METHODS: Immunofluorescence staining was used to observe the structural features of PC12 cells after culturing in medium with nerve growth factor (NGF). After different doses and different durations of alcohol treatment, CCK-8 assay was performed to detect the viability of PC12 cells, flow cytometry assay was carried out to detect the apoptosis rate of PC12 cells, dual-luciferase reporter assay was used to definitude the regulatory relationship between miR-96-5p and Tp73, and western blot was used to detect the protein expression of TAp73.
RESULTS: The result of immunofluorescence staining demonstrated that PC12 cells abundantly expressed Map2, CCK-8 assay illustrated alcohol exposure significantly downregulated the cell viability of PC12 cells, Treatment with miR-96-5p inhibitor induced apoptosis and upregulated the expression of TAp73 in PC12 cells. Contrastingly, miR-96-5p mimic reversed the above effects and downregulation of TAp73 inhibited the apoptosis of PC12 cells.
CONCLUSION: The present study demonstrated that miR-96-5p participates in alcohol-induced apoptosis in PC12 cells via negatively regulating TAp73.
MATERIALS AND METHODS: In mutation screening of CRNN gene, gDNA from OSCC tissues were extracted, amplified, and followed by direct sequencing. OSCC samples were also subjected to fragment analysis on CRNN gene to investigate its microsatellite instability (MSI) and loss of heterozygosity (LOH). Immunohistochemistry was performed to validate CRNN downregulation in OSCC samples.
RESULTS: No pathogenic mutation was found in CRNN gene, while high frequency of allelic imbalances was found at 1q21.3 region. MSI was found more frequent (25.3 %) than LOH (9.3 %). Approximately 22.6 % of cases had high MSI which reflects higher probability of inactivation of DNA mismatch repair genes. MSI showed significant association with no betel quid chewing (p = 0.003) and tongue subsite (p = 0.026). LOH was associated with ethnicity (p = 0.008) and advanced staging (p = 0.039). The LOH at 1q21.3 was identified to be as an independent prognostic marker in OSCC (HRR = 7.15 (95 % CI, 1.41-36.25), p = 0.018). Downregulation of CRNN was found among MSI-positive OSCCs and was associated with poor prognosis (p = 0.044).
CONCLUSION: This study showed a significant correlation between LOH/MSI at 1q21.3 with clinical outcomes and that downregulation of CRNN gene could be considered as a prognostic marker of OSCC.
CLINICAL RELEVANCE: Insights of the downregulation mode of CRNN gene lays the basis of drug development on this gene as well as revealing its prognostic value.
METHODS: In vitro cell model of IDD was established by treating human nucleus pulposus cells (HNPCs) with interleukin-1β (IL-1β). The level of peroxisome proliferator-activated receptor γ (PPARγ) was examined in the IDD cell model by Western blot and quantification real-time reverse transcription-polymerase chain reaction (qRT-PCR). The expression level of miR-96-5p was detected by RT-qPCR. Effects of PPARγ or/and PPARγ agonist on inflammatory factors, extracellular matrix (ECM), apoptosis, and nuclear factor-kappaB (NF-κB) nuclear translocation were examined through enzyme-linked immunosorbent assay (ELISA), Western blot, flow cytometry assay, and immunofluorescence staining. The Starbase database and dual luciferase reporter assay were used to predict and validate the targeting relationship between miR-96-5p and PPARγ, and rescue assay was performed to gain insight into the role of miR-96-5p on IDD through PPARγ/NF-κB signaling.
RESULTS: PPARγ expression reduced with concentration and time under IL-1β stimulation, while miR-96-5p expression showed the reverse trend (P
METHODS: Immunoblotting, immunofluorescence, quantitative real-time PCR and flow cytometry assays investigated the expression of E-cadherin and CXCR3 isoforms. Intrasplenic inoculation of human prostate cancer (PCa) cells with spontaneous metastasis to the liver analyzed E-cadherin and CXCR3-B expression during cancer progression in vivo.
RESULTS: We found reciprocal regulation of E-cadherin and CXCR3 isoforms. E-cadherin surface expression promoted CXCR3-B presentation on the cell membrane, and to a lesser extent increased its mRNA and total protein levels. In turn, forced expression of CXCR3-A reduced E-cadherin expression level, whereas CXCR3-B increased E-cadherin in PCa. Meanwhile, a positive correlation of E-cadherin and CXCR3-B expression was found both in experimental PCa liver micro-metastases and patients' tissue.
CONCLUSIONS: CXCR3-B and E-cadherin positively correlated in vitro and in vivo in PCa cells and liver metastases, whereas CXCR3-A negatively regulated E-cadherin expression. These results suggest that CXCR3 isoforms may play important roles in cancer progression and dissemination via diametrically regulating tumor's phenotype.