Expression Of miRNA Let-7a Research Articles

Expression of miRNA let-7a and HMGA2 and Diagnostic Value of Serum miRNA let-7a Level in Pancreatic Cancer

To investigate the diagnostic value of miRNA let-7a, high mobility group A2 (HMGA2) expression and serum miRNA let-7a level in pancreatic cancer. From January 2014 to January 2019, 60 patients with pancreatic cancer were collected for fresh pancreatic ductal adenocarcinoma tissue and normal pancreatic tissue adjacent to the cancer after the operation. Serum samples before and after operation were also collected, while 60 healthy people were enrolled as the control group. The expression of miRNA let-7a (qRT-PCR) and HMGA2 (qRT-PCR and Western blot) in cancer and adjacent normal tissues were measured. The serum level of miRNA let-7a was detected by qRT-PCR. The relationship between miRNA let-7a and HMGA2 expression and the clinicopathological features of pancreatic cancer was analyzed. The diagnostic value of serum miRNA let-7a pre-operation in patients with pancreatic cancer was also analyzed with ROC curve. Compared with normal tissues adjacent to the cancer, the expression level of miRNA let-7a in pancreatic cancer tissues decreased ( t=20.291, P<0.01), and the expression of HMGA2 mRNA increased ( t=46.681, P<0.01). The expression of HMGA2 protein in cancer tissues was higher than that in normal tissues adjacent to the cancer ( t=22.973, P<0.01). The serum level of miRNA let-7a in pancreatic cancer patients was significantly lower than that in healthy controls ( t=24.854, P<0.01). The relative level of serum miRNA let-7a at 1 week after surgery was significantly lower than that before surgery in pancreatic cancer patients ( t=6.885, P<0.01). There was a positive correlation between cancer tissue and serum miRNA let-7a expression 1 week after surgery ( r=0.411, P=0.000). The relative expression levels of miRNA let-7a and HMGA2 in pancreatic cancer tissues were significantly different in different TNM stages and lymph node metastasis ( P<0.05). The area under curve of pre-operation serum miRNA let-7a for the diagnosis of pancreatic cancer was 0.823 ( 95% confidence interval: 0.665-0.917); when the optimal cut-off value of miRNA let-7a was 0.614, the sensitivity was 82.3%, the specificity was 74.1%. The expression of HMGA2 may be involved in the invasion and metastasis of pancreatic cancer. The level of serum miRNA let-7a may provide a reference for the diagnosis and postoperative monitoring of pancreatic cancer.

Expression of long noncoding RNA in skin exosomes of patients with vitiligo

Background Vitiligo is a relatively common pigmentation disorder in which the skin melanocytes are lost or destroyed. Experimental downregulation of long noncoding RNA H19 (lnc RNA H19) in keratinocytes promoted melanocytes melanogenesis. lnc RNA H19 acts as an important regulator for micro-RNA let7a (miRNA let7a). miRNA let7a has a powerful role in regulation of cell survival and inducing cell apoptosis.Objectives This study was performed to clarify the suggested role of lnc RNA H19 and miRNA let7a derived from human skin exosomes in vitiligo pathogenesis.Patients and methods The study included 50 patients with vitiligo vulgaris and 50 healthy volunteers serving as controls. From all patients, 4-mm punch skin biopsies were taken. Skin biopsies were examined for lnc RNA H19 and miRNA let7a genes expression using RT-PCR technique.Results The expression of lnc RNA H19 and miRNA let7a in vitiligo lesions (1.736±0.84 and 6.7±2.26 pg/mg, respectively) was significantly higher than their expression in controls (0.8462±0.3483 and 1.514± 0.9202 pg/mg, respectively) (P<0.001). A significant positive correlation was found between the expression of lnc RNA H19 and the disease activity (P=0.0382). Additionally, a positive correlation was detected between the expression of miRNA let7a and the disease duration (P<0.0306).Conclusions We can conclude that upregulation of lnc RNA H19 and miRNA let7a gene expression describes their possible role in the pathogenesis of vitiligo.

The Differential Expression Profiles of miRNA-let 7a, 7b, and 7c in Bronchoalveolar Lavage Fluid From Infants With Asthma and Airway Foreign Bodies.

