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Objective:To investigate the expression levels and clinical significance of glioma-associated oncogene homolog 1 (GLI1) and sonic hedgehog signaling molecule (Shh) in the malignant transformation of ovarian endometriosis (EM).Methods:The expressions of GLI1 and Shh were detected by real-time reverse transcription (RT)-polymerase chain reaction (PCR) and EnVision method in 50 cases of ovarian EM tissues, 35 cases of atypical endometriosis (aEM) and 50 cases of endometriosis-associated ovarian cancer (EAOC). The expression differences of two molecular markers in the malignant transformation of ovarian EM were compared, and the relationships between two molecular markers and the clinicopathological features and prognosis of EAOC were analyzed.Results:(1) RT-PCR showed that the expression levels of GLI1 mRNA in EM, aEM and EAOC group were 1.77±0.40, 3.54±0.44, and 7.80±0.24, respectively. The expression levels of Shh mRNA were 0.95±0.21, 3.14±0.35, and 5.41±0.31, respectively. GLI1 and Shh mRNA in EAOC group were significantly higher than those in EM and aEM group (all P<0.01), and there were statistically significant differences between EM and aEM group (all P<0.01). The percentages of GLI1 in ovarian EM, aEM and EAOC were 32% (16/50), 57% (20/35), and 66% (33/50), respectively, meanwhile, the positive expression rates of Shh were 20% (10/50), 49% (17/35), and 54% (27/50), respectively (all P<0.01). GLI1 mRNA expression was positively correlated with Shh mRNA expression in EAOC tissues ( r=0.721, P<0.01). The expressions of GLI1 protein were proportionated to Shh protein in EAOC tissues ( r=0.608, P=0.001). (2) The expression of GLI1 was significantly related to the International Federation of Gynecology and Obstetrics (FIGO) stage, cancer antigen 125 (CA 125) levels, lymph node metastasis, and Platinum resistance in EAOC patients (all P<0.05). The expression of Shh were related to FIGO stage and lymph node metastasis in EAOC patients (all P<0.05). Logistic regression analysis showed that GLI1 expression was an independent risk factor for poor prognosis in EAOC patients ( P<0.05). Kaplan-meier survival analysis showed that the overall survival rate of EAOC patients with high GLI1 expression and low GLI1 expression was 12.1% and 35.3%, respectively, with statistical significance ( χ2=10.73, P<0.01). The overall survival rate of EAOC patients with high and low expression of Shh protein was 11.1% and 30.4%, in which there was statistically significant difference ( χ2=3.96, P=0.047). Conclusion:GLI1 and Shh are highly associated with the malignant transformation of ovarian EM, which may play a role in promoting malignant degeneration of ovarian EM, and the high expression of GLI1 and Shh indicates a poor prognosis in EAOC patients.
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ObjectiveTo investigate the molecular mechanism of cordycepin inhibiting proliferation and promoting apoptosis of human hepatoma cells （HCCs）. MethodGlioma-associated oncogene homolog 1 （Gli1） gene was silenced by small interfering RNA （siRNA） and transfected into SMMC-7721 cells， and then cell proliferation was detected by cell counting kit-8 （CCK-8） assay and cell cloning assay. SMMC-7721 cells were treated with different concentration of cordycepin， and the cell proliferation and apoptosis were examined. The expression of Gli1 and the downstream related genes was determined by Real-time polymerase chain reaction（PCR） and Western blot. ResultThe mRNA and protein expression of Gli1 in SMMC-7721 cells was higher than that in normal liver cells （P<0.01）. The proliferation rate of SMMC-7721 with silenced Gli1 decreased at 72 and 96 h （P<0.05， P<0.01）， and the colony-forming capacity lowered （P<0.01） compared with those in the blank group. Compared with the control， 80 μmol·L-1 and 120 μmol·L-1 cordycepin significantly inhibited the proliferation of SMMC-7721 cells at 72 and 96 h （P<0.05， P<0.01）， and promoted the apoptosis of them （P<0.01）. Moreover， 80 and 120 μmol·L-1 cordycepin restrained the mRNA and protein expression of Gli1 in SMMC7721 cells （P<0.05， P<0.01）. At 120 μmol·L-1， cordycepin led to the decrease in the mRNA and protein levels of B-cell lymphoma-2 （Bcl-2） and c-Myc （P<0.05， P<0.01）， and the increase in the mRNA and protein expression of cysteine-aspartic acid protease-3 （Caspase-3） （P<0.05）. ConclusionGli1 is highly expressed in HCCs， and cordycepin can suppress the proliferation and enhance the apoptosis of HCCs by regulating Gli1 and the downstream apoptosis-related factors.
