MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression at the post-transcriptionallevel. Since aberrant expression of miRNAs has been proposed as usage for blood-based biomarkers, hence reliabletechniques for miRNA isolation as well as stability of miRNAs in various stored conditions needs to be explored. Thispresent study aimed to investigate the efficacy of the Trizol-based isolation technique and the stability of miRNAs instored serum and cDNA derivatives. Total RNA, including miRNAs, was isolated from human serum and a comparisonof the efficiency of the Trizol®LS reagent isolation method against the miRNeasy®mini kit was conducted. Expressionof RNU6, miR-145, and miR-20a was determined by quantitative real-time polymerase chain reaction (qRT-PCR).We showed that Trizol®LS isolation yielded significantly lower RNA concentrations than that of the miRNeasy®minikit by approximately 35%. Purity of the isolated RNAs by both methods was similar. RNU6, miR-145, and miR-20adegraded at room temperature, but all genes were stable at 4ºC, -20ºC and -80ºC for a 72-hrs period, in both serum andcDNA storage conditions. In the stored cDNA derivatives, we observed the stability of RNU6, miR-145, and miR-20afor 3 months at -20ºC, and all genes also resisted 4 repeated freeze-thaw cycles at -20ºC. In conclusion, the Trizol-basedmethod is efficient as well as economical to use for quantification of circulating miRNAs. In addition, we proposedthat the storage of miRNA-derived cDNAs may be an alternative choice to avoid the stability effect.

Prognostic Significance of 14-3-3γ Overexpression in Advanced Non-Small Cell Lung Cancer 14-3-$3{\gamma}$;immunohistochemistry;non-small cell lung cancer;invasion;metastasis; The 14-3-3 protein has been shown to be involved in the cancer process. However, there is no understanding of the relationship between 14-3-$3{\gamma}$ (14-3-3 gamma) expression and prognosis in advanced non-small cell lung cancer. In this study, we therefore investigated the association between protein levels by immunohistochemistry and clinicopathological features of advanced NSCLC patients. Survival curves were estimated using the Kaplan-Meier method and tested by log-rank. Multivariate analysis was conducted with the Cox's regression model to determine independence of factors. p values less than 0.05 were considered significant. A total 153 patients were studied, with 54.3% being stage III and 45.8% stage IV. Fifty-one cases (33.3%) were squamous cell carcinomas, and 98 cases (64.1%) were adenocarcinomas. High 14-3-$3{\gamma}$ expression was seen in 59.5% and significantly correlated with lymph node metastasis (p=0.010) and distant metastasis (p=0.017). On Kaplan-Meier analysis, high 14-3-$3{\gamma}$ expression was associated with poorer survival with a marginal trend toward significance (p=0.055). On multivariate analysis, age, treatment, and 14-3-$3{\gamma}$ expression proved to be independent prognostic parameters. In vitro experiments indicated that 14-3-$3{\gamma}$ overexpression also played a potential role in cancer invasion. In conclusion, our data suggest that 14-3-$3{\gamma}$ overexpression is associated with invasion and a poor prognosis. Therefore, 14-3-$3{\gamma}$ may be a potential prognostic marker of advanced non-small cell lung cancer.

Serum miR-339-3p as a potential diagnostic marker for non-small cell lung cancer

Background/Aim: 14-3-3γ is involved in the metastasis of lung cancer cells. However, its functional roles in tumor cell invasion and the underlying mechanisms are still not understood. In this study, the roles and molecular mechanisms of 14-3-3γ in epithelial–mesenchymal transition (EMT), migration, and invasion were investigated using A549 and H358 non-small cell lung cancer (NSCLC) cell lines. Materials and Methods: siRNA against 14-3-3γ was used to suppress 14-3-3γ expression. Expression levels of proteins were detected by western blotting. Activity of matrix metalloproteinases (MMP)-2 and MMP9 was determined by gelatin zymography. Cell migration and invasion were analyzed using the Transwell assay. Results: Knockdown of 14-3-3γ resulted in a significant reduction of expression of EMT-associated proteins in NSCLC cell lines, and led to significant reduction in invasion and migration by approximately 59% and 39% in A549 cells, and 65% and 62% in H358 cells, respectively. In addition, MMP2 and MMP9 activity was significantly reduced in both NSCLC cell lines after down-regulation of 14-3-3γ. Conclusion: Our results suggest that knockdown of 14-3-3γ may be a potential strategy for suppressing metastasis of lung cancer by inhibiting MMP2 and MMP9 through regulation of EMT.

Detoxification and elimination of permethrin (PM) are mediated by hydrolysis via carboxylesterase (CES). Mitragyna speciosa (kratom) contains mitragynine (MG) and other bioactive alkaloids. Since PM and MG have the same catalytic site and M. speciosa is usually abused by adding other ingredients such as pyrethroid insecticides, the effects of MG and an alkaloid extract (AE) on the elimination of PM were investigated in rats. Rats were subjected to single and multiple pretreatment with MG and AE prior to receiving a single oral dose (460 mg/kg) of PM. Plasma concentrations of trans-PM and its metabolite phenoxybenzylalcohol (PBAlc) were measured. The elimination rate constant (k_el) and the elimination half-life (t_{1/2 el}) of PM were determined, as well as the metabolic ratio (PMR). A single and multiple oral pretreatment with MG and AE altered the plasma concentration-time courses of both trans-PM and PBAlc during 8–22 h, decreased the PMRs, delayed elimination of PM, but enhanced elimination of PBAlc. Results indicated that PM–MG or AE toxicokinetic interactions might have resulted from the MG and AE interfering with PM hydrolysis. The results obtained in rats suggest that in humans using kratom cocktails containing PM, there might be an increased risk of PM toxicity due to inhibition of PM metabolism and elimination.

Wilms tumor (WT) is an embryonal tumor occurring in developing kidney tissue. WT cells showing invasive cancer characteristics, also retain renal stem cell behaviours. In-vitro culture of WT is hampered by limited replicative potential. This study aimed to establish a longterm culture of WT cells to enable the study of molecular events to attempt to explain its cellular senescence. Methods: Primary cell cultures from fresh WT tumor specimen were established. Of 5 cultures tried, only 1 could be propagated for more than 7 passages. One culture, identified as PSU-SK-1, could be maintained > 35 passages and was then subjected to molecular characterization and evaluation for cancer characteristics. The cells consistently harbored concomitant mutations of CTNNB1 (Ser45Pro) and WT1 (Arg413Stop) thorough the cultivation. On Transwell invasion assays, the cells exhibited migration and invasion at 55% and 27% capability of the lung cancer cells, A549. On gelatin zymography, PSU-SK-1 showed high expression of the matrix metaloproteinase. The cells exhibited continuous proliferation with 24-hour doubling time until passages 28-30 when the growth slowed, showing increased cell size, retention of cells in G1/S proportion and positive β-galactosidase staining. As with those evidence of senescence in advanced cell passages, expression of p21 and cyclin D1 increased when the expression of β-catenin and its downstream protein, TCF, declined. There was also loss-of-expression of p53 in this cell line. In conclusion, cellular senescence was responsible for limited proliferation in the primary culture of WT, which was also associated with increased expression of p21 and was independent of p53 expression. Decreased activation of the Wnt signalling might explain the induction of p21 expression.