml),median fPSA value was 14.62 ng/ml(0.12-640.19 ng/ml),median PSA density was 1.15 ng/(ml · cm3) [0.02-62.63 ng/(ml · cm3)].Among these,88 (64.7％)patients were diagnosed Gleason score≥8,39(28.7％) patients with Gleason score 7,while 7 (6.6％) patients Gleason score ＜7.There were 54(39.7％) patients diagnosed T4 stage,57(41.9％)patients T3 stage and 25 (18.4％) patients Tx stage;62 (45.6％) patients were diagnosed N 1 stage,46 (33.8％) N0 stage and 28(20.6％) patients Nx stage;97(71.3％) patients were diagnosed M1 stage,30(22.1％) M0 stage,9 (6.6％) patients Mx stage.Cause-specific Cox regression and Kaplan-Meier Analysis were used to analyze the prognosis risk.Results The median follow-up time was 44 months,ranged 15-71 months.During the surveillance,the disease progression-free survival time ranged from 5-59 month,median 19 months.The overall survival time ranged from 12-61 months,median 31 months.Among these,79(58.1％) patients were AR positive and 26(19.1％) patients were AR-V7 positive,while AR and AR-V7 expression had no significant correlation (Spearman-test r =0.042,P =0.629).The AR-V7 positive patients showed significantly lower CRPC progression free survival (10.8 months vs.25.0 months,P ＜ 0.001) and much lower overall survival (20.3 months vs.42.8 months,P ＜ 0.001).The high AR-V7/AR expression ratio group showed significantly lower CRPC progression free survival (12.0 months vs.24.8 months,P ＜ 0.001) and much lower overall survival (22.5 months vs.42.8 months,P ＜ 0.001).In univariate Cox regression analyses,Gleason score at diagnosis,T stage,tPSA,PSA density and high AR-V7/AR expression ratio could predict the prognosis of hormonal therapy.While in multivariate Cox regression analyses,T stage(HR =2.597,95％ CI 1.351-4.995,P =0.004) and high AR-V7/AR expression ratio(HR =5.788,95％ CI 2.530-13.242,P ＜ 0.001) could effectively and independently predict the prognosis of hormonal therapy.Conclusion High AR-V7/AR HS ratio is the independent predictor of the prognosis of prostate cancer hormone therapy.AR-V7 positive and high AR-V7/AR HS ratio patients may have shorter PFS and overall survival time than AR-V7 negative and low AR-V7/AR HS ratio patients.
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Objective:To investigate the effect of human growth hormone releasing hormone receptor splice variant type 1 (GHRHR SV1) on the proliferation of human liver cancer HepG2 cells,and to clarify the proliferation effect of GHRHR SV1 on the human cancer cells.Methods:The GHRHR SV1 plasmids were transfected into the human HepG2 cells to construct the HepG2-SV1 cell line.HepG2 group(HepG2 cells),HepG2-empty group(HepG2-pcDNA3.0 cell line) and HepG2-SV1 group(HepG2-SV1 cells) were set up.PCR and Western blotting methods were used to identify the HepG2-SV1 cell line;CCK-8 method was used to detect prolifernation rate of cells;colony formation assay was used to detect the colony formation rate of cells;cell wound healing assay was used to evaluate the migration rate of cells.Results:The PCR and Western blotting results showed the HepG2-SV1 cell line expressed GHRHR SV1 steadily.The CCK-8 results showed that the proliferation rate of the HepG2-SV1 cells in HepG2-SV1 group was higher than that of the HepG2-pcDNA3.0 cells in HepG2-empty group(P<0.05).The colony formation assay results showed that the colony formation rate of HepG2-SV1 cells in HepG2-SV1 group was 3.5 times higher than that of the HepG2-pcDNA3.0 cells in HepG2-empty group(P<0.05).The cell wound scratch assay results showed that the migration rate of the HepG2-SV1 cells in HepG2-SV1 group was higher than that of the HepG2-pcDNA3.0 cells in HepG2-empty group(P<0.05).Conclusion:GHRHR SV1 could increase the proliferation of HepG2 cells.