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Objective: To investigate the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSC)induced by simvastatin(SIM)in vitro and then further investigate whether the Hedgehog signaling pathway is involved in the SIM-induced osteogenic differentiation of BMSC using the Hedgehog signaling pathway blocking agent cyclopamine(Cpn). Methods: The rat BMSC was extracted by the whole-bone marrow adherent culture method and cul- tured in the induction medium(control medium,CM)and the induction medium containing SIM or SIM+Cpn. The expression of alkaline phosphatase(ALP)in induced cells was detected by the ALP staining. Immunofluorescence stain- ing was performed to detect the espressions of Gli1 and osteocalcin(OCN)in the induced cells. The Gli1,ALP,colla- gen type(COL)and OCN mRNA expression was detected by real-time quantitative PCR(RT-PCR). Western blot (WB)was used for assessing the expression level of Gli1,Runx2,COLand OCN proteins. The cell calcium nodule for- mation and matrix mineralization ability were detected by alizarin red staining. Results: The ALP expression was signifi- cantly higher in SIM group than in CM group(P<0.05),while the ALP expression was lower in SIM+Cpn group than in the SIM group(P<0.05)but higher than in the CM group. Immunofluorescence assay showed that SIM could promote the expression of Gli1 and OCN in the conditions with or without Cpn. On the 10th and 18th day of intervention,the mRNA expression level of Gli1,ALP,OCN and COLwas significantly higher in the SIM group than in the CM group(P<0.05), and the higher mRNA expression in the SIM group could be completely blocked by Cpn. Further,on the 10th and 18th day of intervention,the expressed Gli1,Runx2,COLand OCN protein level was significantly higher in the SIM group in the CM group(P<0.05),while the level of these expressed proteins in the SIM+Cpn group was significantly lower than in the SIM group(P<0.05). The alizarin red staining showed that SIM group had stronger matrix mineralization ability than the other groups(P<0.05). Conclusion: SIM could up-regulate the expression of Gli1 and the osteogenesis markers ALP,Runx2,COLand OCN to induce osteogenic differentiation of BMSC,and Cpn could not completely block the SIM-induced differentiation. Further study is needed to uncover the underlying mechanism.
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AIM:To investigate the relationship between Sonic Hedgehog(Shh)signaling pathway and cell cycle and radioresistance of esophageal cancer by up-regulating Gli1,a key factor in Shh signaling pathway.METHODS:The human esophageal cancer cell line Eca 109 was transfected with plasmid to induce Gli 1 over-expression,which served as Eca109-ox-Gli1 group.In addition, Eca109 cells transfected with empty plasmid served as negative control group and the untreated Eca109 cells were used as normal control group.The expression of Gli1 was confirmed by real-time PCR and Western blot.The radiosensitivity of the cells in the 3 groups was determined by colony formation assay.The effect of irra-diation on the cell cycle was analyzed by flow cytometry.RESULTS:The expression of Gli1 in Eca109-ox-Gli1 group was higher than that in the other 2 groups(P<0.05).The survival fraction at dose of 2 Gy in Eca109-ox-Gli1 group was high-er than that in normal control group, indicating that the radioresistance of the Eca 109 cells transfected with Gli1 plasmid was increased.The cells in Eca109-ox-Gli1 group showed higher S phase proportion than that in normal control group and negative control group(P<0.01).After irradiation at dose of 6 Gy,all cells in the 3 groups found that the cell cycle was arrested at G2/M phase,while the cells in normal control group showed higher G 2/M phase proportion than that in Eca109-ox-Gli1 group(P<0.01).CONCLUSION: The up-regulation of Gli1 may enhance the radioresistance of esophageal cancer by regulating the cell cycle.
