| Oral cancer is one malignant tumor with high incidence, known as the6th frequent carcinomas in the whole body in which tongue is the easily affected intraoral site. And tongue squamous cell carcinoma (TSCC) is the most common pathological type of tongue cancer. It’s known for his poor prognosis and high mortality rates because of its features such as high invasiveness and metastasis. Although in cases with successful surgery, TSCC frequently leads to severe defects in speech, chewing as well as cancer-related death. The common strategies to treat tongue cancer is head and neck surgery in combination with adjuvant therapy, but the treatment results are still not ideal until now. Recently, more and more researchers are pursuing for new biological methods to treat TSCC in order to get better results. Until now, many molecules has been found very important in the development and prognosis of tongue squamous cell carcinoma as well as in other carcinoma, such as NF-κ B and β-catenin. However, precise mechanism of TSCC is still unknown until now.Four and a half lim domains2(FHL2) is a protein of279amino acids in length containing four full LIM-domains and a half LIM-domain at the amino-terminus. Due to the differential usage of different LIM-domains, FHL2is one transcriptional cofactor, able to interact with many different proteins, such as AP-1, BRCA1, IGFBP and integrin, involving in organ differentiation, development, cell apoptosis and carcinogenesis. Recent studies showed that FHL2could play different roles acted as co-activator or co-repressor in different types of cancers depending on the cell types involved. Researchers found the intriguing phenomenon that FHL2is overexpressed in breast cancer, gastrointestinal cancer, prostate cancer, ovarian cancer, glioma, contributing to their development, but down-regulated in liver cancer which inhibit the carcinogenesis, making the role of FHL2in cancer development elusive.The two sides of FHL2function was so intriguing that we wonder if FHL2could play a role in TSCC. Until now, no report has been found about the relationship between FHL2and TSCC. Therefore, the study aims to investigate the role of FHL2in TSCC and its signaling pathway by in vitro and in vivo experiments. Our study mainly contains three experiments,1) TSCC samples and normal tongue tissues were collected, then FHL2expression was detected and compared by immunohistochemistry and related software. Our results found that FHL2was expressed in TSCC, much stronger positive staining was found with the increase of TSCC tumor grade, and only weak positive staining was detected in normal tissues. This result indicates that FHL2might play an important role in development and carcinogenesis of TSCC.2) TSCC cell line Tca8113was transfected by pcDNA3.0-FHL2-flag plasmid, Tca8113cell lines with pcDNA3.0plasmid and untreated Tca8113were used as controls. Cell biological behaviors including cell proliferation, apoptosis, invasiveness and metastases were investigated by MTT, Flow Cytometry, Transwell and cell scratch methods. And possible related molecules including NF-κ B and β-catenin were investigated by Real-time PCR and Western blot. The results showed that FHL2overexpression could stimulate cell proliferation, invasiveness, metastases and cell apoptosis. Meantime, it could upregulate RNA and protein levels of NF-κ B and β-catenin.3) Nude mice tumorigenisis experiment was performed in this study. Three cell lines were transplanted into nude mice and the tumor volume was investigated at time points6ds,9ds,12ds,15ds,18ds,2Ids,24ds,27ds,3Ods. We examined the tumorigenecity of stably transfected Tca8113-FHL2, Tca8113-G418or parental Tca8113cells in nude mice, and found that tumorigenecity of Tca8113-FHL2group was significantly increased compared with control and parental Tca8113groups.In conclusion, by in vivo and in vitro studies, our results showed that FHL2expression might simulate the growth, proliferation, apoptosis, invasiveness, metastasis of the human tongue carcinoma cells. FHL2function in TSCC might be mediated by the increase of NF-κ B and β-catenin expresson levels. Four and a half lim domains2(FHL2) is a protein of219amino acids (aa) in length containing four full LIM-domains and a half LIM-domain at the amino-terminus. It is one transcriptional cofactor, able to interact with many different proteins, such as AP-1, BRCA1, IGFBP and integrin, involving in organ differentiation, development, cell apoptosis and carcinogenesis. Recent studies showed that FHL2could play different roles acted as co-activator or co-repressor in different types of cancers depending on the cell types involved. Until now, no report has been found about their relationship. Therefore, the study aims to investigate the role of FHL2in TSCC and its signaling pathway by in vitro and in vivo study.Our results found that FHL2was expressed in TSCC, much stronger positive staining was found with the increase of TSCC tumor grade, and only weak positive staining was detected in normal tissues by immunohistochemistry. Meanwhile, TSCC cell line Tca8113was transfected by pcDNAB.0-FHL2-flag plasmid, cell lines with pcDNA3.0plasmid and Tca8113were used controls. Cell biological behaviors including cell proliferation, apoptosis, invasiveness and metastases were investigated by MTT, Flow Cytometry, Transwell and Cell Scratch methods. And RNA and protein levels of possible related molecules including NF-κ B and β-catenin were investigated by Real-time PCR and Western blot. The results showed that FHL2overexpression could stimulate cell proliferation, invasiveness, metastases, apoptosis as well as NF-κ B and β-catenin expression levels both in transcriptional and translational levels. In the experiment of tumor formation in nude mice we examined the tumorigenecity of stably transfected Tca8113-FHL2, Tca8113-G418or parental Tca8113cells in nude mice, and found that tumorigenecity of Tca8113-FHL2group was significantly increased compared with control and parental Tca8113groups. The results indicate that FHL2stable expression may stimulate the growth of the human tongue carcinoma cells in vivo.In conclusion, by in vivo and in vitro studies, our results showed that FHL2expression might simulate the growth, proliferation, apoptosis, invasiveness, metastasis of the human tongue carcinoma cells. FHL2function in TSCC might be mediated by the increase of NF-κ B and β-catenin expresson levels. |
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