Regulatory effects of lncRNA ATB targeting miR-200c on proliferation and apoptosis of colorectal cancer cells

This investigation was intended to elucidate whether long noncoding RNA (lncRNA)-activated by transforming growth factor-β (ATB) interacting with miR-200c could mediate colorectal cancer (CRC) progression, offering potential strategies for diagnosing and treating CRC. Here totally 315 patients with CRC were recruited, and their CRC tissues and adjacent normal tissues were gathered. Concurrently, four colon cancer cell lines (ie, SW620, Lovo, HCT116, and SW480) and the human colon mucosal epithelial cell line (NCM460) were also purchased. Moreover, si-ATB, si-NC, miR-200c mimic, miR-200c inhibitor, and miR-NC were prepared for transfection into the CRC cells, and their effects on CRC cell lines were evaluated based on the conduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony formation assay, and flow cytometry assay. Eventually, the Luciferase reporter gene assay was carried out to judge if there existed a targeted relationship between ATB and miR-200c. The results of Cox regression analyses suggested that overexpressed lncRNA ATB, underexpressed miR-200c, poor tumor differentiation, lymph-vascular invasion, and perineural invasion were symbolic of shortened survival of the patients with CRC (all P < .05). Besides, transfection of pcDNA3.1-ATB and miR-200c inhibitor could boost the viability and proliferation of Lovo and SW620 cell lines (all P < .05). Meanwhile, the expressions of p53 and p21 were also reduced under treatments of pcDNA3.1-ATB and miR-200c inhibitor (P < .05). In addition, CDK2 seemed to reverse the contribution of miR-200c to intensifying viability and proliferation of Lovo and SW420 cell lines (P < .05). Furthermore, ATB might downregulate miR-200c expression by targeting it (P < .05), and CDK2 was subjected to dual regulation of both ATB and miR-200c (P < .05). In conclusion, the lncRNA ATB/miR-200c/CDK2 signaling was responsible for intensified proliferation and prohibited apoptosis of CRC cells, which might provide effective approaches for diagnosing and treating CRC.     

Inhibitory effects of anti-CXCR4 antibodies on human colon cancer cells

Background: CXCR4, the chemokine receptor for CXCL12, has recently been involved in the metastatic process of several neoplasms. Materials and methods: The expression of CXCR4 was evaluated by immunohistochemistry of colorectal tissue samples and by flow cytometry on Caco2, GEO, SW480, SW48, Lovo and SW620 human colon carcinoma cell lines. Correlations with pathological characteristics of the specimens were analysed with chi-square test. To verify the functional status of CXCR4, cell lines were tested in adhesion, migration, and proliferation assays. Results: We studied the expression of CXCR4 in 88 human colorectal tissues and we found that CXCR4 was expressed in >10% of epithelial cells in 50% of normal mucosae (7/14), in 55% of polyps (29/53), in all of carcinomas (16/16) and hepatic metastasis (5/5). Notably, CXCR4 was significantly over-expressed in cancerous lesions (carcinomas and metastasis) compared to non-cancerous lesions (normal mucosa and polyps) (P=0.003) and in adenomatous polyps versus hyperplastic polyps (P=0.009). The diameter of a polyp was also significantly associated with CXCR4 expression (P=0.031). SW480, SW48 and SW620 cell lines showed the highest levels of CXCR4 (60–80% of positive cells). Adhesion, migration, and proliferation increased in response to the CXCL12 chemokine. These effects were abrogated by the addition of anti-CXCR4 antibodies. Further, CXCL12 activated ERK1/2 in SW480 cells. Conclusions: These data suggest that CXCR4 might play a role in colon cancer cell properties and that anti-CXCR4 antibodies could have therapeutic effects against colorectal cancer.Partially presented at the 94th Annual Meeting of the American Association for Cancer Research, Washington, DC, 11–14 July 2003 (abstract LB-132).     