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Objective:To investigate the expression and clinical significance of glioma cancer-related gene homologous protein 1 (Gli-1) and epithelial-mesenchymal transition (EMT)-related proteins, namely, Snail, E-cadherin, and N-cadherin in non-small cell lung cancer (NSCLC). Methods:Immunohistochemical staining was performed to determine the expression levels of Gli-1, Snail, E-cadherin, and N-cadherin in 67 cases of NSCLC and 20 cases of normal lung tissues and its relationship with clinicopathological features and prognosis was explored. Another 20 samples of fresh NSCLC tissues and corresponding normal lung tissues were collected to detect the mRNA level through reverse transcription polymerase chain reaction (RT-PCR). Results:1) The positive rates of Gli-1, Snail, E-cadherin, and N-cadherin in NSCLC were 61.19％(41/67), 50.75％(34/67), 56.72％(38/67), and 53.73％(36/67), respectively;whereas those of normal lung tissues were 20％(4/20), 10％(2/20), 100％(20/20), and 5％(1/20), respectively. These two sets of data have significant statistical difference (P<0.05). 2) The high expression of Gli-1 in tumor tissues was closely related to lymph node metastasis, tumor, node, and metastasis (TNM) stage, and tumor differentiation (P<0.05) but was not associated with gender, age, tumor size, and pathological type. The expression of Gli-1 in NSCLC tissues was negatively correlated with E-cadherin (r=-0.325, P<0.05) and positively correlated with Snail and N-cadherin (r=0.379, r=0.490, P<0.05). 3) RT-PCR results showed that the expression levels of Gli-1, Snail, and N-cad-herin mRNA were significantly higher in NSCLC cases than in normal lung cases (P<0.05). The expression level of E-cadherin mRNA was lower in tumor tissues than in lung tissues (P<0.05). 4) Patients with high expression levels of Gli-1, Snail, and N-cadherin had signifi-cantly worse prognosis and lower survival rate than those with low expression (all P<0.05), whereas patients with low expression lev-els of E-cadherin had significantly better prognosis and lower survival rate than those with high expression (P<0.05). Multivariate Cox's proportional hazard regression analysis indicated that E-cadherin-negative group expression, lymph node metastasis, TNM stage, and tumor differentiation were independent prognostic factors for NSCLC. Conclusion: The abnormal activation of Hedgehog signaling pathway in NSCLC is correlated with EMT. Detecting the expression levels of Gli-1 and EMT-related proteins Snail, E-cadherin, and N-cadherin might be helpful in understanding the clinicopathological features and prognosis of patients with NSCLC.
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Objective:To investigate the expression and the significance of inflammatory cytokine IL-6 through regulating Gli1 in acute pancreatitis.Methods: In this study,C57 mice were randomly divided into three groups:control group,model group,inhibitor group.caerulein intraperitoneal injection induce acute pancreatitis model.Use HE staining and amylase to testify the model successfully.Use RT-qPCR,Western blot to detect the expression of Gli1 in the pancreas,liver,lung,kidney and intestine and ELISA method to detect inflammatory cytokines IL-6.Results: Compared with control group,the expression of Gli1 is higher in model group,then the expression of IL-6 increases in inhibitor group which uses Gant61 to suppress Gli1 compared with model group.Conclusion: Gli1 may involved in the process of the distant tissue injury and repair in acute pancreatitis and through regulate its downstream cytokines like IL-6 to play a protective role in acute pancreatitis.
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Objective: To investigate the effect of oxymatrine (OM) on expression of related molecules in Smad3, Gli1 signaling pathway in PANC-1 cells induced by TGF-β1. Methods: TGF-β1-induced PANC-1 cells were used to establish the pancreatic fibrosis model in vitro, and observe the effects of OM pretreatment on the related molecular expression of Smad3/Gli1. Gli1 and Smad3 RNA interference plasmids were transfected into PANC-1 cells. The protein expression levels of Smad3, Gli1 and α-SMA were measured by Western blotting. The levels of fibronectin (FN) and type I collagen (CoL-I) in the supernatant of cell culture were detected by ELISA. Results: Compared with the control group, the protein expressions of Smad3, Gli1 and α-SMA increased significantly in PANC-1 cells after treated with TGF-β1. The expressions of Gli1, α-SMA, FN, and CoL-I in PANC-1 cells decreased significantly after Gli1 RNA interference plasmid transfection compared with TGF-β1 induced group. The expression of Smad3, Gli1, α-SMA, FN, and CoL-I also decreased significantly in PANC-1 cells after Smad3 RNA interference plasmid transfection compared with TGF-β1 group. Conclusion: OM could prevent pancreatic fibrosis by regulating TGF-β1/Smad3/Gli1 signaling pathway in PANC-1 cells.
