Prediction of Tumor Microenvironment Characteristics and Treatment Response in Lung Squamous Cell Carcinoma by Pseudogene OR7E47P-related Immune Genes

Objective Pseudogenes are initially regarded as nonfunctional genomic sequences,but some pseudogenes regulate tumor initiation and progression by interacting with other genes to modulate their transcriptional activities.Olfactory receptor family 7 subfamily E member 47 pseudogene(OR7E47P)is expressed broadly in lung tissues and has been identified as a positive regulator in the tumor microenvironment(TME)of lung adenocarcinoma(LUAD).This study aimed to elucidate the correlation between OR7E47P and tumor immunity in lung squamous cell carcinoma(LUSC).Methods Clinical and molecular information from The Cancer Genome Atlas(TCGA)LUSC cohort was used to identify OR7E47P-related immune genes(ORIGs)by weighted gene correlation network analysis(WGCNA).Based on the ORIGs,2 OR7E47P clusters were identified using non-negative matrix factorization(NMF)clustering,and the stability of the clustering was tested by an extreme gradient boosting classifier(XGBoost).LASSO-Cox and stepwise regressions were applied to further select prognostic ORIGs and to construct a predictive model(ORPScore)for immunotherapy.The Botling cohorts and 8 immunotherapy cohorts(the Samstein,Braun,Jung,Gide,IMvigor210,Lauss,Van Allen,and Cho cohorts)were included as independent validation cohorts.Results OR7E47P expression was positively correlated with immune cell infiltration and enrichment of immune-related pathways in LUSC.A total of 57 ORIGs were identified to classify the patients into 2 OR7E47P clusters(Cluster 1 and Cluster 2)with distinct immune,mutation,and stromal programs.Compared to Cluster 1,Cluster 2 had more infiltration by immune and stromal cells,lower mutation rates of driver genes,and higher expression of immune-related proteins.The clustering performed well in the internal and 5 external validation cohorts.Based on the 7 ORIGs(HOPX,STX2,WFS,DUSP22,SLFN13,GGCT,and CCSER2),the ORPScore was constructed to predict the prognosis and the treatment response.In addition,the ORPScore was a better prognostic factor and correlated positively with the immunotherapeutic response in cancer patients.The area under the curve values ranged from 0.584 to 0.805 in the 6 independent immunotherapy cohorts.Conclusion Our study suggests a significant correlation between OR7E47P and TME modulation in LUSC.ORIGs can be applied to molecularly stratify patients,and the ORPScore may serve as a biomarker for clinical decision-making regarding individualized prognostication and immunotherapy.

Promoter methylation of tumor suppressor genes in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma(ESCC) is a prevalent and fatal cancer in China and other Asian countries.Epigenetic silencing of key tumor suppressor genes(TSGs) is critical to ESCC initiation and progression.Recently,many novel TSGs silenced by promoter methylation have been identified in ESCC,and these genes further serve as potential tumor markers for high-risk group stratification,early detection,and prognosis prediction.This review summarizes recent discoveries on aberrant promoter methylation of TSGs in ESCC,providing better understanding of the role of disrupted epigenetic regulation in tumorigenesis and insight into diagnostic and prognostic biomarkers for this malignancy.

mi R-382 functions as a tumor suppressor against esophageal squamous cell carcinoma

AIM To explore the effect of miR-382 on esophageal squamous cell carcinoma (ESCC) in vitro and its possible molecular mechanism. METHODS Eca 109 cells derived from human ESCC and Het-1A cells derived from human normal esophageal epithelium were used. Lentivirus-mediated miR-382 was overexpressed in Eca109 cells. The effect of miR-382 on cell proliferation was evaluated by MTT and colony formation assay. For cell cycle analysis, cells were fixed and stained for 30 min with propidium iodide (PI) staining buffer containing 10 mg/mL PI and 100 mg/mL RNase A, and analyzed by BD FACSCalibur (TM) flow cytometer. For cell apoptosis assay, cells were stained with an Annexin V-FITC/PI Apoptosis Detection Kit according to the manufacturer's instructions and analyzed by a dual-laser flow cytometer. Cell invasion and migration abilities were determined through use of transwell chambers, non-coated or pre-coated with matrigel. Levels of proteins related to cell growth and migration were examined by western blotting. RESULTS Endogenous miR-382 was down-regulated in Eca109 cells compared with Het-1A. Introduction of miR-382 not only significantly inhibited proliferation and colony formation, but also arrested cell cycle at the G2/M phase, as well as promoted apoptosis and autophagy in Eca109 cells. Migration, invasion and epithelial-mesenchymal transition of Eca109 cells were suppressed by overexpressing miR-382. Western blotting results showed that miR-382 inhibited the phosphorylation of mTOR and 4E-BP1. CONCLUSION miR-382 functions as a tumor suppressor against ESCC development and metastasis, and could be considered as a potential drug source for the treatment of ESCC patients.

