Suppression Of Survivin Expression By RNA Interference Induces Apoptosis In Keloid Fibroblasts

Background and objectiveKeloids are skin lesions such as wounds, burns or surgery-induced fibrosis changes, a fiber metabolic disease characterized by excessive proliferation of fibroblasts and excessive collagen of extracellular matrix (extracellular matrix, ECM) excessive deposition. Keloid destructs human body's surface, leads to dysfunction, and gives patients tremendous physical and heart pain, therefore, the prevention of keloid has been an important issue in clinical studies. The unique keloid growth characteristics and clinical performance are considered to be benign for tumor formed after wound healing by many scholars. Fibroblasts are key effector cells in the-process of forming scar. Researches in recent years have found that the abnormal proliferation of fibroblasts is an important mechanism of keloid disease, also the imbalance of proliferation and apoptosis lead to continued proliferation of keloid and difficult degrading of cellular. The survivin gene as the strongest ever found apoptosis inhibitory factor is closely related to the formation of keloids. The results show that survivin gene could inhibit fibroblasts'death, cause cells abnormal proliferation and malignant transformation. Because survivin is closely related with keloid formation, anti-survivin therapy is likely to be an effective way to treat keloids.Currently, a large number of studies on keloid are mainly focusing on the diagnoses of etiology, and treatments are also limited to drug injection and the surgical removal of lesion, but they can't completely cure from the root, relapse often occurs after treatment, especially after the proliferation of keloid surgery. Clinics urgently need a method that could inhibit proliferation of keloid fibroblasts during the early stage of wound healing. With the emergence and rapid development of genetic engineering, gene therapy will become an ideal treatment for keloid treatment. Currently, many scholars try to use antisense oligonucleotide, ribozyme gene to inhibit the expression of gene, but these methods have shortcomings such as low transfection efficiency and short duration of action. The discovery of RNA interference (RNA interference) phenomenon provides a new era for the gene therapy of keloid, and RNA interference has already made remarkable progress. RNA interference uses the targeted mRNA that has homologous sequence-specific siRNA to induce transcriptional gene silencing, which resulted in a. similar "knock out" phenotypes, can be effective and specifically inhibit the target gene, and then causes corresponded lack of functionality. This process is post-transcriptional gene silencing (post transcriptional gene silencing, PTGS). Because of its specificity, efficiency and durability it has been widely used. The successful application of RNAi technology knockout disease genes indicates that the technique has opened the door to new therapeutic approaches. RNAi has made great progress in a wide range of areas such as medicine, and been widely used in medical treatment of various diseases, especially relevant aspects of tumor treatment. Although plastic surgery academics and scholars began to apply RNAi technology to inhibit formation of scar-related gene, but none of the research about application of vitro synthesized siRNA on keloid fibroblasts to inhibit survivin expression has been reported domestically and internationally. Thus we assume that if the keloid fibroblasts has high survivin expression and inhibits fibroblast programmed death, then that using RNAi technology to block or inhibit the expression of survivin gene, reduce survivin protein synthesis, and inhibit fibroblast abnormal cell proliferation and malignant transformation, could be able to treat hypertrophic scar. Therefore, we could use this bio-informatics method to design and synthesize siRNA molecules according to a person's survivin Gene, transfect of primary cultured keloid fibroblasts, reduce survivin expression and inhibitthe effective synthesis of survivin, observe its effect to human's survivin expression in keloid fibroblasts and fibroblast cell function, and provide the preliminary theory for the therapy of keloid.Materials and Methods1. SamplesSamples are taken from the scar tissue that removed through Plastic Surgery by the first affilated