[Identification of circular RNA expression profiles in oral lichen planus].

PURPOSE: To construct the expression profile of circular RNA (circRNA) in human oral lichen planus (OLP), and to identify and validate the differentially expressed circRNA in oral lichen planus tissues and provide theoretical basis for the diagnosis and treatment of this disease. METHODS: Six patients newly diagnosed with OLP from September to December 2018 in the Department of Oral Mucosal Diseases, Shanghai Ninth People's Hospital and 6 healthy volunteers were enrolled in this study. RNA sequencing and evaluation in OLP tissues and normal oral mucosa were performed by high-throughput RNA sequencing technology, and the differences between groups were analyzed. qRT-PCR was used to validate the results. Statistical analysis was conducted with SPSS 24.0 software package. Finally, bioinformatics techniques GO (Gene Ontology) enrichment analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway were used to analyze the functions and related pathways of the dysregulated genes. RESULTS: According to the sequencing results, 85 differentially expressed circRNAs with fold change > 2 were identified in OLP tissues compared to the normal oral mucosa, including 66 upregulated circRNAs and 19 downregulated circRNAs. Three circRNAs with the most significant up-regulation and down-regulation were selected for qRT-PCR verification in expanded samples, and the results were consistent with the sequencing results. Bioinformatics analysis suggested that the differentially expressed circRNAs may play an important role in the occurrence and progression of oral lichen planus. CONCLUSIONS: Differentially expressed circRNAs between oral lichen planus tissues and normal oral mucosa were identified, which may be involved in the pathogenic mechanism of oral lichen planus and could be potential biomarkers for diagnosis and treatment of this disease.

[Application of intraoral ultrasonic imaging in diagnosis and treatment of 18 patients with oral leukoplakia in non-masticatory mucosa].

PURPOSE: To explore the sonographic appearance of leukoplakia in non-masticatory oral mucosa, classifying mucosal leukoplakia according to the characteristics of sonogram, and providing reference for clinical diagnosis and treatment. METHODS: Eighteen patients (24 lesions) were diagnosed as oral leukoplakia at the Department of Oral Mucosal Diseases, Shanghai Ninth People's Hospital. The lesions were located in the tongue, floor of mouth, buccal mucosa and libial mucosa. Before the biopsy was taken, intra-oral path ultrasound was performed at the Department of Ultrasound to observe the lesion's extent, continuity, presence or absence of keratinization, the thickness of each layer in the epithelium, and color doppler flow imaging of the lesions. Quantitative analysis software 'Qontraxt' was used to randomly measure the relative echo intensity of the mucosal surface in leukoplakia areas, and summarize the keratinization type. SPSS 25.0 software package was used for statistical analysis of the data, and paired t test was used for inter-group comparison of the data. RESULTS: Oral leukoplakia sonograms showed that the epithelial layer appeared keratinization, the epithelial was thickened, and the echo was enhanced. The stratum intermedium showed a low echo thickening band, and the echo of partial lesions' surface decreased or the blood flow signal in oral mucosa increased. The hyperechoic band in the leukoplakia area was significantly thickened (P<0.001), and the echo was enhanced, with the tongue and buccal mucosa being the most significant. The hypoechoic band was significantly thicker (P<0.001), with the buccal mucosa and labial mucosa being the most significant. The surface and stratum corneum echo intensity values were determined by Qontraxt quantitative analysis software to determine whether there were keratinization and the keratinization types. The echo intensity values was 43.28±9.33 in non-OLK area, 92.88±3.12 in OLK with orthokeratosis, and 84.75±5.76 in OLK with parakeratosis. CONCLUSIONS: Ultrasound imaging can effectively define mucosal leukoplakia and measure the thickness of each layer in the epithelium. In addition, special adjoint changes such as ulcers, infections and cancerous changes can be detected. Intraoral ultrasonic imaging can provide imaging evidence for clinical diagnosis, treatment planning and post-treatment follow-up and contribute to avoid unnecessary mucosal iatrogenic injury or recurrence of disease after treatment.

