Objective： The activation of hedgehog （HH） pathway is implicated in the development of human malignancies including hepatocellular carcinoma （HCC）. However, the clinical impact of HH activation in HCC patients is...Objective： The activation of hedgehog （HH） pathway is implicated in the development of human malignancies including hepatocellular carcinoma （HCC）. However, the clinical impact of HH activation in HCC patients is still unclear. This study was conducted to confirm whether the expression of HH pathway components was associated with HCC progression and clinical outcome. Methods： This study was a sample-expanded and prolonged follow up of one of our previous studies. It included 46 HCC patients who underwent surgical treatment from 2002 to 2005. The expression of sonic HH （SHIq）, patched-1 （PTCHI）, smoothened （SMOH） and glioma-associated oncogene-1 （GLI1） genes in tumor and adjacent normal tissues extracted from the patients were examined by reverse transcription- polymerase chain reaction （RT-PCR） to explore the relationship between these genes and the clinical prognosis of HCC. Results： The expression levels ofSHH, PTCH1, SMOH and GLI1 in HCC tissues were 60.87%, 50.00%, 32.61% and 54.35%, respectively. The expression levels of SHH-related molecules were relatively intense in cancer tissue, but insignificantly correlated with any clinicopathological factors of tumor. Transcriptional factor GLI1 was the only molecule associated with poor prognosis among the HCC patients. The expression of GLI1 gene in tumor tissues was significantly related with disease-free survival （DFS） （P=0.042） and overall survival （OS） （P=0.030）. The simultaneous expression of GLI1 in tumor and adjacent normal liver tissues correlated with DFS （P〈0.029） and OS （P〈0.025）. Conclusions： HH signaling activation is an important event in the development of human HCC. The expression of GLI1 in SHH pathway is possibly involved in HCC progression, which may be a useful prognostic indicator of HCC.展开更多
AIM:To investigate the regulative effect of miRNA-3383p(miR-338-3p) on cell growth in colorectal carcinoma(CRC).METHODS:The lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed.The re...AIM:To investigate the regulative effect of miRNA-3383p(miR-338-3p) on cell growth in colorectal carcinoma(CRC).METHODS:The lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed.The recombinant viral vector encoding the pre-miR338-3p or miR-338-3p-inhibitor and the two packaging plasmids psPAX2 and pMD2.G were cotransfected into human embryonic kidney 293T cells to package lentivirus.The supernatant containing the lentivirus particles was harvested to determine the viral titer,and this supernatant was then used to transduce CRCderived cell line,SW-620.Flow cytometry was utilized for sorting the green fluorescent protein(GFP) + cells to establish the SW-620 cell line stably expressing premiR-338-3p or miR-338-3p-inhibitor.Moreover,the expression of miR-338-3p was determined by real-time reverse transcriptase polymerase chain reaction,andWestern blotting was used to detect the expression of the smoothened(SMO,the possible target of miR-3383p) protein in SW-620 cells.Furthermore,the status of CRC cell proliferation and apoptosis were detected by 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay and flow cytometry,respectively.RESULTS:Restriction enzyme digestion and DNA sequencing demonstrated that the lentiviral vector pLVTHM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed successfully.GFP was expressed after the SW-620 cells were transduced by the lentivirus.Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p was significantly increased(relative expression 3.91 ± 0.51 vs 2.36 ± 0.44,P < 0.01).Furthermore,overexpression of miR-338-3p inhibited the expression of SMO protein in SW-620 cells,which showed obviously suppressed proliferation ability [cellular proliferation inhibition rate(CPIR) 61.9% ± 5.2% vs 41.6% ± 4.8%,P < 0.01].Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p-inhibitor was significantly decreased(relative expression 0.92 ± 0.29 vs 2.36 ± 0.44,P < 0.展开更多
