Keratinization-associated miR-7 and miR-21 regulate tumor suppressor reversion-inducing cysteine-rich protein with kazal motifs (RECK) in oral cancer.

MicroRNAs (miRNAs) are small non-coding RNAs that posttranscriptionally regulate gene expression during many biological processes. Recently, the aberrant expressions of miRNAs have become a major focus in cancer research. The purpose of this study was to identify deregulated miRNAs in oral cancer and further focus on specific miRNAs that were related to patient survival. Here, we report that miRNA expression profiling provided more precise information when oral squamous cell carcinomas were subcategorized on the basis of clinicopathological parameters (tumor primary site, histological subtype, tumor stage, and HPV16 status). An innovative radar chart analysis method was developed to depict subcategories of cancers taking into consideration the expression patterns of multiple miRNAs combined with the clinicopathological parameters. Keratinization of tumors and the high expression of miR-21 were the major factors related to the poor prognosis of patients. Interestingly, a majority of the keratinized tumors expressed high levels of miR-21. Further investigations demonstrated the regulation of the tumor suppressor gene reversion-inducing cysteine-rich protein with kazal motifs (RECK) by two keratinization-associated miRNAs, miR-7 and miR-21. Transfection of miR-7 and miR-21-mimics reduced the expression of RECK through direct miRNA-mediated regulation, and these miRNAs were inversely correlated with RECK in CAL 27 orthotopic xenograft tumors. Furthermore, a similar inverse correlation was demonstrated in CAL 27 cells treated in vitro by different external stimuli such as trypsinization, cell density, and serum concentration. Taken together, our data show that keratinization is associated with poor prognosis of oral cancer patients and keratinization-associated miRNAs mediate deregulation of RECK which may contribute to the aggressiveness of tumors.

Can Retroperitoneal Lymph Node Dissection (RPLND) be feasibly performed to prolong survival in Renal Cell Carcinoma (RCC) with limited lymph node involvement? An Analysis of Recurrence Patterns.

INTRODUCTION: The therapeutic benefit of performing a lymph node dissection (LND) in patients with renal cell carcinoma (RCC) has been controversial. In prior studies, it was thought that a low event rate for nodal metastases affected the ability to draw any conclusions. Here, we opted to select patients that had low burden 1 or 2 nodes positive to study survival outcomes and recurrence patterns based on limited LND or extended LND with a template retroperitoneal lymph node dissection (RPLND). METHODS: We used our single institutional database from 2000 and 2019 and identified 45 patients that had only 1 or 2 nodes positive on final pathology without any other systemic disease. These patients all underwent nephrectomy with limited LND or a template RPLND on the ipsilateral side. RESULTS: We identified 23 patients in the limited LND and 22 in the template RPLND group. Thirty-one patients included in the study had 1 positive lymph node and 14 patients had 2 positive lymph nodes. For patients undergoing a limited LND, a median 4 (IQR 1-11) lymph nodes were resected and for those undergoing template RPLND, 18 (IQR: 13-23) lymph nodes were resected. On Kaplan-Meier analysis, a difference was noted in overall survival (P = 0.04) when comparing limited LND to template RPLND. We also mapped out patterns of recurrence and found that 6 patients had retroperitoneal lymph node recurrences after a limited LND in the ipsilateral node packet. On univariate analysis, pathologic stage was a major factor for survival, but did not remain as significant with the inclusion of template RPLND status and Charlson Comorbidity Index in multivariate analysis. CONCLUSION: We identified specific patients that had RCC with limited lymph node involvement. We found that a select number of patients had durable improvement in survival outcomes with template RPLND. In examining the recurrence patterns, a greater number of patients may have derived benefit for an initial template RPLND.

Overexpression of dicer as a result of reduced let-7 MicroRNA levels contributes to increased cell proliferation of oral cancer cells.