The aim of this study was to investigate the expression patterns of miRNA-let 7a, 7b, and 7c in bronchoalveolar lavage fluid in infants with asthma and airway foreign bodies. Between January 2016 and February 2017, 27 infants were included and divided into observation group (infants with asthma, n = 15) and control group (infants with airway foreign bodies, n = 12). The differential expression profiles of miRNA-let 7a, 7b, and 7c were determined by reverse transcription–polymerase chain reaction in bronchoalveolar lavage fluid (BALF) from infants of the 2 groups. The BALF was collected from infants undergoing flexible bronchoscopy. MiRNA-let 7a, 7b, and 7c increased significantly in infants from observation group as compared with control group (2.72 ± 0.48 vs 1, 8.23 ± 1.64 vs 1, 3.16 ± 0.62 vs 1, respectively). The increased expression of miRNA-let 7a, 7b, and 7c were associated with the asthma of infants.

Calcitonin administration improves endometrial receptivity via regulation of LIF, Muc-1 and microRNA Let-7a in mice.

Calcitonin (CT) is one of the factors affecting the embryo implantation, but its effects on the implantation window have not been fully investigated. The current study investigated the effects of CT on the endometrium receptivity by morphological study and evaluation of leukemia inhibitory factor (LIF), mucin 1 (Muc-1), and microRNA (miRNA) Let-7a in the ovarian stimulation and the normal ovarian cycle. Then the mechanism of the CT effects through the mammalian target of rapamycin (mTOR) signaling pathway was studied by using PP242. A total of 64 BALB/c mice were divided into the normal ovarian cycle and ovarian stimulation groups. Each group consisted of four subgroups: control, calcitonin, PP242, and calcitonin+PP242. CT and PP242 were injected on the fourth of pregnancy into the mice and 24 hr later all the mice were killed. The uterine tissue samples were used for morphological analysis, and endometrial cells were mechanically isolated for evaluation of gene and protein expression. The results showed that ovarian stimulation induced mTOR phosphorylation as well as increased expression of the Let-7a miRNA. In addition, CT injection increased the expression of LIF and miRNA Let-7a in ovarian stimulation similar to that in normal ovarian cycles. However, injection of PP242 reduced expression of miRNA Let-7a and increased Muc-1 expression in ovarian stimulation group. In conclusion, the administration of CT improved endometrial receptivity in mice. This phenomenon occurred by upregulation of LIF, miRNA Let-7a and downregulation of Muc-1 via mTOR signaling pathway.

Upregulation of HB-EGF, Msx.1, and miRNA Let-7a by administration of calcitonin through mTOR and ERK1/2 pathways during a window of implantation in mice.

Successful implantation of embryos requires endometrial receptivity. Calcitonin is one of the factors influencing the implantation window. This study aimed to evaluate calcitonin effects on endometrial receptivity. To this end, the effects of calcitonin on the implantation window in the ovarian stimulation and the normal ovarian cycle were investigated by the morphological study of the endometrium as well as the expression of MSX.1, HB-EGF, and micro-RNA (miRNA) Let-7a; then the mechanisms of calcitonin effects were studied through the mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways. A total of 64 Bulb/c mice were divided into two groups: Normal ovarian cycle and ovarian stimulation. Each group consisted of four subgroups: Ctrl, CT, PP242, and CT + PP242. Calcitonin and PP242 were injected on the fourth day of pregnancy and 24 hr later all the mice were killed. Uterine tissue samples were used for morphological analysis and the endometrial epithelial and the stromal cells were isolated from myometrium for evaluation of gene and protein expression. Ovarian stimulation increased the phosphorylation levels of mTOR and ERK1/2 and the expression of miRNA Let-7a. Calcitonin injection increased the expression of HB-EGF, Msx.1, and miRNA Let-7a in a normal ovarian cycle and in ovarian-stimulated mice. It also increased eukaryotic initiation factor 4E-binding protein 1 and ERK1/2 phosphorylation in normal ovarian cycles. Calcitonin improved the receptivity of the uterine endometrium by upregulation of the HB-EGF, Msx.1, and miRNA Let-7a likely through mTOR and ERK1/2 signaling pathway.