Serum miR‐92a‐1 is a novel diagnostic biomarker for colorectal cancer

Colorectal cancer (CRC) is one of the most common cancers worldwide, with high mortality. Abnormally expressed microRNAs (miRNAs) are considered novel biomarkers in cancer diagnosis. The aim of this study was to investigate the diagnostic value of miR‐92a‐1 in patients with CRC. Serum samples were collected from 148 patients pathologically diagnosed with CRC and 68 gender‐ and age‐matched healthy volunteers. Quantitative real‐time polymerase chain reaction (qRT‐PCR) was used to measure serum miR‐92a‐1 level. Relationship between miR‐92a‐1 and clinicopathological features of CRC cases was analysed via chi‐square test. Receiver operating characteristic (ROC) curve was plotted to estimate the diagnostic value of miR‐92a‐1 in CRC. Serum miR‐92a‐1 was significantly up‐regulated in CRC patients compared with healthy individuals (P < .001). Moreover, miR‐92a‐1 expression was correlated with TNM stage (P = .02), histological stage (P = .003), lymph node metastasis (P = .003) and distant metastasis (P < .001). ROC analysis showed that the area under the ROC curve (AUC) was 0.914, suggesting high diagnostic accuracy of miR‐92a‐1 in ROC. The optimal cut‐off value was 1.485, with a sensitivity of 81.8% and a specificity of 95.6%. MiR‐92a‐1 is increased in CRC patients and correlated with aggressive clinical characteristics. Serum miR‐92a‐1 may be a potential diagnostic biomarker for CRC.     

Characterization of the expression profile of <Emphasis Type="Italic">calpain</Emphasis>-<Emphasis Type="Italic">3</Emphasis> (<Emphasis Type="Italic">CAPN3</Emphasis>) gene in chicken

Calpain-3 is a skeletal muscle-specific protease and participates in the regulation of myogenesis. In this study, we quantified the expression of calpain-3 (CAPN3) mRNA in a Chinese local chicken breed (Sichuan Mountainous Black-boned chicken [MB]), to discern the tissue and ontogenic expression pattern. Meanwhile, we compared the CAPN3 mRNA expression pattern in MB chicken at 10 weeks with a commercial meat type chicken line (S01) of the same age to identify the unique expression pattern under different genetic background. A real time quantitative PCR (qRT-PCR) assay was developed for an accurate measurement of its expression in various tissues from chickens at different ages (0, 2, 4, 6, 8, 10, and 12 weeks). Expression of the CAPN3 mRNA was detected in the selected tissues, regardless of age. The breast muscle and leg muscle tissues had a significantly higher expression than the other tissues from the same individual (P < 0.01). Overall, the CAPN3 mRNA level exhibited a “rise-decline” developmental change in detected tissues except for brain. The S01 chicken had a higher expression of the CAPN3 mRNA in detected tissues than the MB chicken at 10 weeks. The present expression data of chicken CAPN3 gene may provide some information to shed light on the tissue and ontogenic expression pattern during chicken development.     

miR‐148a inhibits early relapsed colorectal cancers and the secretion of VEGF by indirectly targeting HIF‐1α under non‐hypoxia/hypoxia conditions

Vascular endothelial growth factor (VEGF) is correlated with angiogenesis and early relapse of colorectal cancer (CRC). This study investigated the role of miR‐148a in the regulation of VEGF/angiogenesis and early relapse of CRC. We established a stable clone with miR‐148a expression in HCT116 and HT29 cell lines and created a hypoxic condition by using CoCl₂ to determine the underlying mechanism of miR‐148a. The effects of miR‐148a on the phosphoryl‐ERK (pERK)/hypoxia‐inducible factor‐1α (HIF‐1α)/VEGF pathway were evaluated through Western blotting and the inhibitory effect of miR‐148a on angiogenesis was demonstrated through a tube formation assay. Sixty‐three CRC tissues (28 early relapse and 35 non‐early relapse) were analysed to assess the relationship between miR‐148a and HIF‐1α/VEGF. The protein expression of pERK/HIF‐1α/VEGF in HCT116 and HT29 cells was significantly decreased by miR‐148a (all P < 0.05). The protein expression of VEGF/HIF‐1α was strongly inversely associated with the expression of miR‐148a in the 63 CRC tissue samples (all P < 0.05). Tube formation assay demonstrated that miR‐148a significantly obliterated angiogenesis. miR‐148a suppresses VEGF through down‐regulation of the pERK/HIF‐1α/VEGF pathway and might lead to the inhibition of angiogenesis; miR‐148a down‐regulation increased the early relapse rate of CRC. This demonstrates that miR‐148a is a potential diagnostic and therapeutic target.     