Aberrant methylation of the 3q25 tumor suppressor gene PTX3 in human esophageal squamous cell carcinoma

AIM:To identify the novel methylation-silenced gene pentraxin 3(PTX3) in esophageal squamous cell carcinoma(ESCC).METHODS:PTX3 mRNA expression was examined in six human ESCC cell lines,one human immortalized normal esophageal epithelial cell line,primary ESCC tumor tissue,and paired adjacent nontumor tissue using reverse transcription polymerase chain reaction(RTPCR).Semi-quantitative immunohistochemistry was used to examine cellular localisation and protein levels.Methylation specific PCR and bisulphite genomic sequencing were employed to investigate the methylation of the candidate gene.RESULTS:In the majority of ESCC cell lines,we found that PTX3 expression was down-regulated due to gene promoter hypermethylation,which was further confirmed by bisulphite genomic sequencing.Demethylation treatment with 5-aza-2'-deoxycytidine restored PTX3 mRNA expression in ESCC cell lines.Methylation was more common in tumor tissues(85%) than in adjacent nontumor tissues(25%)(P < 0.01).CONCLUSION:PTX3 is down-regulated through promoter hypermethylation in ESCC,and could potentially serve as a biomarker of ESCC.

Tumor invasion front in oral squamous cell carcinoma

Oral squamous cell carcinoma is a neoplasm that originates from the epithelial mucosa.It is usually more frequent between the fifth and sixth decades of life,and more than 90% of carcinomas of the oral cavity are squamous cell carcinoma.It is an invasive neoplasia with a significant recurrence rate;40% of patients present with metastases in the cervical lymph nodes at the time of diagnosis.The tumor invasion front is a characteristic of tumor growth,which can be infiltrative or noninvasive.The histopathological parameters examined include the number of mitoses,depth of the tumor,invasion pattern,degree of keratinization,and nuclear pleomorphism.For the pathologist,these parameters are routinely evaluated but are not reported to the treating physician in all cases,which we consider to be useful information when determining the therapeutic route.

The recurrence and survival of oral squamous cell carcinoma: a report of 275 cases

Oral squamous cell carcinoma(OSCC) is a common malignant tumor of the head and neck, and recurrence is an important prognostic factor in patients with OSCC. We explored the factors associated with recurrence of OSCC and analyzed the survival of patients after recurrence. Clinicopathologic and follow-up data of 275 patients with OSCC treated by surgery in the Cancer Institute and Hospital of Tianjin Medical University between 2002 and 2006 were analyzed. Recurrence factors were analyzed with Chisquare or Fisher′s exact test and multivariate analysis. The prognosis of patients after recurrence was analyzed with the Kaplan-Meier method and log-rank test. The recurrence rate was 32.7%. The recurrence time ranged from 2 to 96 months, with a median of 14 months. Univariate analysis showed that T stage, degree of differentiation, pN stage, flap application, resection margin, and lymphovascular invasion were factors of recurrence (P<0.05). Multivariate analysis showed that T stage, degree of differentiation, and pN stage were independent factors of recurrence (P<0.001). The differences in gender, age, tumor site, region of lymph node metastasis, and perineural invasion between the recurrence and non-recurrence groups were not significant (P>0.05). Kaplan-Meier and log-rank tests showed that the 2- and 5-year survival rates were significantly lower in the recurrence group than in non-recurrence group(67.6% vs. 88.0%, 31.8% vs. 79.9%, P<0.001). Therefore, to improve prognosis, we recommend extended local excision, flap, radical neck dissection, and adjuvant chemoradiotherapy for patients more likely to undergo recurrence.

