Objective: To investigate the relationship between the expression of integrin α 6 (ITGA6), miR-4484 and the pathologic stage of gastric cancer. Methods: Gastric cancer tissues and normal gastric mucosa tissues adjacent to cancer (>5 cm from tumor margin) of 30 patients with primary gastric cancer who underwent direct surgical resection without adjuvant therapy from June to September 2017 in West China Hospital of Sichuan University were selected. Real-time quantitative polymerase chain reaction (PCR) was used to detect the expression levels of miR-4484 and ITGA6, western blot was used to detect the expression level of ITGA6 protein, dual luciferase reporter gene was used to verify the relationship between ITGA6 and miR-4484. Spearman's correlation analysis was used to determine the relationship between miR-4484 and ITGA6 expression levels in gastric cancer tissues. Results: The expression level of ITGΑ6 in gastric cancer (32.30±13.47) was higher than that in matched normal gastric tissues (24.55±10.25, P=0.015), the area under the receiver operating characteristic (ROC) curve was 0.660 and the diagnostic sensitivity and specificity were 43.3% and 96.7%, respectively. The expression level of miR-4484 in gastric cancer (4.11±2.87) was lower than that of matched normal gastric tissues (5.75±2.80, P=0.029), the area under the ROC curve was 0.690 and the diagnostic sensitivity and specificity were 30.0% and 86.7%, respectively. The expression level of miR-4484 was negatively correlated with ITGA6 in gastric cancer tissues (r=-0.621, P<0.001). The expression level of ITGA6 protein in gastric cancer tissues (0.65±0.19) was higher than that in normal adjacent tissues (0.26±0.12, P<0.001). Compared with ITGA6 3'UTR wild-type+ miR-NC group, ITGA6 3'UTR wild-type+ miRNA mimics group had lower luciferase activity (50.69±5.10, 34.00±1.19, P<0.001), while the luciferase activity of ITGA6 3'UTR wild-type+ ASO miR-4484 group was higher than that of ITGA6 3'UTR wild-type+ miR-NC group (82.44±6.37, 50.69±5.10, P<0.001), indicated that ITGA6 was the direct target gene of miR-4484. The expression levels of miR-4484 in T1, T2, T3 and T4 (4a and 4b) gastric cancer tissues were 9.98±2.24, 5.28±2.03, 2.92±2.04 and 4.11±2.87, respectively, with statistical significance (P<0.001). The expression levels of ITGA6 in N0, N1, N2 and N3 gastric cancer tissues were 29.55±8.32, 21.71±3.75, 24.60±8.79 and 40.69±15.83, respectively, with statistical significance (P=0.022). The expression levels of miR-4484 in N0, N1, N2 and N3 gastric cancer tissues were 5.01±3.52, 5.48±2.76, 5.88±1.83 and 2.30±1.56, respectively, with statistical significance (P=0.032). The expression levels of ITGA6 in M0 and M1 gastric cancer tissues were 26.28±7.66 and 52.08±8.12, respectively, with statistical significance (P<0.001). The expression levels of miR-4484 in M0 and M1 gastric cancer tissues were 4.95±2.74 and 1.34±0.80, respectively, with statistical significance (P<0.001). Conclusions: ITGA6 is upregulated in gastric cancer tissues, while miR-4484 is downregulated in the gastric cancer group, and its expression level is related to the clinicopathological features of gastric cancer. ITGA6 is the direct target gene of miR-4484, implicates that miR-4484 may inhibit the invasion and metastasis of gastric cancer by regulating the expression of ITGA6. Both miR-4484 and ITGA6 may be the new prognostic markers and potential therapeutic targets of gastric cancer.
3' Untranslated Regions ; China ; Humans ; Integrin alpha6/genetics* ; MicroRNAs/genetics* ; Stomach Neoplasms/pathology*