Up‐regulation of LIMK1 expression in prostate cancer is correlated with poor pathological features,lymph node metastases and biochemical recurrence

This study aimed to explore the association between LIM domain kinase 1 (LIMK1) expression in prostate cancer (PCa) tissues with advanced pathological features, lymph node metastases and biochemical recurrence. A total of 279 PCa specimens from patients who underwent radical prostatectomy and 50 benign prostatic hyperplasia (BPH) specimens were collected to construct tissue microarray, which were subjected to immunohistochemical staining for LIMK1 expression subsequently. Logistic and Cox regression analysis were used to evaluate the relationship between LIMK1 expression and clinicopathological features of patients with PCa. Immunohistochemical staining assay demonstrated that LIMK1 expression was significantly higher in PCa than BPH specimens (77.1% vs 26.0%; P < .001). LIMK1 expression was significantly higher in positive lymph node specimens than corresponding PCa specimens (P = .002; P < .001). Up‐regulation of LIMK1 was associated with prostate volume, prostate‐specific antigen, prostate‐specific antigen density, Gleason score, T stage, lymph node metastases, extracapsular extension and seminal vesicle invasion, and positive surgical margin. Multivariate logistic regression analysis demonstrated that LIMK1 was an independent risk factor for PCa lymph node metastasis (P < .05). Multivariate Cox regression analysis revealed that the up‐regulation of LIMK1 was an independent risk factor for biochemical recurrence. Kaplan‐Meier analysis indicated that up‐regulation LIMK1 was associated with shortened biochemical‐free survival (BFS) after radical prostatectomy (P < .001). In conclusion, LIMK1 was significantly up‐regulated in PCa and positive lymph node specimens and correlated with lymph node metastasis and shortened BFS of PCa. The underlying molecular mechanism of LIMK1 in PCa should be further evaluated.     

Expression of Duffy antigen receptor for chemokines (DARC) is down-regulated in colorectal cancer

Abstract

Duffy antigen receptor for chemokines (DARC) is a silent chemokine receptor which selectively binds angiogenic chemokines without inducing conventional signaling responses. DARC has been reported to inhibit the development of multiple cancers through clearance of angiogenic chemokines. However, its role in colorectal cancer (CRC) remains unclear. We investigated the expression of DARC in CRC and explored correlation of DARC expression with clinical pathological features and microvessel density (MVD). The protein expression levels of DARC were detected by immunohistochemistry in 90 CRC and 64 paired unaffected tissues. The mRNA levels of DARC were detected by quantitative real-time PCR in 15 CRC and paired unaffected tissues. MVD in CRC was also assessed by immunohistochemistry of CD34. We found that the mRNA and protein expression levels of DARC were significantly lower in CRC than in the unaffected tissues (p?<?0.05). The DARC protein expression levels were positively correlated with DARC mRNA expression levels in both CRC (p?<?0.001) and unaffected tissues (p?<?0.001). We also found that DARC expression was significantly correlated with tumor differentiation (p?<?0.001), lymph node metastasis (p?<?0.01) and TNM stage (p?<?0.05). Moreover, we observed a strong negative relationship between DARC expression and MVD in CRC (p?<?0.001). We showed that DARC expression is down-regulated in CRC and associated with clinical pathological features and MVD of CRC. DARC might be involved in tumorigenesis, progression, angiogenesis, and metastasis of CRC.     