MicroRNA-134 inhibits tumor stem cell migration and invasion in oral squamous cell carcinomas via downregulation of PI3K-Akt signaling pathway by inhibiting LAMC2 expression.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the most common malignant neoplasm of the mouth. Some studies have found that multiple microRNAs (miRs) participate in OSCC physiological and pathological processes. METHODS: We explored the mechanism of action of miR-134 in OSCC involving the PI3K-Akt signaling pathway. Different bioinformatics methods were used to analyze the potential genes and their related miRs in OSCC. Tumor stem cells were separated from OSCCs through magnetic cell sorting. Regulatory pattern between miR-134 and LAMC2 in OSCC was evaluated by ectopic expression, knockdown and reporter assay experiments. The expression of miR-134, LAMC2, genes in PI3K-Akt signaling pathway, and apoptosis-related genes was detected. Cell proliferation was assessed by MTT assay, cell invasion by scratch test, cell migration by Transwell assay, cell cycle and apoptosis by flow cytometry, and cell growth and migration by xenograft tumor in nude mice. LAMC2 was predicted as the crucial factor related to OSCC using different chip data, and miR-134 was predicted to specifically bind LAMC2 in all five databases. RESULTS: Overexpressed miR-134 or silenced LAMC2 was observed to inhibit cell proliferation, migration, invasion of OSCC cells, growth of subcutaneous xenograft in nude mice, as well as promote OSCC cell apoptosis. LAMC2, a target gene of miR-134, decreased following miR-134 promotion, while the PI3K-Akt signaling pathway was inactivated following LAMC2 knockdown. Furthermore, we also observed that the effect of overexpressed miR-134 was enhanced when LAMC2 was knocked down. CONCLUSIONS: Taken together, these findings suggest that miR-134-mediated direct downregulation of LAMC2 inhibits migration and invasion of tumor stem cells in OSCC by suppressing the PI3K-Akt signaling pathway.

Up-regulation of microRNA-136 induces apoptosis and radiosensitivity of esophageal squamous cell carcinoma cells by inhibiting the expression of MUC1.

OBJECTIVE: This present study is performed to figure out the role of microRNA-136 (miR-136) in radiosensitivity of esophageal squamous cell carcinoma (ESCC) cells through the regulation of MUC1. METHODS: Seventy-four ESCC patients were divided into radiotherapy sensitive group and radiotherapy resistance group. Colony formation assay and flow cytometry were used to test the radiosensitivity of radiotherapy resistant strain and parent strain. The expression of miR-136 between radiotherapy resistant strain and parent strain was detected by RT-qPCR, and the expression of miR-136 in Eca109 and TE-1 cells as well as Eca109-R and TE-1-R cells was detected after different doses of X-ray irradiation. Eca109 and TE-1 cells as well as Eca109-R and TE-1-R cells with overexpression of miR-136 or co-overexpression of miR-136 and MUC1 were constructed. Cell proliferation, colony formation and apoptosis was detected by CCK-8 assay, colony formation assay, and flow cytometry, respectively. RESULTS: The expression of miR-136 in ESCC tissues was lower and MUC1 mRNA and protein expression was higher than that in adjacent normal tissues. The expression of miR-136 was negatively correlated with the expression of MUC1 mRNA in ESCC. Low expression of miR-136 and high expression of MUC1 were associated with tumor size, lymph node metastasis and distant metastasis. The expression of miR-136 increased while the expression of MUC1 decreased in the radiotherapy sensitive group of ESCC patients relative to the radiotherapy resistant group. The colony formation ability of radiation resistant cell line was stronger than that of parent cell line, and the apoptosis rate showed an opposite trend. Up-regulation of miR-136 reduced the survival rate, suppressed colony formation ability and induced apoptosis of ESCC cells under irradiation, which was reversed by upregulated MUC1. CONCLUSION: This study demonstrates that up-regulation of miR-136 induces apoptosis and radiosensitivity of ESCC cells by inhibiting the expression of MUC1.

Human papillomavirus genotypes and p16 expression in oral leukoplakia and squamous cell carcinoma.

Several studies have shown a broad variation in the prevalence of human papillomavirus (HPV) in oral leukoplakia (OLK) and oral squamous cell carcinoma (OSCC), whereas the relationship is less well-defined and specific HPV genotypes lack examination in OLK. In the present study, the role of HPV and surrogate p16 expression was investigated to explore the correlation and pathogenesis in OLK and OSCC. Polymerase chain reaction (PCR) and flow-through hybridization technology were utilized to detect HPV genotypes in oral exfoliated cells from 30 healthy volunteers, 103 OLK and 30 OSCC patients. Expression of p16 was assessed by immunohistochemistry (IHC) in biopsies from these OLK and OSCC, in addition to 15 normal oral mucosal tissues as the control group. The healthy controls showed 3.3% (1/30) HPV presence; In OLK and OSCC, the detection rate was 4.9% (5/103), 3.3% (1/30), respectively. No significant relationship between HPV and OLK or OSCC was observed when compared with the control group (P>0.05). All 6 HPV-positive OLK and OSCC cases had p16 overexpression. But the sensitivity of p16 IHC was poor, because 88.4% (38/43) of p16 over-expressed OLK were HPV negative. There was no statistical significance between HPV and the sex, age, site, alcohol consumption, or smoking. These findings suggested HPV had a low prevalence in OLK and OSCC. This suggests the detection of HPV genotypes by PCR in exfoliated cells combined with p16 IHC may be more accurate to represent HPV infection.