PURPOSE: Metastasis and local recurrence are the primary causes of treatment failure and patient death in breast cancer. The aim of this study was to validate a metastasis- and local recurrenceassociated biomarker for prognostic evaluation and planning treatment strategies. METHODS: Formalin-fixed, paraffin-embedded tissues from a cohort of 312 patients (all stage II and III) were used. The prevalence of CD49f⁺ cells in the patients' tumors was analyzed and correlated with clinical characteristics to determine its prognostic and clinical implications. RESULTS: CD49f⁺ tumor cells were found in a minority of tumors, with 62.8% of the samples showing not a single cell of this subtype. In the clinical characteristics analysis, which were performed with t-tests, CD49f⁺ tumors were not associated with age, tumor size, World Health Organization grade, nodal status, human epidermal growth factor receptor 2 status, progesterone receptor status, or estrogen receptor status, although they were significantly associated with disease recurrence (distant metastasis or/and local recurrence). Univariate survival analysis using the Kaplan-Meier method showed that CD49f⁺ tumors were associated with markedly decreased disease-free survival (DFS); the same result was found using multivariate Cox analysis, even when only chemotherapy-treated patients were analyzed. CONCLUSION: Our results indicated that breast tumors with CD49f⁺ cancer cells are associated with an increased risk for disease recurrence after initial surgery with poor clinical outcomes (decreased DFS). Therefore, as it requires testing for only one additional protein, adding CD49f testing to conventional surgical pathology is a strategy that has great potential for prognostic and treatment-guidance purposes.
Breast Neoplasms* ; Breast* ; Cohort Studies ; Disease-Free Survival ; Estrogens ; Humans ; Integrin alpha6 ; Methods ; Neoplasm Metastasis ; Neoplastic Stem Cells ; Pathology, Surgical ; Prevalence ; Prognosis ; Receptor, Epidermal Growth Factor ; Receptors, Progesterone ; Recurrence* ; Treatment Failure ; World Health Organization

OBJECTIVETo explore the clinicopathological significance and relationship of Tspan 1 and Integrin α6 expression in pancreatic ductal adenocarcinoma (PDAC) tissue and pancreatic cancer cell lines.

METHODSImmunohistochemistry was used to detect the expression of Tspan 1 and Integrin α6 in 95 paraffin-embedded PDAC specimens and 55 adjacent non-cancerous pancreatic tissues which were collected from May 2004 to January 2013.Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detected the protein and mRNA expression in 16 paired fresh PDAC specimens of the pancreas and adjacent non-cancerous pancreatic tissues and 6 different pancreatic cancer cell lines.χ(2) test, Spearman-rank correlation analysis, Kaplan-Meier method and multivariate Cox regression analysis were used to analyze the data.

RESULTSTspan 1 and Integrin α6 were significantly over-expressed in PDAC than in adjacent non-cancerous pancreatic tissues (χ(2) = 7.429, P < 0.05; χ(2) = 15.1, P < 0.01). Lymph node metastasis, TNM stage and post-operation recurrence were positively correlated with the expression of Tspan 1 (χ(2) = 6.688, P < 0.01; χ(2) = 13.055, P < 0.01; χ(2) = 6.116, P < 0.05) . TNM stage was positively correlated with the expression of Integrin α6 (χ(2) = 8.896, P < 0.05) . Tspan 1 was correlated with Integrin α6 (r = 0.223, P < 0.05) . The expressions of Tspan 1 and Integrin α6 were negatively correlated with survival time (χ(2) = 5.263, P < 0.05;χ(2) = 10.124, P < 0.01) . Multivariate analysis revealed that Tspan 1 and Integrin α6 expressions were independent prognostic factors in PDAC patients (χ(2) = 6.152, P < 0.05; χ(2) = 9.479, P < 0.01). Western blot (t = 2.278, P < 0.05; t = 3.153, P < 0.05) and qRT-PCR (t = 2.439, P < 0.05; t = 3.258, P < 0.05) showed that Tspan 1 and Integrin α6 expressions were higher in PDAC tissues than in adjacent non-cancerous pancreatic. Tspan 1 and Integrin α6 were expressed in all six pancreatic cancer cell lines.In SW1990 which derived from metastasis PDAC, Tspan 1 and Integrin α6 expressions were higher than the cell lines from primary tumor.

CONCLUSIONTspan 1 and Integrin α6 expression can up-regulate the invasion and metastasis of PDAC and may be used to predict the prognosis of PDAC.

Adenocarcinoma ; pathology ; Carcinoma, Pancreatic Ductal ; pathology ; Cell Line, Tumor ; Humans ; Immunohistochemistry ; Integrin alpha6 ; metabolism ; Kaplan-Meier Estimate ; Lymphatic Metastasis ; Neoplasm Recurrence, Local ; Neoplasm Staging ; Pancreas ; Pancreatic Neoplasms ; pathology ; Prognosis ; Real-Time Polymerase Chain Reaction ; Tetraspanins ; metabolism