CD133<Superscript>+</Superscript>CD44<Superscript>+</Superscript> subgroups may be human small intestinal stem cells

The identification and separation of small intestinal epithelial stem cells are still on the preliminary stage. In this study, we planned to utilize immunohistochemistry, fluorescence-activated cell sorting (FACS) and RT-PCR to investigate the possibility of CD133 and CD44 as markers of human small intestinal epithelial stem cells. The expressions of CD133, CD44 and Lgr5 were studied by immunohistochemistry. Four subgroups of CD133⁺CD44⁺, CD133⁺CD44⁻, CD133⁻CD44⁺, CD133⁻CD44⁻ were sorted out through FACS and the expression level of Lgr5 gene was measured by RT-PCR and polyacrylamide gel electropheresis (PAGE) with sliver stained. Ten cases of samples were available for analyzing. By immunohistochemical staining, few cells with positive expressions of CD133, CD44 and Lgr5 were distributed in the bottom of crypts with the expression locations somewhat overlapped. The average percentage of CD133⁺CD44⁺ cells was 0.0580 ± 0.0403%, while the corresponding contents of CD133⁺CD44⁻ cells, CD133⁻CD44⁺ cells and CD133⁻CD44⁻ cells were 0.4000 ± 0.1225%, 0.7000 ± 0.2646% and 76.5600 ± 3.5529% respectively. Ten times of positive expressions of Lgr5 were detected in the CD133⁺CD44⁺ groups, while 9/10, 8/10 and 4/10 times for CD133⁺CD44⁻, CD133⁻CD44⁺ and CD133⁻CD44⁻ subgroups respectively. With the help of Quantityone 4.62 software, the densities of corresponding place to Lgr5 and reference gene were obtained. The density ratios of corresponding place to Lgr5 to reference gene were significant difference between subgroups (P < 0.001). By means of LSD method, the density ratios in CD133⁺CD44⁺ subgroups had statistical differences from the other subgroups (P < 0.05). We concluded CD133⁺CD44⁺ cells may be human small intestinal epithelial stem cells, which need further researches to confirm.     

Molecular characterization and expression patterns of <Emphasis Type="Italic">Lbx1</Emphasis> in porcine skeletal muscle

Ladybird-like genes were recently identified in mammals. The first member characterized, Lbx1, is expressed in developing skeletal muscle and the nervous system. However, little is known about the porcine Lbx1 gene. In the present study, we cloned and characterized Lbx1 from porcine muscle. RT-PCR analyses showed that Lbx1 was highly expressed in porcine skeletal muscle tissues. And we provide the first evidence that Lbx1 has a certain regulated expression pattern during the postnatal period of the porcine skeletal muscle development. Lbx1 gene expressed at higher levels in biceps femoris muscles compared with masseter, semitendinosus and longissimus dorsi muscles in Meishan pigs. Phylogenetic tree was constructed by aligning the amino acid sequences of different species. Moreover, single nucleotide polymorphism (SNP) scanning in the Lbx1 genomic fragment identified two mutations, g.752A>G and g.−1559C>G. Association analysis in our experimental pig populations showed that the mutation of g.752A>G was significantly associated with loin muscle area (P < 0.05) and internal fat rate (P < 0.05). Our results suggest that the Lbx1 gene might be a candidate gene of carcass traits and provide useful information for further studies on its roles in porcine skeletal muscle.     