Differential mRNA expression profiling of oral squamous cell carcinoma by high-throughput RNA sequencing

Differentially expressed genes are thought to regulate the development and progression of oral squamous cell carcinomas （OSCC）. The purpose of this study was to screen differentially expressed mRNAs in OSCC and matched paraneoplastic normal tissues, and to explore the intrinsic mechanism of OSCC development and progres- sion. We obtained the differentially expressed mRNA expression profiles in 10 pairs of fresh-frozen OSCC tissue specimens and matched paraneoplastic normal tissue specimens by high-throughput RNA sequencing. By using Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analyses, the functional significance of the differentially expressed genes were analyzed. We identified 1,120 sig- nificantly up-regulated mRNAs and 178 significantly down-regulated mRNAs in OSCC, compared to normal tissue. The differentially expressed mRNAs were involved in 20 biological processes and 68 signal pathways. Compared to adjacent normal tissue, the expression of MAGEAll was up-regulated; TCHH was down-regulated. These find- ings were verified by real-time PCR. These differentially expressed mRNAs may function as oncogenes or tumor suppressors in the development and progression of OSCC. This study provides novel insights into OSCC. However, further work is needed to determine if these differentially expressed mRNAs have potential roles as diagnostic bio- markers and candidate therapeutic targets for OSCC.

Black phosphorous nanosheets−gold nanoparticles−cisplatin for photothermal/photodynamic treatment of oral squamous cell carcinoma

To improve five-year survival rate of oral squamous cell carcinoma(OSCC),the development of a novel composite material of black phosphorus nanosheets(BPNSs)and gold nanoparticles(AuNPs)for tumor treatment was carried out.The purpose of this study is to evaluate the cytostatic effects of BPNSs,AuNPs loaded with cisplatin(CDDP)on human tongue squamous cell carcinoma cells lines(SCC-9),and 7,12-dimethylbenz anthracene induced cheek squamous cell carcinoma was validated in golden hamsters animal models.The results showed that BPNSs could efficiently inhibit the metastasis and growth of OSCC compared with CDDP and AuNPs.And a combination composite of AuNPs−BPNSs loaded with CDDP could more effectively inhibit the metastasis and growth of OSCC,which might be due to the high drug-loading capacity,excellent photothermal properties and the combination of photodynamic and photothermal therapy of BPNSs and AuNPs,as well as the synergistic effects of AuNPs,BPNSs and CDDP.

Identification of microRNA-21 as a valuable diagnostic marker of oral squamous cell carcinoma and potential target

Objective The aim of the study was to summarize the diagnostic value of miR-21 as a biomarker in oral squamous cell carcinoma(OSCC)using a review of the literature and data from the cancer genome atlas(TCGA)database.Methods Data from TCGA database was sorted and analyzed by bioinformatics to determine the expression level of miR-21 in OSCC.Further,we searched for relevant articles in Embase,PubMed/Medline,Scopus,and Web of Science published before March 2021,extracted the data,and conducted quality assessment.The bivariate meta-analysis model with Stata 16.0 was used to analyze the diagnostic value of miR-21 for OSCC.Results A total of 304 related articles were identified,and seven were selected for meta-analysis.The diagnostic results after analysis were as follows:sensitivity 0.76[95%confidence interval(CI),0.57-0.88];specificity 0.77(95%CI,0.58-0.89);positive likelihood ratio 3.34(95%CI,1.58-7.08);negative likelihood ratio 0.31(95%CI,0.15-0.63);diagnostic odds ratio 10.75(95%CI,2.85-40.51);and area under the curve 0.83(95%CI,0.80-0.86).The Deeks’funnel chart showed that there was no potential bias(P=0.54).Prediction analysis of the potential target genes of miR-21 was performed via the biological website,and DAVID was used to cross target genes for gene ontology(GO)annotation function analysis.Conclusion The results showed that miR-21-3p and miR-21-5p were significantly more highly expressed in OSCC tissues than in normal tissues(P<0.05),and the results of the meta-analysis indicated that they could be used as potential biomarkers in the diagnosis of OSCC.In addition,58 potential target genes of miR-21 were significantly enriched in 28 GO annotation functional pathways,which provided a biological basis for further clinical diagnostic value research.