Recent reports have demonstrated that Dicer, an RNase III endonuclease required for microRNA (miRNA) maturation, is aberrantly expressed in different types of cancer. Furthermore, Dicer has been reported to be regulated by the let-7 family of miRNA genes. We hypothesize that Dicer is aberrantly expressed in oral cancer cells due to altered expressions of let-7 and that Dicer contributes to the development and progression of the disease. Western blot examination of Dicer protein levels in four head and neck squamous cell carcinoma (HNSCC) cell lines, including two oral cancer cell lines, demonstrated that Dicer had between 4- and 24-fold higher expression levels when compared to normal human primary gingival epithelial cells. Furthermore, five of six oral cancer tissues analyzed by indirect immunofluorescence had increased Dicer protein expression, compared to normal gingival epithelial tissue. The Dicer mRNA levels were not found to correlate well with protein expression in the HNSCC cell lines, suggesting that Dicer protein expression was post-transcriptionally regulated. Analysis of let-7a and let-7b levels in HNSCC cell lines by real-time PCR demonstrated that let-7b, but not let-7a, was significantly reduced in the HNSCC cell lines compared to control cells. Lastly, transfection of oral cancer cells with chemically synthesized let-7b and small interfering RNAs targeting Dicer significantly inhibited cell proliferation up to 83% and >100%, respectively, as early as 3 days post-transfection. Together, these data demonstrate that elevated expression levels of Dicer in oral cancer cells correlate with downregulation of let-7b and increased cell proliferation.

Tumor suppressor miR-375 regulates MYC expression via repression of CIP2A coding sequence through multiple miRNA-mRNA interactions.

MicroRNAs (miRNAs) are small, noncoding RNAs involved in posttranscriptional regulation of protein-coding genes in various biological processes. In our preliminary miRNA microarray analysis, miR-375 was identified as the most underexpressed in human oral tumor versus controls. The purpose of the present study is to examine the function of miR-375 as a candidate tumor suppressor miRNA in oral cancer. Cancerous inhibitor of PP2A (CIP2A), a guardian of oncoprotein MYC, is identified as a candidate miR-375 target based on bioinformatics. Luciferase assay accompanied by target sequence mutagenesis elucidates five functional miR-375-binding sites clustered in the CIP2A coding sequence close to the C-terminal domain. Overexpression of CIP2A is clearly demonstrated in oral cancers, and inverse correlation between miR-375 and CIP2A is observed in the tumors, as well as in NCI-60 cell lines, indicating the potential generalized involvement of the miR-375-CIP2A relationship in many other cancers. Transient transfection of miR-375 in oral cancer cells reduces the expression of CIP2A, resulting in decrease of MYC protein levels and leading to reduced proliferation, colony formation, migration, and invasion. Therefore this study shows that underexpression of tumor suppressor miR-375 could lead to uncontrolled CIP2A expression and extended stability of MYC, which contributes to promoting cancerous phenotypes.

High resolution of microRNA signatures in human whole saliva.

OBJECTIVE: Identifying discriminatory human salivary RNA biomarkers reflective of disease in a low-cost non-invasive screening assay is crucial to salivary diagnostics. Recent studies have reported both mRNA and microRNA (miRNA) in saliva, but little information has been documented on the quality and yield of RNA collected. Therefore, the aim of the present study was to develop an improved RNA isolation method from saliva and to identify major miRNA species in human whole saliva. DESIGN: RNA samples were isolated from normal human saliva using a combined protocol based on the Oragene RNA collection kit and the mirVana miRNA isolation kit in tandem. RNA samples were analysed for quality and subjected to miRNA array analysis. RESULTS: RNA samples isolated from twenty healthy donors ranged from 2.59 to 29.4 µg/ml saliva and with 1.92-2.16OD(260/280 nm) ratios. RNA yield and concentration of saliva samples were observed to be stable over 48 h at room temperature. Analysis of total salivary RNA isolated from these twenty donors showed no statistical significance between sexes; however, the presence of high-, medium-, and low-yield salivary RNA producers was detected. MiRNA array analysis of salivary RNA detected five abundantly expressed miRNAs, miR-223, miR-191, miR-16, miR-203, and miR-24, that were similarly described in other published reports. Additionally, many previously undetected miRNAs were also identified. CONCLUSION: High quality miRNAs can be isolated from saliva using available commercial kits, and in future studies, the availability of this isolation protocol may allow specific changes in their levels to be measured accurately in various relevant diseases.