Zhaojian Gong

Role of tumor microenvironment in tumorigenesis

Tumorigenesis is a complex and dynamic process, consisting of three stages: initiation, progression, and metastasis. Tumors are encircled by extracellular matrix (ECM) and stromal cells, and the physiological state of the tumor microenvironment (TME) is closely connected to every step of tumorigenesis. Evidence suggests that the vital components of the TME are fibroblasts and myofibroblasts, neuroendocrine cells, adipose cells, immune and inflammatory cells, the blood and lymphatic vascular networks, and ECM. This manuscript, based on the current studies of the TME, offers a more comprehensive overview of the primary functions of each component of the TME in cancer initiation, progression, and invasion. The manuscript also includes primary therapeutic targeting markers for each player, which may be helpful in treating tumors.

Circular RNAs in human cancer

CircRNAs are a novel type of RNAs. With the newly developed technology of next-generation sequencing (NGS), especially RNA-seq technology, over 30,000 circRNAs have already been found. Owing to their unique structure, they are more stable than linear RNAs. CircRNAs play important roles in the carcinogenesis of cancer. The expression of circRNAs is correlated with patients’ clinical characteristics, and circRNAs play a vital role in many aspects of malignant phenotypes, including cell cycle, apoptosis, vascularization, and invasion; metastasis as a RNA sponge, binding to RBP; or translation. Therefore, it is meaningful to further study the mechanism of interactions between circRNAs and tumors. The role of circRNAs as molecular markers or potential targets will provide promising application perspectives, such as early tumor diagnosis, therapeutic evaluation, prognosis prediction, and even gene therapy for tumors.

LOC401317, a p53-regulated long non-coding RNA, inhibits cell proliferation and induces apoptosis in the nasopharyngeal carcinoma cell line HNE2

Recent studies have revealed that long non-coding RNAs participate in all steps of cancer initiation and progression by regulating protein-coding genes at the epigenetic, transcriptional, and post-transcriptional levels. Long non-coding RNAs are in turn regulated by other genes, forming a complex regulatory network. The regulation networks between the p53 tumor suppressor and these RNAs in nasopharyngeal carcinoma remains unclear. The aims of this study were to investigate the regulatory roles of the TP53 gene in regulating long non-coding RNA expression profiles and to study the function of a TP53-regulated long non-coding RNA (LOC401317) in the nasopharyngeal carcinoma cell line HNE2. Long non-coding RNA expression profiling indicated that 133 long non-coding RNAs were upregulated in the human NPC cell line HNE2 cells following TP53 overexpression, while 1057 were downregulated. Among these aberrantly expressed long non-coding RNAs, LOC401317 was the most significantly upregulated one. Further studies indicated that LOC401317 is directly regulated by p53 and that ectopic expression of LOC401317 inhibits HNE2 cell proliferation in vitro and in vivo by inducing cell cycle arrest and apoptosis. LOC401317 inhibited cell cycle progression by increasing p21 expression and decreasing cyclin D1 and cyclin E1 expression and promoted apoptosis through the induction of poly(ADP-ribose) polymerase and caspase-3 cleavage. Collectively, these results suggest that LOC401317 is directly regulated by p53 and exerts antitumor effects in HNE2 nasopharyngeal carcinoma cells.

Epstein-Barr virus-encoded miR-BART6-3p inhibits cancer cell metastasis and invasion by targeting long non-coding RNA LOC553103

Epstein-Barr virus (EBV) infection is causatively related to a variety of human cancers, including nasopharyngeal carcinoma (NPC) and gastric cancer (GC). EBV encodes 44 mature miRNAs, a number of which have been proven to promote carcinogenesis by targeting host genes or self-viral genes. However, in this study, we found that an EBV-encoded microRNA, termed EBV-miR-BART6-3p, inhibited EBV-associated cancer cell migration and invasion including NPC and GC by reversing the epithelial–mesenchymal transition (EMT) process. Using microarray analysis, we identified and validated that a novel long non-coding RNA (lncRNA) LOC553103 was downregulated by EBV-miR-BART6-3p, and LOC553103 knockdown by specific siRNAs phenocopied the effect of EBV-miR-BART6-3p, while LOC553103 overexpression promoted cancer cell migration and invasion to facilitate EMT. In conclusion, we determined that EBV-miR-BART6-3p, a microRNA encoded by oncogenic EBV, inhibited EBV-associated cancer cell migration and invasion by targeting and downregulating a novel lncRNA LOC553103. Thus, our study presents an unreported mechanism underlying EBV infection in EBV-associated cancer carcinogenesis, and provides a potential novel diagnosis and treatment biomarker for NPC and other EBV-related cancers.

