A pair of long intergenic non-coding RNA LINC00887 variants act antagonistically to control Carbonic Anhydrase IX transcription upon hypoxia in tongue squamous carcinoma progression

Background Long noncoding RNAs (lncRNAs) are important regulators in tumor progression. However, their biological functions and underlying mechanisms in hypoxia adaptation remain largely unclear. Results Here, we established a correlation between a Chr3q29-derived lncRNA gene and tongue squamous carcinoma (TSCC) by genome-wide analyses. Using RACE, we determined that two novel variants of this lncRNA gene are generated in TSCC, namely LINC00887_TSCC_short (887S) and LINC00887_TSCC_long (887L). RNA-sequencing in 887S or 887L loss-of-function cells identified their common downstream target as Carbonic Anhydrase IX (CA9), a gene known to be upregulated by hypoxia during tumor progression. Mechanistically, our results showed that the hypoxia-augmented 887S and constitutively expressed 887L functioned in opposite directions on tumor progression through the common target CA9. Upon normoxia, 887S and 887L interacted. Upon hypoxia, the two variants were separated. Each RNA recognized and bound to their responsive DNA cis-acting elements on CA9 promoter: 887L activated CA9’s transcription through recruiting HIF1α, while 887S suppressed CA9 through DNMT1-mediated DNA methylation. Conclusions We provided hypoxia-permitted functions of two antagonistic lncRNA variants to fine control the hypoxia adaptation through CA9.

7SK Acts as an Anti-tumor Factor in Tongue Squamous Cell Carcinoma

Increasing evidence has shown the mechanistic insights about non-coding RNA 7SK in controlling the transcription. However, the biological function and mechanism of 7SK in cancer are largely unclear. Here, we show that 7SK is down-regulated in human tongue squamous carcinoma (TSCC) and acts as a TSCC suppressor through multiple cell-based assays including a migration assay and a xenograft mouse model. The expression level of 7SK was negatively correlated with the size of tumors in the 73 in-house collected TSCC patients. Through combined analysis of 7SK knockdown of RNA-Seq and available published 7SK ChIRP-seq data, we identified 27 of 7SK-regulated genes that were involved in tumor regulation and whose upstream regulatory regions were bound by 7SK. Motif analysis showed that the regulatory sequences of these genes were enriched for transcription factors FOXJ3 and THRA, suggesting a potential involvement of FOXJ3 and THRA in 7SK-regulated genes. Interestingly, the augmented level of FOXJ3 in TSCC patients and previous reports on THRA in other cancers have suggested that these two factors may promote TSCC progression. In support of this idea, we found that 21 out of 27 aforementioned 7SK-associated genes were regulated by FOXJ3 and THRA, and 12 of them were oppositely regulated by 7SK and FOXJ3/THRA. We also found that FOXJ3 and THRA dramatically promoted migration in SCC15 cells. Collectively, we identified 7SK as an antitumor factor and suggested a potential involvement of FOXJ3 and THRA in 7SK-mediated TSCC progression.

Model of human tongue squamous cell line stably transfected with human papillomavirus（HPV) 16 E6 and E7 genes and biological characteristic analysis

Human tongue squamous carcinoma cell lines transfected with HPV16E6E7 gene were established to provide a model for further study of HPV16 E6E7-related human tongue squamous carcinoma cell lines .Plasmid pEGFR/HPV16 of E6E7 and plasmid pEGFR/HPV16 of No E6E7 were constructed.Human tongue squamous carcinoma cell lines including SCC9 and SCC15 were infected by liposome transfection and would be highly selected by antibiotic .Fluorescence imaging, RT-PCR and Westernblot were used to detect the expression of HPV16 E6E7 in cells.The biological characteristics of human tongue squamous carcinoma cell lines infected with HPV16 E6E7 were detected by CCK-8 and wound healing assay. The human tongue squamous carcinoma cell lines transfected with HPV16 E6E7 gene were successfully established and identified, and the proliferation and migration ability of the human tongue squamous carcinoma cell lines infected with HPV16 E6E7 gene was significantly stronger than that of the blank group.Human tongue squamous carcinoma cell lines infected with HPV16 E6E7 were more malignant, and their proliferation and migration ability were higher than those not infected with HPV16 E6E7.