Expression patterns of cancer stem cell markers ALDH1 and CD133 correlate with a high risk of malignant transformation of oral leukoplakia.

Molecular markers for predicting oral cancer development in premalignant oral leukoplakia (OL) are urgently needed. The objective of this study was to examine the expression patterns of cancer stem cell markers ALDH1 and CD133 in samples from patients with OL, and determine their prognostic values for subsequent development of oral cancer. Immunohistochemistry for ALDH1 and CD133 was performed in samples from a cohort of 141 patients with biopsy-proven OL who received a mean follow-up of 5.5 years. Patient clinicopathologic and follow-up data were analyzed. Expression of ALDH1 and CD133 was observed in 54 (38.3%) and 32 (22.7%) of 141 patients with OL, respectively. Kaplan-Meier analysis showed that 48.1% patients with ALDH1-positivity developed oral cancer compared with 12.6% those with ALDH1-negativity (p < 0.001). Meanwhile, 59.4% patients with CD133-positivity developed oral cancer compared with 16.5% those with CD133-negativity (p < 0.001). Multivariate analysis revealed that ALDH1 and CD133 expression was associated with 4.17-fold [95% confidence interval (CI), 1.96-8.90; p < 0.001] and 2.86-fold (95% CI, 1.48-5.55; p = 0.002) increased risk of OL transformation, respectively. Collectively, these data demonstrated for the first time that the expression of ALDH1 and CD133 correlated with malignant transformation in a large series of patients with OL who received a long-term follow-up, which suggests that they may serve as predictors to identify OL with a high risk of oral cancer development.

Two stem cell markers, ATP-binding cassette, G2 subfamily (ABCG2) and BMI-1, predict the transformation of oral leukoplakia to cancer: a long-term follow-up study.

BACKGROUND: Although oral leukoplakia (OL) is the best-known potentially malignant disorder, the risk of OL malignant transformation is difficult to assess. ATP-binding cassette, G2 subfamily (ABCG2) and BMI-1 are stem cell markers that have been found to be associated with head and neck tumorigenesis. The objective of the current study was to evaluate the usefulness of ABCG2 and BMI-1 in predicting OL transformation. METHODS: In a retrospective cohort of 135 patients with OL from the study institution who had a mean follow-up of 5.5 years, 32 developed cancer between 1985 and 2008. The expression of ABCG2 and BMI-1 was determined using immunohistochemistry in samples from these patients, and included untransformed OL (n = 103) and malignant-transformed OL (n = 32). The association between protein expression and clinicopathological parameters and transformation was analyzed. RESULTS: Expression of ABCG2 and BMI-1 was observed in 58 (43.0%) and 44 (32.6%) of 135 patients, respectively. The correlation between ABCG2 and BMI-1 expression was significant (P = .024). Kaplan-Meier analysis revealed that 37.9% of patients with ABCG2 positivity developed cancer compared with 13.0% of patients with ABCG2 negativity (P = .014, log-rank test). Approximately 40.9% of patients with BMI-1 positivity developed cancer compared with 15.4% of patients with BMI-1 negativity (P = .029, log-rank test). Multivariate analysis revealed that ABCG2 and BMI-1 expression was associated with a 3.24-fold (95% confidence interval [95% CI], 1.31-7.98; P = .011) and 4.03-fold (95% CI, 1.59-10.26; P = .003) increased the risk of transformation, respectively. CONCLUSIONS: ABCG2 and BMI-1 expression was found to be associated with the development of oral cancer in a large cohort of patients with OL for whom long-term follow-up was available, which suggests that ABCG2 and BMI-1 may be used as predictors of OL transformation.

The high expression level of programmed death-1 ligand 2 in oral lichen planus and the possible costimulatory effect on human T cells.

