| 一Expression and function of miR-483-3p in pancreatic ductal adenocarcinomaBACKGROUND:MicroRNAs (miRNAs) are a set of small endogenous non-coding RNA, which involve in cell proliferation, differentiation, apoptosis and other biological processes through post-transcriptional repression or target mRNA cleavage. The human genome has identified more than1000miRNAs, which are tissue-specific expression. Differential expression of miRNA may play a role of oncogenes or tumor suppressor genes. Researches have revealed that there was a close relationship between miR-483-3p and a variety of human tumors. And expression of miR-483-3p also showed tissue specificity, and it even plays opposite roles in various tumors. For example, it is highly expressed in gastric cancer and plays the oncogene role, but it plays the role of tumor suppressor genes in non small cell lung cancer. The purpose of this study is to clarify the miR-483-3p expression pattern in invasive pancreatic ductal adenocarcinoma and non-invasive precancerous lesions and its correlation with clinical pathological characteristics and prognosis, and to further explore the roles it plays in the pancreatic ductal adenocarcinoma carcinogenesis.METHODS:1. MiR-483-3p levels were evaluated by Locked Nucleic Acid-In Situ Hybridization in a tissue microarray (63PDAC tumors and10normal pancreatic tissues).2. MiR-483-3p expression patterns were verified in an independent set of117pairs of matched PDAC tumors and adjacent tumor-free pancreatic tissues, including65(58.8%) male and51(41.2%) female cases. The correlations between miR-483-3p expression and the clinicopathological factors were also analyzed. And survival analysis was performed in the94cases of patients with complete follow-up data using log-rank method, univariate and multivariate Cox proportional hazard regression model, respectively.3. Expression of miR-483-3p was further evaluated in82pancreatic intra-epithelial neoplasias (PanINs)(39PanINls,27PanIN2s and16PanIN3s) and11chronic pancreatitis (CPs).4. CCK-8assay, Edu assay, soft agar colony formation assay, PI staining, and PI/FITC staining were used for observing the impact of miR-483-3p on cell proliferation, cell cycle, and apoptosis in PANC-1and MIA PaCa-2cell lines, in which miR-483-3p were highly expressed. A subcutaneous transplantation tumor model was examined to test the role of miR-483-3p in vivo.RESULTS:1. Results of tissue microarray showed miR-483-3p was positively expressed in61(96.8%) PDAC samples and in all normal pancreatic acinar tissue, but negatively stained in normal pancreatic duct tissue.2. In the117pancreatic ductal adenocarcinoma cases, miR-483-3p was100%positive, with55.6%(65/117) strong positive staining. The corresponding non-tumor normal pancreatic acinar all showed strong positive staining of miR-483-3p. However, miR-483-3p was only weakly positive in13.7%(16/117) adjacent normal pancreatic duct tissues. The relationships between miR-483-3p and clinicopathological features of pancreatic ductal adenocarcinoma were analyzed, and miR-483-3p expression was found correlated with tumor differentiation (p<0.05). The strong positivity rate was81.3%(26/32) in well-differentiated,75.5%(37/49) in moderated-differentiated, and33.3%(12/36) in poor-differentiated tumors, respectively. The correlations between miR-483-3p expression and survival were also analyzed. The group of patients with high miR-483-3p expression had significantly shorter survival time than that with low miR-483-3p expression (median overall survival:22.0±4.2versus34.0±9.3months; P=0.040), according to the log-rank analysis. Univariate Cox regression model analysis revealed high miR-483-3p expression was correlated with poor prognosis (HR=1.807; CI=1.012-3.22795%; P=0.045). Multivariate Cox proportional hazard regression model analysis also showed that high expression of miR-483-3p was an independent factor indicating poor prognosis (HR=2.584; CI=1.268-5.26495%; P=0.009).3. Pancreatic intraepithelial neoplasias (PanINs) are the most common noninvasive precurious lesions of pancreatic ductal adenocarcinoma (PDAC). Among the39PanIN-1cases, miR483-3p was weakly positive (1+) in19(48.7%) cases, and negative (-) in the other20(51.3%) cases. Correspondingly, among the27PanIN-2cases,4cases (14.8%) showed strong positivity (2+), and the other23(85.2%) cases showed weak positivity (1+). As to the16PanIN-3cases,14(87.5%) cases were strong positive staining(2+), and2(12.5%) cases were weak positive staining (1+). From PanIN-1to PanIN-3, the strong positive rate of miR-483-3p was0/39(0%),4/27(14.8%) and14/16(87.5%), respectively. Obviously, the expression of miR-483-3p increased from PanIN-1to PanIN-3.