Different Expression of Occludin and ZO-1 in Primary and Metastatic Liver Tumors

Tight junction (TJ) components were found to be correlated with carcinogenesis and tumor development. TJs are composed of three main integral membrane proteins; occludin, claudins and JAMs. Alteration of the TJ protein expression may play an important role in the process of cell dissociation, which is among the first steps of tumor invasion and metastasis. Reduced expression of ZO-1 has been reported to be associated with invasion of several tumors. The aim of the present study was to detect differences between occludin and ZO-1 expression in normal liver samples, HCCs and colorectal liver metastases. Expression of occludin and ZO-1 was analysed in 25 surgically removed human hepatocellular carcinomas (HCC) and 25 human colorectal liver metastases. Gene expression levels were measured by real-time RT PCR, protein expression was determined by immunohistochemistry, comparing tumors with the surrounding nontumorous parenchyma and with seven normal liver samples. Occludin and ZO-1 mRNAs showed significant downregulation in HCCs in comparison with normal liver and were also downregulated in the metastases when compared with normal liver. Occludin and ZO-1 proteins were weakly expressed on hepatocytes in normal liver, while strong expression was found on bile canaliculi. In HCCs occludin and ZO-1 did not show immunopositivity on tumor cells, while colorectal metastatic tumors revealed high levels of these molecules. HCCs and metastases are characterized by markedly different protein expression pattern of occludin and ZO-1, which phenomenon might be attributed to the different histogenesis of these tumors.     

卵巢上皮恶性肿瘤侵袭转移相关miRNA的筛选与鉴定

目的 探索与卵巢上皮恶性肿瘤侵袭转移可能相关的miRNA.方法 采用miRNA芯片,筛选SKOV-3ip和SKOV-3细胞差异表达miRNA.利用生物信息学软件TargetScan、MicroCosm、PicTar和GO,预测差异表达miRNA的靶基因及其功能.采用实时逆转录聚合酶链反应(real-time RT-PCR)技术,验证与卵巢癌侵袭转移可能相关的5种miRNA(let-7a、let-7e、let-7f、miR-22和miR-886-5p)在SKOV-3ip和SKOV-3细胞的表达.同时,检测这5种miRNA在另外一组侵袭转移能力不同的卵巢癌细胞株HO8910和HO-8910PM中的表达,并进行统计学分析.结果 基因芯片筛选显示,42种miRNA在SKOV-3ip和SKOV-3细胞株表达差异明显.进一步分析显示,let-7a、let-7e、let-7f、miR-22和miR-886-5p等5种miRNA可能与卵巢癌的侵袭转移密切相关.real-time RT-PCR结果证实,let-7f和miR-22在两组侵袭转移能力不同的卵巢癌细胞(SKOV-3和SKOV-3ip细胞、HO-8910和HO-8910PM细胞)表达差异有统计学意义(均P＜0.05).结论 let-7f和miR-22在侵袭转移能力强的卵巢癌细胞中低表达,可能具有抑癌基因的作用.     

Distinct microRNA expression profiles in prostate cancer stem/progenitor cells and tumor-suppressive functions of let-7

MiRNAs regulate cancer cells, but their potential effects on cancer stem/progenitor cells are still being explored. In this study, we used quantitative real-time-PCR to define miRNA expression patterns in various stem/progenitor cell populations in prostate cancer, including CD44+, CD133+, integrin α2β1+, and side population cells. We identified distinct and common patterns in these different tumorigenic cell subsets. Multiple tumor-suppressive miRNAs were downregulated coordinately in several prostate cancer stem/progenitor cell populations, namely, miR-34a, let-7b, miR-106a, and miR-141, whereas miR-301 and miR-452 were commonly overexpressed. The let-7 overexpression inhibited prostate cancer cell proliferation and clonal expansion in vitro and tumor regeneration in vivo. In addition, let-7 and miR-34a exerted differential inhibitory effects in prostate cancer cells, with miR-34a inducing G1 phase cell-cycle arrest accompanied by cell senescence and let-7 inducing G2-M phase cell-cycle arrest without senescence. Taken together, our findings define distinct miRNA expression patterns that coordinately regulate the tumorigenicity of prostate cancer cells.     