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Objective To investigate the effect of lentivirus carrying shRNA-VDR vector on GLi1 in pros-tate cancer PC-3 cells. Methods The cells were cultured according to the culture conditions of PC-3 cells. Expression of VDR and GLi1 in PC-3 cells was detected by fluorescence quantitative PCR and immunocytochemistry SP method.The efficiency of PC-3 cell virus infection was evaluated.The effect of VDR gene interference and GLi1 transcription level on PC-3 cells was detected by RT-PCR.Results Cell culture,cell status was recorded and PC-3 cells were in good condition and the passages was 4 days. Fluorescence quantitative and immunocytochemi-cal SP showed that VDR and GLi1 were expressed in PC-3 cells.The virus infection efficiency showed that the in-fection efficiency was about 95% when adding LV3-NC lentivirus to PC-3 cells at 1:10 ratio. RT-PCR showed that VDR-shRNA lentivirus successfully disturbed VDR expression and decreased by 85%(P < 0.05)compared with the control group after 72 days of VDR-shRNA lentivirus transfection. Transcription level of GLi1 gene in the experimental group increased by 9% compared with the control group(P < 0.05). The transcription level of GLi1 gene in the experimental group increased by 248% compared with the control group(P < 0.05). Conclusion The cultured PC-3 cells were in good condition. VDR and GLi1 genes were expressed in PC-3 cells. Lentivirus showed highest efficiency in infecting PC-3 at 1:10 ratio. When VDR was disturbed,the expression of GLi1 in-creased.In prostate cancer cells,vitamin D can inhibit the Hh signaling pathway and cause GLi1 expression down expression.
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Objective To investigate the expression of Gli1 protein in colorectal cancer (CRC) tissues,and evaluate its association with the prognoses of the patients.Methods A total of 106 patients were enrolled and their clinicopathological characteristics were analyzed.The expression of Gli1 proteins were detected by immunohistochemical staining,and its association with time to recurrence/metastasis (TTR) and overall survival (OS) of the patients were analyzed.Results The positive rate and expression intensity of Gli1 protein in recurrent or metastatic tumor tissues were higher than those in non-recurrent and non-metastatic tumor tissues [86.84 ％ (33/38) vs.58.82 ％ (40/68),1.32 scores vs.0.71 scores,both P ＜ 0.01),while the positive rate and expression intensity of Ki-67 protein remained no difference between both groups [97.37 ％ (37/38) vs.91.18 ％ (62/68),1.89 scores vs.1.75 scores,both P ＞ 0.05).The positive rates and expression intensities of Gli1 and Ki-67 proteins in CRC tissues were higher than those in adjacent tissues [26.42 ％ (28/106) and 0.27 scores;4.72 ％(5/106),0.05 scores] and normal tissues [3.33 ％ (1/106),0.03 scores;0,0.00 scores] (all P ＜ 0.01).Results of univariate analysis showed that the expression of Gli1 protein,tumor grade and lymph node involvement were significantly associated with TTR,but all of the clinicopathological factors had no obvious association with OS.The association remained significant between the expression of Gli1 protein and TTR in multivariate analysis (P ＜ 0.01).Conclusion The expression of Gli1 protein is an independent prognostic marker of recurrence or metastasis in CRC patients,its high expression implicates a high risk of CRC recurrence or metastasis.