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Objective To solve the problem of castration resistant prostate cancer (castrationresistant prostate cancer,CRPC) the problem of drug resistance by studing the expression of the CUDC-101 inhibitor of castration resistant prostate cancer androgen receptor splice variant 7.Methods In this study,the expression of AR-V7 protein in prostate cancer cell lines PC-3,VCaP,22Rv1,LNCap was detected by imnmunoblotting between April 2015 and April 2016,and the highest expression level of cell lines was selected follow-up experiments.Through the cell proliferation and activity experiments,the epigenetic inhibitors:histone deacetylase inhibitor CUDC-101,histone methylation inhibitor DZNeP,DNA methylation inhibitor gemcitabine,histone acetyltransferase inhibitor MG149 to select an inhibitor that reduces the expression of AR-V7 protein in CRPC cells.22Rv1 cells were treated with 30 nmol and 300 nmol of CUDC101 and 1,10 and 20 μmol of Enzalutamide (MDV3100),respectively,and their inhibitory effects on the growth of 22Rv1 cells were examined.Results The results of immunoblotting showed that AR-V7 protein was only expressed in CRPC cell line 22Rv1 and negative in non-CRPC cell line.The expression of AR-V7 in 22Rv1 cells treated with CUDC-101 was significantly lower than that of negative control.While other inhibitors had no effect on the expression of AR-V7.In the cell proliferation and activity assay,the inhibitory rates of 30 nmol CUDC-101 and 1,10 and 20 μmol MDV3100 were 10％,35％ and 45％,respectively,higher than that of MDV3100 alone.28％ and 42％,the difference was statistically significant (P ＜ 0.05).The inhibitory rates of 300 nmol CUDC-101 and MDV3100 were 30％,60％ and 65％,respectively,which were significantly higher than those of MDV3100 alone (P ＜ 0.05).Conclusions CUDC-101 can inhibit the castration resistant prostate cancer androgen receptor splice variant 7 expression,and solved the resistance problem of CRPC.
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AIM:To explore the effect of androgen receptor splice variant 7 ( AR-V7 ) on endocrine therapy resistance of prostate cancer cells and the resistance mechanisms .METHODS:Four prostate cancer cell lines were trans-fected with AR-V7 siRNA ( siAR-V7) using Lipofectamine 2000 kit, and the transfected cells were named as PC 3-siAR-V7, DU145-siAR-V7, LNCaP-siAR-V7 and ArCaP-siAR-V7 cells.The prostate cancer cells transfected with negative con-trol ( NC) siRNA served as negative controls .The expression of AR-V7 at mRNA and protein levels was detected by real-time PCR and Western blot , respectively .The cell viability and cell migration rate were measured by MTT assay and Tran-swell method, respectively.The promoter activity of AR and the protein levels of targeted prostate-specific antigen (PSA) and FK506-binding protein 5 (FKBP5) were monitored by luciferase reporter gene assay and Western blot , respectively. The bicalutamide-resistant cell line, LNCaP-DR, was constructed, and the subcellular localization of AR and AR-V7 pro-teins in LNCaP, LNCaP-siAR-V7 and LNCaP-DR cells was observed by the method of immunofluorescence .The protein in-teraction of AR-V7 and heat shock protein 90 ( HSP90) was determined by co-immunoprecipitation .RESULTS:The mR-NA level of AR-V7 in the 4 prostate cancer cell lines was significantly higher than that in normal prostate epithelial cell line RWPE-1 (P<0.05).The AR-V7 level, cell viability and cell migration rate in the cells transfected with siAR-V7 were notablely lowered compared with the NC siRNA-transfected cells (P<0.05).The cell viability was gradually decreased following with the increase in bicalutamide dose , and down-regulation of AR-V7 expression significantly enhanced the sensi-tivity to bicalutamide (P<0.05).Down-regulation of AR-V7 expression significantly inhibited AR promoter activity and reduced the protein levels of PSA and FKBP5 (P<0.05).The results of immunofluorescence observation showed that most AR and AR-V7 were mainly located in the nucleus , a few AR was located in the cytoplasm , and down-regulation of AR-V7 expression inhibited AR nuclear transport .AR was entirely located in the nucleus and the protein levels of AR-V7 was sig-nificantly increased in the bicalutamide-resistant cells .The interaction of endogenous AR-V7 with HSP90 was found in the prostate cancer cells .CONCLUSION: High AR-V7 level is found in the prostate cancer cells , and down-regulation of AR-V7 expression inhibits the cell viability and migration .High AR-V7 level is related to the bicalutamide resistance .The possible mechanism is that AR nuclear transport is mediated by the interaction of AR -V7 with HSP90 to activate AR signal pathway and regulate targeted gene transcriptional activity , thus resulting in drug resistance .