Fibroblast-like synoviocytes （FLSs） contribute to synovial hyperplasia in rheumatoid arthritis （RA）. Smoothened （Smo） is a key component of sonic hedgehog （Shh） signaling and contributes to tumor cell prolifer...Fibroblast-like synoviocytes （FLSs） contribute to synovial hyperplasia in rheumatoid arthritis （RA）. Smoothened （Smo） is a key component of sonic hedgehog （Shh） signaling and contributes to tumor cell proliferation. The objective of this study was to investigate the role of Smo in RA synoviocyte proliferation. FLSs were isolated from RA synovium. Shh signaling was studied using a Smo antagonist （GDC-0449） and small interfering RNA （siRNA） targeting the Smo gene in FLSs. Cell proliferation was quantified by using kit-8 assay and cell cycle distribution and apoptosis were evaluated by flow cytometry. Cell cycle-related genes and proteins were detected by real-time PCR and western blot. FLSs treated with GDC-0449 or Smo-siRNA showed significantly decreased proliferation compared to controls （P〈0.05）. Incubation with GDC-0449 or transfection with Smo-siRNA resulted in a significant increase of G1 phase cells compared to controls （P 〈 0.05）. Cell cycle arrest was validated by the significant increase in cyclin D1 and E1 mRNA expression, decrease in cyclin-dependent kinase p21 mRNA expression in Smo-siRNA transfected cells （P 〈 0.05）. Protein expression of cyclin D1 was also downregulated after Smo gene knockdown （P 〈0.05）.The results suggest that Shh signaling plays an important role in RA-FLSs proliferation in a Smo-dependent manner and may contribute to synovial hyperplasia. Targeting Shh signaling may help control joint damage in patients with RA.展开更多
Hedgehog was first described in Drosophila melanogaster by the Nobel laureates Eric Wieschaus and Christiane Nüsslein-Volhard. The hedgehog (Hh) pathway is a major regulator of cell differentiation, proliferation...Hedgehog was first described in Drosophila melanogaster by the Nobel laureates Eric Wieschaus and Christiane Nüsslein-Volhard. The hedgehog (Hh) pathway is a major regulator of cell differentiation, proliferation, tissue polarity, stem cell maintenance, and carcinogenesis. The first link of Hh signaling to cancer was established through studies of a rare familial disease, Gorlin syndrome, in 1996. Follow-up studies revealed activation of this pathway in basal cell carcinoma, medulloblastoma and, leukemia as well as in gastrointestinal, lung, ovarian, breast, and prostate cancer. Targeted inhibition of Hh signaling is now believed to be effective in the treatment and prevention of human cancer. The discovery and synthesis of specific inhibitors for this pathway are even more exciting. In this review, we summarize major advances in the understanding of Hh signaling pathway activation in human cancer, mouse models for studying Hh- mediated carcinogenesis, the roles of Hh signaling in tumor development and metastasis, antagonists for Hh signaling and their clinical implications.展开更多
