| Oral squamous cell carcinoma is the major type of head and neck cancer, with275,000new occurrences annually in the worldwide. High malignancy and mortality are its major characteristics. Patients survivable are often under surgical resection, and then accompanied with tissue defects on head and neck and deficiency on pronunciation, mastication and facial expression.30years have passed clinical development, including operation, reconstruction, infection control, adjuvant radio-and chemotherapy are achieved as well as that of development in basic research including tumorigenesis, pathogenesis, disease development, biomedical and target therapy. However, the5-year overall survival rate is still less than50%, not changed much. The major reasons accounted for this phenomenon is the unknown of cancer initiation, pivotal regulation and high specific markers. Hence, continual investigation on the pathogenesis, development and metastasis of oral squamous cell carcinoma (OSCC), and on therapeutic strategies and targets, as well as the development on clinical management, will be of great significance for patient survival and prognosis.LIN-28is a chronic timing regulated gene during the development of Caenorhabditis elegans, and the homologues gene in human are Lin28A and Lin28B. Recent studies show Lin28as a reprogramming factor which could induct pluripotent cells from stromal cells by using transcriptional factor cocktails with Oct4, Sox2, and Nanog. Lin28protein also shows abilities to bind mRNAs and miRNAs. By inhibitions on the maturing of let-7miRNAs and other potential miRNAs, and inhibitions on the transcription of mRNAs that related to cell growth and metabolism, Lin28hence functions as a pluripotent factor and participates in the process of body development, stem cell renewal, tumorigenesis and metabolism. In the time thesis proposal, the expression and function of Lin28in OSCC was not clear, so we performed the study. The expression and location of Lin28A and Lin28B were detected by immunohistochemistry and immunofluorescence. The relation between the expression level and tumor clinical patho-features and patient prognosis were statistically analyzed, and further investigated by in vitro and in vivo function assays. Results show Lin28B plays important role in the malignant transformation of OSCC, and the work has been published in the journal of Plos one. Though the exactly molecular mechanism is till need to be further studied, this study first investigates the preliminary role of the role of reprogramming factor Lin28in OSCC, and hence will promote the study between OSCC and cell reprogramming.Part One Expression and clinical significance of reprogramming factor Lin28A and Lin28B in OSCCObjective: Oncogenes, stem cell markers and oncogenesis emerged during the generation of iPS cells show helpful reference to cancer research. The functions of reprogramming factors in cancer field are not fully studied. The purpose for this part was to confirm the expression of known roles of reprogramming factors of Oct4, Sox2, Klf4, c-Myc, Nanog and to investigate the expression of unknown role of Lin28in OSCC, as well as their subcellular locations and the clinical significance. Methods&Materials:Expressions of Oct4, Sox2, Klf4, c-Myc, Nanog, Lin28A and Lin28B were detected in OSCC tissues by immunohistochemistry; subcellular location of Lin28A and Lin28B were detected by immunohistochemistry; association between Lin28A and Lin28B that with tumor clinical pathological features and patient prognosis were analyzed by SPSS based statistics. Result: Reprogramming factors Oct4, Sox2, Klf4, c-Myc, Nanog, Lin28A and Lin28B were all detected in OSCC tissues. Lin28A was high expressed in all specimens, while Lin28B high expressed in69/72(95.08%) specimens. Comparing with normal control tissues, Lin28A and Lin28B were highly expressed in OSCC (p<0.001). In all specimens,61%had Lin28A in cytoplasm, while70%had Lin28B in cell nucleus. Single factor analysis showed Lin28B was associated with patients’gender (p=0.005), cancer cell differentiation (p=0.024). Kaplan-Meier survival analysis showed patients with high expression of Lin28A and Lin28B had trends of earlier disease recurrence (p=0.16, p=0.06). Multivariate analysis by Cox proportional hazard regression model showed Lin28B was associated with patient poor prognosis (HR=2.5, p=0.001), suggesting Lin28B could be an independent prognostic factor. Conclusion:Reprogramming factors have associations with OSCC; Lin28A and Lin28B overexpressed in OSCC tissues with different subcellular location, suggesting difference in their regulation mechanisms. Lin28B but not Lin28A could be an independent prognostic factor, proteins and pathways related are worth to be further studied to find potential therapeutic values.Part Two Expression of Lin28B in oral cancer cell lines, selection of Lin28B stable expression cell lines and the influence of Lin28B on cellular biology of cancer cellsObjective: As abovementioned showed, Lin28B associates with patients’ poor prognosis, but the expression of Lin28B in oral cancer cell lines, for instance, SCC9, SCC15and SCC25were not known. The purpose for this part was to detect the expression of Lin28B in oral cancer cell line, and to observe the influence of overexpression Lin28in cancer-related cell behavior. Methods&Materials: Transcriptional and translational level of Lin28B in SCC9, SCC15and SCC25were detected by qRT-PCR and by Western blot. Cells with stable overexpression of Lin28B were constructed by lentiviral based vector. The influence of Lin28on cancer colony formation, migration and invasion were observed. Influence of the expression of inflammation, epithelial-mesenchymal transition and angiogenesis related genes including IL-6, STAT3, HMAG2, Snail, Twist, E-cadherin, Survivin and VEGF were then tested. Results:Lin28B low expressed in oral cancer cell lines of SCC9, SCC15and SCC25; Lin28B promotes oral cancer cell migration, invasion and colony formation (p<0.05, for all); Lin28B significantly up-regulated the pro-inflammation factor IL-6, STAT3and epithelial-mesenchymal transition markers Snail, Twist and angiogenesis marker VEGF, and the expression of HMGA2. The expression of E-cadherin was a bit low regulated. Conclusion:Characteristics of oral cancer cell line might be different from oral cancer tissues, as the expression of Lin28B is significantly different. Lin28B may promote cell malignancy through promotions of topical inflammation, EMT, angiogenesis and the inhibition of cell apoptosis.Part three Influence of Lin28B on in vivo tumor formation and developmentObjective: In vitro studies showed that Lin28B promoted expression of inflammation, EMT and apoptosis resistance markers, however, its role on in vivo tumor development was still unknown. The purpose of this part was to analyze the influence of Lin28B on in vivo tumor formation. Methods&Materials:oral cancer cell lines with stable expression of Lin28B were constructed by lentiviral based vector infection; selected Lin28B-overexpressed cell line, empty vector cell line and cell line that without dispose were injected into dorsal part of BALB/c-nu mice; continuous observation and measurement were performed every second day and finally analyzed statistically; expression of reprogramming factors Oct4, Sox2, Lin28A, Lin28B and Klf4in the xenograft tumor were detected by immunohistochemistry. Results: oral cancer cell line with stable expression of Lin28B was successfully established; SCC25-Lin28B had the medium expression of Lin28B comparing with SCC9-Lin28B and SCC15-Lin28B cells; expression of Oct4, Sox2, E-cadherin, N-cadherin and Lin28A and Lin28B could be detected in the xenograft tumor tissues; tumor tissues generated from Lin28B-overexpression cell had worse basal infiltration. Conclusion: Lin28B promotes oral cancer growth and development and might function through pathways related to topical inflammation, EMT and apoptosis resistance as abovementioned. |
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