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AIM:To explore the effect of androgen receptor splice variant 7 ( AR-V7 ) on endocrine therapy resistance of prostate cancer cells and the resistance mechanisms .METHODS:Four prostate cancer cell lines were trans-fected with AR-V7 siRNA ( siAR-V7) using Lipofectamine 2000 kit, and the transfected cells were named as PC 3-siAR-V7, DU145-siAR-V7, LNCaP-siAR-V7 and ArCaP-siAR-V7 cells.The prostate cancer cells transfected with negative con-trol ( NC) siRNA served as negative controls .The expression of AR-V7 at mRNA and protein levels was detected by real-time PCR and Western blot , respectively .The cell viability and cell migration rate were measured by MTT assay and Tran-swell method, respectively.The promoter activity of AR and the protein levels of targeted prostate-specific antigen (PSA) and FK506-binding protein 5 (FKBP5) were monitored by luciferase reporter gene assay and Western blot , respectively. The bicalutamide-resistant cell line, LNCaP-DR, was constructed, and the subcellular localization of AR and AR-V7 pro-teins in LNCaP, LNCaP-siAR-V7 and LNCaP-DR cells was observed by the method of immunofluorescence .The protein in-teraction of AR-V7 and heat shock protein 90 ( HSP90) was determined by co-immunoprecipitation .RESULTS:The mR-NA level of AR-V7 in the 4 prostate cancer cell lines was significantly higher than that in normal prostate epithelial cell line RWPE-1 (P<0.05).The AR-V7 level, cell viability and cell migration rate in the cells transfected with siAR-V7 were notablely lowered compared with the NC siRNA-transfected cells (P<0.05).The cell viability was gradually decreased following with the increase in bicalutamide dose , and down-regulation of AR-V7 expression significantly enhanced the sensi-tivity to bicalutamide (P<0.05).Down-regulation of AR-V7 expression significantly inhibited AR promoter activity and reduced the protein levels of PSA and FKBP5 (P<0.05).The results of immunofluorescence observation showed that most AR and AR-V7 were mainly located in the nucleus , a few AR was located in the cytoplasm , and down-regulation of AR-V7 expression inhibited AR nuclear transport .AR was entirely located in the nucleus and the protein levels of AR-V7 was sig-nificantly increased in the bicalutamide-resistant cells .The interaction of endogenous AR-V7 with HSP90 was found in the prostate cancer cells .CONCLUSION: High AR-V7 level is found in the prostate cancer cells , and down-regulation of AR-V7 expression inhibits the cell viability and migration .High AR-V7 level is related to the bicalutamide resistance .The possible mechanism is that AR nuclear transport is mediated by the interaction of AR -V7 with HSP90 to activate AR signal pathway and regulate targeted gene transcriptional activity , thus resulting in drug resistance .
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Background and purpose:Inhibitor of apoptosis-stimulating protein of p53 (iASPP) is one of the ASPP family. It binds to p53 to inhibit the transcriptional activity of p53-target genes and cell apoptosis, which is asso-ciated with tumor formation. Previously, we found a new subtype of iASPP, iASPP splice variant (iASPP-SV), which is a nuclear protein containing 407 amino acid residues and can bind to p53, inhibiting p53 transcriptional activity. However, the relationship of iASPP-SV and breast cancer is still obscure. Therefore, the purpose of this research was to study the role of iASPP-SV on breast cancer tumorigenesis and progression.Methods:5’-rapid ampliifcation of cDNA ends (RACE) was used to identify the 5’-end of iASPP-SV mRNA in MCF-7 cells. HEK 293 cells were transfected with pFLAG-iASPP-SV and pFLAG-iASPP (828). Then Western blot was used to identify whether endogenous iASPP-SV was expressed in HEK 293 cells and 8 types of human tumor cell lines. This study established the stable clones of NIH 3T3 expressing FLAG-iASPP-SV and FLAG-iASPP (828). Cell proliferation assay, colony formation and soft agar colony formation assay were used to identify whether iASPP-SV and iASPP (828) can promote cell proliferation and iASPP-SV is an oncogene. Real-time lfuorescent quantitative polymerase chain reactive (RTFQ-PCR) was used to de-tect the levels of iASPP-SV and iASPP (828) mRNA in primary breast cancers. Luciferase assays were used to identify the relationships between iASPP-SV, iASPP (828), p53 and NF-κB p65.Results:The study identiifed that iASPP-SV was encoded by previously reported NF-κB p65 subunit (RelA)-associated inhibitor (RAI), and endogenously expressed in many human cancer cell lines. Analysis of cell proliferation, colony formation assay and soft agar assay for colony formation identiifed that similarly to iASPP (828), iASPP-SV promoted tumor cell proliferation and acted as an onco-gene. RTFQ-PCR result showed that the median values of iASPP-SV and iASPP (828) in breast cancers with wild-type p53 were more signiifcantly over-expressed than those of mutant p53. Luciferase assays showed that iASPP-SV and iASPP (828) could suppress NF-κB p65 transcriptional activity. Thus iASPP family may participate in the regulation of p53 and NF-κB activity, which imply that iASPP perhaps shows pro- or anti-survival activities when it interacts with different proteins.Conclusion:These ifndings indicate that iASPP-SV may be a potential target for breast cancer thera-py.