Background miR-338-3p is a recently discovered miRNA and is involved in cell differentiation.However,few data are yet available on the aberrant expression of miR-338-3p in human colorectal carcinoma （CRC）.This work ...Background miR-338-3p is a recently discovered miRNA and is involved in cell differentiation.However,few data are yet available on the aberrant expression of miR-338-3p in human colorectal carcinoma （CRC）.This work aimed to investigate the relationship between miR-338-3p expression pattern and clinicopathological features of human CRC and the possible regulative mechanisms.Methods The 40 CRC,adjacent nontumorous tissues and 2 human CRC-derived cell lines （SW-480 and SW-620） were collected,respectively,and the total RNA and protein were isolated routinely.The miR-338-3p expression pattern was detected by real-time reverse transcription-polymerase chain reaction （RT-PCR） and Northern blotting.Smoothened （SMO,possible target of miR-338-3p） mRNA and corresponding protein expression pattern were detected by semiquantitative RT-PCR and Western blotting.miR-338-3p expression patterns were compared between nontumor mucosa and CRC samples,graded by progression-related factors.Disease outcome was calculated by Kaplan-Meier survival analysis to determine whether miR-338-3p was related to disease-free survival （DFS） and overall survival （OS） of patients.Moreover,SMO 3＇-UTR fragment was PCR amplified from genome DNA of human colon and inserted into a luciferase reporter plasmid.The luciferase reporter plasmid construct was then transfected into CRC cells together with pre-miR-338-3p or anti-miR-338-3p and the luciferase activity in the transfected cells was detected.Results The expression of miR-338-3p was significantly downregulated in CRCs than those in the adjacent nontumorous tissues,and the value was negatively related to advanced TNM stage and local invasion （P ＜0.01）.Furthermore,miR338-3p value was decreased markedly in SW-620 cell line relative to SW-480 （P ＜0.01）.Low expression of miR-338-3p was associated with unfavorable outcome in DFS but not in OS independent of clinical covariates.Moreover,RT-PCR and Western blotting analysis demonstrated that there was no significant differ展开更多
Emerging evidence suggests that neuro-inflammation begins early and drives the pathogenesis of Alzheimer＇s disease （AD）, and anti-inflammatory therapies are under clinical development. However, several anti-inflamm...Emerging evidence suggests that neuro-inflammation begins early and drives the pathogenesis of Alzheimer＇s disease （AD）, and anti-inflammatory therapies are under clinical development. However, several anti-inflammatory compounds failed to improve memory in clinical trials, indicating that reducing inflammation alone might not be enough. On the other hand, neuro-inflammation is implicated in a number of mental disorders which share the same therapeutic targets. Based on these observations, we screened a batch of genes related with mental disorder and neuro-inflammation in a classical olfactory conditioning in an amyloid beta （Aβ） overexpression fly model. A Smoothened （SMO） mutant was identified as a genetic modifier of Aβ toxicity in 3-min memory and downregulation of SMO rescued Aβ- induced 3-min and 1-h memory deficiency. Also, Aβ activated innate inflammatory response in fly by increasing the expression of antimicrobial peptides, which were alleviated by downregulating SMO. Furthermore, pharmaceutical administration of a SMO antagonist LDE rescued Aβ-induced upregulation of SMO in astrocytes of mouse hippocampus, improved memory in Morris water maze （MWM）, and reduced expression ofastrocyte secreting pro-inflammatory factors IL-1β, TNFα and the microglia marker IBA-1 in an APP/PS1 transgenic mouse model. Our study suggests that SMO is an important conserved modulator of Aβ toxicity in both flv and mouse models of AD.展开更多