Expression of serum miRNAlet-7a in patients with renal clear cell carcinoma and its significance

Objective To investigate the changes of serum miRNAlet-7a expression in clear renal cell carcinoma (cRCC) patients and the diagnostic value of let-7a for cRCC. Methods Forty patients with cRCC who were hospitalized in our hospital from November 2015 to February 2017, and Forty-two healthy subjects were enrolled in this study. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to determine the levels of let-7a in serum samples. The association between serum let-7a levels and the clinical and pathological parameters of cRCC were analyzed, and the diagnostic efficacy of let-7a for cRCC was analyzed. Results ①The relative expression levels of let-7a in the experimental group and normal control group was(0.519±0.114)and(1.622±0.131), respectively. There was significant difference between the two groups (P 0.05). ③We used the receiver's characteristic curve (ROC) for analysis.The sensitivity was 81.4%, the specificity was 73.9%, the AUC was 0.794, and the 95% confidence interval was 0.657~0.912 for the optimal threshold of the relative expression of let-7a for cRCC. Conclusions The serum let-7a levels in patients with cRCC are down-regulated and the determination of let-7a has certain reference value for the diagnosis of cRCC and the judgment of the formation of tumor thrombus. PCR technology is mature, easy to operate, high amplification efficiency, has a certain clinical value. Key words: Kidney Neoplasms; Adenocarcinoma, Clear Cell; MicroRNAs

MiRNALet-7a mediates prostate cancer PC-3 cell invasion, migration by inducing epithelial-mesenchymal transition through CCR7/MAPK pathway.

Prostate cancer is one of the most common malignancies in older men. Recent evidence has demonstrated microRNA (miRNA) Let-7a expression decreased in prostate cancer, while the expression of CC chemokine receptor type 7 (CCR7) increased. In this study, we investigated whether CCR7 overexpression was associated with a decrease in the expression of miRNA Let-7a in invasion and metastasis of prostate cancer cell. Synthetic Let-7a mimics and Let-7a inhibitors were transfected into prostate cancer PC-3 cells, respectively. Then Western blot was used to detect the expression of CCR7, ERK, p38, MMP-9, and Epithelial-Mesenchymal Transition (EMT)-related proteins. Matrigel invasion assays were performed to assess the migratory and invasive activities of PC3 cells. To confirm the fact that 3'UTR of CCR7 is a direct target of Let-7a, a luciferase assay for the reporter gene expressing the Let-7a binding sites of CCR7 3'UTR was used. Synthetic Let-7a mimics decreased prostate cancer cell migration and invasion, as well as the expression of CCR7, phospho-p38, phospho-ERK1/2, MMP-9, N-cadherin, and Snail in PC-3 cells. The Let-7a inhibitors reversed the effects of Let-7a on PC-3 cells. The 3'UTR of CCR7 was confirmed as a direct target of Let-7a by using the luciferase assay. All findings demonstrated that Let-7a/CCR7 axis regulated EMT progress in prostate cancer cells and mediated the tumor cell invasion and migration process via activation of P38/ERK signal pathway. Our results suggested that the therapeutic potential of Let-7a as an antitumor and antimetastatic manager in prostate cancer and CCR7 may be regarded as a therapeutic target for the prostate cancer treatment.

Effects of long non-coding RNA H19 and microRNA let7a expression on thyroid cancer prognosis

This study aims to explore the effects of long non-coding RNA H19 (lncRNA H19) and microRNA let7a (miRNA let7a) expression on the prognosis of thyroid cancer (TC). This may aid in the discovery of more effective treatment and prognosis approaches for TC. Between January 2008 and January 2011, 131 TC tissues and adjacent tissues were obtained from TC patients. An additional 122 normal thyroid tissues were also collected as normal controls from patients with benign thyroid lesions. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect lncRNA H19 and miRNA let7a mRNA expression. Five-year follow-ups were conducted. A Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic values of lncRNA H19 and miRNA let7a in TC. The Kaplan-Meier method was applied to analyze the 5-year survival rate of TC patients. Univariate and multivariate factor analyses were employed to analyze the prognostic factors of TC. The lncRNA H19 mRNA expression was higher while the miRNA let7a mRNA expression was lower in TC tissues than, in the normal thyroid tissues and adjacent tissues. The area under the ROC curve (AUC) of lncRNA H19 and miRNA let7a were 0.801 and 0.116, with sensitivity at 72.5% and 84%, as well as specificity 75.4% and 77%, respectively. In TC patients with tumor diameters≥1.0cm, lncRNA H19 mRNA expression was elevated, but miRNA let7a mRNA expression was reduced. This was also evident in TC patients with TNM stages III+IV and those with lymph node metastasis. TC patients with a lower 5-year survival rate showed upregulated levels of lncRNA H19 expression and, downregulated levels of miRNA let7a expression. LncRNA H19 and miRNA let7a expression, tumor diameter, TNM stage and lymph node metastasis were independent prognostic factors of TC. This study demonstrated that increased lncRNA H19 and decreased miRNA let7a expression levels are associated with poor prognosis in TC patients. An inverse relationship between lncRNA H19 and miRNA let7a expression levels was exhibited.

