Papel do Receptor Lgr5 no Prognóstico e na Biologia de Sarcomas de Partes Moles / Role of the Lgr5 Receptor in the Prognosis and Biology of Soft Tissue Sarcomas

INTRODUÇÃO: Os sarcomas de partes moles (SPM) de alto grau são neoplasias heterogêneas, de prognóstico ruim e que apresentam poucas alternativas de tratamento. A identificação de marcadores de resposta tumoral ao tratamento, prognóstico, e até ao desenvolvimento de novas drogas, é uma busca incessante para um melhor tratamento dos sarcomas. Neste aspecto, o receptor Lgr5 tem um grande potencial em ser um novo alvo molecular, sendo um marcador de células-tronco das criptas intestinais e glândulas mamárias que também atua como um modulador negativo da sinalização da via Wnt/ß-catenina, uma das mais importantes na biologia de sarcomas emerge como um promissor candidato para estudos pré-clínicos, uma vez que já foi demonstrada sua importância em tumores do trato gastrointestinal. Para isso, os modelos de tumor de xenoenxerto derivado do paciente (PDX) representam uma plataforma valiosa para identificar novos biomarcadores e novos alvos, assim como o Lgr5 para avaliar a resposta à terapia e os mecanismos de resistência. OBJETIVO: Este estudo teve como objetivo estabelecer, caracterizar e testar a proteína Lgr5 através de ensaios in vitro e in vivo para desvendar a importância de Lgr5 na biologia de SPM e estabelecer uma estrutura integrada, convergente e translacional para o estudo deste tipo de tumor. MATERIAIS E MÉTODOS: Para a determinação da expressão de Lgr5 foi estabelecida duas coortes de estudo, uma retrospectiva oriunda do Registro Institucional de Sarcomas e uma prospectiva, onde foram convidados pacientes operados na Instituição com o intuito de gerar Patient-derived xenografts (PDX), um modelo-pré-clínico que possui a capacidade de manter as características moleculares dos tumores dos pacientes. Para isso, foram utilizados fragmentos implantados em camundongos imunossuprimidos para gerar esses modelos tumorais derivados de pacientes, além dos estudos funcionais in vitro utilizando linhagens de SPM para análise de perfil de expressão da proteína Lgr5 através de ensaios com imunofluorescência para verificar a capacidade de expressão de Lgr5, citometria de fluxo para verificar o padrão e quantidade de proteína nas amostras analisadas e western blotting para obter um padrão de marcação da proteína Lgr5. Além dos ensaios funcionais para avaliar a participação da proteína na proliferação, se a expressão da proteína interfere no poder migratório das células e tumores de SPM e capacidade de auto renovação, bem como sua associação com os dados clínicos e dados de sobrevida. RESULTADOS: O Registro Institucional retrospectivo conta com mais de 300 pacientes, já o Registro prospectivo com 70 pacientes que derivaram a geração de 33 PDX. Foi observado que pacientes com H-score superior a 20 apresentaram sobrevida global menor em 5 anos em comparação com o H-score de pacientes com valores inferiores a 20. Agora na outra análise feita, o H-score de pacientes com valores superiores a 25 é pior em comparação com os que apresentaram valores inferiores a 25 nos dados de sobrevida livre de doença. Além disso, células que superexpressam a proteína Lgr5 tem maior capacidade migratória (p= 0.02) e uma tendência de aumento na proliferação e auto renovação. Realizamos o teste de implante dessas populações positivas e negativas de Lgr5, separadas previamente por cell sorting. Para isso foram utilizados animais Balb/c Nude. Sugerindo que a expressão da proteína transduzida pode ser modulada por mecanismos compensatórios que precisam ser explorados. CONCLUSÃO: A construção do Registro Institucional de SPM é um grande passo para o melhor compreendimento da biologia dos Sarcomas, além da possibilidade de estudar novos alvos terapêuticos desse tumor raro, uma vez que os estudos e artigos científicos ainda são muito escassos. A geração dos modelos PDX também foi uma estratégia implantada muito bem executada com a geração de 33 PDX de diversos subtipos histológicos. Além da proteína Lgr5 induzir a migração celular a sua expressão está relacionada a um pior prognóstico, uma vez que, quanto maior a expressão de Lgr5 menor é a sobrevida global do paciente.

