Chloroquine promotes gefitinib­induced apoptosis by inhibiting protective autophagy in cutaneous squamous cell carcinoma.

Aberrant expression of the epidermal growth factor receptor (EGFR) plays vital roles in tumor development and progression. In the present study, ultraviolet irradiation induced the upregulation of EGFR in skin­derived keratinocytes, which may contribute to the development of cutaneous squamous cell carcinoma (CSCC). This was supported by the high expression of EGFR in CSCC clinical samples. Treating A431 CSCC cells with gefitinib, a tyrosine kinase inhibitor, activated the intrinsic mitochondrial apoptotic pathway while inducing protective autophagy. Combined application of chloroquine with gefitinib enhanced the treatment efficacy of gefitinib against CSCC by inhibiting autophagic flux. These findings demonstrated that autophagy inhibition may be an effective strategy for enhancing the sensitivity of EGFR­expressing cells to tyrosine kinase inhibitor treatment. Manipulating pro­survival autophagy by the combined application of chloroquine with gefitinib is a promising approach for improving the efficacy of EGFR inhibitors in cancer treatment. This may contribute to novel EGFR­targeted therapeutic strategies in the near future.

Loss of MiR-664 Expression Enhances Cutaneous Malignant Melanoma Proliferation by Upregulating PLP2.

Proteolipid protein 2 (PLP2) has been shown to be upregulated in several cancers, including breast cancer, hepatocellular carcinoma, osteosarcoma, and melanoma. PLP2 specifically binds to phosphatidylinositol 3 kinase to activate the protein kinase B pathway to enhance cell proliferation, adhesion, and invasion in melanoma cells. Therefore, we speculated that PLP2 exhibits oncogenic potential. However, the regulatory mechanisms of PLP2 in cancer cells remain unclear.Herein, we found that microRNA (miR)-664 expression was significantly downregulated in cutaneous malignant melanoma (CMM) cells and tissues compared with normal human melanocytes and benign melanocytic naevi. MiR-664 expression level was significantly correlated with patient survival. Ectopic expression of miR-664 reduced CMM cell proliferation and anchorage-independent growth, whereas the inhibition of miR-664 induced these effects. Furthermore, inhibition of miR-664 in CMM cells resulted in modulation of their entry into the G1/S transitional phase, which was caused by downregulation of the cyclin-dependent kinase inhibitor P21 and upregulation of the cell-cycle regulator cyclin D1. Moreover, we demonstrated that miR-664 downregulated PLP2 expression by directly targeting the PLP2 untranslated region.Taken together, our results suggest that miR-664 may play an important role in suppressing proliferation of CMM cells and present a novel mechanism of miR-mediated direct suppression of PLP2 expression in cancer cells.

microRNA-365-targeted nuclear factor I/B transcriptionally represses cyclin-dependent kinase 6 and 4 to inhibit the progression of cutaneous squamous cell carcinoma.

Cyclin-dependent kinases are either post-transcriptionally regulated by interacting with cyclins and cyclin-dependent kinase inhibitors or are transcriptionally regulated by transcription factors, but the latter mechanism has not been extensively investigated. Dysregulated transcription factors resulting from aberrantly expressed microRNAs play critical roles in tumor development and progression. Our previous work identified miR-365 as an oncogenic microRNA that promotes the development of cutaneous squamous cell carcinoma via repression of cyclin-dependent kinase 6, while miR-365 also targets nuclear factor I/B. However, the underlying mechanism(s) of the interaction between nuclear factor I/B and cyclin-dependent kinase 6 are unclear. In this work, we demonstrate that miR-365-regulated nuclear factor I/B transcriptionally inhibits cyclin-dependent kinases 6 and 4 by binding to their promoter regions. In vivo and in vitro experiments demonstrate that the loss of nuclear factor I/B after miR-365 expression or treatment with small interfering RNAs results in the upregulation of cyclin-dependent kinases 6 and 4. This upregulation, in turn, enhances the phosphorylation of retinoblastoma protein and tumor progression. Characterizing this transcriptional repression of cyclin-dependent kinases 6 and 4 by nuclear factor I/B contributes to the understanding of the transcriptional regulation of cyclin-dependent kinases by transcription factors and also facilitates the development of new therapeutic regimens to improve the clinical treatment of cutaneous squamous cell carcinoma.

miR-365 promotes cutaneous squamous cell carcinoma (CSCC) through targeting nuclear factor I/B (NFIB).