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@#HH (hedgehog) signal pathway consists the hedgehoge ligand (Shh、Ihh and Dhh), the Patch and Smo membrane protein complex and Zinc finger transcription factor Gli (Gli1, Gli2, Gli3). In HH (hedgehog) signaling pathway, Gli1 not only plays a dominant and decisive role in the zinc finger transcription factor Gli (Glioma-associated oncogene homolog) family, but also includes epithelial-mesenchymal transition (EMT) and oral squamous cell carcinoma, tumor invasion and metastasis. The research on oral squamous cell carcinoma and Shh/Gli1 signal axis is rare. In this paper, the squamous cell carcinoma of oral epithelial mesenchymal transformation and correlation study of quality Gli1 is reviewed.
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Objective To study the relationship between expression of Shh and Gli1 in Hedgehog(Hh) signal pathway and esophageal squamous cell carcinoma (ESCC).Methods Expressions of Shh andGli1 were detected in 64 cases primary tumor tissues and 24 cases normal esophageal mucosa with SP immunohistochemistal method.Results There were significant differences of Shh and Gli1 expressions between esophageal carcinoma and normal mucosa epithelium (67.19％ (43/64) vs.4.17％ (1/24), 60.94％ (39/64) vs.4.17％ (1/24);x2 =27.729,22.689;P＜0.01).There were significant differences of Shh expression between different degrees of differentiation in esophageal squamous cell carcinoma, metastasis of lymph node and different clinical staging group (x2 =3.873, 11.349,6.429;P ＜ 0.05 or P＜ 0.01).The positive expression rate of Gli1 showed significant differences between different degrees of differentiation in esophageal squamous cell carcinoma, metastasis of lymph node and different clinical staging group (x2=12.598, 9.741,26.341;P ＜0.01).There was a positive relationship between Shh and Gli1 expression(r =0.259, P＜0.05).Conclusion Hh signaling pathway is abnormally activated in ESCC, so that Shh and Gli1 play important roles in carcinogenesis and progression ofesophageal carcinoma.The Hh signaling pathway may be a useful target in ESCC.
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AIM: To explore whether strontium ranelate ( Sr ) promotes osteogenic differentiation of rat bone mesenchymal stem cells (BMSCs) through the Hedgehog/Gli1 signaling pathway.METHODS:BMSCs were isolated from 4-week-old rats by adherent culture and induced to differentiate into osteoblasts .According to the experimental purposes , the cells were exposed to different concentrations of Sr , cyclopamine ( Cy, an inhibitor of Hedgehog receptor ) or Gli1-siR-NA.The expression of Gli1 and Runx2 in the cells was detected by Western blotting .The activity of alkaline phosphatase ( ALP) was measured by the method of colorimetry , and the mineralized nodules were observed under microscope with aliz-arin red staining .RESULTS:Exposure to Sr at concentrations of 0.1 to 5 mmol/L for 7 d markedly increased the expres-sion of Gli1 in the BMSCs , and the increase in Gli1 expression was the most obvious following Sr exposure at concentration of 3 mmol/L.Cy at concentration of 10 μmol/L inhibited Sr-induced up-regulation of Gli1 expression.Transfection of the BMSCs with Gli1-siRNA not only obviously inhibited Sr-induced up-regulation of Gli1 and Runx2 ( a downstream protein of Gli1) expression, but also antagonized Sr-induced enhancement of ALP activity and the formation of mineralized nodules . CONCLUSION:The Hedgehog/Gli1 pathway is involved in Sr-induced osteogenic differentiation of rat BMSCs .
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Objective To evaluate the clinical value of Gli1 expression in breast cancer.Methods Immunohistochemistry was used to analyze the protein expression of Glil in breast cancer tissues,adjacent cancer tissues and metastatic lymph node.Results ①Breast cancer tissues expressed high levels of Gli1 compared to paracancerous tissues.Gli1 overexpression was found in 93.3％ of breast cancer tissues with recurrence and metastasis and 81.2％ in breast cancer tissues without metastasis.Gli1 expression could be detected either in interstitial tissues of cancer or in cancer tissues,or in both.The rate of Gli1 overexpression was 89.3％ in the inter stitial tissues of the primary cancer with recurrence and metastasis and 13.4％ in interstitial tissues of breast cancer tissues without metastasis.The difference had statistical significance(P ＜ 0.001).②High expression of Gli1 in breast cancer tissues was closely related to early recurrence and metastasis of breast cancer.Gli1 expression displayed a significant correlation with relapse-free survival (RFS) (P =0.046).The recurrence rate was 17.8％ in the high expression group in breast cancer tissue,whereas it was only 6.2％ in the low expression group(P =0.046),while the recurrence rate was 31.2％ in the high expression group in interstitial tissues,whereas it was only 11.1％ in the low expression group(P ＜ 0.001).Conclusion The high expression of Gli1 in breast primary cancer tissues,metastasis tissues and interstitial tissues is associated with recurrence and metastasis of breast cancer.