INTRODUCTION: High-grade soft tissue sarcomas (STS) are heterogeneous neoplasms with a poor prognosis and few treatment alternatives. The identification of tumor response markers to treatment, prognosis, and even the development of new drugs, is an incessant search for a better treatment of sarcomas. In this aspect, the Lgr5 receptor has great potential to be a new molecular target, being a marker of stem cells of the intestinal crypts and mammary glands that also acts as a negative modulator of the signaling of the Wnt/ß-catenin pathway, one of the most important in the biology of sarcomas emerges as a promising candidate for preclinical studies, since its importance in tumors of the gastrointestinal tract has already been demonstrated. To that end, patient-derived xenograft (PDX) tumor models represent a valuable platform to identify new biomarkers and new targets, as does Lgr5 to assess therapy response and resistance mechanisms. OBJECTIVE: This study aimed to establish, characterize, and test the Lgr5 protein through in vitro and in vivo assays to unravel the importance of Lgr5 in the biology of PMS and to establish an integrated, convergent and translational framework for the study of this type of tumor. MATERIALS AND METHODS: To determine the expression of Lgr5, two study cohorts were established, a retrospective one from the Institutional Registry of Sarcomas and a prospective one, in which patients operated on at the Institution were invited to generate Patient-derived xenografts (PDX), a pre-model -clinical that has the ability to maintain the molecular characteristics of patients' tumors. For this, fragments implanted in immunosuppressed mice were used to generate these tumor models derived from patients, in addition to in vitro functional studies using SPM strains to analyze the expression profile of the Lgr5 protein through immunofluorescence assays to verify the ability to express Lgr5, flow cytometry to verify the pattern and amount of protein in the analyzed samples and western blotting to obtain a pattern of labeling of the Lgr5 protein. In addition to functional assays to assess the protein's participation in proliferation, whether protein expression interferes with the migratory power of SPM cells and tumors and self-renewal capacity, as well as its association with clinical data and survival data. RESULTS: The Institutional Retrospective Registry has more than 300 patients, while the Prospective Registry has 70 patients who derived the generation of 33 PDX. It was observed that patients with an H-score greater than 20 had a lower overall survival at 5 years compared to the H-score of patients with values below 20. Now in the other analysis performed, the H-score of patients with values greater than 25 it is worse compared to those who had values less than 25 in the disease-free survival data. Furthermore, cells that overexpress the Lgr5 protein have greater migratory capacity (p=0.02) and a tendency to increase proliferation and self-renewal. We performed the implant test of these positive and negative populations of Lgr5, previously separated by cell sorting. For this, Balb/c Nude animals were used. Suggesting that the expression of the transduced protein can be modulated by compensatory mechanisms that need to be explored. CONCLUSION: The construction of the Institutional Registry of PMS is a big step towards a better understanding of the biology of Sarcomas, in addition to the possibility of studying new therapeutic targets for this rare tumor, since studies and scientific articles are still very scarce. The generation of PDX models was also an implemented strategy very well executed with the generation of 33 PDX of several histological subtypes. In addition to the Lgr5 protein inducing cell migration, its expression is related to a worse prognosis, since the higher the Lgr5 expression, the lower the overall survival of the patient

