Characterization Of Basigin Alternative Splicing Variants And Their Functions In Proliferation And Invasion Of Hepatocellular Carcinoma

Alternative splicing is a key molecule mechanism to generate a large number of mRNA variants and protein isoforms from the surprisingly low number of human gene, which expands transcript and protein diversity to a great extent. In addition to alternative splicing, an increased number of recent studies report the existence of alternative promoter for genes, demonstrating it is another important source for generating protein and regulatory diversity. Alternative splicing and alternative promoter are common phenomenon in mammalian genomes. They could regulate tissue/cell-specific transcription pattern and translation efficiency, produce protein isoforms with additional or deleted protein domains, resulting in the change of protein functions, such as receptor-ligand binding, protein-protein or protein-nucleic acid interactions, intracellular localization, post-translational modification and signaling activities. Numerous reports have shown that alternative splicing patterns are changed in cancer. The expression of alternative or even tumor-specific splice variants significantly affect many cellular events critical for cancer biology such as cell proliferation, motility and drug response.Basigin, also called EMMPRIN, CD147 and HAb18G/CD147, is a highly glycosylated type I transmembrane protein belonging to the immunoglobulin superfamily. Basigin is encoded by BSG gene, which located at human chromosome 19p13.3. Several functions of basigin have been described in both physiological and pathological processes. For examples, basigin could regulate energy metabolism, reproduction and development. In cancer, basigin is obviously overexpressed and promote tumor progression, invasion and metastasis through stimulating MMPs secretion, which is the best characterized function for this gene. Due to the pivotal role of basigin in cancer, this molecule has been termed a cancer associated biomarker and served as a target for cancer therapy.Up to the present, four basigin AS transcript variants encoding different isoforms have been reported. Among them, basigin-2 is the most predominant splicing pattern, just encoding the well-known basigin/CD147/EMMPRIN and functioning as mentioned above. Basigin-1 encodes a long, retina-specific isoform, distinguished by an additional Ig-like domain. The other two less widely found variants, basigin-3 and basigin-4, are firstly identified in human endometrial stromal cells and cervical carcinoma cell lines. Basigin-3 is a short isoform, comprising only one Ig-like domain, and could interact with the internalized basigin receptor-ligand complex. However, detailed characteristics of basigin isoforms are not known, such as transcriptional regulation, expression profile and their functions distinguished from basigin-2.In order to further understand the role of alternative splicing in basigin expression regulation as well as the biological functions of basigin isoforms, a series of approaches were employed in this work. We firstly determined the expression of basigin transcript variants in human cell lines. Then we cloned the full-length cDNAs of basigin-3 and basigin-4, the most abundant AS transcript variants except basigin-2, and analyzed their exon arrangement and splicing patterns. Using luciferase reporter assay, an alternative promoter in the 5'-flanking regulatory region of basigin-3 and basigin-4 was identified that promotes both variants gene transcription. Mapping the expression profiles by real-time quantitative reverse transcription PCR, basigin-3 and basigin-4 were found expressed in all twenty kinds of human normal tissues at mRNA level and up-regulated in tumor tissues compared to normal tissues. Using GFP fusion protein over-expression cell model, we determined basigin-3 and basigin-4 mRNA variants could lead to expression of protein isoforms in glycosylated state, which could be localized to the plasma membrane. However, in cultured cell lines, only native basigin-3 but not basigin-4 could be detected at protein level using antibodies against different epitopes on CD147 and variant-specific RNA interference. Using over-expression cell models and orthotopic transplantation tumor models, we found that over-expression of basigin-3 could inhibit hepatocellular carcinoma cell proliferation, MMPs induction and cell invasion both in vitro and in vivo. Confocal microscopy imaging, bimolecular fluorescence complementation assay and NMR analysis indicated that basigin-3 could interact with basigin-2 and form hetero-oligomerization complex. Taken together, basigin-3 is the one of most important isoforms of basigin family, and it may be involved in regulating the functional activity of basigin-2 as an endogenous inhibitor probably via hetero-oligomerization. The inhibitory function of basigin-3 could be used as a novel strategy for cancer biotherapy.