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To investigate the role of Gli1 in DDP-resistance in lung cancer.Methods:A DDP-resistant human lung adenocarcinoma cell line A 549/DDP was cultured and transfected with Gli 1 siRNA.The mRNA and protein expression levels of Gli 1 were evaluated by qPT-PCR,Western blot analysis and immunofluorescence microscopy.The expression of Bcl-2、caspases-3、cyclinD1 was evaluated by Western blot analysis.Hoechst 33258 staining and flow cytometry were used to detect spontaneous cell apoptosis and cell cycle.The cell inhibition rate and DDP-induced cell death were examined by MTT and Annexin V-FITC/propidium iodide staining.Results:Gli1-knockdown by using Gli 1-specific siRNA led to a markedly decrease in Gli 1 mRNA and protein expression levels,when compared to negative siRNA transfected cells and untreated control cells (P=0.001).The downstream effectors of Gli1, Bcl-2 and cyclinD1 proteins were also inhibited (P=0.003),the expression of caspase-3 was increased (P=0.001).Hoechst 33258 staining showed that Gli1 depletion by Gli1 siRNA in A549/DDP cells could induce spontaneous apoptosis.The result of cell cycle showed Gli1 siRNA could lead more cells arrest in G1 phase.The IC50 of A549/DDP cells on DDP was (12.63 ±1.11 )μg/ml /(13.81±1.14)μg/ml,which decreased to (2.65±0.85)μg/ml after being transfected with Gli1 siRNA (P=0.000).Annexin V-FITC/propidium iodide staining showed that DDP-induced apoptosis rate in Gli 1-silencing cells was higher than that in negative siRNA or untreated control cells ( 35.19%±3.92% vs 6.43%±0.11%/5.01%±0.77%, P=0.000 ).Conclusion: Application of RNA interference can restrain the expression of Gli 1 mRNA and protein observably in A549/DDP cells,and increase the sensitivity of A549/DDP cells to cisplatin.Maybe Gli1 will become a new target to reverse the cisplatin resistance for lung cancer.
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BACKGROUND: Certain epidermal appendage tumors, including hyperplasias (hamartomas), adenomas, benign epitheliomas, primordial epitheliomas, and malignant tumors, can exhibit any stage of differentiation. Several molecules associated with tumorigenesis, such as Gli-1, pleckstrin homology-like domain, family A, member 1 (PHLDA-1), transforming growth factor (TGF)-beta1, TGF-beta2, and p63, are associated with tumor grade and aggressive behavior in follicular and sebaceous tumors in ways that are not well understood. OBJECTIVE: The aim of this study was to elucidate the expression of Gli-1, PHLDA-1, TGF-beta1/beta2, and p63 in benign and malignant tumors of the hair and sebaceous glands and to determine their importance in the degree of tumor differentiation. METHODS: Immunohistochemistry was performed in follicular and sebaceous tumors using antibodies against Gli-1 (sebaceous tumor marker), PHLDA-1 (hair follicle outer root sheath [ORS] cell marker), p63, TGF-beta1, and TGF-beta2. RESULTS: Gli-1 was expressed in basaloid cells, sebocytes, and sebaceous carcinoma cells, and expression levels decreased as differentiation progressed. PHLDA-1 was expressed in ORS cells and some follicular tumor cells. Expression of p63 was observed in the nuclei of the outermost basaloid cells (seboblasts), poorly differentiated sebaceous carcinoma cells, and tumor cells toward the direction of the hair. Remarkably, TGF-beta1 was expressed exclusively in the nuclei of benign and malignant follicular (hair) tumors, but not in sebaceous tumors, at levels that correlated with the degree of differentiation. CONCLUSION: We propose that p63 and/or TGF-beta1 are useful for predicting the degree of differentiation and malignant potential of sebaceous and follicular tumors and for distinguishing trichilemmal carcinoma from sebaceous carcinoma.
