Synchronous detection of miRNAs, their targets and downstream proteins in transferred FFPE sections: applications in clinical and basic research.

After discovering new miRNAs, it is often difficult to determine their targets and effects on downstream protein expression. In situ hybridization (ISH) and immunohistochemistry (IHC) are two commonly used methods for clinical diagnosis and basic research. We used an optimized technique that simultaneously detects miRNAs, their binding targets and corresponding proteins on transferred serial formalin fixed paraffin embedded (FFPE) sections from patients. Combined with bioinformatics, this method was used to validate the reciprocal expression of specific miRNAs and targets that were detected by ISH, as well as the expression of downstream proteins that were detected by IHC. A complete analysis was performed using a limited number of transferred serial FFPE sections that had been stored for 1-4 years at room temperature. Some sections had even been previously stained with H&E. We identified a miRNA that regulates epithelial ovarian cancer, along with its candidate target and related downstream protein. These findings were directly validated using sub-cellular components obtained from the same patient sample. In addition, the expression of Nephrin (a podocyte marker) and Stmn1 (a recently identified marker related to glomerular development) were confirmed in transferred FFPE sections of mouse kidney. This procedure may be adapted for clinical diagnosis and basic research, providing a qualitative and efficient method to dissect the detailed spatial expression patterns of miRNA pathways in FFPE tissue, especially in cases where only a small biopsy sample can be obtained.

[Growth-inhibiting effect of psoralen plus ultraviolet-A light therapy on K562 cells].

OBJECTIVE: To observe the effects of psoralen plus ultraviolet-A light (PUVA) on K562 cells and the relative mechanism. METHODS: The effects of psoralen, ultraviolet-A light and PUVA on K562 cells were assayed by monotetrazolium test (MTT). DNA content was analyzed by flow cytometry (FCM). The apoptotic rates of K562 cells treated with 40 and 80 microg/ml psoralen for 24 and 48 hours were assayed by Annexin-V-FITC/PI reagent kit on FCM respectively. The ultrastructures of apoptotic cells were observed by a transmission electron microscope (TEM). RESULTS: Either single psoralen therapy or single ultraviolet-A irradiation had inhibiting effect on K562 cells. The inhibiting effect of PUVA on K562 cells was stronger than that of the single psoralen therapy or single ultraviolet-A light irradiation (P<0.05). Apoptotic peak (AP) was detected by FCM. TEM test showed that K562 cells treated with PUVA were smaller, having condensed cell nucleus, assembled chromatin, disintegrated nucleus body and the majority of the cells appeared to be apoptotic conformation. CONCLUSION: Psoralen has inhibiting effect on K562 cells, and the effect of PUVA is more significant. It is suggested that 10 min irradiation and 40 microg/ml terminal concentration of psoralen be probably the best choice for PUVA. The inhibiting effect of PUVA is due to apoptosis.

[Expression of aurora-A kinase in human lung cancer cell lines PG, A549, and NCI-H460].

BACKGROUND & OBJECTIVE: Recent researches found that Aurora-A overexpresses in various malignancies. This study was to detect the expression of Aurora-A in lung cancer cell lines PG (highly-metastatic giant cell lung cancer), A549 (lung adenocarcinoma), and NCI-H460 (large cell lung cancer) and explore its correlation to DNA content, provide a theoretical basis for screening tumor marker and molecular therapeutic target of lung cancer. METHODS: mRNA and protein levels of Aurora-A in PG, A549, and NCI-H460 cells were detected by reverse transcription-polymerase chain reaction(RT-PCR) and Western blot. Flow cytometry was used to analyze DNA contents in cell cycles of PG, A549, and NCI-H460 cells. RESULTS: mRNA level of Aurora-A was 1.14 in PG cells, 1.16 in A549 cells, and 0.84 in NCI-H460 cells, respectively; protein level of Aurora-A was 8.96 in PG cells, 21.13 in A549 cells, and 6.43 in NCI-H460 cells, respectively. The proportion of cells with tetraploid DNA was 19.88% in PG cells, 14.97% in A549 cells, and 10.6% in NCI-H460 cells, respectively (P<0.01); the proportion of cells with polyploid DNA was 2.66% in PG cells, 3.59% in A549 cells, and 2.30% in NCI-H460 cells, respectively. CONCLUSION: Aurora-A is overexpressed in the 3 lung cancer cell lines, but the mRNA levels are different.

[Effect of tanshinone microemulsion on reversing MDR in human tumor cells].

OBJECTIVE: To study the effect of tanshinone microemulsion (Tan-M) on the cytotoxicity to human leukemia-cell-line (K562/ADM) and the reversion of MDR in vitro. METHOD: Microemulsion being supposed as the control group, MT method is adopted to test cytotoxicity and the reverse of MDR. RESULT: Obvious cytotoxicity to K562/ADM was observed for tan-M. Cell non-toxic dosage (growth quotiety > 95%) of Tan-M is 0.2 microg x mL(-1). Low toxic dosage (growth quotiety 85-90%) was 0.7 microg x mL(-1). Cell non-toxic dosage of was 0.7 microg x mL(-1) and low toxic dosage was 1.2 microg x mL(-1). Cell non-toxic dosage of Tan-M (0.2 microg x mL(-1)) significantly lowered the IC50 of K562/ADM by ADM (P < 0.01), and reversed MDR was 3.88 times. Low toxic dosage of Tan-M reversed MDR was 3.97 times. E-M (0.2 microg x mL(-1)) reversed MDR was 2.62 times. CONCLUSION: The result indicates that tanshinone microemulsion possesses cell-toxic effects on human leukemia cell-line and may reverse MDR of tumor cells.