Changes in microRNA (miRNA) expression during pancreatic cancer development and progression in a genetically engineered KrasG12D;Pdx1-Cre mouse (KC) model

Differential expression of microRNAs (miRNAs) has been demonstrated in various cancers, including pancreatic cancer (PC). Due to the lack of tissue samples from early-stages of PC, the stage-specific alteration of miRNAs during PC initiation and progression is largely unknown. In this study, we investigated the global miRNA expression profile and their processing machinery during PC progression using the Kras^G12D;Pdx1-Cre (KC) mouse model. At 25 weeks, the miRNA microarray analysis revealed significant downregulation of miR-150, miR-494, miR-138, miR-148a, miR-216a, and miR-217 and upregulation of miR-146b, miR-205, miR-31, miR-192, and miR-21 in KC mice compared to controls. Further, expression of miRNA biosynthetic machinery including Dicer, Exportin-5, TRKRA, and TARBP2 were downregulated, while DGCR8 and Ago2 were upregulated in KC mice. In addition, from 10 to 50 weeks of age, stage-specific expression profiling of miRNA in KC mice revealed downregulation of miR-216, miR-217, miR-100, miR-345, miR-141, miR-483-3p, miR-26b, miR-150, miR-195, Let-7b and Let-96 and upregulation of miR-21, miR-205, miR-146b, miR-34c, miR-1273, miR-223 and miR-195 compared to control mice. Interestingly, the differential expression of miRNA in mice also corroborated with the miRNA expression in human PC cell lines and tissue samples; ectopic expression of Let-7b in CD18/HPAF and Capan1 cells resulted in downregulation of KRAS and MSST1 expression. Overall, the present study aids an understanding of miRNA expression patterns during PC pathogenesis and helps to facilitate the identification of promising and novel early diagnostic/prognostic markers and therapeutic targets.     

microRNAs in uterine sarcomas and mixed epithelial–mesenchymal uterine tumors: a preliminary report

Uterine sarcomas and mixed epithelial–mesenchymal uterine tumors are a heterogeneous group of rare tumors for which there are very few diagnostic markers available. As aberrant microRNA (miRNA) expression patterns represent putative diagnostic cancer markers, we aimed to identify miRNA expression profiles of the major uterine sarcoma subtypes and mixed epithelial–mesenchymal tumors of the uterus. Eighty-eight miRNAs were assessed by quantitative RT-PCR in cancerous and non-cancerous tissue samples collected from 29 patients with endometrial sarcoma, leiomyosarcoma, and mixed epithelial–mesenchymal tumors. Tumor and control samples significantly (P?<?0.05) differed in the expression of miR-23b, miR-1, let-7f, and let-7c in endometrial sarcomas, and miR-1, let-7c, miR-133b, let-7b, miR-143, let-7a, let-7d, let-7e, let-7g, miR-222, let-7i, and miR-214 in mixed epithelial–mesenchymal tumors. All the significantly changed miRNAs were down-regulated in the malignant tissues as compared to their normal counterparts. This may suggest their tumor suppressor role in these malignancies. No statistically significant changes in miRNA expression levels were found between leiomyosarcoma tumors and controls. The identified miRNAs warrant further studies as valuable candidate markers for the differential diagnosis of uterine sarcomas from benign uterine lesions and between uterine sarcoma subtypes.     

MicroRNA expression signatures of stage,grade, and progression in clear cell RCC

Clear cell RCC is the most common, and more likely to metastasize, of the three main histological types of RCC. Pathologic stage is the most important prognostic indicator and nuclear grade can predict outcome within stages of localized RCC. Epithelial tumors are thought to accumulate a series of genetic and epigenetic changes as they progress through well-defined clinical and histopathological changes. MicroRNAs (miRNAs) are involved in the regulation of mRNA expression from many human genes and miRNA expression is dysregulated in cancer. To better understand the contribution of dysregulated miRNA expression to the progression and biology of ccRCC, we examined the differences in expression levels of 723 human miRNAs through a series of analyses by stage, grade, and disease progression status in a large series of 94 ccRCC. We found a consistent signature that included significant upregulation of miR-21-5p, 142-3p, let-7g-5p, let-7i-5p and 424-5p, as well as downregulation of miR-204-5p, to be associated with ccRCC of high stage, or high grade, or progression. Discrete signatures associated with each of stage, grade, or progression were also identified. The let-7 family was significantly downregulated in ccRCC compared with normal renal parenchyma. Expression of the 6 most significantly differentially expressed miRNAs between ccRCC was verified by stem-loop qRT-PCR. Pathways predicted as targets of the most significantly dysregulated miRNAs included signaling, epithelial cancers, metabolism, and epithelial to mesenchymal transition. Our studies help to further elucidate the biology underlying the progression of ccRCC and identify miRNAs for potential translational application.     

microRNA-320a inhibits tumor invasion by targeting neuropilin 1 and is associated with liver metastasis in colorectal cancer