Over-expression of CD151 was found to be associated with metastasis and poor prognosis of prostatic carcinoma. This study was designed to examine the mechanism by which CD151 promotes the proliferation and migration of prostatic cancer cells. The pAAV-CD151, pAAV-GFP and pAAV-CD151-AAA mutant plasmids were constructed and used to transiently transfect PC3 cells (a prostatic carcinoma 3 cell line) by the mediation of Fugene HD. Then, the cells were assigned to control group, pAAV-GFP group, pAAV-CD151 group, and pAAV-CD151-AAA group respectively. Cell proliferation was evaluated by using the 3-[4,5-dimet-hylthiazol-2-yl]-2,5, diphenyltetrazolium bromide (MTT) method. Cell migration assay was performed by using Boyden chambers. The formation of CD151-integrin α3/α6 complex was determined by the method of co-immunoprecipitation. The protein expression levels of CD151 and extracellular signal-regulated kinase (ERK) were measured by Western blotting. The results showed that transfection of pAAV-CD151 or pAAV-CD151-AAA mutant increased the expression of CD151 protein in PC3 cells. Co-immunoprecipitation showed that more CD151-integrin α3/α6 complex was formed in the pAAV-CD151 group than in the control group, the pAAV-GFP group and the pAAV-CD151-AAA mutant group. Furthermore, the proliferative and migrating capacity of PC3 cells was substantially increased in the pAAV-CD151 group but inhibited in the pAAV-CD151-AAA mutant group. CD151 transfection increased the expression of phospho-ERK. Taken together, it was concluded that CD151 promotes the proliferation and migration of PC3 cells through the formation of CD151-integrin complex and the activation of phosphorylated ERK.
Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Humans ; Integrin alpha3 ; metabolism ; Integrin alpha6 ; metabolism ; Male ; Prostatic Neoplasms ; metabolism ; pathology ; Tetraspanin 24 ; metabolism

Breast cancer stem cells are a group of undifferentiated cells with self-renewal and multidifferentiation potential. Chemotherapeutic and radiotherapeutic resistance, hypoxic resistance, high tumorigenicity, high cell invasion, and metastatic abilities are characteristics of these cells, which are responsible for breast cancer recurrence. Therefore, the correct sorting and identification of breast cancer stem cells is a primary step for research in this field. This article briefly describes the recent progress on sorting and identification technologies for breast cancer stem cells. Sorting technologies include the side population technique, technologies that depend on cell surface markers, ALDEFLUOR assays, and in situ detection. Identification technologies include mammosphere cultures, limited dilution in vitro, and in-vivo animal models. This review provides an important reference for breast cancer stem cell research, which will explore new methods for the treatment of patients with breast cancer.
AC133 Antigen ; ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; metabolism ; Aldehyde Dehydrogenase ; metabolism ; Animals ; Antigens, CD ; metabolism ; Breast Neoplasms ; pathology ; Female ; Flow Cytometry ; methods ; Glycoproteins ; metabolism ; Humans ; Hyaluronan Receptors ; metabolism ; Integrin alpha6 ; metabolism ; Integrin beta1 ; metabolism ; Integrin beta3 ; metabolism ; Isoenzymes ; metabolism ; Membrane Proteins ; metabolism ; Neoplasm Proteins ; metabolism ; Neoplastic Stem Cells ; metabolism ; pathology ; Peptides ; metabolism ; Retinal Dehydrogenase ; metabolism ; Side-Population Cells ; cytology ; metabolism

The adaptor protein, LAD/TSAd, plays essential roles in T cell activation. To further understand the functions of this protein, we performed yeast two-hybrid screening using TSAd as bait and identified 67 kDa laminin binding protein (LBP) as the interacting partner. Subsequently, TSAd-LBP interaction was confirmed in D1.1 T cell line. Upon costimulation by T cell receptor (TCR) plus laminin crosslinking or TCR plus integrin alpha6 crosslinking, LBP was coimmunoprecipitated with TSAd. Moreover, TCR plus laminin costimulation-dependent T cell migration was enhanced in D1.1 T cells overexpressing TSAd but was disrupted in D1.1 cells overexpressing dominant negative form of TSAd or TSAd shRNA. These data show that, upon TCR plus integrin costimulation, TSAd associates with LBP and mediates T lymphocyte migration.
Adaptor Proteins, Signal Transducing/genetics/*metabolism ; Amino Acids, Diamino/metabolism ; Carrier Proteins/*metabolism ; *Cell Movement ; Humans ; Integrin alpha6/metabolism ; Jurkat Cells ; Mutation ; Protein Binding ; RNA, Small Interfering/genetics ; *Receptor Cross-Talk ; Receptors, Antigen, T-Cell/metabolism ; T-Lymphocytes/*metabolism ; Transgenes ; Two-Hybrid System Techniques

OBJECTIVETo explore the specific surface markers for the isolation and purification of human spermatogonial stem cells (SSC).