LPLUNC1 Inhibits Nasopharyngeal Carcinoma Cell Growth via Down-Regulation of the MAP Kinase and Cyclin D1/E2F Pathways

Long-palate, lung and nasal epithelium clone 1 (LPLUNC1) gene expression is relatively tissue specific. It is highly expressed in nontumor nasopharyngeal epithelial tissues, but its expression is reduced in nasopharyngeal carcinoma (NPC), indicating that LPLUNC1 may be associated with the tumorigenesis of NPC. To study the effects of LPLUNC1 on NPC tumorigenesis, a full-length LPLUNC1 expression plasmid was stably transfected into the NPC cell line, 5-8F. Our data indicated that LPLUNC1 inhibited NPC cell proliferation in vitro and tumor formation in vivo. LPLUNC1 also delayed cell cycle progression from G1 to S phase and inhibited the expression of cyclin D1, cyclin-dependent kinase 4 (CDK4) and phosphorylated Rb. To further investigate the molecular mechanisms underlying the suppressive effects of LPLUNC1 on NPC tumorigenesis, cDNA microarray was performed. These studies revealed that LPLUNC1 inhibited the expression of certain mitogen-activated protein (MAP) kinases (MAPK) kinases and cell cycle-related molecules. Western blotting confirmed that the expression of MEK1, phosphorylated ERK1/2, phosphorylated JNK1/2, c-Myc and c-Jun were inhibited by LPLUNC1. Furthermore, the transcriptional activity of AP-1 was down-regulated by LPLUNC1, suggesting that the MAPK signaling pathway is regulated by LPLUNC1. Taken together, the present study indicates that LPLUNC1 delays NPC cell growth by inhibiting the MAPK and cyclin D1/E2F pathways and suggests that LPLUNC1 may represent a promising candidate tumor suppressor gene associated with NPC.

Upregulated long non-coding RNA LINC00152 expression is associated with progression and poor prognosis of tongue squamous cell carcinoma

Altered expression of long non-coding RNAs (lncRNAs) associated with human carcinogenesis and might be used as diagnosis and prognosis biomarkers. However, the expression of lncRNAs in tongue squamous cell carcinoma (TSCC) and their relevance on the diagnosis, progression and prognosis of TSCC have not been thoroughly elucidated. To discover novel TSCC-related lncRNAs, we analyzed the lncRNA expression patterns in two sets of previously published TSCC gene expression profile data (GSE30784 and GSE9844), and found that long intergenic non-coding RNA 152 (LINC00152) was significantly upregulated in TSCC samples. We then detected LINC00152 expression in two other cohorts of TSCC samples. Quantitative Real time PCR (qRT-PCR) results indicated that LINC00152 was highly expressed in 15 primary TSCC biopsies when compared with 14 adjacent non-tumor tongue squamous cell epithelium samples. The expression of LINC00152 was also measured in 182 paraffin-embedded human TSCC tissues by in situ hybridization, increased expression of LINC00152 was significantly correlated with TSCC progression, such as T stage (p = 0.009), N stage (p = 0.036), TNM stage (p = 0.017), and associated with relapse (p < 0.001), and invasion (p < 0.001). Kaplan-Meier analysis demonstrated that increased LINC00152 expression contributed to both poor overall survival (p = 0.006) and disease-free survival (p = 0.007) of TSCC patients. These findings suggest that LINC00152 might serve as a potential biomarker for early detection and prognosis prediction of TSCC.

Epstein-Barr virus encoded miR-BART11 promotes inflammation-induced carcinogenesis by targeting FOXP1