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Humans , Adenoma , Antibodies , Carcinogenesis , Carcinoma , Hair , Hyperplasia , Immunohistochemistry , Sebaceous Glands , Transforming Growth Factor beta1 , Transforming Growth Factor beta2 , Transforming Growth FactorsABSTRACT
Objective To construct RNAi eukaryotic expressing vectors of human transcription factor glioma-associated oncogene homolog 1 (GLI1) with pGCsi-U6-GFP plasmid and to identify its activity in interfering GLI1.Methods Three GLI1siRNA targeting GLI1 were designed and synthesized according to the GLI1cDNA sequence in GeneBank,and then were cloned into pGCsi-U6-GFP to construct the recombinant plasmids,and transformed into E.coli DH5a,then it was amplified and plasmids were extracted,which were further confirmed by PCR reaction and DNA sequencing,pGCsi-U6-siRNA-C was negative as control wector.Then recombinant plasmids pGCs-U6-GLI1siRNA-1,pGCs-U6-GLI1 siRNA-2,pGCs-U6-GLI1siRNA-3 pGCsi-U6-siRNA-C and a eukaryotic over-expression vector pEGFP-N1-GLI1 were co-transfected into HEK293 cells by Lipofectamine 2000 respectively.The ceils were collected at 48 h after transfection.Semi-quantitative RTPCR and Western Blot were performed to detect the expression of GLI1 mRNA and protein to screen the optimal vector which had the best interfering effect.Results A 369 bp fragment was amplified from all three recombinant plasmids,(pGCs-U6-GLI1 siRNA-1,pGCs-U6-GLI1 siRNA-2,pGCs-U6-GLIlsiRNA-3),showing that synthesized shRNA oligonucleotide fragments were correctly inserted into three recombinant plasmids,which were further confirmed by sequencing.Expression levels of GLIlmRNA and protein in cells in pGCs-U6-GLI1 siRNA-1,pGCs-U6-GLI1 siRNA-2,pGCs-U6-GLI1 siRNA-3 were 0.290 ± 0.011,0.421 ± 0.018,0.373 ±0.018,and 0.318 ± 0.026,0.443 ± 0.021,0.381 ± 0.018,which were significantly lower than those in negative control group (0.834 ± 0.022,0.818 ± 0.024,P =0.000),the inhibitory rates were 65.8 ％,50.7％,55.7％,and 63.9％,48.3％,53.9％.The interfering efficacy of pGCs-U6-GLIlsiRNA-1 was the strongest among the three recombinant plasmids.Conclusions RNAi eukaryotic vectors pGCs-U6-GLIlsiRNA-1,pGCs-U6-GLI1 siRNA-2,pGCs-U6-GLI1 siRNA-3 are successfully constructed and the optimal vector is identified,and this can provide a solid experimental foundation for further functional study of GLI1 gene.
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Objective This study analyzes the expression and clinical significance of Gli1 and Gli3 proteins in hepatocellular carcinoma.Methods 36 patients with hepatocellular carcinoma were studied.The expressions of Gli1 and Gli3 in the carcinoma and adjacent non-tumor tissues were detected with immunohistochemical assay,and their correlations with clinicopathological factors were statistically analyzed.Results Expression rates of Gli1 in hepatocellular carcinoma and adjacent nontumor tissues were 75 ％ and 36.1％,respectively.Expression rates of Gli3 in hepatocellular carcinoma and adjacent non-tumor tissues were 58.3％ and 30.6％,respectively.Expression rates of Gli1 and Gli3 in hepatocellular carcinoma were significantly higher than in adjacent non-tumor tissues (P＜0.05),and a positive correlation was found between the expression of Gli1 and Gli3 (r=0.423,P＜0.05).There was no association between the expression of Gli3 and clinicopathological factors such as age,tumor size,tumor differentiation and lymphatic metastasis.The expression of Gll1 was not related witha patient's age and tumor size,hut there were significant associations with tumor differentiation and lymphatic metastasis.Conclusions Therefore,the expression rate of Gli1 was positively correlated with tumor malignancy,which makes the detection of Gli1 and Gli3 valuable for the diagnosis and prognosis of hepatocellular carcinoma.
