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HCCS1 translocation relevant regional and functional studies to determine

Author: KongWei
Tutor: ZhaoXinTai
School: Fudan University
Course: Immunology
Keywords: Tumor suppressor gene Functional sequences Translocation HCCS1 Hepatic carcinoma
CLC: R392
Type: Master's thesis
Year: 2008
Downloads: 23
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Abstract


[Objective] HCCS1 is achieved by patients with hepatocellular carcinoma chromosome 17p13.3 region of loss of heterozygosity analysis found a new tumor suppressor gene. HCCS1 in intracellular protein sorting transport plays an important role in its tumor suppressor role may be through the function of its protein transport play. This experiment aimed at finding HCCS1 sequence associated with translocation of the area and to further explore this area and their sequence features and tumor suppressor role in protein transport links. [Method] pEGFP-C2 as the carrier, constructed with fragments of different lengths HCCS1cDNA subclones transfected subclones will build cervical cancer HeLa, by immunofluorescence confocal microscopy of different lengths HCCS1 subcellular localization, and with a 6 - phosphate mannose receptor (M6PR) co-localization. The constructed subclones transfected HeLa cervical cancer and liver cancer cells BEL7404, through G418 selection after a certain time to do crystal violet staining of cells transfected with different subclones clone formation. The constructed subclones transfected cervical cancer HeLa, hepatoma cell BEL7404, SMMC7721 cells as well as cancer cells SGC7901, G418 screening after a certain time by MTT assay, was observed on the four kinds of different subclones of tumor cell growth conditions. [Results] successfully constructed with pEGFP-C2 as the carrier of 10 kinds of different lengths HCCS1cDNA containing fragments were subcloned; HCCS1 protein subcellular localization and colocalization with M6PR showed: pEGFP-C2-HCCS1 (2100 bp), pEGFP- C2-HCCS1ΔE5 (1861 bp), pEGFP-C2-HCCS1-S1 (1835bp), pEGFP-C2-HCCS1-S22 (1732 bp) and pEGFP-C2-HCCS1-S39 (1571 bp) encoding proteins showed different lengths HCCS1 granular, polar distributed in the perinuclear cytoplasm and colocalization with M6PR there; while pEGFP-C2-HCCS1ΔBH (1120 bp), pEGFP-C2-HCCS1ΔBB (884 bp) and pEGFP-C2-HCCS1-TR3 ( 778 bp) encoding proteins of different lengths HCCS1 although grainy distributed in the nucleus, but the disappearance of polar distribution, that is, when deletion subclones pEGFP-C2-HCCS1-S39 in the fragment HCCS1-S39 (1571 bp) 3 'end of the 451bp sequence, HCCS1 protein specific localization within the cell begins to change, and with the co-localization of M6PR disappeared; subcloned into pEGFP-C2-HCCS1-TR2 (678 bp), pEGFP-C2-HCCS1-TR1 (578 bp), pEGFP -C2-HCCS1ΔBS (478 bp) and pEGFP-C2-HCCS1ΔBE (339 bp) encoding proteins of different lengths HCCS1 were scattered punctate dispersed in the cytoplasm and nucleus. Cell colony formation assay showed that in cervical cancer HeLa and hepatoma cell BEL7404 in transfected with pEGFP-C2-HCCS1 (2100 bp), pEGFP-C2-HCCS1ΔE5 (1861 bp), pEGFP-C2-HCCS1-S1 (1835 bp), pEGFP-C2-HCCS1-S22 (1732bp) and pEGFP-C2-HCCS1-S39 (1571 bp) was subcloned cells, formed by the number of clones transfected with empty vector pEGFP-C2 as compared with the number of clones formed , significantly reduced (P <0.05), and transfected with pEGFP-C2-HCCS 1ΔBH (1120 bp), pEGFP-C2-HCCS1ΔBB (884 bp) and pEGFP-C2-HCCS1-TR3 (778 bp) subcloned cells number of clones formed with cells transfected with empty vector pEGFP-C2 colonies formed was no significant difference (P> 0.05). MTT assay showed that, pEGFP-C2-HCCS1 (2100 bp), pEGFP-C2-HCCS1ΔE5 (1861 bp), pEGFP-C2-HCCS1-S1 (1835bp), pEGFP-C2-HCCS1-S22 (1732 bp) and pEGFP-C2 -HCCS1-S39 (1571 bp) was subcloned for cervical cancer HeLa, hepatoma cell BEL7404, SMMC7721 cells and inhibited the growth of gastric cancer cell SGC7901; while pEGFP-C2-HCCS1ΔBH (1120 bp), pEGFP-C2-HCCS1ΔBB ( 884 bp) and pEGFP-C2-HCCS1-TR3 (778 bp) was subcloned to the above four kinds of cancer cells without inhibition. [Conclusion] initially identified HCCS1cDNA 1571 bp fragment of the 3 'end of the 451 bp of sequence HCCS1 protein translocation associated minimum regional context. Deletion of the region, HCCS1 protein transport function disappear; Further experiments showed that deletion of the region can lead HCCS1 inhibit tumor cell growth disappeared, suggesting HCCS1 play the role of tumor suppressor proteins may be related to transport function are linked.

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