The Hedgehog （Hh） signaling pathway plays important roles in developmental processes including pattern formation and tissue homeostasis. The seven-pass transmembrane receptor Smoothened （Smo） is the pivotal transd...The Hedgehog （Hh） signaling pathway plays important roles in developmental processes including pattern formation and tissue homeostasis. The seven-pass transmembrane receptor Smoothened （Smo） is the pivotal transducer in the pathway; it, and thus the pathway overall, is regulated by ubiquitin-mediated degradation, which occurs in the absence of Hh. In the presence of Hh, the ubiquitination levels of Smo are decreased, but the molecular basis for this outcome is not well understood. Here, we identify the deubiquitinase UCHL5 as a positive regulator of the Hh pathway. We provide both genetic and biochemical evidence that UCHL5 interacts with and deubiquitinates Smo, increasing stability and promoting accumulation at the cell membrane. Strikingly, we find that Hh enhances the interaction between UCHL5 and Smo, thereby stabilizing Smo. We also find that proteasome subunit RPN13, an activator of UCHL5, could enhance the effect of UCHL5 on Smo protein level. More importantly, we find that the mammalian counterpart of UCHL5, UCH37, plays the same role in the regulation of Hh signaling by modulating hSmo ubiquitination and stability. Our findings thus identify UCHL5/UCH37 as a critical regulator of Hh signaling and potential therapeutic target for cancers.展开更多
目的 Hedgehog信号通路参与了肿瘤的发生发展,本研究探讨该通路关键信号分子Smoothened(SMO)在肾透明细胞癌中的表达及对肾癌细胞增殖和凋亡的影响。方法选取2012-01-01-2013-06-30青岛大学附属医院泌尿外科,行手术治疗的80例肾透明细...目的 Hedgehog信号通路参与了肿瘤的发生发展,本研究探讨该通路关键信号分子Smoothened(SMO)在肾透明细胞癌中的表达及对肾癌细胞增殖和凋亡的影响。方法选取2012-01-01-2013-06-30青岛大学附属医院泌尿外科,行手术治疗的80例肾透明细胞癌患者的临床及病理资料,采用免疫组织化学方法检测SMO在肾透明细胞癌组织中的表达并分析其与临床病理特征间的关系。采用小干扰RNA下调SMO在人肾癌细胞786-O中的表达,分别应用CCK-8法、流式细胞术及蛋白质印迹法检测下调SMO表达对细胞增殖、凋亡及Gli1和Gli2表达的影响。结果 SMO在73例(91.25%)肾透明细胞癌组织中有表达,其在高级别肾癌中表达(80.49%)较低级别(56.41%)显著升高,χ2=5.39,P=0.02。RT-PCR检测结果显示,SMO在人肾癌细胞系786-O中表达量为0.704±0.059;蛋白质印迹结果显示,SMO在786-O中表达量为0.651±0.074。在786-O细胞中应用小干扰RNA沉默SMO表达后,实验组细胞相较于对照组其活力百分比在48、72和96h分别为92.7%、80.9%和79.9%,3个时间点差异有统计学意义(P=0.003),细胞凋亡显著增加,t=-29.2,P<0.001;与空白对照组和阴性对照组相比,其下游分子Gli1和Gli2蛋白表达明显减少(Gli1:3.2 vs 2.9 vs 1;Gli2:2.5 vs 2.1vs 1)。结论 SMO可能通过调控细胞增殖和凋亡,以及调节Gli蛋白表达参与了肾癌的发生发展。展开更多
基金supported by the Project 2009CB521700 of the National Basic Research Program of China ("973"Program)the Capital Development Grant (2007-2053)
文摘Objective： The activation of hedgehog （HH） pathway is implicated in the development of human malignancies including hepatocellular carcinoma （HCC）. However, the clinical impact of HH activation in HCC patients is still unclear. This study was conducted to confirm whether the expression of HH pathway components was associated with HCC progression and clinical outcome. Methods： This study was a sample-expanded and prolonged follow up of one of our previous studies. It included 46 HCC patients who underwent surgical treatment from 2002 to 2005. The expression of sonic HH （SHIq）, patched-1 （PTCHI）, smoothened （SMOH） and glioma-associated oncogene-1 （GLI1） genes in tumor and adjacent normal tissues extracted from the patients were examined by reverse transcription- polymerase chain reaction （RT-PCR） to explore the relationship between these genes and the clinical prognosis of HCC. Results： The expression levels ofSHH, PTCH1, SMOH and GLI1 in HCC tissues were 60.87%, 50.00%, 32.61% and 54.35%, respectively. The expression levels of SHH-related molecules were relatively intense in cancer tissue, but insignificantly correlated with any clinicopathological factors of tumor. Transcriptional factor GLI1 was the only molecule associated with poor prognosis among the HCC patients. The expression of GLI1 gene in tumor tissues was significantly related with disease-free survival （DFS） （P=0.042） and overall survival （OS） （P=0.030）. The simultaneous expression of GLI1 in tumor and adjacent normal liver tissues correlated with DFS （P〈0.029） and OS （P〈0.025）. Conclusions： HH signaling activation is an important event in the development of human HCC. The expression of GLI1 in SHH pathway is possibly involved in HCC progression, which may be a useful prognostic indicator of HCC.
