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Furthermore, a subset of miRNAs was found to be generated by a DICER1-dependent but DROSHA -independent mechanism, explaining the distinct and non-overlapping phenotypes caused by loss of DROSHA and DICER1

Furthermore, a subset of miRNAs was found to be generated by a DICER1-dependent but DROSHA -independent mechanism, explaining the distinct and non-overlapping phenotypes caused by loss of DROSHA and DICER1.32 Reduced DROSHA, but not DICER1, expression in human mesenchymal stem cells was demonstrated to significantly reduce proliferation rate.33 In addition, unlike DICER1 knockdown, DROSHA knockdown human mesenchymal stem cells contained an increased number of G1 phase cells, with a reduced level of cells in S phase, accompanied by decreased pRB, 28S and 18S rRNA expression and increased p16 and p15 (two key regulators of the G1/S phase transition) expression, indicating that DROSHA modifies human mesenchymal stem cells proliferation through a miRNA independent mechanism.33 Our data Bax-activator-106 demonstrating further reduction in survival rate of the melanoma cases with negative DICER1-negative and DROSHA expression compared with that of the cases with loss of either DICER1 or DROSHA (Figure 5) also indicate the possible existence of at least some non-overlapping functions of these two factors. pattern of DROSHA varies from that of DICER1 and concomitant loss of expression of both DICER1 and DROSHA confers the worse outcome for melanoma patients. Our results demonstrate a reduced nuclear expression of DROSHA which further highlights a perturbed miRNA biogenesis pathway in melanoma. In addition, the aberrant subcellular localization of DROSHA indicates possible deregulation in the mechanisms responsible for its proper localization in the nucleus. value of less than 0.05 was considered significant. Results Reduced nuclear DROSHA staining correlates with melanoma progression We used a polyclonal rabbit antibody raised against the N-terminal (residues 1C100) of human DROSHA protein to investigate its expression pattern in 409 melanocytic lesions (29 normal nevi, 43 dysplastic nevi, 226 primary melanomas, and 111 metastatic melanomas). We observed a predominant nuclear DROSHA staining in different samples (Figure 1a). We also detected some cytoplasmic signal with this antibody in melanocytic lesions in all stages. A significant difference in nuclear DROSHA staining was observed between different stages of melanoma. Kruskal-Wallis test revealed a clear reduction in the expression of nuclear DROSHA during melanoma progression (= 0.0001; Figure 1b). We found significant reduction in expression of nuclear DROSHA from normal nevi to dysplastic nevi (= 0.002) and from dysplastic nevi to primary melanomas (= 0.0001) but not between primary melanomas and Bax-activator-106 metastatic melanomas (= 0.052). Similarly, when we divided the samples from each stage into two groups based on expression of nuclear DROSHA, we observed an increase in percentage of samples with no nuclear DROSHA staining during melanoma progression (= 0.0001; Figure 1c). Accordingly, while 82.7% of normal nevi and 62.7% of dysplastic nevi had Bax-activator-106 positive nuclear staining for DROSHA, only 26.1% of primary melanomas and 17.1% of metastatic melanomas stained positive for nuclear DROSHA. Open in a separate window Figure 1 Reduced expression of nuclear Drosha correlates with melanoma progression. (a) Representative images of normal nevi (NN) and dysplastic nevi (DN) with strong nuclear Drosha staining, primary melanoma (PM) with weak staining, and metastatic melanoma (MM) with negative nuclear Bax-activator-106 Drosha staining. Scale bar = 50 m. (b) Kruskal-Wallis test for differences in nuclear Drosha staining among NN, DN, PM, and MM. Median is depicted as a horizontal line in each group (= 0.0001). (c) Chi-square test for differences in nuclear Drosha staining in NN, DN, PM, and MM. Significant difference was found between DN Rabbit polyclonal to RB1 and PM (0.0001) but not NN and DN (0.068) or PM and MM (0.066). Inverse correlation between nuclear DROSHA staining and tumor thickness, AJCC staging and ulceration status To assess whether reduced nuclear DROSHA staining correlates with clinicopathologic variables of the patients, we examined the expression pattern of nuclear DROSHA in 226 primary melanoma samples (Table 1). Although DROSHA staining did not have a significant correlation with patients age or sex, location of tumor and lymphocytic response, it showed a significant inverse correlation with tumor thickness (Breslows depth of invasion). Accordingly, percentage of samples with positive staining for nuclear DROSHA reduced from 41.0% in tumors 1 mm thick to 18.2% in tumors thicker than 1 mm (= 0.0001, 2 test; Figure 2a). We also observed an inverse correlation between expression of nuclear DROSHA and ulceration status of the melanoma patients. While 30.6% of the samples without ulceration stained positive for nuclear DROSHA, only 6.9% of those with ulceration had positive nuclear DROSHA staining (= 0.002, 2 Bax-activator-106 test; Table 1). In addition, when compared the nuclear DROSHA staining between different subtypes of melanoma, we found that lentigo maligna and superficial spreading subtypes express less DROSHA than other subtypes (=.