CD57 as a routine neuroendocrine marker for liver metastasis

Background: The characterization of hepatic metastases as having neuroendocrine origins is essential and the main markers currently used are chromogranin A (CgA) and synaptophysin (Syn). However, these markers may exhibit certain limitations, and the use of CD56 and CD57 can also be considered, although, due to low specificity, their use is discouraged. Aim: This study sought to compare the immunohistochemical expression of these markers in hepatic metastases of neuroendocrine neoplasms (NEN). Materials and Methods: Eighteen samples, were used for immunohistochemical staining with CgA, Syn, CD56, and CD57 antibodies. The immunostaining reactions were compared according to its intensity (I), the percentage of labeled cells (P), and a final score (I × P). Statistical agreement between the markers was also evaluated. Results: CD57 was expressed in the highest number of cases and also showed the most intense expression. CgA showed the highest number of cases with more than 80% positively stained area (72.2%), followed by CD57 (61.1%). The highest average score (I × P) was obtained for CD57 (9.1 ± 4.1). The best indices of agreement were between CgA and CD57 with respect to positivity (P = 0.021) and score (P = 0.014). According to the primary site, stomach/duodenum, lungs, and undetermined subgroups showed the highest average scores for CD57, followed by CgA. For the small bowel subgroup, the highest average score was obtained for CgA, followed by CD57. Conclusion: Our results highlight the importance of CD57 in the evaluation of hepatic metastases of NEN and indicate that this marker should be included with CgA and Syn in routine diagnostic panels.

The role of tumor budding in colorectal adenocarcinoma: Possible involvement of the intestinal cancer stem cell marker Lgr5

Background: Tumor budding (TB) is a promising prognostic factor in colorectal cancer (CRC) that is independent of tumor-node-metastasis (TNM) staging. Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) is a stem cell marker and a member of the canonical Wnt-signaling cascade. It is involved in colorectal carcinogenesis. However, its role in CRC progression and TB needs to be clarified. Materials and Methods: TB was assessed in both H and E and CK immunostained sections of 92 CRC cases. Associations between TB grade and different clinicopathological parameters were evaluated. Lgr5 expression in CRC cases and its association with TB grade and other clinicopathological features was also evaluated. Results: H and E stained sections revealed low- and high-grade budding in 55 (59.8%) and 37 (40.2%) tumors, respectively, whereas Cytokeratin Immunohistochemistry (CK-IHC) showed low- and high-grade budding in 31 (33.7%) and 61 (66.3%) tumors, respectively. TB grade (in H and E and CK stained sections) was significantly associated with adverse pathological prognostic variables including vascular invasion (P = 0.03 and 0.001), lymph node metastasis (P = 0.001 and 0,001), advanced Dukes (P = 0.000 and 0.000), and TNM (P = 0.001 and 0.000) stages and inversely associated with Tumor infiltrating lymphocytes (TILS) (P = 0.02 and 0.0001) which is known to be a good prognostic indicator. Lgr5 protein was positively expressed in 52.2% (48/92) of the CRCs. Immunoreactivity of Lgr5 was significantly associated with histological grade (P = 0.01), lymph node metastasis (P = 0.002), vascular invasion (P = 0.02), TNM stage (P = 0.000), Dukes stage (P = 0.000), and TILS (P = 0.03). Furthermore, Lgr5 was found to be significantly associated with TB estimated in both H and E and CK stained tumors (P = 0.003 and 0.001 respectively). Conclusion: This study supported the relevance of TB in the assessment of CRC aggressiveness. It also revealed that Lgr5 expression is related to morphologic features in the invasive front of CRC. Lgr5 could have an important role in forming a morphologic feature at the invasive front associated with the aggressiveness of the tumor.