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Prostate cancer cells demonstrate a remarkable "addiction" to androgen receptor (AR) signaling in all stages of disease progression. As such, suppression of AR signaling remains the therapeutic goal in systemic treatment of prostate cancer. A number of molecular alterations arise in patients treated with AR-directed therapies. These molecular alterations may indicate the emergence of treatment resistance and may be targeted for the development of novel agents for prostate cancer. The presence of functional androgen receptor splice variants may represent a potential explanation for resistance to abiraterone and enzalutamide, newer AR-directed agents developed to treat metastatic castration-resistant prostate cancer (mCRPC). In the last 8 years, many androgen receptor splice variants have been identified and characterized. Among these, androgen receptor splice variant-7 (AR-V7) has been investigated extensively. In AR-V7, the entire COOH-terminal ligand-binding domain of the canonical AR is truncated and replaced with a variant-specific peptide of 16 amino acids. Functionally, AR-V7 is capable of mediating constitutive nuclear localization and androgen receptor signaling in the absence of androgens, or in the presence of enzalutamide. In this review, we will focus on clinical translational studies involving detection/measurement of AR-V7. Methods have been developed to detect AR-V7 in clinical mCRPC specimens. AR-V7 can be reliably measured in both tissue and circulating tumor cells derived from mCRPC patients, making it possible to conduct both cross-sectional and longitudinal clinical correlative studies. Current evidence derived from studies focusing on detection of AR-V7 in mCRPC support its potential clinical utility as a treatment selection marker.
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Objective To investigate the expression of marrow-derived thyroid stimulating hormone β (TSHβ) splice variant in thyroid of mouse with autoimmune thyroiditis induced by thyroglobulin(Tg) immunization,and to analyze whether TSHβ splice variant participated in the pathological process of autoimmune thyroiditis.Methods Using random number table,forty-eight mice(24 females and 24 males) of 7 to 8 weeks old with body mass 20 to 25 g were randomly divided into 4 groups(12 females and 12 males in each group) based on body weight and gender.The control group:fed with deionized water; the Tg-treated group(TG):fed with deionized water,and immunized subcutaneously with 0.1 mg Tg at 8 weeks old,boost immunized twice at 11 and 15 weeks old,respectively; the high iodine-treated group (HI):fed with deionized water containing 0.05％ sodium iodide (NaI); the Tg combined with HI group (TG + HI):fed with deionized water containing 0.05％ NaI,and immunized the same way as the TG group did.Peripheral blood was collected after 8 weeks treatment,which was used for determination of total tetraiodothyronine (TT4),free tetraiothyronine (FT4),total triiodothyronine (TT3) and free triiodothyronine (FT3) with chemiluminescence immunoassay (CIA); thyroid glands were collected to examine the expression of TSHβ splice variant with SYBR Green fluorescent quantitative real-time PCR,and frozen sections were HE stained for observation of histopathological changes of thyroid cells under light microscopy.Results Under naked eyes,the thyroid gland enlarged significantly,and looked dark red in HI and TG + HI groups.Under an optical microscope,thyroid follicular epithelial cells presented cuboidal,with abundant cytoplasm,presented abundant glial in follicular cavity,without lymphocyte infiltration in the control group; in TG group,the thyroid follicular epithelial cells presented cuboidal,with some single scattered lymphocytes; in HI group,colloid volume expansion appeared in thyroid follicles,thyroid follicular epithelial cells presented low cuboidal or flat,with few single scattered lymphocytes; in TG + HI group,most colloid accumulative large follicles presented in thyroid,thyroid follicular epithelial cells presented flat,some destructive thyroid follicular structure and infiltrating lymphocytes appeared.The differences of FT3,TT4,FT4 and TSHβ splice variant between groups were statistically significant(F =4.00,12.54,31.92,214.29,all P ＜ 0.05).Compared to the control group,the serum TT3(nmol/L:0.92 ± 0.07 vs.1.30 ± 0.33,t =-2.24),TT4(nmol/L:1.30 ± 0.33 vs.95.60 ± 14.10,t =-3.02),FT4(pmoL/L:54.07 ± 3.67 vs.154.80 ± 0.01,t =-54.87) and the thyroids' TSHβ(× 10-3:4.11±0.32 vs.8.38 ± 0.22,t =-19.11) were higher in TG group(all P ＜ 0.05) ; the serum TT4(nmol/L:67.75 ± 11.91 vs.45.50± 3.85,t =3.55,P ＜ 0.05) was lower in HI group; the serum FT4(pmol/L:54.07 ± 3.67 vs.139.46 ± 30.00,t =-5.65) and the thyroids' TSHβ splice variant (× 10-3:4.11 ± 0.32 vs.5.33 ± 1.47,t =-5.95) were higher in TG + HI group (all P ＜ 0.05).Conclusions High iodine has aggravated thyroiditis of BALB/c mice induced by Tg immunization; the level of thyroid TSHβ in mice with autoimmune thyroiditis is higher; all of these results indicated that TSHβ is involved in the pathogenesis of autoimmune thyroiditis.