Expression of microRNA let-7a, miR-155, and miR-205 in tumor and tumor-adjacent histologically normal tissue in patients with non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is a main group of lung malignancies. Epigenetic changes are as important as genome structural changes in carcinogenesis. MicroRNA (miRNA) is a class of non-coding single-stranded RNAs that play an important role in the regulation of matrix RNA (mRNA) translation and degradation. MicroRNA expression changes occur in many cancers. According to the field cancerization theory, tumor-adjacent histologically normal tissue takes part in tumor progression by triggering cell transformation. The important clinical implication is that the fields may serve as the basis for a recurrence after surgery. Thus, the aim of our study was to determine the expression levels of miRNAs let-7a, miR-155, and miR-205 in tumor and tumor-adjacent apparently normal tissues to evaluate these changes as potential prognostic markers in NSCLC patients. The expression of miRNAs let-7a, miR-155, and miR-205 in tumor and tumor-adjacent apparently normal tissues at 2 and 5 cm was determined by real-time PCR with subsequent quantification using a 2-ΔΔСt method. The findings were then analyzed to reveal possible associations with clinical and morphological parameters, such as age, cancer stage, and tumor grade. The expression of miRNA let-7a was found to be significantly lower in tumor than that in tumor-adjacent apparently normal tissue at 2 and 5 cm. In groups of patients older than 63 years with Stage III-IV NSCLC, the expressions of microRNA let-7a and miR-155 in tumor tissue were substantially lower than that in the adjacent normal tissue. Beyond that point, patients with high-grade tumors had also a significantly lower expression of miRNA let-7a in relatively adjacent apparently normal tissue. The findings suggest that miRNA let-7a and miR-155 may be used as poor prognostic markers for patients with NSCLC.

SDF-1 Inhibition Using Spiegelmer® Nox-A12 As a Novel Strategy For Targeting AML Cells Within Their BM Microenvironment

Bone marrow (BM) mesenchymal stromal cells (MSC) protect leukemia cells from chemotherapy-induced apoptosis. CXCR4, the receptor for the stromal-derived factor 1 (SDF-1/CXCL12), is involved in modulating leukemic progenitor cell homing and it is essential for the migration of these cells to the BM niche. As the BM niche imparts favorable survival advantage to leukemia cells, a strategy to disrupt leukemic cell homing to the BM microenvironment may represent a novel way to effectively kill these malignant cells. Strategies to disrupt the SDF-1/CXCR4 axis have relied on inhibition of the CXCR4 receptor using various drugs including plerixafor, which is potent at mobilizing normal progenitor cells. We previously found that plerixafor antagonized SDF-1 and MSC-induced migration of leukemic cells, inhibited SDF-1-induced AKT and ERK activation, and decreased stroma-mediated protection from AraC-induced apoptosis in 11 out of 18 primary AML samples (Zeng et al Blood 2009; 113: 6215). We also found that the inhibition of CXCR4 induces miRNA let-7a expression via the transcription factor Yin Yang1 (Chen et al, JCI, 2013; 123:2395), and induces chemosensitization irrespective of cell mobilization. While inhibition of CXCR4 has shown promise for use in AML therapy (Andreeff, et al ASH, Blood 120(21) (#142), 11/2012), a more effective strategy might be to target the SDF-1 ligand itself since SDF-1 can bind to other receptors (i.e. CXCR7) that are likely not recognized by CXCR4 antibodies. NOXXON (Berlin, Germany) has recently identified a potent inhibitor to SDF-1 that is based on a so-called Spiegelmer®. The Spiegelmer NOX-A12 is a structured l-RNA oligonucleotide, PEGylated at the 5'-end to improve the pharmacokinetics. In healthy volunteers, NOX-A12 displays a half-life of∼38 hours and has demonstrated a long-term and dose-dependent mobilization of WBCs and CD34+ cells. The drug is currently tested in clinical trials in multiple myeloma and chronic lymphocytic leukemia patients (Gobbi et al., ASH 2012 120: 4593). In the current study, the ability of NOX-A12 to suppress survival signaling in leukemic and MSC cells was examined. In addition, we tested the ability of the Spiegelmer to mobilize leukemia cells in a murine xenograft model. NOX-A12 was found to suppress the phosphorylation of ERK in AML cells, and suppress the phosphorylation of ERK, FAK, and STAT3 in MSC. These results suggested that SDF-1 affected diverse survival signaling pathways in the leukemic cells and the critical supporting stromal niche. In vivo activity was examined in NOD/SCID/-g- mice injected intravenously with murine leukemia Baf/ITDluc/GFP cells and monitored by a non-invasive bioluminescent imaging. The in vivo mobilization assay was assessed when the majority of mice had approximately 10% tumor burden. Plerixafor, NOX-A12, or a combination of both agents was subcutaneously administrated and leukemic GFP+ cells were detected by analytical flow cytometry. Plerixafor and NOX-A12 administered separately increased total WBC count by 2- to 3-fold and concomitantly increased the percentage of circulating GFP+ cells in all mice (to 14.2% and 18.5%, respectively). Importantly, the combination of both targeted agents further increased the percentage of circulating GFP+ cells in an approximately additive fashion (to 26.4%). Conclusionwe demonstrate that the novel SDF-1 Spiegelmer NOX-A12 can suppress survival signaling in both leukemia and supportive MSC cells. Furthermore, increased mobilization of leukemic cells in vivo was observed when NOX-A12 and plerixafor were used in combination. These findings suggest the possibility that targeted disruption of leukemia/stroma interactions by optimized blockade of the pro-survival SDF-1/CXCR4 axis will not only increase leukemia cell mobilization, but also enhance the sensitivity to chemotherapy of both mobilized and non-mobilized the leukemic cells and leukemic stem cells. Experiments are ongoing to further validate this hypothesis. Disclosures:Kruschinski:Noxxon: Employment.

