검색 상세

miRNA-199b-5p suppresses oral squamous cell carcinoma progression by regulating apical-basolateral polarity through the Scribble/Lgl1 axis

  • Shihyun Kim (김시현, 강원대학교 일반대학원)

초록/요약 도움말

Oral cancer refers to malignant tumors that occur throughout the oral cavity, including the tongue, lips, gums, and palate. Although the incidence of oral cancer is relatively low, accounting for 3–5% of overall cancers, it has been steadily increasing by more than 30% over the past decade due to the trend of younger people smoking and drinking. In particular, oral squamous cell carcinoma （OSCC）, which accounts for approximately 80–90% of all oral cancers, develops from the oral cavity mucosa or epithelium and is characterized by high invasiveness and lymph node metastasis. Although surgical resection, radiation therapy, and chemotherapy are currently utilized for OSCC treatment, there is a limitation in that advanced-stage cases involve functional and aesthetic damage due to extensive resection. Therefore, identifying new regulatory mechanisms from a molecular biological perspective on OSCC progression is critical. However, research on the key molecular mechanisms underlying OSCC progression remains insufficient to this day. In epithelial cells, cell-cell junctions and apico-basal polarity （ABP） play a crucial role in maintaining cell morphology and homeostasis, whereas the loss of polarity is known to be closely associated with the progression of various types of cancer. Scribble is a conserved gene that regulates cell polarity, reported for the first time in Drosophila. In mammals, it is reported to involve in maintaining cell junctions and regulating the stability of epithelial structures by forming the Scribble polarity complex and has been shown to function as a tumor suppressor in various cancers. In particular, hypoxia is known to significantly impact the survival and progression of tumor cells; however, the relationship between hypoxia and cell polarity regulation in OSCC has not yet been sufficiently elucidated. Therefore, this study investigated the potential role of the specific microRNA miR-199b-5p and Scribble in the progression and metastasis of OSCC and analyzed their correlation and the underlying mechanisms regarding cell polarity under hypoxic conditions. First, we analyzed an OSCC patient dataset and performed immunohistochemical （IHC） staining of tissue samples to verify the expression of Scribble and miR-199b-5p. The results showed that both Scribble and miR-199b-5p were expressed at lower levels in OSCC tissue compared to normal tissue. Next, we selected the OSCC cell lines SCC-9 and HSC-2 to observe changes in intracellular protein localization and cell morphology under Scribble knockdown and hypoxia conditions using confocal microscopy. As a result, we found that reduced Scribble expression decreased the expression of Lgl1, a component of the Scribble polarity module, and E-cadherin, an adhesion junction marker, and the co-localization of cell junctions was significantly disrupted. Significantly, these results led to increased cell motility and proliferation in OSCC cells, as confirmed by Boyden chamber and EdU assays. Under hypoxic conditions, Scribble expression decreased and cell polarity became disrupted. This suggests that the hypoxic microenvironment may influence the maintenance of polarity. Subsequently, treatment with hsa-miR-199b not only increased the mRNA and protein expression of Scribble but also partially reversed the co-localization with Lgl1 and E-cadherin in OSCC cell lines. Using Boyden chamber and EdU assays, we confirmed that treatment with hsa-miR-199b effectively reduced the migration and proliferation of OSCC cell lines. Furthermore, in vivo xenograft and lung metastasis models, we observed that reduced Scribble expression was associated with decreased expression and tissue localization of polarity-associated proteins, and that these changes were partially reversed by inducing miR-199b-5p expression. In summary, this study suggests that miR-199b-5p potentially participates in Scribble-mediated polarity regulation within the hypoxic tumor microenvironment and suggests a significant role in the maintenance of cellular polarity during the OSCC progression. Moreover, by advancing our understanding of the relationship between hypoxic environments and the regulation of cell polarity, our study is expected to serve as a foundation for clarifying the mechanisms associated with OSCC progression and metastasis.

more

목차 도움말

1. INTRODUCTION 2
2. MATERIALS AND METHODS 6
2.1 Clinical specimen and human tissue samples 6
2.2 Cell culture and transfection 6
2.3 Immunoblot assays 7
2.4 Co-immunoprecipitation (Co-IP) assays 8
2.5 Transwell migration and invasion assays 8
2.6 EdU cell proliferation assay 9
2.7 Cloning of the vector construct 9
2.8 Luciferase reporter assay 10
2.9 In vivo tumor growth and metastasis 11
2.10 Real-time PCR 11
2.11 Immunofluorescence (IF) 12
2.12 Proximity ligation assay (PLA) 13
2.13 Histological analysis 13
2.14 Statistical analysis 14
3. RESULTS 16
3.1 Scribble interacts with Lgl1 in OSCC cell lines 16
3.2 Scribble cooperatively regulates the metastatic and proliferative properties of OSCC with Lgl1 22
3.3 Hypoxia modulates the localization of Scrib/Lgl1/E-cadherin in OSCC cell lines 32
3.4 miRNA-199b-5p suppresses the metastatic and proliferative properties of OSCC 38
3.5 Suppression of Scribble induced the tumor growth and metastasis of OSCC in vivo 48
3.6 miR-199b-5p inhibits the growth and metastasis of OSCC in vivo 54
4. DISCUSSION 58
5. CONCLUSIONS 65
REFERENCES 68
ABSTRACT IN KOREAN 74

more
반출 Meta View 목록