IR780 loaded hollow MnO_(2) nanoparticles for dual-mode imaging and enhanced photodynamic therapy of oral squamous cell carcinoma

Photodynamic therapy(PDT)has emerged as a novel therapeutic modality for cancer treatment,but its therapeutic efficacy is severely limited by the hypoxic tumor microenvironment(TME).Here we designed an innovative multifunctional nano-platform which consists of a hollow MnO_(2) shell and internal photosensitizer IR780.It is not only used for multimodal imaging of oral squamous cell carcinoma(OSCC),but also for adjustment hypoxic TME to enhance cancer treatment.Hollow MnO_(2) can promote decomposition of tumor endogenous H2O2 to relieve tumor hypoxia,thereby enhancing the effect of photodynamic therapy.Photosensitizer IR780 generates singlet oxygen under laser irradiation to kill tumor cells,playing photodynamic effect,can also act as the contrast agents for photoacoustic and fluorescence multiple imaging,providing potential imaging capability for cancer therapeutic guidance and monitoring.Our research results in this article show that HMnO_(2)-IR780 nanocomposite exhibits good biocompatibility and nontoxicity,strong PA/FL imaging contrast,excellent oxygen production capacity and outstanding photodynamic therapy effect.This finding provides a new idea for multimodal imaging-guided nanotherapy for OSCC.

EXPRESSION OF P53 GENE IN ORAL SQUAMOUS CELL CARCINOMA AND ITS RELATION WITH CLINICAL AND PATHOLOGICAL PARAMETERS AND PROGNOSIS OF PATIENTS

One hundred and eleven cases of oral squamous cell carcinoma (OSCC) were examined for overexpression of p53 protein by using immunohistochemical technique. Association between p53 protein overexpression and clinical and pathological parameters as well as prognosis of patients were also analyzed. p53 protein overexpression was commonly observed (69. 4%) in OSCC and may be used as a marker of carcinogenesis of OSCC. The level of p53 protein overexpression is correlated with the lower three and five-year survival rate of OSCC. The presence or absence of p53 overexpression was not correlated with sex, age, site of tumor, size of tumor, degree of differentiation,node status,and clinical stage in OSCC. Single factor COX proportional hazards regression model analysis indicated that there was no significant association between p53 overexpression and prognosis of OSCC. Multivariable COX model analysis failed to establish effective life function or risk rate function. These showed that all the parameters analyzed in this study as well as p53 overexpression were not significant and effective risk factors of prognosis for patients with OSCC.

Epigenetic inactivation of secreted frizzled-related protein 2 in esophageal squamous cell carcinoma

AIM： To investigate the expression and methylation status of the secreted frizzled-related protein 2 （SFRP2） in esophageal squamous cell carcinoma （ESCC） and ex- plore its role in ESCC carcinogenesis.METHODS： Seven ESCC cell lines （KYSE 30, KYSE150, KYSE410, KYSE510, EC109, EC9706 and TE-1） and one immortalized human esophageal epithelial cell line （Het- 1A）, 20 ESCC tissue samples and 20 paired adjacent non-tumor esophageal epithelial tissues were analyzed in this study. Reverse-transcription polymerase chain reaction （RT-PCR） was employed to investigate the expression of SFRP2 in cell lines, primary ESCC tumor tissue, and paired adjacent normal tissue. Methylation status was evaluated by methylation-specific PCR and bisulfite sequencing. The correlation between expres- sion and promoter methylation of the SFRP2 gene was confirmed with treatment of 5-aza-2＇-deoxycytidine. To assess the potential role of SFRP2 in ESCC, we es-tablished stable SFRP2-transfected cells and examined them with regard to cell proliferation, colony formation, apoptosis and cell cycle in vivo and in vitro.RESULTS： SFRP2 mRNA was expressed in the im- mortalized normal esophageal epithelial cell line but not in seven ESCC cell lines. By methylation-specific PCR, complete methylation was detected in three cell lines with silenced SFRP2 expression, and extensive methylation was observed in the other four ESCC cell lines. 5-aza-2＇-deoxycytidine could restore the expres- sion of SFRP2 mRNA in the three ESCC cell lines lack- ing SFRP2 expression. SFRP2 mRNA expression was obviously lower in primary ESCC tissue than in adjacent normal tissue （0.939 ± 0.398 vs 1.51 ± 0.399, P 〈 0.01）. SFRP2 methylation was higher in tumor tissue than in paired normal tissue （95% vs 65%, P 〈 0.05）. The DNA methylation status of the SFRP2 correlated inversely with the SFRP2 expression. To assess the potential role of SFRP2 in ESCC, we established stable SFRP2 transfectants and control counterparts by in- troducing pcDNA3.1/v5 hisA -SFRP2 or pcDNA3.1/v5 hisA -empty vector into KYSE30 cells lacking SFRP2 expression. After transfection, the forced-expression of SFRP2 was confirmed by the RT-PCR. In comparison with the control groups, stably-expressed SFRP2 in KYSE 30 cells significantly reduced colony formation in vitro （47.17% 4± 15.61% vs 17% ：1： 3.6%, P = 0.031） and tumor growth in nude mice （917.86：1：249.35 mm3 vs 337.23 ± 124.43 mm3, P 〈 0.05）. Using flow cytom- etry analysis, we found a significantly higher number of early apoptotic cells in SFRP2-transfected cells than in the control cells （P = 0.025）. The mean cell number in the S and G2-M phases of the cell cycle was also significantly lower in SFRP2-transfected KYSE30 cells compared with mock transfected counterparts. CONCLUSION： Silencing of SFRP2 expression through promoter hypermethylation may be a factor in ESCC carcinogenesis through loss of its tumor-suppressive activity.