基金Supported by National Natural Science Foundation of China,No.81101896
文摘AIM:To investigate the regulative effect of miRNA-3383p(miR-338-3p) on cell growth in colorectal carcinoma(CRC).METHODS:The lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed.The recombinant viral vector encoding the pre-miR338-3p or miR-338-3p-inhibitor and the two packaging plasmids psPAX2 and pMD2.G were cotransfected into human embryonic kidney 293T cells to package lentivirus.The supernatant containing the lentivirus particles was harvested to determine the viral titer,and this supernatant was then used to transduce CRCderived cell line,SW-620.Flow cytometry was utilized for sorting the green fluorescent protein(GFP) + cells to establish the SW-620 cell line stably expressing premiR-338-3p or miR-338-3p-inhibitor.Moreover,the expression of miR-338-3p was determined by real-time reverse transcriptase polymerase chain reaction,andWestern blotting was used to detect the expression of the smoothened(SMO,the possible target of miR-3383p) protein in SW-620 cells.Furthermore,the status of CRC cell proliferation and apoptosis were detected by 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay and flow cytometry,respectively.RESULTS:Restriction enzyme digestion and DNA sequencing demonstrated that the lentiviral vector pLVTHM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed successfully.GFP was expressed after the SW-620 cells were transduced by the lentivirus.Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p was significantly increased(relative expression 3.91 ± 0.51 vs 2.36 ± 0.44,P < 0.01).Furthermore,overexpression of miR-338-3p inhibited the expression of SMO protein in SW-620 cells,which showed obviously suppressed proliferation ability [cellular proliferation inhibition rate(CPIR) 61.9% ± 5.2% vs 41.6% ± 4.8%,P < 0.01].Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p-inhibitor was significantly decreased(relative expression 0.92 ± 0.29 vs 2.36 ± 0.44,P < 0.
基金This work was supported in part by grants from the National Natural Science Foundation of China （81072480）, from the Natural Science Foundation of Guangdong Province （$2012020010927）, and from the Science and Technology Program of Guangdong Province （2013B021800076） （Jian-lin Huang） the major projects from Science and Technology Program of Guangzhou City, from the National Natural Science Foundation of Guangdong Province, from the Department of Education of Guangdong Province, and grants from NIH AR059103 and NIH AI084359 （Song Guo Zheng）.
文摘Fibroblast-like synoviocytes （FLSs） contribute to synovial hyperplasia in rheumatoid arthritis （RA）. Smoothened （Smo） is a key component of sonic hedgehog （Shh） signaling and contributes to tumor cell proliferation. The objective of this study was to investigate the role of Smo in RA synoviocyte proliferation. FLSs were isolated from RA synovium. Shh signaling was studied using a Smo antagonist （GDC-0449） and small interfering RNA （siRNA） targeting the Smo gene in FLSs. Cell proliferation was quantified by using kit-8 assay and cell cycle distribution and apoptosis were evaluated by flow cytometry. Cell cycle-related genes and proteins were detected by real-time PCR and western blot. FLSs treated with GDC-0449 or Smo-siRNA showed significantly decreased proliferation compared to controls （P〈0.05）. Incubation with GDC-0449 or transfection with Smo-siRNA resulted in a significant increase of G1 phase cells compared to controls （P 〈 0.05）. Cell cycle arrest was validated by the significant increase in cyclin D1 and E1 mRNA expression, decrease in cyclin-dependent kinase p21 mRNA expression in Smo-siRNA transfected cells （P 〈 0.05）. Protein expression of cyclin D1 was also downregulated after Smo gene knockdown （P 〈0.05）.The results suggest that Shh signaling plays an important role in RA-FLSs proliferation in a Smo-dependent manner and may contribute to synovial hyperplasia. Targeting Shh signaling may help control joint damage in patients with RA.