Insights into Insulin-Mediated Regulation of CYP2E1: miR-132/-212 Targeting of CYP2E1 and Role of Phosphatidylinositol 3-Kinase, Akt (Protein Kinase B), Mammalian Target of Rapamycin Signaling in Regulating miR-132/-212 and miR-122/-181a Expression in Primary Cultured Rat Hepatocytes

Several microRNAs (miRNAs) were selected for characterization of their response to insulin signaling based on in silico predictions of targeting CYP2E1 mRNA and previous reports implicating their role in hepatic metabolism and disease. CYP2E1 expression decreases with increasing insulin concentration and has been shown to be regulated by the phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. In primary cultured rat hepatocytes, insulin at 0.1, 1.0, and 10 nM elevated miRNA-132 and -212 expression ∼2- and 1.8-fold, respectively, whereas expression of miRNA-181a and -122 increased ∼1.6- and 1.4-fold, respectively. In contrast, insulin failed to alter significantly the expression of miRNA let-7a. Mechanistic studies using inhibitors of PI3-K, Akt, and mTOR were used to examine the role of the insulin signaling pathway on miR expression and resulted in significant suppression of the insulin-mediated elevation of miR-132, miR-212, and miR-122 levels, with a lesser effect observed for miR-181a. Targeting of the rat CYP2E1 3'-untranslated region (UTR) by miR-132 and -212 was demonstrated with an in vitro luciferase reporter assay. These data show that insulin, which regulates CYP2E1 through the PI3-K, Akt, mTOR signaling pathway, also regulates the expression of miRs that target the 3'-UTR of CYP 2E1 mRNA and are involved in the regulation of hepatic metabolism and disease.

Abstract 5281: Insulin-mediated regulation of the miR132/212 cluster and miR 122/181a expression through PI3K, Akt, mTOR signaling in primary cultured rat hepatocytes.