Enforced C-Src Activation Causes Compartmental Dysregulation of PI3K and PTEN Molecules in Lipid Rafts of Tongue Squamous Carcinoma Cells by Attenuating Rac1-Akt-GLUT-1-Mediated Sphingolipid Synthesis

Pharmacologic intervention to affect the membrane lipid homeostasis of lipid rafts is a potent therapeutic strategy for cancer. Here we showed that gallic acid (GA) caused the complex formation of inactive Ras-related C3 botulinum toxin substrate 1 (Rac1)-phospho (p)-casein kinase 2 α (CK2α) (Tyr 255) in human tongue squamous carcinoma (TSC) cells, which disturbed the lipid raft membrane-targeting of phosphatidylinositol 3-kinase (PI3K)-Rac1-protein kinase B (Akt) signal molecules by inducing the association of p110α-free p85α with unphosphorylated phosphatase tensin homolog deleted on chromosome 10 (PTEN) in lipid rafts. The effects on induction of inactive Rac1-p-CK2α (Tyr 255) complex formation and attenuation of p-Akt (Ser 473), GTP-Rac1, glucose transporter-1 (GLUT-1) lipid raft membrane-targeting, and cell invasive activity by GA were counteracted either by CK2α short hairpin RNA or cellular-Src (c-Src) inhibitor PP1. PP1 treatment, GLUT-1 or constitutively active Rac1 ectopic-expression blocked GA-induced decreases in cellular glucose, sphingolipid and cholesterol of lipid raft membranes, p85α-p110α-GTP-Rac1 complexes, glucosylceramide synthase activity and increase in ceramide and p110α-free p85α-PTEN complex levels of lipid raft membranes, which reversed the inhibition on matrix metalloproteinase (MMP)-2/-9-mediated cell invasion induced by GA. Using transient ectopic expression of nuclear factor-kappa B (NF-κB) p65, MMP-2/-9 promoter-driven luciferase, and NF-κB-dependent luciferase reporter genes and NF-κB specific inhibitors or Rac1 specific inhibitor NSC23766, we confirmed that an attenuation of Rac1 activity by GA confers inhibition of NF-κB-mediated MMP-2/-9 expression and cell invasion. In conclusion, GA-induced c-Src activation is a key inductive event for the formation of inactive Rac1-p-CK2α (Tyr 255) complexes, which disturbed lipid raft compartment of PI3K and PTEN molecules by impairing Akt-regulated GLUT-1-mediated sphingolipid synthesis, and finally resulting in inhibition of TSC cell invasion.

Electrochemical Assessment of Anticancer Compounds on the Human Tongue Squamous Carcinoma Cells

The most common oral cancer is squamous cell carcinoma (SCC) and its highest occurrence is in the tongue. Almost 30% of patients with one primary head and neck tumor will have a second primary malignancy. In recent studies, two novel plant extracts, andrographolide and cannabidiol (CBD), have been exploited for their anticancer effects. Here, we investigated the cytotoxic effects of these two compounds on SCC-25 cells, a human tongue squamous carcinoma cell line, and compared the outcomes with two chemotherapeutic drugs, cisplatin and fluorouracil. Electric cell substrate impedance sensing (ECIS) system was applied to measure frequency- and time-dependent impedance of SCC-25 cell-covered electrodes and to further assess subtle changes in cell morphology and micromotion in response to different concentrations (0, 10, 30, 100, and 300 µM) of these compounds. AlamarBlue and Annexin V/7-AAD binding assays were used to measure the concentration dependent changes in viability and apoptosis of SCC-25 cells. Our results demonstrate that 24 hours after exposure to 30 µM CBD can significantly decrease the micromotion rate, damage the integrity of cell morphology, reduce cell viability, and induce higher apoptosis in treated SCC-25 cells, while the other three drugs attain similar effects at the concentration of 100 µM or higher. The apoptosis-induced changes in cell morphology and micromotion monitored by ECIS correlate well with biochemical assays. Thus, both frequency- and time-dependent impedance measurements using ECIS can be used to real-time follow cancer cell activities in response to anticancer drugs with different temporal cytotoxicity profiles.