Aberrant expression of microRNAs plays vital roles in tumor development and progression. As transcription factors (TFs) are the critical components of signaling cascades, specific targeting effects of microRNAs to specific TFs may determine the role of microRNAs in different cancers. In this study, we identified Nuclear Factor I/B (NFIB) as one of the targets of miR-365 which was previously verified as an onco-miR in cutaneous squamous cell carcinoma (CSCC). Down-regulation of NFIB was a general feature in both CSCC cell lines and tumors from patients which show drastically up-regulated miR-365 expression levels. The siRNA-based knockdown of NFIB mimic the carcinogenic transformation of normal cells by ectopically expression of miR-365 which indicates depletion of NFIB is necessary for miR-365 to exert its pro-carcinogenic function. NFIB may represent a functional barrier targeted by miR-365 to the development of CSCC. Further studies also discovered a conserved feedback regulatory circuitry formed by NFIB and miR-365 in CSCC development which may be potentially utilized as therapeutic target to improve the clinical CSCC treatment.

A novel onco-miR-365 induces cutaneous squamous cell carcinoma.

The expression levels of miR-365 vary in different malignancies. Herein, we found that miR-365 was overexpressed in both cells and clinical specimens of cutaneous squamous cell carcinoma (SCC). We demonstrated that the HaCaT(pre-miR-365-2) cell line, which overexpressed miR-365, could induce subcutaneous tumors in vivo. Antagomir-365, an anti-miR-365 oligonucleotide, inhibited cutaneous tumor formation in vivo, along with G1 phase arrest and apoptosis of cancer cells. These findings suggest that miR-365 may act as an onco-miR in cutaneous SCC both in vitro and in vivo. The present study provides valuable insight into the role of miR-365 in cutaneous SCC formation, which can help develop new drug and miR-365 target-based therapies for cutaneous SCC.

Differential expression profiles of microRNAs in NIH3T3 cells in response to UVB irradiation.

MicroRNAs (miRNAs) are known as a kind of small, noncoding RNA, which play an important role in mediating many biological processes such as development, cell proliferation and differentiation in plants and animals. Here we report the differential expression profiles of miRNAs and characterized putative target genes in NIH3T3 cells at a series of different time points after UVB irradiation (compared with no UVB irradiation). The relative expression of mature miRNA genes was determined by miRNA microarray technique and the results were confirmed by real-time reverse transcriptase polymerase chain reaction (qRT-PCR). Potential target genes of these miRNAs were classified into different function categories with the GOstat software (http://gostat.wehi.edu.au/cgi-bin/goStat.pl). Several miRNAs in this study expressed highly at different time points, especially mmu-miR-365 and mmu-miR-21. Three miRNAs were lowly expressed, of which mmu-miR-465 showed low levels of expression at all time points, whereas after 50 J m(-2) UVB irradiation mmu-miR-296 and mmu-miR-376c showed low levels of expression at 6 and 12 h, respectively. Our study provided a basis for the global characterization of UV-regulated miRNA expression.

[Effects of radiation injury on peripheral blood and liver NO concentration in mice].

OBJECTIVE: To study the effect of radiation injury on nitric oxide (NO) concentration in mouse peripheral blood and liver. METHODS: NIH mice were subjected to gamma-ray exposure at 9.0 Gy and transferred immediately in room temperature condition. NO concentrations in the liver and peripheral blood were examined before and at different time points after the exposure. RESULTS: Compared to that before exposure, NO concentration in the peripheral blood and liver significantly increased after gamma-ray exposure. NO concentration in the peripheral blood began to increase 3 h after the exposure, but that in the liver increased till 6 h after the exposure. CONCLUSION: Radiation can cause the increase of NO concentration in the peripheral blood and liver, but different tissues may exhibit different response intensities to radiation.

[Functional changes of dendritic cells after infection by recombinant retrovirus carrying human telomerase reverse transcriptase gene fragment].

OBJECTIVE: To observe the functional changes of dendritic cells (DCs) after infection by recombinant retrovirus carrying human telomerase reverse transcriptase (hTERT) gene fragment. METHODS: Interleukin-12 (IL-12) levels in DC culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA). The abilities of DCs infected with recombinant retrovirus carrying hTERT gene (hTERT-DCs) and non-infected DCs (N-DCs) to stimulate allogeneic lymphocyte proliferation were evaluated with mixed leukocytes reaction (MLR), and the surface markers of DCs including CD80, CD83, CD86 and HLA-DR were detected by flow cytometry. Cytotoxic T lymphocyte (CTL) assay was performed with CytoTox 96 non-radioactive cytoxicity assay. RESULTS: Compared with N-DCs, hTERT-DCs showed no significant changes in IL-12 secretion and capacity to stimulate allogeneic lymphocytes reaction, but had significantly lower CD83 expression. Specific CTLs induced by hTERT-DCs resulted in higher cytotoxicity against telomerase-positive target cells than that against the negative target cells. CONCLUSION: Infection with the recombinant retrovirus carrying hTERT fragment may jeopardize the maturation of DCs, which, however, still retain their capacity to activate and stimulate lymphocyte proliferation and to prime autologous T lymphocytes to generate specific CTL against hTERT.