Calcium ameliorates obesity induced by high-fat diet and its potential correlation with p38 MAPK pathway

To investigate whether and on which pathway dietary calcium influence the obesity induced by high-fat diet, thirty male Kunming mice were fed in six groups for 4 weeks and mouse preadipocytes were divided into eight groups for different treatment. Body weight gain was measured each week. Calcium in serum and tissues, intracellular free Ca²⁺ concentration ([Ca²⁺]i), blood fat and intracellular lipid content were also measured. The expression of Lipid metabolism-related genes were measured by q RT-PCR. Compared with control group, body weight gain (P < 0.05) and fat pad weight (P < 0.01) in Low calcium group decreased. Triglycerides (TG) and total Cholesterol (TC) level decreased (P < 0.01), while HDL-Cholesterol (HDL) level increased (P < 0.01). And calcium supply increased calcium content in blood serum and tissues. In tissues, adipogenesis and vitamin D receptor (VDR) genes expression decreased but lipoclasis genes expression increased. These anti-obesity effects were more obvious when supplying with 2.8% calcium, but the effects were reduced while supplying Nifedipine at the same time. The results in preadipocytes indicated that calcium-treated can reduce intracellular lipid content, along with adipogenesis and lipoclasis genes expression decrease, promoted the expression levels of p38 MAPK pathway upstream gene MKK6 (P < 0.01) and downstream gene MAPKAPK2 (P < 0.01). Treated with SB203580 could increase adipogenesis genes expression, decrease lipoclasis genes expression and ([Ca²⁺]i) (P < 0.01). These results implied that dietary calcium had remarkable effect on anti-obesity effect and p38 MAPK pathway potentially participated in calcium-mediated lipid accumulation and lipolysis in mouse preadipocytes.     

Epigenetic silencing of DACH1 induces the invasion and metastasis of gastric cancer by activating TGF‐β signalling

Gastric cancer (GC) is the fourth most common malignancy in males and the fifth most common malignancy in females worldwide. DACH1 is frequently methylated in hepatic and colorectal cancer. To further understand the regulation and mechanism of DACH1 in GC, eight GC cell lines, eight cases of normal gastric mucosa, 98 cases of primary GC and 50 cases of adjacent non‐tumour tissues were examined. Methylation‐specific PCR, western blot, transwell assay and xenograft mice were used in this study. Loss of DACH1 expression correlated with promoter region methylation in GC cells, and re‐expression was induced by 5‐Aza‐2′‐deoxyazacytidine. DACH1 is methylated in 63.3% (62/98) of primary GC and 38% (19/50) of adjacent non‐tumour tissues, while no methylation was found in normal gastric mucosa. Methylation of DACH1 correlated with reduced expression of DACH1 (P < 0.01), late tumour stage (stage III/IV) (P < 0.01) and lymph node metastasis (P < 0.05). DACH1 expression inhibited epithelial–mesenchymal transition and metastasis by inhibiting transforming growth factor (TGF)‐β signalling and suppressed GC cell proliferation through inducing G2/M phase arrest. The tumour size is smaller in DACH1‐expressed BGC823 cell xenograft mice than in unexpressed group (P < 0.01). Restoration of DACH1 expression also sensitized GC cells to docetaxel. These studies suggest that DACH1 is frequently methylated in human GC and expression of DACH1 was controlled by promoter region methylation. DACH1 suppresses GC proliferation, invasion and metastasis by inhibiting TGF‐β signalling pathways both in vitro and in vivo. Epigenetic silencing DACH1 may induce GC cells' resistance to docetaxel.     