(p<0.001). In the11cases of chronic pancreatitis, only6(54.5%) cases showed weak positive expression (1+) of miR-483-3p, the others were all negative(-). According to the above results, the aberrant expression of miR-483-3p occurred in the early stage in the carcinogenesis of pancreatic ductal adenocarcinoma4. Inhibition of miR-483-3p expression can affect pancreatic cancer cell growth, proliferation, cell cycle, and apoptosis in vitro. Intratumoral injection of miR-483-3p antagomir was found significantly suppressed tumor growth compared to the control group with injection of control antagomir, the difference was statistically significant (P<0.05).CONCLUSIONS:The aberrant expression of miR-483-3p is an early event in pancreatic ductal adenocarcinoma tumorigenesis. MiR-483-3p aberrant expression is associated with differentiation, and its high expression indicates a poor prognosis. Inhibition of miR-483-3p expression suppressed the growth of pancreatic cancer cells in vitro and in vivo, therefore it may be a potential therapeutic target in pancreatic ductal adenocarcinoma. 二.The study of miR-483-3p in diagnosis and prognosis of adrenal cortical tumorsBACKGROUND:The treatment and prognosis of adrenal cortical carcinoma are very different from adrenal cortical adenoma. However, the differential diagnosis of the two entities is very difficult. The purpose of this study is to determine clinicopathological criteria and molecular markers helpful in distinguishing adrenocortical carcinomas (ACCs) from adrenocortical adenomas (ACAs).METHODS:Samples of adrenal tumor were collected from patients undergoing adrenal ectomy at Peking X X Hospital, Chinese Academy of Medical Sciences, from July2001to July2012.Twenty-five malignant tumor were available and used as experimental samples, and25benign tumor selected randomly from1564patients with adrenocortical adenomas (ACAs) were used as control samples. All50tumor had full clinical and pathological data. We retrospectively analyzed the clinical and pathological features of50adrenal cortical tumors, and tested the expression of miR-483-3p by in-situ hybridization as well as the expression of IGF2and Smad4by immunohistochemistry. The relationships between the expression of miR-483-3p and IGF2, Smad4were respectively analyzed. And correlations of the three markers with clinicopathological characteristics were also analyzed. Furthermore, the expression of MIB-1index was also validated in the same50adrenal samples, and the relationships between MIB-1and the above three indicators were also analyzed. To further validate the differential diagnostic efficacy of the molecular markers, their expression levels were evaluated in an independent set of15borderline tumors (Weiss score=2or3).RESULTS:The retrospective analysis of clinical data and WEISS system evaluation revealed that tumor size, tumor weight, hormonal function and the Weiss system are all high-efficacy criteria for differentiating malignant from benign tumors (P <0.001). MiR-483-3p was overexpressed in68%(17of25) of ACCs compared to12%(3of25) of ACAs (P<0.05). Moreover, the relationships between the expression of miR-483-3p, IGF2and Smad4were also analyzed. MiR-483-3p expression was found positively correlated with the expression of IGF2(P<0.05), but no significant relationship with Smad4expression (P>0.05). Using a combination of miR-483-3p and Smad4improved diagnostic accuracy. Moreover, overexpression of Smad4is a prognostic indicator of well disease outcome. MIB-1was highly expressed in64%(16/25)ACCs, but only in4%(1/25) ACAs. However, there were no statistical correlations between the expression of MIB-1and the above three biomarkers. These molecular markers were then tested in an independent set of15borderline tumors, confirming that the combined use of miR-483-3p and Smad4can complement the Weiss score in the diagnosis of ACC in cases that display borderline histology.CONCLUSIONS:Tumor size, tumor weight, hormonal function and the Weiss system are useful clinicopathological criteria that can result in accurate diagnosis of most ACCs and ACAs. In challenging cases, miR-483-3p and Smad4expression may help in distinguishing these two entities. |
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