基金supported by grants from the National Cancer InstituteCA94160 and Wells Center for Pediatric Research
文摘Hedgehog was first described in Drosophila melanogaster by the Nobel laureates Eric Wieschaus and Christiane Nüsslein-Volhard. The hedgehog (Hh) pathway is a major regulator of cell differentiation, proliferation, tissue polarity, stem cell maintenance, and carcinogenesis. The first link of Hh signaling to cancer was established through studies of a rare familial disease, Gorlin syndrome, in 1996. Follow-up studies revealed activation of this pathway in basal cell carcinoma, medulloblastoma and, leukemia as well as in gastrointestinal, lung, ovarian, breast, and prostate cancer. Targeted inhibition of Hh signaling is now believed to be effective in the treatment and prevention of human cancer. The discovery and synthesis of specific inhibitors for this pathway are even more exciting. In this review, we summarize major advances in the understanding of Hh signaling pathway activation in human cancer, mouse models for studying Hh- mediated carcinogenesis, the roles of Hh signaling in tumor development and metastasis, antagonists for Hh signaling and their clinical implications.
基金This work was supported by a grant from the National Natural Science Foundation of China （No. 81101896）.
文摘Background miR-338-3p is a recently discovered miRNA and is involved in cell differentiation.However,few data are yet available on the aberrant expression of miR-338-3p in human colorectal carcinoma （CRC）.This work aimed to investigate the relationship between miR-338-3p expression pattern and clinicopathological features of human CRC and the possible regulative mechanisms.Methods The 40 CRC,adjacent nontumorous tissues and 2 human CRC-derived cell lines （SW-480 and SW-620） were collected,respectively,and the total RNA and protein were isolated routinely.The miR-338-3p expression pattern was detected by real-time reverse transcription-polymerase chain reaction （RT-PCR） and Northern blotting.Smoothened （SMO,possible target of miR-338-3p） mRNA and corresponding protein expression pattern were detected by semiquantitative RT-PCR and Western blotting.miR-338-3p expression patterns were compared between nontumor mucosa and CRC samples,graded by progression-related factors.Disease outcome was calculated by Kaplan-Meier survival analysis to determine whether miR-338-3p was related to disease-free survival （DFS） and overall survival （OS） of patients.Moreover,SMO 3＇-UTR fragment was PCR amplified from genome DNA of human colon and inserted into a luciferase reporter plasmid.The luciferase reporter plasmid construct was then transfected into CRC cells together with pre-miR-338-3p or anti-miR-338-3p and the luciferase activity in the transfected cells was detected.Results The expression of miR-338-3p was significantly downregulated in CRCs than those in the adjacent nontumorous tissues,and the value was negatively related to advanced TNM stage and local invasion （P ＜0.01）.Furthermore,miR338-3p value was decreased markedly in SW-620 cell line relative to SW-480 （P ＜0.01）.Low expression of miR-338-3p was associated with unfavorable outcome in DFS but not in OS independent of clinical covariates.Moreover,RT-PCR and Western blotting analysis demonstrated that there was no significant differ
基金supported by grants from the National Science Foundation of China(Nos.91332207 and 91632301,to Yi Zhong)the Beijing Municipal Science and Technology Commission(Z161100002616010,to Yi Zhong)+1 种基金the National Basic Research Project(973 program)of the Ministry of Science and Technology of China(2013cb835100,to Yi Zhong)the Tsinghua-Peking Joint Center for Life Sciences
文摘Emerging evidence suggests that neuro-inflammation begins early and drives the pathogenesis of Alzheimer＇s disease （AD）, and anti-inflammatory therapies are under clinical development. However, several anti-inflammatory compounds failed to improve memory in clinical trials, indicating that reducing inflammation alone might not be enough. On the other hand, neuro-inflammation is implicated in a number of mental disorders which share the same therapeutic targets. Based on these observations, we screened a batch of genes related with mental disorder and neuro-inflammation in a classical olfactory conditioning in an amyloid beta （Aβ） overexpression fly model. A Smoothened （SMO） mutant was identified as a genetic modifier of Aβ toxicity in 3-min memory and downregulation of SMO rescued Aβ- induced 3-min and 1-h memory deficiency. Also, Aβ activated innate inflammatory response in fly by increasing the expression of antimicrobial peptides, which were alleviated by downregulating SMO. Furthermore, pharmaceutical administration of a SMO antagonist LDE rescued Aβ-induced upregulation of SMO in astrocytes of mouse hippocampus, improved memory in Morris water maze （MWM）, and reduced expression ofastrocyte secreting pro-inflammatory factors IL-1β, TNFα and the microglia marker IBA-1 in an APP/PS1 transgenic mouse model. Our study suggests that SMO is an important conserved modulator of Aβ toxicity in both flv and mouse models of AD.