Expression of macrophage-derived chemokine (MDC)/CCL22 in human lung cancer

Background: Ligands for CXCR3 chemokines [IFN-γ-inducible protein of 10 kD (IP-10/CXCL10), monokine induced by IFN-γ (Mig/CXCL9), IFN-inducible T cell α chemoattractant (I-TAC/CXCL11)] and those for CCR4 [macrophage-derived chemokine (MDC/CCL22), thymus- and activation-regulated chemokine (TARC/CCL17)] have been shown to play the central roles for T helper-cell recruitment into the tissues. To examine the role of these chemokines in tumor progression of lung cancer, we investigated their expression in human lung cancer tissues to determine the possible relationship between their expression and the prognosis of patients. Methods: Total RNA was prepared from lung cancer tissues of 40 patients (24 adenocarcinoma and 16 squamous cell carcinoma). We measured gene expression levels of chemokines (IP-10, Mig, I-TAC, MDC and TARC) by real-time quantitative RT-PCR. Results: Higher gene expression of MDC in lung cancer was significantly correlated with longer disease-free survival time and lower risk of recurrence after tumor resection. We could not find any significant relationship of IP-10, Mig, I-TAC and TARC gene expression with disease-free survival or lower risk of recurrence after surgery. Conclusions: These results suggest that increased gene expression of MDC in tumor tissues may be a predictive marker for improving the prognosis of lung cancer.Toru Nakanishi and Kazuyoshi Imaizumi equally contributed to this work.     

Basal expression studies of cystatins during specific growth stages of wheat spikes for defining their possible role in differential and stage dependent immunity against Karnal bunt (<Emphasis Type="Italic">Tilletia indica</Emphasis>)

Two genotypes showing differential immunity against Karnal bunt (Tilletia indica) were used to investigate the role of three members of cystatin gene family in growth stage dependent immunity in wheat (Triticum aestivum L.). Three members of cystatin gene family (WC1, WC2, and WC4) were cloned and sequenced. Analysis of sequenced data showed that there was 76–99% nucleotide and protein sequence identity between different genes of the wheat cystatin. In silico amino acid sequence analysis revealed the presence of a conserved signature pattern of residues and also the functional domains were presumed to be actively involved in imparting cysteine protease inhibition capability. The semi-quantitative and quantitative levels of these members were measured by means of RT-PCR, northern blotting, western blotting, and by ELISA techniques. The members of cystatin gene family were expressed in both resistant (HD 29) and susceptible genotypes (WH 542); however, the expression level was significantly (P < 0.001) higher in resistant compared to susceptible genotype at all the stages of wheat spikes. The patterns of expression of WC2, WC4 were similar except in the levels in S₁ and S₂ stages as it remained constant (P > 0.05) in contrary to WC1 family whose expression gradually increased from S_v to S₂ stage. According to the intensity of the detected band in RT PCR, northern blot and western blot, WC1 family seems to be expressed more than the other gene families. The immunoassay results further showed that WC1 protein was abundantly expressed in resistant genotype and high expression was observed at the S2 stage as compared to susceptible genotype (P < 0.001) suggesting that low level of expression of WC1 in S2 stage is responsible for KB infection. The results of the present study clearly indicate the role of cystatin gene family in differential and stage dependent immunity against KB.     

Identification of three novel SNPs and association with carcass traits in porcine <Emphasis Type="Italic">TNNI1</Emphasis> and <Emphasis Type="Italic">TNNI2</Emphasis>

In this study, two novel SNPs (EU743939:g.5174T>C in intron 4 and EU743939:g.8350C>A in intron 7) in TNNI1 and one SNP (EU696779:g.1167C>T in intron 3) in TNNI2 were identified by PCR–RFLP (PCR restriction fragment length polymorphism) using XbaI, MspI and SmaI restriction enzyme, respectively. The allele frequencies of three novel SNPs were determined in the genetically diverse pig breeds including ten Chinese indigenous pigs and three Western commercial pig breeds. Association analysis of the SNPs with the carcass traits were conducted in a Large White × Meishan F₂ pig population. The linkage of two SNPs (g.5174T>C and g.8350C>A) in TNNI1 gene had significant effect on fat percentage. Besides these, the g.5174T>C polymorphism was also significantly associated with skin percentage (P < 0.05), shoulder fat thickness (P < 0.05) and backfat thickness between sixth and seventh ribs (P < 0.05). The significant effects of g.1167C>T polymorphism in TNNI2 gene on fat percentage (P < 0.01), lean meat percentage (P < 0.05), lion eye area (P < 0.05), thorax–waist backfat thickness (P < 0.01) and average backfat thickness (P < 0.05) were also found.