The Study Of T-cadherin Gene Expression And Transfection In Malignant Melanoma

Melanoma is a kind of cancer that arises from melanocytes. Melanoma is the most aggressive form of skin cancer and advantage stages are inevitably resistant to conventional radiotherapeutic and chemotherapeutic agents and other external stimuli poses a selective advantage for tumor progression, metastasis formation. Malignant melanoma affects approximately 40,000 new patients each year in the United States and an estimated 100,000 people worldwide. There is no satisfactory treatment for patients with metastatic melanoma that have an estimated 5-year survival of 6%.So scholars all over the world pay much attention to the malignant melanoma . And novel strategies that conquer malignant melanoma are clearly needed .Cadherins are a superfamily of adhesion molecules that mediate Ca2+-dependent cell-cell adhesion in all solid tissues of the organism .Cadherins play a key role in the regulation of organ and tissue development during embryogenesis and in maintenance of normal tissue structure in the adult organism .Moreover, changes in expression of caherin are associated with the tumorigenesis , invasion and metastasis .T-cadherin is one of the new members of the cadherin superfamily . Numerous recent data indicate that malignant tumor development is associated with the changes of T-cadherin expression . The loss expression of T-cadherin correlates with the development of breast , parastata, liver, lung, stomach, pancreas, ovary, B lymphocyte, skin cancers and so on . The expression of T-cadherin was reduced in numerous types of cancers and it indicates that it may play important role in tumor invasiveness, metastasis and metaining the phynotype of nomal cells .However, there are no reports about the expression of T-cadherin in malignant melanoma and if T-cadherin play a role in the development of malignant melanoma. For these reasons ,we tested the expression of T-cadherin in malignant melanoma tissue and cell lines with immunohistochemistry and cloned the gene of T-cadherin from SMC of human ,construct its eukaryiotic expression vector and transfect it into malignant melanoma cell lines .The results demonstrated in present study showed that the expression of T-cadherin in malignant melanoma tissue and cell lines obvious downregulated or be loss .And the transfection of T-cadherin cDNA has been proved successful .The content of present thesis consists of following parts :Part one : The expression of T-cadherin in naevus tissue of normal skin and in malignant melanoma tissue,cell lines .Objective: To investigate the difference of the expression of T-cadherin in naevus tissue of normal skin and in malignant melanoma tissue,cell lines . Methods Collect specimens of inpatients and outpatients including naevus tissue of normal skin 32 cases( cryoslice 20 cases and paraffin slice 12 cases ) and malignant melanoma tissue(cryoslice 8 cases and paraffin slice 6 cases),cell lines(M14 and A375) and test the expression of T-cadherin in naevus tissue of normal skin and in malignant melanoma tissue,cell lines with SP immunohistochemistry and immunocytochemistry .Results The expression of T-cadherin in the tissue of malignant melanoma compared with melanocyte of naevus is trace or loss However ,we found that there is light expression of T-cadherin in cell lines of A375 and M14 . The expression of HMB-45 is strong .Conclusion T-cadherin as a member of adhesive molecular and its expression decreased from naevus tissue of normal skin and maliganant melanoma tissue . It suggests that T-cadherin might play an important role in the tumorigenesis, progress and metastasis .Part Two: T-cadherin gene cloning and its eukaryotic expression vector constructingObjective :To clone T-cadherin gene from human SMC , reconstruct eukaryotic expression vector pEGFP-N1/T-cadherin.Method : 1 Cloning T-cadherin cDNA: Total RNA was extracted from human SMC .The full length of T-cadherin was obtained from RT-PCR method with Superscriptâ…¢reverse transcriptase and PfxDNA polymerase after RNA was verified there was no obvious degradation . 2 Constructing pEGFP-N1/T-cadherin : Plasmids pEGFP-N1/T-cadherin were extracted and digested with EcoRâ… and BamHâ… .The linear fragments were purified and retrieved then ligated by T4 DNA ligationase . of cDNA was ligated to eukaryotic expression vector pEGFP-N1 by T4 DNA ligationase . Ligation production was transferred, miniprepared and identified by EcoRâ… and BamHâ… .The recombinant vector with 2142bp insert fragment was sequenced by sangon of ShangHai. Results 1 Extracting Total RNA :28s, 18s, 5s RNA bands were observed clearly . 2 Acquiring T-cadherin cDNA fragment : About 2142bp band was acquired by RT-PCR . 3 Cloning PCR production into pEGFP-N1 vertor and sequencing:After identified by EcoRâ… and BamHâ… , the clone with 2142bp insert fragment was further to be identified by restricted enaymes located in vector and fragment . This sequence was proved to be correct compared with that in GeneBnak(NM₀01257) 4 Constructing eukaryotic expression plasmid pEGFP-N1/T-cadherin: After being transferred ,miniprpated and digested by EcoRâ… and BamHâ… , the finding of 2142bp insert fragment showed pEGFP-N1/T-cadherin vector was reconstructed successfully. Conclusion: Total RNA was got from human SMC, the cloned pEGFP-N1/T-cadherin was proved correctly ,and eukaryotic expression vector was constructed successfully .Part Three : Transfection of pEGFP-N1/T-cadherin into M14 and A375Objective To investigate whether T-cadherin gene can be expressed or not .Methods Transfecting M14 and A375 cells: When the confluence of M14 and A375 cells are up to 90%, the complex of pEGFP-N1/T-cadherin with LipofectamineTM2000 was added . selected by G418 or by green fluorescence potein .Results After 24h of transfection , we can investigate the green fluorescence in cultured human malignant melanoma cells of with pEGFP-N1/T-cadherin , pEGFP-N1 respectively under the fluorescent microscope. pEGFP-N1 excitation/emission=488/507. pBudCE4.1 excitation/emission ) =575/630nm .However, the wave length of the fluorescent microscope in our lab is 510～560 nm,so we can not investigate the red fluorescence of pBudCE4.1/ T-cadherin . The efficacy of transfection for blank vector is about 70～80% ,and the vector inserted T-cadherin is a little lower than the blank vector. Conclusion Transfection of pEGFP-N1/T-cadherin into M14 and A375 is successful .