Epstein-Barr virus (EBV) infection and chronic inflammation are closely associated with the development and progression of nasopharyngeal carcinoma (NPC) and gastric cancer (GC), and the infiltration of inflammatory cells, including tumor-associated macrophages (TAMs), is often observed in these cancers. EBV encodes 44 mature micro RNAs (miRNAs), but the roles of only a few EBV-encoded miRNA targets are known in cancer development, and here, our aim was to elucidate the effects of EBV-miR-BART11 on FOXP1 expression, and potential involvement in inflammation-induced carcinogenesis. We constructed an EBV miRNA-dependent gene regulatory network and predicted that EBV-miR-BART11 is able to target forkhead box P1 (FOXP1), a key molecule involved in monocyte to macrophage differentiation. Here, using luciferase reporter assay, we confirmed that EBV-miR-BART11 directly targets the 3′-untranslated region of FOXP1 gene, inhibits FOXP1 induction of TAM differentiation, and the secretion of inflammatory cytokines into the tumor microenvironment, inducing the proliferation of NPC and GC cells. FOXP1 overexpression hindered monocyte differentiation and inhibited NPC and GC cells growth. Our results demonstrated that EBV-miR-BART11 plays a crucial role in the promotion of inflammation-induced NPC and GC carcinogenesis by inhibiting FOXP1 tumor-suppressive effects. We showed a novel EBV-dependent mechanism that may induce the carcinogenesis of NPC and GC, which may help define new potential biomarkers and targets for NPC and GC diagnosis and treatment.

Co-expression of AFAP1-AS1 and PD-1 predicts poor prognosis in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) carries a high potential for metastasis and immune escape, with a great risk of relapse after primary treatment. Through analysis of whole genome expression profiling data in NPC samples, we found that the expression of a long non-coding RNA (lncRNA), actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1), is significantly correlated with the immune escape marker programmed death 1 (PD-1). We therefore assessed the expression of AFAP1-AS1 and PD-1 in a cohort of 96 paraffin-embedded NPC samples and confirmed that AFAP1-AS1 and PD-1 are co-expressed in infiltrating lymphocytes in NPC tissue. Moreover, patients with high expression of AFAP1-AS1 or PD-1 in infiltrating lymphocytes were more prone to distant metastasis, and NPC patients with positive expression of both AFAP1-AS1 and PD-1 had the poorest prognosis. This study suggests that AFAP1-AS1 and PD-1 may be potential therapeutic targets in NPC and that patients with co-expression of AFAP1-AS1 and PD-1 may be ideal candidates for future clinical trials of anti-PD-1 immune therapy.

Antifibrotic Effect of Curcumin in TGF-β1-Induced Myofibroblasts from Human Oral Mucosa

BACKGROUND Myofibroblasts play an important role in the development of oral submucous fibrosis (OSF). In the current study, we investigate the effect of curcumin on growth and apoptosis of myofibroblasts derived from human oral mucosa. METHODS Myofibroblasts were generated by incubating fibroblasts, obtained from human oral mucosa, with transforming growth factor-β 1 (TGF-β 1). MTT, PI staining, and FACS assays were used to investigate curcumins effect on proliferation and cell cycle of fibroblasts and myofibroblasts. Annexin V/PI binding and FACS assays were used to examine apoptosis of myofibroblasts, Western blotting to determine the levels of Bcl-2 and Bax, and enzyme-linked immunosorbant assay was employed to examine the levels of collagen type I and III in the supernatants of myofibroblasts. RESULTS Curcumin inhibits proliferation of fibroblasts and myofibroblasts; it also disturbs the cell cycle, induces apoptosis and decreases the generation of collagen type I and III in myofibroblasts, which are more sensitive to its effects than fibroblasts. Curcumin induces apoptosis in myofibroblasts by down-regulating the Bcl-2/ Bax ratio. CONCLUSION Our results demonstrate the antifibrotic effect of curcumin in vitro. It may therefore be a candidate for the treatment of OSF.

Overexpression long non-coding RNA LINC00673 is associated with poor prognosis and promotes invasion and metastasis in tongue squamous cell carcinoma

Long non-coding RNAs (lncRNAs) associated with the tumorigenesis of human cancers. However, the relevance of lncRNAs in tongue squamous cell carcinoma (TSCC) is still unclear. To discover novel TSCC-related lncRNAs, we analyzed the lncRNA expression patterns in two sets of TSCC gene expression profile data, and found that long intergenic non-coding RNA 673 (LINC00673) was significantly upregulated in TSCC samples. Then we examined LINC00673 expression in 202 TSCC tissue specimens, LINC00673 is highly expressed in a significant proportion of human TSCC biopsies and correlates with poor prognosis. Knockdown LINC00673 significantly inhibited the cell invasion and migration capability in TSCC cells. Our findings suggest that LINC00673 may play an essential role in TSCC progression and might serve as a potential biomarker for early detection and prognosis prediction of TSCC.