LGR5 and Downstream Intracellular Signaling Proteins Play Critical Roles in the Cell Proliferation of Neuroblastoma, Meningioma and Pituitary Adenoma

Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) has been reported to play critical roles in the proliferation of various cancer cells. However, the roles of LGR5 in brain tumors and the specific intracellular signaling proteins directly associated with it remain unknown. Expression of LGR5 was first measured in normal brain tissue, meningioma, and pituitary adenoma of humans. To identify the downstream signaling pathways of LGR5, siRNA-mediated knockdown of LGR5 was performed in SH-SY5Y neuroblastoma cells followed by proteomics analysis with 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE). In addition, the expression of LGR5-associated proteins was evaluated in LGR5-inhibited neuroblastoma cells and in human normal brain, meningioma, and pituitary adenoma tissue. Proteomics analysis showed 12 protein spots were significantly different in expression level (more than two-fold change) and subsequently identified by peptide mass fingerprinting. A protein association network was constructed from the 12 identified proteins altered by LGR5 knockdown. Direct and indirect interactions were identified among the 12 proteins. HSP 90-beta was one of the proteins whose expression was altered by LGR5 knockdown. Likewise, we observed decreased expression of proteins in the hnRNP subfamily following LGR5 knockdown. In addition, we have for the first time identified significantly higher hnRNP family expression in meningioma and pituitary adenoma compared to normal brain tissue. Taken together, LGR5 and its downstream signaling play critical roles in neuroblastoma and brain tumors such as meningioma and pituitary adenoma.

Clinical relevance of Lgr5 expression in colorectal cancer patients

PURPOSE: Lgr5 is a well-known stem cell marker in colorectal cancer (CRC). This retrospective study evaluated the expressions of Lgr5 in CRC specimens, and examined whether these expressions were associated with survival outcomes.METHODS: We used immunohistochemistry to retrospectively examine expressions of Lgr5 in paraffin-embedded specimens from 337 patients with CRC between January 2009 and December 2013. All clinicopathologic data were collected by retrospective review based on medical records. The correlation between its expression and clinicopathological data as well as clinical outcomes of patients was analyzed.RESULTS: Low expression and high expression of Lgr5 in 337 patients were 175 (51.9%) and 162 (48.1%), respectively. There was no statistically significant difference in the association of Lgr5 expression with clinicopathologic factors (age, tumor location, lymphatic invasion, vascular invasion, perineural invasion, TNM stage, and differentiation). In the survival analysis, the high expression group of Lgr5 showed a better prognosis than the low expression group in disease-free survival (P=0.044). However, overall survival was not significantly different between the two groups (P=0.087). In multivariate analysis, we found that high expression of Lgr5 was independent prognostic factor for tumor relapse (hazard ratio, 0.601; 95% confidence interval, 0.388–0.929; P=0.022).CONCLUSION: In present study, high expression of Lgr5 is an independent predictor of favorable prognosis in patients with CRC. So, further well designed, prospective, large scale studies are needed to examine the value of Lgr5 as a prognostic biomarker for CRC.

Expression of Vasohibin-1 and LGR5 in oral squamous cell carcinoma and their clinical significances / 临床与实验病理学杂志

Purpose To detect the expression of vasohibin-1 (VASH-1) and LGR5 protein in oral squamous cell carcinoma(OSCC) and their clinical significances. Methods Expression of VASH-1 and LGR5 protein in 223 cases of OSCC tissues and 80 cases of adjacent normal oral mucosa tissues was examined by the immunohistochemicalEliVision method. Results The positive rate of VASH-1 and LGR5 protein in OSCC tissues and the control tissues was 59.6％, 55.6％ and 12.5％, 13.8％, respectively. There was a significant difference between the two groups (P＜ 0.05 ). The expression of VASH-1 and LGR5 protein was significantly related with grades of tumors, size of tumors, lymph node metastasis, and TNM stages (all P＜0.05). The positive expression of VASH-1 and LGR5 was not relevant to age, gender, location of tumor, smoking, and alcohol (all P＞ 0.05). Spearman correlation analysis showed that there was a positive association between the VASH-1 expression and LGR5 expression (rs =0.718, P＜0.001). Conclusion The expression of VASH-1 and LGR5 in OSCC is high and may promote the initiation and development of the cancer, which play an important role in the invasion, metastasis, and prognosis of OSCC.