基金This work was supported by grants from the National Basic Research Program of China （2011CB943902）, the National Natural Science Foundation of China （30971679, 31071264, and 31271531）, and the Fundamental Research Funds for the Central Universities （090314380019）.
文摘The Hedgehog （Hh） signaling pathway plays important roles in developmental processes including pattern formation and tissue homeostasis. The seven-pass transmembrane receptor Smoothened （Smo） is the pivotal transducer in the pathway; it, and thus the pathway overall, is regulated by ubiquitin-mediated degradation, which occurs in the absence of Hh. In the presence of Hh, the ubiquitination levels of Smo are decreased, but the molecular basis for this outcome is not well understood. Here, we identify the deubiquitinase UCHL5 as a positive regulator of the Hh pathway. We provide both genetic and biochemical evidence that UCHL5 interacts with and deubiquitinates Smo, increasing stability and promoting accumulation at the cell membrane. Strikingly, we find that Hh enhances the interaction between UCHL5 and Smo, thereby stabilizing Smo. We also find that proteasome subunit RPN13, an activator of UCHL5, could enhance the effect of UCHL5 on Smo protein level. More importantly, we find that the mammalian counterpart of UCHL5, UCH37, plays the same role in the regulation of Hh signaling by modulating hSmo ubiquitination and stability. Our findings thus identify UCHL5/UCH37 as a critical regulator of Hh signaling and potential therapeutic target for cancers.
文摘目的 Hedgehog信号通路参与了肿瘤的发生发展,本研究探讨该通路关键信号分子Smoothened(SMO)在肾透明细胞癌中的表达及对肾癌细胞增殖和凋亡的影响。方法选取2012-01-01-2013-06-30青岛大学附属医院泌尿外科,行手术治疗的80例肾透明细胞癌患者的临床及病理资料,采用免疫组织化学方法检测SMO在肾透明细胞癌组织中的表达并分析其与临床病理特征间的关系。采用小干扰RNA下调SMO在人肾癌细胞786-O中的表达,分别应用CCK-8法、流式细胞术及蛋白质印迹法检测下调SMO表达对细胞增殖、凋亡及Gli1和Gli2表达的影响。结果 SMO在73例(91.25%)肾透明细胞癌组织中有表达,其在高级别肾癌中表达(80.49%)较低级别(56.41%)显著升高,χ2=5.39,P=0.02。RT-PCR检测结果显示,SMO在人肾癌细胞系786-O中表达量为0.704±0.059;蛋白质印迹结果显示,SMO在786-O中表达量为0.651±0.074。在786-O细胞中应用小干扰RNA沉默SMO表达后,实验组细胞相较于对照组其活力百分比在48、72和96h分别为92.7%、80.9%和79.9%,3个时间点差异有统计学意义(P=0.003),细胞凋亡显著增加,t=-29.2,P<0.001;与空白对照组和阴性对照组相比,其下游分子Gli1和Gli2蛋白表达明显减少(Gli1:3.2 vs 2.9 vs 1;Gli2:2.5 vs 2.1vs 1)。结论 SMO可能通过调控细胞增殖和凋亡,以及调节Gli蛋白表达参与了肾癌的发生发展。