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Splice variants are transcripts that exhibit various functions or activities and are obtained from alternative splicing of precursor mRNA. An increasing number of tumor-specific splice variants are being discovered and their roles in cancer development and progression are being investigated. In this study, the expression patterns and activities of tumor-specific splice variants were charac-terized. The results suggest that a variant-specific gene therapy that targets cancer cells would be more specific and would have minimal effect on healthy tissues. Using antisense oligonucleotides, mRNA that contributed to cancer cell survival and altered splicing patterns was downregulated in a clinical trial. The results provide a new appreciation for the gene therapy of cancer. Identification of novel tu-mor-specific splice variants and constant improvement of therapeutic strategies by modifying splicing patterns can lead to the develop-ment of more effective and safer cancer therapies.
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ObjectiveTo find out if the immune system derived thyroid stimulating hormone(TSH) β splice variant(TSHβ-Ⅴ) would be regulated by circulating thyroid hormone levels to get a further understanding of the function and mechanism of this TSHβ-Ⅴ in thyroid homeostasis.MethodsA total of 20 weaning Balb/c mice (half male and half female) were selected and randomly divided into two groups according to their body mass and gender(n =10).Mice of control group were fed with common diet and deionized water.Mice of the low-iodine(LI) group were fed with low-iodine diet(containing iodine 20 - 40 μg/kg,iodine-intake about 0.25 μg/d) and deionized water.The experimental period was 3 months.At the end of the experiment,mice were executed and the blood was collected to observe the levels of TSH and thyroid hormone by chemiluminescence immunoassay (CIA) ; bone marrow (BM),peripheral blood(PBL),thyroid gland and pituitary were collected to assay the TSHβ-Ⅴ mRNA expression by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR).ResultsThe serum free thyroxine(FT4) and total thyroxine(TT4) levels in LI group of mice[(0.47 ± 0.70)nmol/L,(2.41 ± 0.28)pmol/L] were significantly lower than that of the control group of mice [(55.2 ± 3.68) nmol/L, (32.72 ± 1.02) pmol/L,t =43.81,86.04 、all P ＜ 0.01 ] and the serum total triiodothyronine(TT3) and free triiodothyronine(FT3) reduction in LI group of mice[ (0.76 ± 0.08)nmol/L,(4.01 ± 0.40)pmol/L] were significantly lower than that of the control group of mice [ (1.10 ± 0.06)nmol/L,(5.40 ± 0.38)pmol/L,t =9.81,7.5 1,P ＜ 0.01 ].Iodine insufficiency strongly elevated the serum TSH in LI group of mice[ (35.67 ± 17.39)mU/L] than that in control group of mice[ (0.24 ± 0.10)mU/L,t =- 6.11,P ＜ 0.01 ].The mRNA levels of TSH β-Ⅴ in BM (9.62 ± 0.60) and in PBL( 9.25 ± 0.83 ) of LI group of mice were lower than those in control group of mice (7.69 ± 0.36,7.11 ± 0.41,t =6.77,5.64,P ＜ 0.01),while the mRNA level of TSH β-Ⅴ in pituitary of LI group of mice (1.99 ± 0.61) was increased compared with that in control group of mice (5.75 ± 0.98,t =- 8.02,P＜ 0.01).Compared with control group of mice(9.12 ± 0.62),the level of thyroid TSH β-Ⅴ mRNA in LI group of mice (9.32 ± 0.91 ) was not significantly changed (t =0.45,P ＞ 0.05).There was no detectable native TSHβ in BM,PBL and thyroid.The mRNA level of native TSHβ in pituitary in LI group of mice( - 7.17 ± 1.78) was dramatically elevated compared to that in control group of mice( - 1.43 ± 0.51,t =- 7.60,P ＜ 0.01 ).ConclusionsThe mRNA levels of TSHβ-Ⅴ are suppressed in BM and PBL in low iodinediet induced hypothyroidism mice,which suggest that immune system derived TSHβ-Ⅴ may be more important thannative TSHβ in immune-thyroid regulation.