Primary Neuroendocrine Carcinoma Combined with Squamous Cell Carcinoma of the Soft Palate: A Case Report and Review of Literature

Background: Neuroendocrine carcinomas (NECs) are rare neoplasms that widely occur in various organs. They are heterogeneous and vary from low to high grade malignant. NEC presenting with a squamous cell carcinoma (SCC) component is referred to as a composite tumor. Thus far, few cases of this composite tumor in the oral cavity have been reported in the literature;thus, the histogenesis remains unclear. ase Presentation: We encountered a rare case of a primary NEC combined with SCC, occurring at the soft palate in a 59-year-old man. A resected specimen of the tumor was composed of two components: NEC and SCC. The NEC area contained small round to oval atypical cells arranged in nests with a glandular-like-pattern, hyperchromatic molded nuclei, a high nuclear-to-cytoplasmic ratio, and a scant eosinophilic cytoplasm. The SCC area was composed of non-keratotic, dysplastic oval to spindle-shaped squamous cells with indistinct cell borders and large nuclei that were hyperchromatic and pleomorphic. Immunohistochemically, the tumor cells of the NEC component were positive for chromagranin A, synaptophysin, CD56, and p16, whereas those of the SCC component were positive for 34βE12, p63, and p16. Conclusion: In consideration of the morphological and immunohistochemical results, the final diagnosis was a primary NEC combined with SCC of the soft palate.

Cisplatin-based miRNA delivery strategy inspired by the circCPNE1/miR-330-3p pathway for oral squamous cell carcinoma

Circular RNAs(circRNAs)are ideal biomarkers of oral squamous cell carcinoma(OSCC)because of their highly stable closed-loop structure,and they can act as microRNA(miRNA)sponges to regulate OSCC progression.By analyzing clinical samples,we identified circCPNE1,a dysregulated circRNA in OSCC,and its expression level was negatively correlated with the clinical stage of OSCC patients.Gain-of-function assays revealed the tumor-suppressive effect of circCPNE1,which was then identified as a miR-330-3p sponge.MiR-330-3p was recognized as a tumor promoter in multiple studies,consistent with our finding that it could promote the proliferation,migration,and invasion of OSCC cells.These results indicated that selective inhibition of miR-330-3p could be an effective strategy to inhibit OSCC progression.Therefore,we designed cationic polylysine-cisplatin prodrugs to deliver antagomiR-330-3p(a miRNA inhibitory analog)via electrostatic interactions to form PP@miR nanoparticles(NPs).Paratumoral administration results revealed that PP@miR NPs effectively inhibited subcutaneous tumor progression and achieved partial tumor elimination(2/5),which confirmed the critical role of miR-330-3p in OSCC development.These findings provide a new perspective for the development of OSCC treatments.

