Let-7d microRNA affects mesenchymal phenotypic properties of lung fibroblasts.

MicroRNAs are small noncoding RNAs that inhibit protein expression. We have previously shown that the inhibition of the microRNA let-7d in epithelial cells caused changes consistent with epithelial-to-mesenchymal transition (EMT) both in vitro and in vivo. The aim of this study was to determine whether the introduction of let-7d into fibroblasts alters their mesenchymal properties. Transfection of primary fibroblasts with let-7d caused a decrease in expression of the mesenchymal markers α-smooth muscle actin, N-cadherin, fibroblast-specific protein-1, and fibronectin, as well as an increase in the epithelial markers tight junction protein-1 and keratin 19. Phenotypic changes were also present, including a delay in wound healing, reduced motility, and proliferation of fibroblasts following transfection. In addition, we examined the effects of transfection on fibroblast responsiveness to TGF-ß, an important factor in many fibrotic processes such as lung fibrosis and found that let-7d transfection significantly attenuated high-mobility group-A2 protein induction by TGF-ß. Our results indicate that administration of the epithelial microRNA let-7d can significantly alter the phenotype of primary fibroblasts.

Nucleic acids absorbance in Mid IR and its effect on diagnostic variates during cell division: a case study with lymphoblastic cells.

The differences in the absorbance in the mid infrared region (Mid IR) between normal and abnormal tissues have been shown to be a possible criterion for the detection of different forms of cancer. The present work aimed to study the effects of cell growth and DNA synthesis on the RNA/DNA ratio, which is a promising parameter for cancer diagnosis by inducing synthesis of DNA. Lymphoblastic cell lines were synchronized by methotrexate and harvested for Fourier transform infrared microscopy (FTIR-MSP) at 30-min interval after thymidine addition. The distribution of DNA in the cells/nuclei and variation of nuclear size was studied using the fluorescent dye propidium iodide (PI) and the nuclear volume was calculated from microscopy. We analyzed the ratio of RNA and DNA to quantify the relative amounts during the process by taking the ratios I(1121)/I(1020) (symmetric) and the I(1244)/I(1230) (antisymmetric) phosphate vibrations. The pattern of changes in the ratios overtime obtained in the symmetric and antisymmetric vibrations were similar though the magnitudes were different. Only a minor effect on the RNA/DNA ratio (due to nuclear volume changes) was observed. The effect due to the unfolding of DNA is greatly masked due to RNA absorbance. The effectiveness of this ratio may lie in the increased transcriptional levels in carcinogenic tissues rather than changes occurring due to unfolding and folding of DNA.

[Numerical aberrations of chromosomes 11 and 17 detected by fish--fluorescence in situ hybridization combined with cytology in exfoliated cells from voided urine in patients with urothelial carcinoma of the bladder].

OBJECTIVES: To study the use of FISH analysis for detecting the presence of numerical alterations of chromosomes 11 and 17 combined with cytology in exfoliated cells from voided urine as a method for diagnosis and follow-up in patients with urothelial carcinoma (UC) of the bladder. MATERIALS AND METHODS: During the period April 2005 till June 2006, three groups were studied. The first group included 15 patients without UC. The second group included 25 patients undergoing evaluation for suspected UC. The third group included 25 patients enrolled in cystoscopy follow-up for previous UC. All the patients underwent cystoscopy, cytologic examination and FISH analysis for centromeric probes 11 and 17 performed on voided urine. After diagnosing the bladder UC, the tumor was staged and graded according to the pathologic findings. The sensitivity and specificity of FISH and Cytology were assessed. Data were analyzed with t-test when comparing two groups, and using ANOVA test when comparing more than two groups. These statistical analyses were executed with statistical software PRISM version 4.03. RESULTS: The sensitivity of FISH when using the centromeric probes of chromosomes 11 and 17 was 95.2%. The specificity was approximately 94.4%. The monosomy, trisomy, and polysomy in the patients with UC were 95.2%, 78.6% and 35.7% (p < 0.05) respectively. FISH was positive in 92.3% (24/26) in low grade tumors and in 100% (16/16) in high grade tumors (p > 0.05). The sensitivity of cytology was 31%. The cytology was positive in 23% (6/26) in low grade tumors and in 43.8% (7/16) in high grade tumors (p < 0.05). CONCLUSIONS: FISH analysis using centromeric probes of chromosomes 11 and 17 is an effective noninvasive method for the detection of altered chromosome numbers in bladder cancer cells in urine exfoliated cells. The sensitivity of FISH is higher than that of cytology in detecting UC. The combined analysis of FISH and cytology, does improve the accuracy of cytology but does not improve the specificity. Monosomy is the most prevalent numerical aberration found in patients with UC of the bladder. FISH analysis might give better results especially when cytology is negative. This method may help to decrease the frequency of cystoscopies in the follow-up of patients with confirmed bladder UC. Using multi-probe FISH test may improve the sensitivity. However, further studies are needed to confirm our results and conclusions.

Fluorescence in situ hybridization performed on exfoliated urothelial cells in patients with transitional cell carcinoma of the bladder.

OBJECTIVES: To evaluate comparatively fluorescence in situ hybridization (FISH) and cytology performed on exfoliated urothelial cells obtained from voided urine and bladder washings as a method of diagnosis and follow-up in patients with transitional cell carcinoma (TCC) of the bladder. METHODS: Thirty patients with confirmed bladder TCC, 10 patients enrolled in cystoscopy follow-up for previous bladder tumors, and 10 patients with bladders free of tumor without a previous history of bladder TCC underwent cytologic examination and FISH performed on voided urine and bladder washing specimens. The FISH probes were targeted to chromosomes 7 and 9. RESULTS: FISH had a sensitivity of 92% for high-grade tumors in both voided urine and bladder washing specimens, significantly greater than that of cytology at a sensitivity of 64% in voided urine and 67% in the bladder washing specimens (P = 0.02). The sensitivity of FISH and cytology were both low and not significantly different statistically from each other for the low-grade tumors. Monosomy of chromosome 9 correlated with early tumor recurrence. Polysomy of chromosomes 7 and 9 correlated with high-grade tumors (80% and 92%, respectively). CONCLUSIONS: According to our results, with the local cytopathology expertise, FISH performed on urothelial cells from voided urine has a sensitivity that supersedes that of cytology, making the former a valuable complementary method in the diagnosis and follow-up of patients with bladder TCC.