MicroRNAs (miRNAs) are noncoding small RNAs that regulate gene expression at the translational level by mainly interacting with 3′ UTRs of their target mRNAs. Archived formalin-fixed paraffin-embedded (FFPE) specimens represent excellent resources for biomarker discovery. Currently there is a lack of systematic analysis on the stability of miRNAs and optimized conditions for expression analysis using FFPE samples. In this study, the expression of miRNAs from FFPE samples was analyzed using high-throughput locked nucleic acid-based miRNA arrays. The effect of formalin fixation on the stability of miRNAs was also investigated using miRNA real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The stability of miRNAs of archived colorectal cancer FFPE specimens was characterized with samples dating back up to 10 yr. Our results showed that the expression profiles of miRNAs were in good correlation between 1 μg of fresh frozen and 1–5 μg of FFPE samples (correlation coefficient R² = 0.86–0.89). Different formalin fixation times did not change the stability of miRNAs based on real-time qRT-PCR analysis. There are no significant differences of representative miRNA expression among 40 colorectal cancer FFPE specimens. This study provides a foundation for miRNA investigation using FFPE samples in cancer and other types of diseases.

The process of malignant transformation, progression and metastasis of melanoma is poorly understood. Gene expression profiling of human cancer has allowed for a unique insight into the genes that are involved in these processes. Thus, we have attempted to utilize this approach through the analysis of a series of primary, non-metastatic cutaneous tumors and metastatic melanoma samples. We have utilized gene microarray analysis and a variety of molecular techniques to compare 40 metastatic melanoma (MM) samples, composed of 22 bulky, macroscopic (replaced) lymph node metastases, 16 subcutaneous and 2 distant metastases (adrenal and brain), to 42 primary cutaneous cancers, comprised of 16 melanoma, 11 squamous cell, 15 basal cell skin cancers. A Human Genome U133 Plus 2.0 array from Affymetrix, Inc. was utilized for each sample. A variety of statistical software, including the Affymetrix MAS 5.0 analysis software, was utilized to compare primary cancers to metastatic melanomas. Separate analyses were performed to directly compare only primary melanoma to metastatic melanoma samples. The expression levels of putative oncogenes and tumor suppressor genes were analyzed by semi- and real-time quantitative RT-PCR (qPCR) and Western blot analysis was performed on select genes. We find that primary basal cell carcinomas, squamous cell carcinomas and thin melanomas express dramatically higher levels of many genes, including SPRR1A/B, KRT16/17, CD24, LOR, GATA3, MUC15, and TMPRSS4, than metastatic melanoma. In contrast, the metastatic melanomas express higher levels of genes such as MAGE, GPR19, BCL2A1, MMP14, SOX5, BUB1, RGS20, and more. The transition from non-metastatic expression levels to metastatic expression levels occurs as melanoma tumors thicken. We further evaluated primary melanomas of varying Breslow's tumor thickness to determine that the transition in expression occurs at different thicknesses for different genes suggesting that the "transition zone" represents a critical time for the emergence of the metastatic phenotype. Several putative tumor oncogenes (SPP-1, MITF, CITED-1, GDF-15, c-Met, HOX loci) and suppressor genes (PITX-1, CST-6, PDGFRL, DSC-3, POU2F3, CLCA2, ST7L), were identified and validated by quantitative PCR as changing expression during this transition period. These are strong candidates for genes involved in the progression or suppression of the metastatic phenotype. The gene expression profiling of primary, non-metastatic cutaneous tumors and metastatic melanoma has resulted in the identification of several genes that may be centrally involved in the progression and metastatic potential of melanoma. This has very important implications as we continue to develop an improved understanding of the metastatic process, allowing us to identify specific genes for prognostic markers and possibly for targeted therapeutic approaches.

