Abstract:

Ni Nana, Wen Sijian, Zhang Wei, Jiang Yiqun, Sun Jianfang Department of Pathology, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China Corresponding author: Sun Jianfang, Email: Sunjf57@163.com 【Abstract】 Objective To construct a eukaryotic plasmid carrying the PKCI-1/HINT1 gene, to investigate its in A375 melanoma cells after transfection, and to evaluate its effects on apoptosis and autophagy of A375 cells. Methods The PKCI-1/HINT1 gene sequence was amplified by reverse transcription PCR （RT-PCR） with total RNA extracted from A375 cells as the template, then inserted into the eukaryotic plasmid PCDNA3.1（+） to construct a recombinant plasmid, PCDNA3.1（+）-PKCI-1/HINT1. Some A375 cells were classified into two groups to be transiently transfected with the recombinant plasmid （PCDNA3.1（+）-PKCI-1/HINT1 group） or the empty plasmid PCDNA3.1（+） （control group）. After additional 48-hour culture, RT-PCR and Western blot analysis were performed to quantify the mRNA and protein s of PKCI-1/HINT1 respectively, Hoechst 33342 staining was conducted to detect apoptosis of A375 cells, Western blot analysis to detect the s of intracellular caspase-3 and autophagy-associated protein beclin1, and cell autophagy was observed by using the green fluorescent protein （GFP）-microtubule-associated protein 1 light chain 3 （LC3） labelling method combined with confocal laser scanning microscopy. Methyl thiazolyl tetrazolium （MTT） assay was performed to evaluate the proliferative activity of A375 cells at 24, 48, 72 and 96 hours after transfection. Results Enzyme digestion and sequencing analysis confirmed that the eukaryotic plasmid PCDNA3.1（+）-PKCI-1/HINT1 was successfully constructed and effectively expressed in the transfected A375 cells. MTT assay showed that PKCI-1/HINT1 could obviously inhibit the proliferation of A375 cells, and the number of live cells was decreased by 17.0%, 25.6% and 29.4% in the PCDNA3.1（+）-PKCI-1/HINT1 group at 48, 72 and 96 hours, respectively, compared with the control group （all P < 0.05）. Hoechest 33258 staining revealed that PKCI-1/HINT1 could promote the formation of apoptotic bodies in A375 cells. Confocal laser scanning microscopy demonstrated that the over of PKCI-1/HINT1 increased GFP-LC3 puncta formation in A375 cells. In addition, Western blot analysis indicated that PKCI-1/HINT1 up-regulated the protein s of caspase-3 and beclin1 in A375 cells. Conclusions The eukaryotic plasmid PCDNA3.1（+）-PKCI-1/HINT1 was successfully constructed, and PKCI-1/HINT1 could be effectively expressed in A375 cells. High-level of PKCI-1/HINT1 could suppress cellular proliferation, promote apoptosis, and induce autophagy, of A375 cells.