New perspectives on biology,disease progression,and therapy response of head and neck cancer gained from single cell RNA sequencing and spatial transcriptomics

Head and neck squamous cell carcinoma(HNSCC)is one of the most frequent cancers worldwide.The main risk factors are consumption of tobacco products and alcohol,as well as infection with human papilloma virus.Approved therapeutic options comprise surgery,radiation,chemotherapy,targeted therapy through epidermal growth factor receptor inhibition,and immunotherapy,but outcome has remained unsatisfactory due to recurrence rates of~50%and the frequent occurrence of second primaries.The availability of the human genome sequence at the beginning of the millennium heralded the omics era,in which rapid technological progress has advanced our knowledge of the molecular biology of malignant diseases,including HNSCC,at an unprecedented pace.Initially,microarray-based methods,followed by approaches based on next-generation sequencing,were applied to study the genetics,epigenetics,and gene expression patterns of bulk tumors.More recently,the advent of single-cell RNA sequencing(scRNAseq)and spatial transcriptomics methods has facilitated the investigation of the heterogeneity between and within different cell populations in the tumor microenvironment(e.g.,cancer cells,fibroblasts,immune cells,endothelial cells),led to the discovery of novel cell types,and advanced the discovery of cell-cell communication within tumors.This review provides an overview of scRNAseq,spatial transcriptomics,and the associated bioinformatics methods,and summarizes how their application has promoted our understanding of the emergence,composition,progression,and therapy responsiveness of,and intercellular signaling within,HNSCC.

Frequent Down-regulation and Deletion of KLF6 in Primary Hepatocellular Carcinoma

Kruppel-like factor 6 (KLF6) was reported as tumor suppressor in multiple cancers. However, loss of chromosomal locus spanning KLF6 is relatively infrequent in previous published studies. To explore the role of KLF6 in hepatocellular carcinoma (HCC), we examined the gene for expression change, loss of heterozygosity (LOH) and mutation in 26 HCC samples. The expression levels of KLF6 were significantly down-regulated in HCCs, as detected by qRT-PCR. LOH occurred in 11 (52%) of 21 tumors, and all the samples with LOH showed KLF6 down-regulation. The mutational frequency was 24%, and sequence changes located in activation domain of KLF6. Furthermore, MTT assay showed a significant antiproliferative effect of the wt KLF6 transfected in HepG2 hepatoblastoma cells. Fluorescence-activated cell sorting analysis revealed that KLF6 could induce apoptosis. These findings indicate that deregulation of KLF6, together with genetic abnormalities of allelic imbalance and mutations, may play a role in HCC pathogenesis.

基于生物信息学构建口腔鳞状细胞癌免疫基因的预后模型

目的旨在构建免疫相关基因(IRGs)的风险预测模型,以预测口腔鳞状细胞癌(OSCC)患者的预后。方法应用生物信息学技术分析OSCC的转录组测序数据,鉴定出差异表达的IRGs(DEIRGs)。通过Cox回归分析构建DEIRGs的风险预测模型,并对其预测能力进行评估。分析该模型与临床病理和免疫细胞浸润的相关性。结果通过比较OSCC和正常样本共鉴定出3634个差异表达基因,其中包括330个DEIRGs(FDR<0.05,|logFC|>1)。单因素Cox回归分析筛选出与预后相关的20个DEIRGs(P<0.05),多因素Cox回归分析筛选出其中15个DEIRGs用于构建风险预测模型。该模型可作为OSCC患者的独立预后因素(P<0.001),预测患者预后的能力具有较高的准确性(AUC=0.732),并与临床分期(t=-3.484,P<0.001)、B细胞(Cor=-0.180,P=0.002)和CD4^(+)T细胞(Cor=-0.127,P=0.026)密切相关。结论基于15个预后相关DEIRGs构建的风险预测模型能够有效地预测OSCC患者的预后,可帮助临床医生为不同风险的OSCC患者选择个性化的治疗策略。

