Effects Of InnVit Gene On The Biological Behavior Of Melanocytes And Its Molecμlar Mechanisms

Vitiligo is a common autoimmune, frequent family clustering depigmentary disorder of skin that results from destruction melanocytes. To date, the pathogenesis of vitiligo is unknown although there are many theories about its etiology, including autoimmune, neural and genetic hypotheses. In recent years, genetic contributions were revealed as the most important causative factor of vitiligo. Moreover, scientists holding the other hypotheses of vitoligo began to seek supporting evidence from genetics.InnVit, a novel gene, was reportedly downregulated in vitiligo melanocytes. Sequence analysis revealed that the InnVit gene lacking its intron is the FBXO11 gene. To analyze the effect of InnVit gene on MC biological behavior further investigates molecular mechanisms of InnVit gene on Tyrosinase. We designed and synthesized siRNA and expression vectors for the InnVit gene, Here, we further investigate the effects of inhibition and over-expression of InnVit gene on the biological behavior of melanocytes, and on the export of Tyrosinase from ER.First, transfection efficiency was estimated by RT-PCR and western blot analysis at post-transfection, Silencing of InnVit gene remarkly decreased the expression of mRNA and protein of InnVit,while over-expression of InnVit gene increased the expression of mRNA and protein of InnVit .Second,We detected biological behavior of melanocytes on cell proliferation, apoptosis, transwell and melanin synthesis. The results demonstrated that the silencing of InnVit gene inhibited cell proliferation and induced cell apoptosis efficiently, while the over-expression of InnVit gene could promote cell proliferation and suppress cell apoptosis. Silencing of InnVit gene resulted in a decreased ability to progress from G0/G1 phase to S phase, eventually inhibiting cell proliferation, which was confirmed by MTT assay. Silencing or over-expression of InnVit gene has little effect on cell migration (as determined by transwell assay) or MMP9 and MMP2 activity.Last, our study further identified the effects of InnVit gene on melanocytes and the relationship of between dilated ER and Tyrosinase by inhibition and over-expression of InnVit gene.By electron microscopicy, obvious swelling of the endoplasmic reticulum (ER) was found in the cells transfected by siRNA for InnVit. Protein levels of Tyrosinase were extraordinarily high following inhibition of InnVit. Further examination revealed Tyrosinase and Calreticulin were co-localized in ER of the transfected cells following siRNA of InnVit, indicating that Tyrosinase could not export from ER effectively, consequently in the abnormity of biological behavior of melanocytes.Collectively, this study provides support that InnVit plays an important role in regulating the proliferation and apoptosis of melanocytes, and revealed the probably mechanisms of functional export of Tyrosinase from ER in vitiligo melanocytes.