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Objective To establish a human pancreatic cancer cell line stably transfected with siRNA expression vector targeting GLU gene and examine the interference efficiency. Methods The expression of GLI1 gene in five human pancreatic cancer cell lineswas detected by quantitative real-time PCR (qRT-PCR); the one with the highest expression level of GLI1 was selected as the target cell line and was transfected with three recombinant plasmids pGCti-U6-GLIlsiRNA-1,-2, and -3. The positive cloneswere screened by G418, and the transfection rate was observed by fluorescence microscope. The expression of GLI1 mRNA and protein was analyzed by qRT-PCR and Western blotting analysis, respectively. Results Panc-1 cell line was found to have the highest GLU expression and was selected as the target cell line for transfection. Plasmids pGCti-U6-GLIlsiRNA-1, -2, and-3 were successfully transfected into Panc-1 cells separately. After 4 weeks of G418 screening, three stably transfected cell lines named Panc-1/GIU1siRNA-1, -2, and -3 were obtained, with the transfection rates all higher than 80%. qRT-PCR and Western blotting analysis showed that the expression levels of GLI1 in Panc-1/GIUlsiRNA-1, -2, and -3 cellswere all significantly lower than those in Panc-1/siControl cells and the blank control cells(P<0. 05), with the lowest expression found in Panc-1/GLIlsiRNA-1 cels. Conclusion We have successfully constructed a cell line Panc-1/GLI1siRNA-1 with GLU gene stably silenced, which paving a way for future research.
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ObjectiveTo investigate the association between R405Q polymorphism of GLI1 gene and tetralogy of fallot(TOF).Methods In the case-control study,the R405Q polymorphism of GLI1 gene in 112 children with TOF and 200 healthy controls were detected with polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP).The distribution of genotype and allele frequency at R405Q polymorphism site were analyzed and to investigate its relationship with the risk of TOF.ResultsThe distribution of genotype frequency at R405Q polymorphism site was not different between TOF group and the healthy control group(x2 =5.317 ,P = 0.07) .However, the distribution of allele frequency at R405Q polymorphism site was significantly different between TOF group and the healthy control group (x2 = 6.790, P = 0.009) , and the relative risk for TOF in A allele carriers was higher than that in G allele carriers (OR = 1.561,95％ CI 1.116 ～ 2.185) Conclusion The R405Q polymorphism of GLI1 gene is associated with TOF and people with A allele have higher risk with TOF.GLI1 gene might be the genetic susceptibility gene of TOF.
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Objective To define the expression of Shh/Gli1,the key elements of Hedgehog signaling pathway in papillary thyroid carcinoma(PTC) and to explore the relationship between the expression of Shh/Gli1 and clinical significance.Methods The expression of Shh and Gli1 was examined in 142 cases of PTC tissues and adjacent tumor thyroid tissues as control by immunohistochemistry.The relationship between the expression of Shh/Gli1 and clinical characteristics of PTC patients was analyzed.Results The positive rate of the cytoplasm Shh expression and the nuclear Gli1 expression was 64.1% and 47.9% ,respectively.Significant difference was found between normal thyroid tissues and PTC.The research showed that the expression of Shh/Gli1 was related to the tumor size,clinical stages and lymph node metastasis,Shh was more significantly related to the tumor size(P ＜0.01) and Gli1 was more significantly related to the lymph node metastasis (P ＜ 0.01).Conclusions Varying expression of the main ligand Shh and transcription factor Gli1 in Hedgehog signaling pathway was found in PTC.The expression of Shh/Gli1 was related to the tumor size,clinical stage and lymph node metastasis,indicating that the aberrant activation of Shh signaling pathway plays some roles in PTC.Shh/Gli1 may be indicators for prognosis and ideal targets for therapy against PTC.