基于网络药理学和分子对接的白藜芦醇治疗口腔鳞状细胞癌的机制研究

目的通过网络药理学及分子对接等生物学信息方法,探讨白藜芦醇治疗口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)的分子机制,为白藜芦醇治疗OSCC的临床应用提供参考。方法利用Swiss Target Prediction(http://www.swisstargetprediction.ch)、SEA数据库(http://sea.bkslab.org)、Pharm mapper数据库(http://lilab-ecust.cn)检索获得白藜芦醇的相关靶点,以DISGENET(www.disgenet.org)、OMIM(https://omim.org)、GeneCards(https://www.genecards.org)数据库筛选OSCC疾病靶点,取药物与疾病靶点的交集,再采用Cytoscape 3.7.2软件构建“药物-疾病-靶点-通路”网络,String数据库构建靶蛋白相互作用网络,采用DAVID数据库对关键蛋白进行富集分析,最后通过AutoDock及PyMOL对关键蛋白进行分子对接验证,结合富集分析和分子对接结果预测白藜芦醇治疗OSCC可能的分子作用机制;细胞水平采用Western blot检测不同浓度(50、100μmol/L)白藜芦醇对OSCC细胞株HSC-3细胞Src酪氨酸激酶(Src tyro-sine kinase,SRC)、表皮生长因子受体(epidermal growth factor receptor,EGFR)、雌激素受体基因1(estrogen receptor gene 1,ESR1)及磷脂酰肌醇三激酶/蛋白激酶B(phosphatidylinositol 3 kinase/protein kinase B,PI3K/AKT)信号通路蛋白表达的影响。结果数据库得到白藜芦醇药物靶点243个,OSCC疾病靶点6094个,将药物与疾病的靶点进行交集获得116个潜在靶点,潜在靶点主要集中参与体内蛋白质自磷酸化、肽基酪氨酸磷酸化、跨膜受体蛋白酪氨酸激酶信号通路、RNA聚合酶Ⅱ启动子转录的正调控等生物过程,干预PI3K/AKT信号通路发挥抗OSCC的作用。分子对接结果表明白藜芦醇与EGFR、ESR1、SRC等OSCC关键靶点具有较好的结合活性。细胞实验结果表明,白藜芦醇药物干预以剂量依赖的方式抑制了HSC-3细胞中SRC、EGFR、ESR1及p-PI3K和p-AKT的蛋白表达。结论白藜芦醇对OSCC细胞SRC、EGFR、ESR1、p-PI3K、p-AKT靶点具有抑制作用。

RhoC基因沉默对口腔鳞状细胞癌迁移和侵袭的影响

目的:分析RhoC对口腔鳞状细胞癌(OSCC)细胞迁移和侵袭能力的影响及其潜在机制。方法:利用UALCAN和K-M plotter在线数据库预测分析RhoC在OSCC中的表达及其与病理分期分生存率的关系,并结合IHC实验检测OSCC组织中RhoC的表达水平。按照人RhoC基因构建两条小干扰RNA(siRNA)并将细胞进行实验分组。RT-qPCR检测转染后各组细胞RhoC的mRNA表达水平,Western blot法分析实验分组细胞中RhoC、FAK、MAPK、p-FAK、p-MAPK、基质金属蛋白酶2(MMP-2)和MMP-9蛋白表达;此外,比较各实验分组细胞的穿膜细胞数和划痕愈合面积分析细胞的侵袭和迁移活动。最终通过构建裸鼠肺转移模型对上述实验进行验证。结果:RhoC在OSCC中高表达,并和病理分期和病理分级等关联性大。RhoC的mRNA和蛋白相对表达量在OSCC中增高(P<0.01);RT-qPCR及Western blot结果显示,敲减RhoC表达后,RhoC的mRNA和蛋白表达水平显著下降(P<0.01),Western blot结果显示p-FAK、p-MAPK、MMP-2和MMP-9蛋白水平显著下降(P<0.05),FAK和MAPK蛋白表达无明显变化(P>0.05);细胞迁移和侵袭能力显著下降(P<0.01);实验组在动物活体荧光成像系统下发出的荧光强度显著低于对照组,且HE染色结果显示实验组裸鼠肺结节数量明显减少(P<0.05)。结论:RhoC基因可影响OSCC细胞的迁移和侵袭能力,其潜在机制可能是通过活化FAK和MAPK信号分子参与OSCC细胞的迁移和侵袭过程。

肿瘤相关成纤维细胞对口腔鳞状细胞癌生物学行为影响的研究进展

口腔鳞状细胞癌(oral squamous cell carcinoma, OSCC)是全球第六大常见恶性肿瘤,其发生、发展与肿瘤微环境(tumor microenvironment, TME)密切相关。肿瘤相关成纤维细胞(cancer-associated fibroblasts, CAFs)作为TME中重要的组成成分,通过分泌多种生长因子、细胞因子、炎性因子、外泌体等参与上皮-间充质转化、重塑细胞外基质,激活多种生物学途径,对OSCC的发生、发展产生影响。本文对CAFs的来源、特点、异质性以及CAFs对OSCC的生物学行为的影响做一综述。