METHODSSpecific markers of human SSC were screened and identified in fetal and adult testes by immunohistochemical assay, using HSC markers c-kit, Thy-1 and human ES integrins.

RESULTSIn human adult testes, the alpha6 integrin extensively and significantly expressed on the surface of most of the germ cells in the seminiferous tubule, and beta1 integrin mainly expressed on the surface of the germ cells residing on or near the basal membrane in the seminiferous tubule. Thy-1 scattering expressed on the surface of some cells of the basal membrane, and on some Leydig cells as well. The three antigen markers expressed on the SSC of human adult testes specifically to some extent. SSEA-1 specifically expressed on the surface of the gonocytes in the fetal testes.

CONCLUSIONThe alpha6 and beta1 integrins and Thy-1 may be used for the SSC isolation as positive markers. SSEA-1 can be used as an identification marker for the fetus SSC.

Adult ; Biomarkers ; analysis ; Cell Differentiation ; Fetus ; cytology ; Humans ; Immunohistochemistry ; Integrin alpha6 ; analysis ; Integrin beta1 ; analysis ; Lewis X Antigen ; analysis ; Male ; Spermatogonia ; cytology ; Stem Cells ; cytology ; Testis ; cytology ; Thy-1 Antigens ; analysis

In postmenopausal osteoporosis, estrogen deficiency leads to unbalance of bone metabolism, decreased bone formation and increased bone resorption, and the result is reduced bone mineral density (BMD) and bone stiffness. The processes of bone formation and resorption involves the expression of integrins in anchoragedependent cells, such as osteoblast and osteoclast. The osteoporosis-induced rats frequently demonstrated the loss of trabecular bone volume in the tibia, vertebra and mandible due to estrogen depletion. However, in maxilla, study has been rare because of its anatomical limits. So the objective of this study was to investigate bony change and property of integrin expression in maxilla of osteoporosis-induced rats. 12-week-old female Sprague-Dawley rats were bilaterally ovariectomized (OVX). At 1, 2, 3, 4, 8 and 12 weeks, control and OVX group rats were sacrificed respectively. BMD of maxilla of the rats was measured using dual-energy X-ray absorptiometry (DEXA). And then the histopathologic observation, histomorphometric analysis and immunohistochemistry with CD44, alpha2 integrin, alpha5 integrin, alpha6 integrin, alphav integrin and beta3 integrin were done. BMD of alveolar bone in maxilla was decreased with significance statistically after OVX 4 weeks and was decreased 18.15% at OVX 12 weeks group compared to control group. From OVX 4 to 12 weeks, the thickness of periodontal ligament space was decreased, the number of osteoclast and the size of marrow stroma were increased than control group. By histomorphometric analysis, the size of marrow stroma of alveolar bone in maxilla was increased 86.42% at OVX 12 weeks group compared to control group. CD44 was widely expressed throughout the odontoblast, cementoblast, dental pulp, preiodontal ligament, osteocyte, osteoclast and perivascular tissue at control group, and CD44 immunoreactivity was increased the odontoblast, osteoblast and osteoclast at OVX groups. alpha2 integrin was expressed the odontoblast and osteoblast at control group, but alpha2 integrin immunoreactivity was decreased the osteoblast at OVX 12 weeks group. alpha5 integrin was expressed the cementoblast, osteoblast and osteoclast at control group, and alpha5 integrin immunoreactivity was decreased the osteoblast and was increased the osteoclast from OVX 4 weeks group. alpha6 integrin was weakly expressed the odontoblast, cementoblast, osteoblast and osteoclast at control group, and alpha6 integrin immunoreactivity was weakly increased the osteoclast from OVX 4 weeks. alphav integrin was expressed the odontoblast and osteoclast at control group, and alphav integrin immunoreactivity was strongly increased the osteoclast from OVX 4 weeks. beta3 integrin was expressed the osteocyte and osteoclast at control group, and beta3 integrin immunoreactivity was strongly increased the osteoclast from OVX 4 weeks. From these results, alveolar bone in maxilla of OVX rats was decreased BMD gradually. Moreover, alpha2 and alpha5 integrin expression of osteoblast was decreased, and alpha5, alphav and beta3 integrin expression of osteoclast was increased in OVX rats. Thus, this study indicates that consideration of reduced BMD is necessary in dental procedure of postmenopausal women.
Absorptiometry, Photon ; Animals ; Bone Density* ; Bone Marrow ; Bone Resorption ; Dental Cementum ; Dental Pulp ; Estrogens ; Female ; Humans ; Immunohistochemistry ; Integrin alpha2 ; Integrin alpha5 ; Integrin alpha6 ; Integrin alphaV ; Integrin beta3 ; Integrins ; Ligaments ; Mandible ; Maxilla* ; Metabolism ; Odontoblasts ; Osteoblasts ; Osteoclasts ; Osteocytes ; Osteogenesis ; Osteoporosis, Postmenopausal ; Ovariectomy* ; Periodontal Ligament ; Rats* ; Rats, Sprague-Dawley ; Spine ; Tibia