The tankyrase inhibitor G007-LK inhibits small intestine LGR5+ stem cell proliferation without altering tissue morphology

Abstract Background The WNT pathway regulates intestinal stem cells and is frequently disrupted in intestinal adenomas. The pathway contains several potential biotargets for interference, including the poly-ADP ribosyltransferase enzymes tankyrase1 and 2. LGR5 is a known WNT pathway target gene and marker of intestinal stem cells. The LGR5+ stem cells are located in the crypt base and capable of regenerating all intestinal epithelial cell lineages. Results We treated Lgr5-EGFP-Ires-CreERT2;R26R-Confetti mice with the tankyrase inhibitor G007-LK for up to 3 weeks to assess the effect on duodenal stem cell homeostasis and on the integrity of intestinal epithelium. At the administered doses, G007-LK treatment inhibited WNT signalling in LGR5+ stem cells and reduced the number and distribution of cells traced from duodenal LGR5+ stem cells. However, the gross morphology of the duodenum remained unaltered and G007-LK-treated mice showed no signs of weight loss or any other visible morphological changes. The inhibitory effect on LGR5+ stem cell proliferation was reversible. Conclusion We show that the tankyrase inhibitor G007-LK is well tolerated by the mice, although proliferation of the LGR5+ intestinal stem cells was inhibited. Our observations suggest the presence of a tankyrase inhibitor-resistant cell population in the duodenum, able to rescue tissue integrity in the presence of G007-LK-mediated inhibition of the WNT signalling dependent LGR5+ intestinal epithelial stem cells.

5-LOX inhibitor MK886 suppresses the stemness of human colon cancer HT-29 cells / 医学研究生学报

Objective It remains a controversy whether 5-lipoxygenase (5-LOX) is associated with colon cancer stem cells.This study was to investigate the effect of the 5-LOX inhibitor MK886 in maintaining the stemness of the human colon cancer cell line HT-29.Methods Using CCK-8 assay, we examined the inhibitory effects of different concentrations of MK886 (12.5, 25, 50, 75, 100, and 200 μmol/L) on the colon cancer HT-29 cells cultured in vitro and calculated its half-inhibitory concentration (IC50).Then, we detected the effects of MK886 IC50 on the clone-and sphere-forming abilities of the cells, determined the mRNA expressions of the stemness markers CD133, Lgr5, Oct4 and Ascl2 by real-time PCR after 24 and 48 hours of MK886 IC50 intervention, and measured their protein expressions by Western blotting after 24, 48 and 72 hours of MK886 IC50 intervention.Results The inhibition rates of MK886 on the HT-29 cells at 24 and 48 hours were significantly increased in a time-and dose-dependent manner ([14.99±3.06] and [19.98±0.57]％ at 12.5 μmol/L, [20.46±1.14] and [34.97±6.02]％ at 25 μmol/L, [50.76±5.94] and [66.90±5.74]％ at 50 μmol/L, [66.84±1.77] and [73.11±2.48]％ at 75 μmol/L, [72.67±2.36] and [77.78±3.30]％ at 100 μmol/L, [83.67±0.24] and [84.69±2.24] ％ at 200 μmol/L) as compared with the blank control (0％ and 0％) (P<0.05).The clone-forming rate and number of spheres formed were remarkably lower in the MK886 intervention than in the control group ([10.60±1.71] vs [44.67±3.21]％, P<0.05;6.00±1.60 vs 19.07±2.89, P<0.05).After 24 and 48 hours of MK886 intervention, the mRNA expression of CD133 in the HT-29 cells was markedly up-regulated in comparison with that at 0 hour (0.72±0.10 and 0.39±0.07 vs 1.66±0.33, P<0.05), and so were those of Lgr5, Oct4 and Ascl2 (P<0.05).Conclusion The 5-LOX inhibitor MK886 can inhibit the proliferation and clone-and sphere-forming abilities of human colon cancer HT-29 cells by down-regulating the expressions of the stemness markers and thus suppressing the stemness of the colon cancer stem cells.