Abstract Background and Objective: Several microRNAs were selected for characterization of their response to insulin signaling based on previous reports implicating their role in the regulation of hepatic metabolism, diabetes, hepatocellular carcinoma, and predictions of targeting CYP2E1. CYP2E1 catalyzes the metabolic activation of carcinogens, increases cellular oxidative stress, and is elevated in diabetes and at low insulin levels (0.1nM) in primary cultured hepatocytes. In addition, aberrant miR-122 expression was reported to be associated with hepatocellular carcinoma in rats, as well as in human tissue, and may be involved with hepatocarcinogenesis. In view of the critical role of the PI3K, Akt and mTOR signaling pathway in pathogenesis, mechanistic studies were conducted to examine the role of this pathway in regulation miR 132/212, 122 and 181a expression. Methods: Insulin effects on miRNA (miRs) levels were monitored using qPCR, and mechanistic studies employing inhibitors of PI3K, Akt, and mTOR were used to examine the role of this insulin signaling pathway on miR expression. The results were compared to miR let-7a, which is not implicated in the regulation of hepatic metabolism or diabetes and does not target CYP2E1 mRNA. Results: Insulin at concentrations of 0.1, 1.0, and 10 nM increased the expression of miRNA-132 and 212 ∼2- and 1.8-fold respectively, whereas expression of miRNAs 181a and 122 increased ∼1.6- and 1.4-fold, respectively. In contrast, insulin failed to significantly alter the expression of miRNA let-7a. Inhibition of PI3K with LY294002, Akt with A-443654 and mTOR with rapamycin resulted in significant suppression of the insulin-mediated elevation of miR-132, miR-212, and miR-122 levels monitored over the insulin concentrations examined. Inhibition of PI3K with LY294002, or Akt with A-443654 decreased the insulin-mediated increase in miR-181a levels by ∼20 to 50% relative to the respective insulin-concentration. Rapamycin inhibition of mTOR produced a marginal (&amp;lt;10%) decrease in miR-181a levels. Inhibition of PI3K or Akt did not show any significant effect on miR let-7a. In summary, seminal evidence is provided for insulin regulation of microRNA expression in primary cultured hepatocytes through PI3K, Akt and mTOR (TORC1). Citation Format: Upasana Shukla, Nithin Tumma, Theresa Gratsch, Alan Dombkowski, Raymond Novak. Insulin-mediated regulation of the miR132/212 cluster and miR 122/181a expression through PI3K, Akt, mTOR signaling in primary cultured rat hepatocytes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5281. doi:10.1158/1538-7445.AM2013-5281

Abstract 4032: Elevated level of let-7a and mir-30c microRNAs in human prostate cancer

Abstract MicroRNAs (miRNAs) are endogenously expressed non-coding RNAs that negatively regulate the expression of protein coding genes at the translational level. Evidence from recent studies suggests that cancer development is associated with deregulation of specific set of miRNAs that determines the cancer characteristics. Recently, miRNA profiling has been shown to identify cancer specific miRNA signatures that are specific to different cellular origin. However, the functional significance of specific miRNAs in prostate cancer developmental stages remains to be investigated. Further, identification and characterization of tumor derived miRNAs using advanced and reliable techniques may benefit early detection and prevention strategies. In this study, to identify potential miRNAs in human prostate cancer tissue specimens, we used 15 human prostate cancer tissue microarrays (TMAs) with 36 cores in each representing different stages of the cancer. We detected the expression of miRNA let-7a and mir-30c by performing locked nucleic acid-in-situ hybridization (LNA-ISH) (EXIQON, Vedbaek, Denmark). To obtain maximal sensitivity of miRNA expression signal, we used double (3′ and 5″) digoxigenin-labeled LNA probes (MiRCURY LNA) of 20uM each after optimizing with lower and higher dilutions. Oversaturation was avoided by reducing the temperature (30-35°C) and the time of exposure (24h). As a negative control the tissues were hybridized either with mismatch or no probe. We determined the expression level of the miRNAs in (a) precancerous single acinus or small group of acini and (b) in the regions of prostatic adenocarcinoma or high grade PIN. Our findings indicated abundant expression of both let-7a and mir-30c localized only in the nucleus of the cells in HGPIN, in contrary to that in the early PIN lesions, where let-7 and mir-30c miRNAs were localized both in the cytoplasm, as well as in the nucleus. Interestingly, not all the acini showed a positive staining for let-7, suggesting the possibility of down regulation of let-7 in the non-neoplastic regions. Finally, the difference in the expression of let-7a and mir-30c in the stroma vs. tumor acini were compared and quantified to predict their functional significance related to tumor proliferation. Although the findings on the expression of let-7a and mir-30c were consistent in more than 20% of the TMAs examined, we observed weak or no signal in several of the tumor acini (5%) suggesting a heterogeneous expression pattern. Overall, our findings suggest that examining the expression level of miRNAs in the nucleus vs. cytoplasm of tumor cells at different stages of prostate cancer is critical to determine the functional status of let-7a and mir30c. Most importantly, monitoring a shift in their expression pattern will provide significant opportunities to define diagnostically useful miRNA as biomarkers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4032.