OBJECTIVEThe very nature of spermatogonial stem cells (SSC) is still poorly understood. The objective of this study is to explore the specific markers of human SSC and search for the suitable method for their isolation and functional identification.

METHODSAdults testicular cell suspensions were sorted by immunomagnetic beads method using alpha6,beta1 integrin and Thy-1 markers. The light-scattering properties and DNA Ploidy of the resultant subpopulations were analyzed by flow cytometry. The efficacies of the sorting on human SSC were evaluated by germ cell transplantation.

RESULTS(1) The alpha6+ Thy-1+ c-kit- and beta1+ Thy-1+ c-kit- cells were relatively uniform subpopulations in size and morphology, which represented about 2%-3% and 0.5%-1% of the unsorted testis cells, respectively. The analysis of light-scattering properties showed that both of the subpopulations had low side light-scattering properties. The DNA Ploidy analysis showed significant changes of these two cell subpopulations in DNA Ploidy. The percentage of diploid cells in alpha6+ Thy-1+ c-kit- cell subpopulation significantly increased to 51.2% and synthesis phase and tetraploid cells disappeared. (2) The functional evaluation showed that the SSC in the alpha6+ Thy-1+ c-kit- cells were enriched 40 times and the SSC in the beta1+ Thy-1+ c-kit- cells 20 times that of the unsorted cells.

CONCLUSIONThe alpha6,beta1 integrin and Thy-1 may be used for the SSC isolation as positive markers. The immunomagnetic beads sorting using alpha6,beta1 integrin and Thy-1 markers can result in significant enrichment of human SSC. It will open up a wide prospect for the researches on the biology of human SSC and the treatment of male sterility.

Adult ; Animals ; Cells, Cultured ; Flow Cytometry ; Humans ; Immunomagnetic Separation ; methods ; Integrin alpha6 ; analysis ; Integrin beta1 ; analysis ; Male ; Mice ; Spermatogonia ; cytology ; Stem Cell Transplantation ; Stem Cells ; cytology ; Testis ; cytology ; Thy-1 Antigens ; analysis

OBJECTIVETo study the role of integrin alpha 6 in cell attachment, spread, survival/proliferation and differentiation of human hepatocellular carcinoma (HCC) BEL-7402 cells on various substrates by monoclonal antibody against the extracellular domain of alpha 6 subunit (IA6ED McAb).

METHODSThe effect of McAb on attachment and spread of BEL-7402 cells on LN or FN substrate was examined. MTT analysis was used to examine the cell survival/proliferation, gelatin zymography to the matrix metalloproteinases (MMPs) secreted by BEL-7402 cells, and microparticle immunoabsorbent kit to AFP secretion while the cells were cultured on LN-, FN- or Matrigel-coated substrates.

RESULTSThe cell attachment, spread and survival/proliferation were inhibited. Moreover importantly, the malignant cell dedifferentiation and abnormal differentiation on LN-coated substrate were also strongly inhibited by IA6ED McAb.

CONCLUSIONLN and integrin alpha 6 regulate human HCC cell phenotypes of survival/proliferation and differentiation. The phenotypes of dedifferentiation and abnormal differentiation can be reversed by blocking the interaction between integrin alpha 6 and LN using IA6ED McAb, which may lower metastatic potency of tumor cells.

Antibodies, Monoclonal ; pharmacology ; Carcinoma, Hepatocellular ; pathology ; Cell Adhesion ; drug effects ; Humans ; Integrin alpha6 ; immunology ; physiology ; Laminin ; physiology ; Liver Neoplasms ; pathology ; Phenotype ; Receptors, Laminin ; physiology ; Tumor Cells, Cultured