Effect of LGR5 knockdown by shRNA on the proliferation and drug resistance of HeLa cells / 西安交通大学学报(医学版)

Objective To investigate the effect of leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5 )on the proliferation and drug resistance of HeLa cells and its possible mechanism.Methods LGR5 expression was interfered using shRNA,and LGR5 knockdown HeLa cells were constructed.The effect of LGR5 on the proliferation and drug resistance of HeLa cell was evaluated by cell count,clone formation and MTT;the expressions of LGR5 and β-catenin in HeLa cells were detected by Western blot method.Results LGR5 knockdown HeLa cell line was successfully constructed;the cell growth rate and clone formation rate in shLGR5 group were markedly decreased compared to those in shCon group (P<0 .01 ).Drug resistance of HeLa to cisplatin differed significantly between shLGR5 group and shCon group (P<0.01 ).Moreover,the LGR5 knockdown inhibited the expression ofβ-catenin in HeLa cells.Conclusion LGR5 plays an important role in cell proliferation and drug resistance of HeLa cells,and its mechanism is related to Wnt/β-catenin signaling pathway.

Expression of Lgr5 in cervical squamous cell carcinoma and its significance / 临床与实验病理学杂志

Purpose To investigate the expression of Lgr5 in cervical squamous cell carcinoma (CSCC) and its significance.Methods Lgr5 protein expression was evaluated by immunohistochemistry of EnVision two-step in 83 paraffin-embedded CSCC specimens,56 normal cervical tissues specimens and 32 cervical intraepithelial neoplasia (CIN) specimens.The expression of Lgr5 mRNA was evaluated by RT-PCR in 13 pairs of surgically removed CSCC and adjacent normal cervical tissues.The correlation between Lgr5 expression and clinicopathological features were statistically analyzed.Results The positive proportion of Lgr5 protein in CSCC tissues was significantly higher than those in adjacent normal cervix (P ＜ 0.01) and CIN (P ＜ 0.05).The expression of Lgr5 in CSCC tissues was associated with grade of tumors (P ＜ 0.01) and depth of invasion (P ＜ 0.05),but not associated with age,tumor size,clinical stage and lymph node metastasis (P ＞ 0.05).Kaplan-Meier analysis showed that the 5-year survival rate of patients with Lgr5 overexpression was significantly lower than that of patients with lower expression (P ＜ 0.01).Conclusion The expression of Lgr5 in CSCC tissues may associated with grade of tumors and depth of invasion and may be involved in initiation and development of CSCC,and it may be a new assistant marker for prognosis of CSCC.

Progress on the cell-surface markers and signaling pathways of colorectal cancer stem cells / 基础医学与临床

Colorectal stem cells have many bio-markers, including Lgr5 which expression is associated with THE stage of disease , also regulating the cell cycle , anothers is +4 stem cell , which is associated with tumor heteroge-neity, also expressed Bmi1, arresting cell cycle.Besides there is Msi1.Many studies show that those markers are highly expressed in colorectal cancer , which activate Notch and Wnt signaling pathway , and can promote the pro-gress of tumor .

Expression of tumor stem cell markers Lgr5 and CD44 in esophageal carcinoma and its clinical significance / 中国生化药物杂志

Objective To analyze the expressions of tumor stem cell markers Lgr5 and CD44 in esophageal carcinoma and its clinical pathological significance.Methods A total of 108 cases of patients with esophageal cancer, 35 cases of stage Ⅱ, 40 cases of stage Ⅲ, 4 cases of stage Ⅳ. After tumor specimens were stained, the expressions of Lgr5 and CD44 in esophageal cancer tissue were detected and its relationship with the clinical features were analyzed.Results There were relations between expressions of Lgr5 and CD44 in esophageal cancer tissue and clinical staging, the later the stageing, the higher expression rate of Lgr5 and CD44, but there was no significant differece with Chi-square test. The Spearman correlation analysis showed there was no correlation between Lgr5 or CD44 and esophageal cancer condition. Conclusion CD44 and Lgr5 are highly expressed in esophageal cancer tissue, but the correlation between CD44 and Lgr5 expression and esophageal cancer condition is uncertain.