BACKGROUND: Oral lichen planus (OLP) is a T-cell-mediated chronic autoimmune disease whose precise etiology is unknown. The recently identified costimulatory programmed death-1 (PD-1) molecule and its ligands, PD-L1 and PD-L2, have been identified as CD28-B7 family molecules and constitute a regulatory pathway of potential therapeutic use in immune-mediated diseases. METHODS: We examined the expression of two ligands of PD-1 at both the protein and gene level in the focal mucosa and peripheral blood of OLP patients using immunohistochemistry and real-time PCR. Next, we used the PD-L2.Ig fusion protein and observed its effects on T cells, which were co-cultured with IFN-γ-treated keratinocytes (KCs) in the presence of PHA. RESULTS: We found that the expression of PD-L2 at both the gene and protein level was statistically different in peripheral blood and local lesion tissue of patients with OLP compared to the normal controls. The proliferation ability of T cells and the expression level of IFN-γ in the supernatant of the above co-culture model were significantly augmented (P < 0.05). PD-L2.Ig fusion protein significantly aggravated the apoptosis of T cells, inhibited the proliferation of T cells and decreased the release levels of IL-2 and IFN-γ in the model (P < 0.05). CONCLUSION: These data show that the increased expression of PD-L2, as a costimulatory molecule, may have an important modulatory function on the local immune responses of OLP in vivo.

[T cell receptor BV gene preferential usage in oral lichen planus].

PURPOSE: To identify the T cell receptor (TCR) structural characteristics of infiltrating T cells derived from the lesional tissues of oral lichen planus (OLP) patients. METHODS: BV gene distribution analysis was performed quantitatively in OLP patients using real-time polymerase chain reaction. The data was analyzed by Mann-Whitney U test using GraphPad Prism version 5.00 software. RESULTS: The usage toward BV4, BV14 and BV18 in the lesional tissues from 43 OLP patients was significantly higher than the usage toward these BV genes in the peripheral blood from 39 OLP patients. CONCLUSION: Infiltrating T cells derived from the lesional tissues in a cohort of Chinese OLP patients display preferential usage toward BV4, BV14 and BV18. It indicates that the T cells bearing BV4, BV14 and BV18 genes play a very important role in the etiology and pathogenesis of OLP. Supported by National Natural Science Foundation of China (30872888), Research Fund of Science and Technology Commission of Shanghai Municipality (06ZR14060) and Shanghai Leading Academic Discipline Project (S30206).

Malignant transformation of oral verrucous leukoplakia: a clinicopathologic study of 53 cases.

BACKGROUND: Oral verrucous leukoplakia (VL) is one of the non-homogenous oral leukoplakias. The objective of this study was to investigate the clinicopathologic features of VL and identify the clinicopathologic risk factors that might be associated with VL malignant transformation from China. METHODS: Among 1541 patients with oral leukoplakia, a total of 53 patients with clinical and histopathologic diagnosis of VL between 1996 and 2009 were reviewed retrospectively in our hospital. RESULTS: Of the 53 patients, 11 (20.8%) with VL were observed to develop cancer in the study period. The average age at diagnosis was 59.8 years with a male/female ratio of 1.7:1. Tongue was the predominant site (41.5%). Multivariate regression analysis revealed that the elderly patients (>65 years old) were associated with 8.36-fold [95% confidence interval (95% CI), 1.45-48.09; P = 0.017] increased risk of malignant transformation compared with the non-elderly patients. The lesion located on gingiva was associated with 20.81-fold (95% CI, 1.94-222.80; P = 0.012) increased risk of malignant transformation compared with tongue. However, the gender, smoking, alcohol intake, and epithelial dysplasia were not risk factors. CONCLUSION: Clinicopathologic features of VL in China were elucidated. The utilization of age and lesion site at diagnosis as significant factors for evaluating malignant transformation risk in patients with VL was suggested. Further studies are required to investigate the roles of the potential risk factors in the VL malignant transformation.

[Study on the expression and potential effect of PD-L1mRNA and PD-L2mRNA in oral lichen planus].

PURPOSE: To study the expression of PD-L1 and PD-L2mRNA in oral lichen planus(OLP) patients' peripheral blood and focal mucosa, and the different expression of target molecules in gene level in different clinical and pathological OLP groups. METHODS: The expression of PD-L1 and PD-L2 mRNA in OLP patients, which included 35 reticular and 25 atrophic-erosive OLP patients, 38 cases without dysplasia and 22 cases with dysplasia, was examined by real-time PCR. Peripheral blood and mucosa from 10 volunteers were used as control. All the results were analysed with Wilcoxon test by SAS.6.12 software package. RESULTS: The expression of PD-L2mRAN, but not PD-L1, was significantly higher in oral mucosa of OLP patients (P<0.01), while decreased in OLP patients' peripheral blood (P=0.0415). The gene expression of PD-L2 differed between different clinical types, and had highly significant correlation between the OLP patients' focal mucosa and peripheral blood(r=0.6976, P<0.0001). CONCLUSIONS: PD-L2 may have some potential effect on the pathogenesis of OLP at systemic and local level.