MicroRNAs (miRNAs) have been implicated in regulating diverse cellular pathways. Although there is emerging evidence that various miRNAs function as oncogenes or tumor suppressors in colorectal cancer (CRC), the role of miRNAs in mediating liver metastasis remains unexplored. The expression profile of miRNAs in liver metastasis and primary CRC tissues was analyzed by miRNA microarrays and verified by real-time polymerase chain reaction (PCR). In 62 CRC patients, the expression levels of miR-320a were determined by real-time PCR, and the effects on migration and invasion of miR-320a were determined using a transwell assay. miR-320a target genes were confirmed by luciferase assay, real-time PCR and western blot analysis. A set of miRNAs was found to be dysregulated in the liver metastasis tissues compared to matched primary CRC tissues, and the expression levels of miR-320a were significantly decreased in the liver metastasis tissues examined. miR-320a was correlated with tumor progression in CRC. miR-320a was downregulated in liver metastatic colon cancer cells and inhibited liver metastatic colon cancer cell migration and invasion. miR-320a directly binds to the 3'UTR of neuropilin 1 (NRP-1), a protein that functions as a co-receptor of vascular epithelial growth factor. miR-320a downregulated the expression of NRP-1 at both the mRNA and protein levels. These data demonstrated that miR-320a may be useful for identifying CRC patients that are at an elevated risk for developing liver metastasis. Our findings suggest that miR-320a may be a novel therapeutic candidate for the treatment of colorectal cancer.     

MicroRNA-320 family is downregulated in colorectal adenoma and affects tumor proliferation by targeting CDK6

AIM: To investigate the microRNA (miRNA) expression during histological progression from colorectal normal mucosa through adenoma to carcinoma within a lesion. METHODS: Using microarray, the sequential changes in miRNA expression profiles were compared in colonic lesions from matched samples; histologically, non-neoplastic mucosa, adenoma, and submucosal invasive carcinoma were microdissected from a tissue sample. Cell proliferation assay was performed to observe the effect of miRNA, and its target genes were predicted using bioinformatics approaches and the expression profile of SW480 transfected with the miRNA mimics. mRNA and protein levels of the target gene in colon cancer cell lines with a mimic control or miRNA mimics were measured using qRT-PCR and Western blotting. The expression levels of miRNA and target gene in colorectal tissue samples were also measured. RESULTS: Microarray analysis identified that the miR-320 family, including miR-320a, miR-320b, miR-320c, miR-320d and miR-320e, were differentially expressed in adenoma and submucosal invasive carcinoma. The miR-320 family, which inhibits cell proliferation, is frequently downregulated in colorectal adenoma and submucosal invasive carcinoma tissues. Seven genes including CDK6 were identified to be common in the results of gene expression array and bioinformatics analyses performed to find the target gene of the miR-320 family. We confirmed that mRNA and protein levels of CDK6 were significantly suppressed in colon cancer cell lines with miR-320 family mimics. CDK6 expression was found to increase from non-neoplastic mucosa through adenoma to submucosal invasive carcinoma tissues and showed an inverse correlation with miR-320 family expression. CONCLUSION: MiR-320 family affects colorectal tumor proliferation by targeting CDK6, plays important role in its growth, and is considered to be a biomarker for its early detection.     

Differential Expression of Non-Coding RNA Signatures in Thyroid Cancer between Two Ethnic Groups

Filipino Americans show higher thyroid cancer recurrence rates compared to European Americans. Although they are likely to die of this malignancy, the molecular mechanism has not yet been determined. Recent studies demonstrated that small non-coding RNAs could be utilized to assess thyroid cancer prognosis in tumor models. The goal of this study is to determine whether microRNA (miRNA) signatures are differentially expressed in thyroid cancer in two different ethnic groups. We also determined whether these miRNA signatures are related to cancer staging. This is a retrospective study of archival samples from patients with thyroid cancer (both sexes) in the pathology division from the last ten years at Loma Linda University School of Medicine, California. Deidentified patient demographics were extracted from the patient chart. Discarded formalin-fixed paraffin-embedded tissues were collected post-surgeries. We determined the differential expressions of microRNA in archival samples from Filipino Americans compared to European Americans using the state-of-the-art technique, HiSeq4000. By ingenuity pathway analysis, we determined miRNA targets and the pathways that those targets are involved in. We validated their expressions by real-time quantitative PCR and correlated them with the clinicopathological status in a larger cohort of miRNA samples from both ethnicities. We identified the differentially upregulated/downregulated miRNA clusters in Filipino Americans compared to European Americans. Some of these miRNA clusters are known to target genes that are linked to cancer invasion and metastasis. In univariate analysis, ethnicity and tumor staging were significant factors predicting miR-4633-5p upregulation. When including these factors in a multivariate logistic regression model, ethnicity and tumor staging remained significant independent predictors of miRNA upregulation, whereas the interaction of ethnicity and tumor staging was not significant. In contrast, ethnicity remained an independent predictor of significantly downregulated miR-491-5p and let-7 family. We provide evidence that Filipino Americans showed differentially expressed tumor-tissue-derived microRNA clusters. The functional implications of these miRNAs are under investigation.     