Expression of Leucine-rich Repeat-containing G-protein Coupled Receptor 5 and CD44: Potential Implications for Gastric Cancer Stem Cell Marker

BACKGROUND: The human leucine-rich repeat-containing G-protein coupled receptor (LGR) 5 and CD44 are one of the candidates for the marker of gastric cancer stem cells. We compared the expressions of two genes among control, dysplasia and cancer groups. METHODS: We compared the mRNA expression of LGR5, CD44 and CD44v8–10 and immunohistochemistry (IHC) of LGR5 and CD44 in gastric antral mucosa of 45 controls, 36 patients with gastric dysplasia, and 39 patients with early gastric cancer. Additionally, IHC of LGR5 in gastric body mucosa was analyzed. Normal mucosa adjacent to dysplastic or cancer lesions was used for the quantitative real-time–PCR and IHC. RESULTS: Immunoreactivity of LGR5 in base of antral mucosa was higher in non-cancerous tissues of cancer than those of control (P = 0.006), whereas the expression of LGR5 mRNA was not different among the three groups. Immunostaining of LGR5 was much stronger in the antrum than in the body of stomach (P < 0.001). Although there was no difference in antral immunointensity of LGR5 according to the severity of intestinal metaplasia, stronger immunostaining was found in the body with an aggravation of intestinal metaplasia (P trend < 0.001). The expression of CD44v8–10 mRNA was higher in cancer patients than control subjects and patients with dysplasia (P = 0.018 and 0.009) while the expression of CD44 mRNA was higher in the control groups than the others. CONCLUSIONS: IHC of LGR5 in crypt base and CD44 may be used for gastric CSC markers. LGR5 expression may be associated with the developing of corporal intestinal metaplasia. The expression of CD44v8–10 mRNA would be more suitable for gastric cancer stem cell marker than CD44 or LGR5 mRNA.

Effects of Weipixiao (胃痞消) on Wnt pathway-associated proteins in gastric mucosal epithelial cells from rats with gastric precancerous lesions / 中国结合医学杂志

<p><b>OBJECTIVE</b>To study the effects of Weipixiao (胃痞消, WPX) on Wnt pathway-associated proteins in gastric mucosal epithelial cells from rats with gastric precancerous lesions (GPL).</p><p><b>METHODS</b>Sprague Dawley rats were randomly divided into control, model, vitacoenzyme (0.2 g·kg(-1)·day(-1)), WPX high-dose (H-WPX, 15 g·kg(-1)·day(-1)), WPX medium-dose (M-WPX, 7.5 g·kg(-1)·day(-1)) and WPX low-dose (L-WPX, 3.75 g·kg(-1)·day(-1)) groups. After successfully establishing the GPL model, the rats were consecutively administered WPX or vitacoenzyme by gastrogavage for 10 weeks. Differential expression of Leucine-rich repeat-containing G-proteincoupled receptor 5 (Lgr5), matrix metalloproteinase-7 (MMP-7), Wnt1, Wnt3a, and β-catenin in gastric mucosal epithelial cells in all groups were immunohistochemically detected, and the images were taken and analyzed semiquantitatively by image pro plus 6.0 software.</p><p><b>RESULTS</b>Gastric epithelium in the model group showed significantly higher expression levels of Lgr5, MMP-7, Wnt1, Wnt3a and β-catenin than those of the control group(P<0.01). Interestingly, we also observed Lgr5+ cells, which generally located at the base of the gastric glandular unit, migrated to the luminal side of gastric epithelium with GPL. The expression levels of Lgr5, MMP-7, Wnt1, and β-catenin were all down-regulated in the L-WPX group as compared with those of both model and vitacoenzyme groups (P<0.05). A similar, but nonsignificant down-regulation in expression level of Wnt3a was noted in all WPX groups (P>0.05).</p><p><b>CONCLUSION</b>Our findings suggested that the therapeutic mechanisms of WPX in treating GPL might be related with its inhibitory effects on the expressions of Lgr5, MMP-7, Wnt1, β-catenin and the aberrant activation of Wnt/β-catenin pathway.</p>