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AIM: To prepare and identify mouse polyclonal antibody against protein Hlb, which is the variant of major subunit of human ASGPR. METHODS: Hlb specific peptide was synthesized and coupled with keyhole limpet hemocyanin (KLH) for immunization. Then H1b-KLH conjugation was injected into mouse subcutaneously to produce polyclonal antibody. ELISA assay was used to detect the titer of the antibody. Antibody was also identified by Western blot and immunohistochemistry assays. RESULTS: Mouse antibody against Hlb was prepared after injection of H1bKLH conjugation. The titer of H1b antibody was about 1:10~5.Western blot confirmed its high specificity. This antibody could also be used for immunohistochemistry analysis. CONCLUSION: The successful preparation of the polyclonal antibody against protein H1b, which can discriminate the two variants of the major subunit of ASGPR with high specificity, will provide an efficient reagent for further study of the physiologic functions of H1b and its role in the pathogenesis of human disease.
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The unique phenomenon of alternative splicing is gathering concern due to its promising therapeutic potential. The human genome sequencing project suggests approximately 20,000-25,000 genes. Among these, about 35-60% of genes generate multiple mRNAs by alternative splicing mechanism and contribute to the diversity of the proteomic world. This 'gene shortfall' has ignited considerable interest in alternative RNA splicing. This process leads to expression of a single gene responsible for the transcription of different mRNA isoforms that might have multiple biological functions. The disruption of splicing pattern can produce aberrant splice variants, which are implicated in more than 50% of genetic disorders including cancer. Altered splice sites in neoplastic cell contribute to the development, progression and/or maintenance of tumorous growth. The repertoire of tumor-specific variant represents a potential marker in pharmacogenomic diagnostic relevance. Alternative splice isoforms have been analyzed serendipitously by qualitative gene profiling with in silico gene prediction software. Computational approach in identifying exonic splicing enhancers in genomic DNA and focus on microarray technology will elucidate differential expression of alternative splice variants. The antisense oligonucleotides modulate alternative splicing and engender the production of therapeutic gene products. Oligonucleotides have the potential to silence the mutations caused by aberrant splicing. The efficacy of the antisense oligonucleotides lies in the chemical configuration, affinity and delivery strategies. Hence the therapeutic potential of antisense oligonucleotides as modulators of aberrant alternative splicing would be a major challenge to the upcoming proteomic era.
ABSTRACT
Latent transforming growth factor (TGF)-beta-binding protein (LTBP) is required for the assembly, secretion, matrix association, and activation of latent TGF-beta complex. To elucidate the cell specific expression of the genes of LTBP-1 and their splice variants and the factors that regulate the gene expression, we cultured primary human glomerular endothelial cells (HGEC) under different conditions. Basal expression of LTBP-1 mRNA was suppressed in HGEC compared to WI-38 human embryonic lung fibroblasts. High glucose, H2O2, and TGF-beta1 upregulated and vascular endothelial growth factor (VEGF) further downregulated LTBP-1 mRNA in HGEC. RT-PCR with a primer set for LTBP-1S produced many clones but no clone was gained with a primer set for LTBP-1L. Of 12 clones selected randomly, Sca I mapping and DNA sequencing revealed that only one was LTBP-1S and all the others were LTBP-1S delta 53. TGF-beta1, but not high glucose, H2O2 or VEGF, tended to increase LTBP-1S delta 53 mRNA. In conclusion, HGEC express LTBP-1 mRNA which is suppressed at basal state but upregulated by high glucose, H2O2, and TGF-beta1 and downregulated by VEGF. Major splice variant of LTBP-1 in HGEC was LTBP-1S delta 53. Modification of LTBP-1S delta 53 gene in HGEC may abrogate fibrotic action of TGF-beta1 but this requires confirmation.