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The mapping between reads (ENSEMBL identifiers) and GO identifier codes was downloaded from EBI. Gene Ontology (GO) mapping was performed using custom scripts. The reads per kilo base of exon model per million mapped reads (RPKM) were calculated, and statistical analysis was performed to determine differential gene expression among different groups.
#Densitometry was done with imagej software software
The CLC Genomics Workbench 10.0 software was used for additional data analysis. TopHat and BowTie software installed in Illumina BaseSpace was used for assembling contigs. Total tissue RNA was extracted from frozen tongue samples by TRIzol reagent and subjected to Next Generation RNA Sequencing (IlluminaNextSeq 500 1x75) at MOgene, LC.
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One part of tissue was fixed in formalin for histopathologic analysis, and the other part was snap frozen in liquid nitrogen for biochemical analysis. Mice were sacrificed at 22 weeks, tongue tissue was macroscopically examined, and the number of macroscopic lesions was counted.
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Experimental design is summarized in Fig. The dose of the BME for animal experimentation was selected on the basis of our previous studies. Briefly, BME was extracted from bitter melon without seeds using a household juicer, centrifuged at 15,000 × g at 4☌ for 30 minutes, and stored at −80☌. The BME was prepared from the Chinese variety of young bitter melons (raw and green) as described previously ( 7). The other group of mice ( n = 10) received BME (30% v/v, 600 mg/mouse) in the drinking water along with 4-NQO treatment for 16 weeks, and then continued with only BME until the end of the experiment. ©2017 AACR.įor oral cancer development, mice ( n = 10) were given 4-NQO (50 μg/mL) in their drinking water for 16 weeks, then only water until the end of the experiment. Together, our data strongly suggest the potential clinical benefits of BME as a chemopreventive agent in the control or delay of carcinogen-induced HNSCC development and progression. Identification of pathways involved in carcinogen-induced oral cancer provides useful information for prevention strategies. Our study demonstrates the preventive effect of BME in 4-NQO–induced carcinogenesis. Enhancement of MMP9 (“ossification” pathway) was noted during carcinogenesis, which was reduced in BME-fed mouse tongue tissues. Interestingly, BME treatment significantly reduced their expression.
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We identified elevated expression of proinflammatory genes, s100a9, IL23a, IL1β and immune checkpoint gene PDCD1/PD1, during oral cancer development. Gene ontology and pathway analyses revealed a signature of biological processes including “immune system process” that is significantly dysregulated in 4-NQO–induced oral cancer. We also examined the global transcriptome changes in normal versus carcinogen-induced tongue cancer tissues, and following BME feeding. Histologic analysis suggested control 4-NQO–treated mouse tongues showed neoplastic changes ranging from moderate dysplasia to invasive squamous cell carcinoma, whereas no significant dysplasia was observed in the BME-fed mouse tongues. We observed that BME feeding significantly reduced the incidence of 4-NQO–induced oral cancer in a mouse model. In this study, we investigated the preventive role of BME in 4-nitroquinoline 1-oxide (4-NQO) carcinogen-induced HNSCC. We have previously observed that bitter melon ( Momordica charantia) extract (BME) exerts antiproliferative activity against several cancers including HNSCC. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and tobacco is one of the most common factors for HNSCC of the oral cavity.