Expression of Lgr5 in chronic superficial gastritis / 中国病理生理杂志

[ ABSTRACT] AIM: To investigate the expression and significance of Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) in chronic superficial gastritis.METHODS:The Wistar rats (n=30) were randomly divided into blank group, model group and control group.The Wistar rat model of chronic superficial gastritis was established by in-tragastric administration of 0.02%ammonia and long-term irregular diet.All rats were sacrificed, and gastric tissues were harvested and stained with hematoxylin and eosin.The expression of Lgr5 at mRNA and protein levels was analyzed by re-verse transcriptional polymerase chain reaction, Western blotting and immunohistochemistry.RESULTS:Lgr5 was mainly expressed in the cell membrane and cytoplasm.Lgr5 showed high expression in model group compared with blank group and control group.No obvious difference between blank group and control group was observed.CONCLUSION:Persistent in-flammation leads to increased expression of Lgr5.Lgr5 may be a proinflammatory tumor promoting factor.

Expression and its significance of stem cells marker leucine-rich repeat containing G protein coupled receptor 5 gene in human colorectal cancer / 中华消化杂志

Objective To investigate the expression of stem cell marker leucine-rich repeat containing G protein coupled receptor 5 (lgr5) gene in human colorectal cancer tissues and peripheral blood and its correlation with clinical pathological characteristics.Methods The expression of lgr5 at mRNA level was detected by SYBR Green quantitative real-time polymerase chain reaction (PCR) in 27 human colorectal cancer tissues and corresponding non-cancerous tissues as well as in peripheral blood of 17 patients and eight healthy controls.The differences of lgr5 mRNA expression in different tissues and clinical pathology parameters were analyzed by Wilcoxon test.Results The expression of lgr5 at mRNA level in colorectal cancer tissues was 1.000 (0.012,496.353),which was higher than that of corresponding non-cancerous tissues 0.147 (0.004,73.002),the difference was statistically significant (Z=8.029,P＜0.01).The lgr5 expression at mRNA level in peripheral blood of colorectal cancer patients was 0.742 (0.077,456.566),which was higher than that of healthy controls 0.104 (0.034,0.274) and the difference was statistically significant (Z=2.048,P＜0.05).There were no statistically significant differences between lgr5 expression at mRNA level and gender,age,primary location of tumor,tumor size and pathological type (all P＞0.05).However,the expression of lgr5 at mRNA level in group with lymph node metastasis was higher than that in group without lymph node metastasis (Z=2.066,P＜0 05).Conclusion The up-regulation of lgr5 gene expression in colorectal cancer tissues and peripheral blood may be involved in the growth and metastasis of colorectal cancer.

Cancer stem cell markers as potential targets for epithelial cancers.

In recent years, the role of tumor-initiating cells (popularly known as cancer stem cells) in tumor development and availability of novel cancer stem cell/tumor initiating cell markers promises a new arena in understanding their role in developing novel targeted molecules. It is important to identify and understand the relevance of cancer stem cells (CSC)/tumor initiating cells (TIC) in tumor development and to design appropriate strategies for CSCs and TICs elimination, which is crucial to future cancer prevention and treatment. In this review, we attempt to define various potential markers of cancer stem cells and potential exploration as therapeutic targets for epithelial cancer prevention and treatment.

Researches on Lgr5 the newest biological marker of stem cells and the relationship with tumour / 国际肿瘤学杂志

Lgr5 is one of the G protein-coupled receptors families, the newest research displays that it has the potency as the special marker of stem cell, it also has a great relationship with tumor formation and development. Lgr5 may be a marker of tumor stem cells, and can be the new treatment target.