Paclitaxel (Taxol) is an effective chemotherapeutic agent for treatment of cancer patients. Despite impressive initial clinical responses, the majority of patients eventually develop some degree of resistance to Taxol-based therapy. The mechanisms underlying cancer cells resistance to Taxol are not fully understood. MicroRNA (miRNA) has emerged to play important roles in tumorigenesis and drug resistance. However, the interaction between the development of Taxol resistance and miRNA has not been previously explored. In this study we utilized a miRNA array to compare the differentially expressed miRNAs in Taxol-resistant and their Taxol-sensitive parental cells. We verified that miR-125b, miR-221, miR-222, and miR-923 were up-regulated in Taxol-resistant cancer cells by real-time PCR. We further investigated the role and mechanisms of miR-125b in Taxol resistance. We found that miR-125b was up-regulated in Taxol-resistant cells, causing a marked inhibition of Taxol-induced cytotoxicity and apoptosis and a subsequent increase in the resistance to Taxol in cancer cells. Moreover, we demonstrated that the pro-apoptotic Bcl-2 antagonist killer 1 (Bak1) is a direct target of miR-125b. Down-regulation of Bak1 suppressed Taxol-induced apoptosis and led to an increased resistance to Taxol. Restoring Bak1 expression by either miR-125b inhibitor or re-expression of Bak1 in miR-125b-overexpressing cells recovered Taxol sensitivity, overcoming miR-125-mediated Taxol resistance. Taken together, our data strongly support a central role for miR-125b in conferring Taxol resistance through the suppression of Bak1 expression. This finding has important implications in the development of targeted therapeutics for overcoming Taxol resistance in a number of different tumor histologies.

MicroRNAs are small and non-coding RNA molecules with the master role in regulation of gene expression at post-transcriptional/translational levels. Many methods have been developed for microRNA loss-of-function study, such as antisense inhibitors and sponges; however, the robustness, specificity, and stability of these traditional strategies are not highly satisfied. CRISPR/cas9 system is emerging as a novel genome editing tool in biology/medicine research, but its indication in microRNA research has not been studied exclusively. In this study, we clone CRISPR/cas9 constructs with single-guide RNAs specifically targeting biogenesis processing sites of selected microRNAs; and we find that CRISPR/cas9 can robustly and specifically reduce the expression of these microRNAs up to 96%. CRISPR/cas9 also shows an exclusive benefit in control of crossing off-target effect on microRNAs in the same family or with highly conserved sequences. More significantly, for the first time, we demonstrate the long term stability of microRNA knockdown phenotype by CRISPR/cas9 in both in vitro and in vivo models.

Purpose: The aim of this study was to investigate the role of p53 in regulating micro-RNA (miRNA) expression due to its function as a transcription factor. In addition, p53 may also affect other cellular mRNA gene expression at the translational level either via its mediated miRNAs or due to its RNA-binding function. Experimental Design: The possible interaction between p53 and miRNAs in regulating gene expression was investigated using human colon cancer HCT-116 (wt-p53) and HCT-116 (null-p53) cell lines. The effect of p53 on the expression of miRNAs was investigated using miRNA expression array and real-time quantitative reverse transcription-PCR analysis. Results: Our investigation indicated that the expression levels of a number of miRNAs were affected by wt-p53. Down-regulation of wt-p53 via small interfering RNA abolished the effect of wt-p53 in regulating miRNAs in HCT-116 (wt-p53) cells. Global sequence analysis revealed that over 46% of the 326 miRNA putative promoters contain potential p53-binding sites, suggesting that some of these miRNAs were potentially regulated directly by wt-p53. In addition, the expression levels of steady-state total mRNAs and actively translated mRNA transcripts were quantified by high-density microarray gene expression analysis. The results indicated that nearly 200 cellular mRNA transcripts were regulated at the posttranscriptional level, and sequence analysis revealed that some of these mRNAs may be potential targets of miRNAs, including translation initiation factor eIF-5A, eIF-4A, and protein phosphatase 1. Conclusion: To the best of our knowledge, this is the first report demonstrating that wt-p53 and miRNAs interact in influencing gene expression and providing insights of how p53 regulates genes at multiple levels via unique mechanisms.