The regulatory mechanism of CCR7 gene expression and its involvement in the metastasis and progression of gastric cancer

Gastric cancer is the second leading cause of cancer mortality, but the molecular mechanisms underlying its progression and metastasis remain unclear. CCR7 and Dicer 1 protein expression in 80 gastric adenocarcinomas and 40 peritumoral tissues were measured by immunohistochemical staining. The expression of let-7a miRNA in serum, tumor tissues, and peritumoral tissues was measured by real-time PCR. The role of let-7a in CCR7 protein expression, migration, and invasion of gastric cancer cells was tested in vitro. Dicer 1 protein expression was found to be significantly reduced, whereas CCR7 protein expression was significantly increased in gastric adenocarcinomas compared to peritumoral tissues. The let-7a miRNA levels in the serum and tumor tissues of gastric adenocarcinoma patients were significantly lower than in the serum of healthy controls and peritumoral tissues, respectively. Dicer 1 protein positively correlated with let-7a miRNA level, but negatively correlated with CCR7 protein level in gastric adenocarcinoma. Negative Dicer 1 protein and let-7a miRNA expression and positive CCR7 protein expression significantly correlated with lymph node metastasis, depth of invasion, high clinical TNM stage, and larger tumor size. Let-7a transfection significantly inhibited CCR7 protein expression, migration, and invasion of MNK-45 cells in vitro. High expression of CCR7 protein and low expression of Dicer 1 protein and let-7a miRNA are significantly associated with the metastasis and progression of gastric cancer. High CCR7 protein expression may be caused by the loss of Dicer 1 protein expression and reduced let-7a miRNA level in gastric cancer. The serum let-7a level might be a marker for the diagnosis of gastric cancer.     

microRNA expression profiling of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is posing a serious health problem worldwide. The association between its pathogenesis and microRNAs (miRNA) has not been elucidated. In this study, miRNA expression profiling was performed to screen the miRNA expression changes in 8 NPC tissues and 4 normal nasopharyngeal tissues. Thirty-four miRNAs were identified to be differentially expressed; of these, one miRNA (miR-18a) was overexpressed and 33 miRNAs (miR-34b, miR-34c, let-7 family, etc.) were underexpressed in NPC tissues compared to the normal samples. Validation was performed by real-time quantitative PCR for two altered miRNAs (miR-34b and let-7g) and one non-differentially expressed miRNA (miR-30c). Unsupervised hierarchical clustering analysis showed that the aberrant miRNAs were correlated with the clinical stage of NPC patients. In addition to several biological pathways that are well characterised in NPC and which were significantly targeted by the underexpressed miRNAs, two novel pathways, nervous system development and sensory perception of sound, were identified to be strongly associated with NPC development. Furthermore, a c-Myc centered miRNA regulatory network was inferred in NPC. Our study reveals that aberrantly expressed miRNAs play important roles in NPC tumorigenesis and may serve as potential targets for novel therapeutic strategies in the future.     

microRNA Expression Profile in Patients with Stage II Colorectal Cancer: A Turkish Referral Center Study

Background: There are increasing data about microRNAs (miRNA) in the literature, providing abundantevidence that they play important roles in pathogenesis and development of colorectal cancer. In this study, weaimed to investigate the miRNA expression profiles in surgically resected specimens of patients with recurrentand non-recurrent colorectal cancer. Materials and Methods: The study population included 40 patients withstage II colorectal cancer (20 patients with recurrent tumors, and 20 sex and age matched patients withoutrecurrence), who underwent curative colectomy between 2004 and 2011 without adjuvant therapy. Expression of16 miRNAs (miRNA-9, 21, 30d, 31, 106a, 127, 133a, 133b, 135b, 143, 145, 155, 182, 200a, 200c, 362) was verifiedby quantitative real-time polymerase chain reaction (qRT-PCR) in all resected colon cancer tissue samples andin corresponding normal colonic tissues. Data analyses were carried out using SPSS 15 software. Values werestatistically significantly changed in 40 cancer tissues when compared to the corresponding 40 normal colonictissues (p<0.001). MiR-30d, miR-133a, miR-143, miR-145 and miR-362 expression was statistically significantlydownregulated in 40 resected colorectal cancer tissue samples (p<0.001). When we compared subgroups,miRNA expression profiles of 20 recurrent cancer tissues were similar to all 40 cancer tissues. However in 20non-recurrent cancer tissues, miR-133a expression was not significantly downregulated, moreover miR-133bexpression was significantly upregulated (p<0.05). Conclusions: Our study revealed dysregulation of expressionof ten miRNAs in Turkish colon cancer patients. These miRNAs may be used as potential biomarkers for earlydetection, screening and surveillance of colorectal cancer, with functional effects on tumor cell behavior.