hospital of kunming medical college. None of the patients had skin diseases, connective tissue disease or any other major organ diseases; and had used steroids, or anti-penicillamine oncology; none of the specimens were treated by radiotherapy, laser therapy or immunotherapy; all samples were confirmed by pathology. Primary fibroblast culture is carried out by digestion. Normal skin was taken from the donor site of patients for surgery after taking the residual skin leather trim.2. Methods(1) Immunohistochemical detection of survivin gene in normal skin, the differences in the expression among hypertrophic scar, keloid tissue of survivin and CD34, microvessel density (MVD), analysis the correlation of survivin expression in abnormal scars and angiogenesis;(2) The expression of immunocytochemical detection between survivin gene in keloid and normal skin fibroblasts in primary culture cells;(3) The molecular design and synthesis of siRNA-survivin;(4) Group testing and transfected siRNA-survivin molecular;(5) Use RT-PCR to assay the survivin mRNA expression after transfection in keloid fibroblasts;(6) Use Westernblot to assay the expression of survivin protein after transfection in keloid fibroblasts(7) Use flow cytometry to analyze cell cycle;(8) Use MTT to assay fibroblasts;(9) Use Tunel to detect apoptotic cells;(10) Data processing:using SPSS12.0 to statistically analyze data, measure mean±standard deviation (X±S), perform T test and variance analysis, use RMANOVA (repeated-measures anova) to analyze cell growth curve, P<0.05 to have significant differences.3. Results(1) In normal skin and keloid survivin expresses positive rate of 0 (0/10) and 66.67%(20/30); keloid survivin expression is significantly higher than in that of hypertrophic scars; with survivin expression in keloid enhanced, MVD increased gradually, survivin gene in primary culture of keloid fibroblasts expresses as positive, but primary cultured normal skin fibroblasts have no significant expression.(2) Plasmid pSIREN-siRNA/survivin has been successfully recombined. The coding sequence of recombinant plasmid shRNA tested by gene sequencing and the nucleotide sequences that we designed shRNA targeting survivin are identical.(3) Construction of eukaryotic expression vector transiently transfected cells stained fibers, fluorescence microscopy up to 67% transfection rate. Through RT-PCR detection of survivin mRNA expression in fibroblasts, survivin mRNA was inhibited 24.48 hours after transfection, the inhibitory rates were 54% and 79.3%; Westerm-blot analyzed survivin protein expression that survivin protein expression were reduced to 50.34% and 82.30% after 24 and 28 hours accordingly.(4) Cell cycle by flow cytometry:the interference plasmid S phase cells decreased; the proportions during the G2/M phase increased, and stagnate phenomenon appeared in of G2/M phase. The apoptosis rates of interfering plasmid 24h,48h fibroblast were 8.8%,19.6%, significantly higher than the negative and the untreated groups, with time prolonged, apoptotic cells gradually increased.(5) Detected by flow cytometry Tunel Apoptosis:after transfection of survivin-shRNA, the apoptosis rates of 24h and 28h were 11.9±1.96%, and 21.2±2.14% according, having significant difference which is (P<0.05) compared with the negative (3.60±0.83%) and treatment group (2.40±0.63%)(6) MTT tests showed that after transfection of plasmid pSIREN-siRNA/survivin keloid fibroblasts proliferation was inhibited.Conclusions1. survivin protein expression increased in keloid, and is closely related with angiogenesis, suggesting that survivin is relevant to the hypertrophic scar development process.2. That siRNA interference plasmids transfecting Keloid fibroblasts can inhabit survivin mRNA expression, reduce survivin protein, suggests that the gene can be used as targets for gene therapy.3. siRNA-survivin molecule greatly inhibits the target gene survivin, inhibiting of fibroblast cells from Gl phase to S phase, but staying in the G2/M phase. Cells underwent apoptosis over time, and the number of fibroblasts is reduced.4. The growth curve of fibroblast become mild after survivin—hRNA transfer fibroblast of keloid. Survivin—hRNA obviously restrain growth of fibroblast of keloid. 5. This study demonstrated that siRNA-survivin molecules can interfere with survivin gene expression, thereby inhibiting abnormal proliferation of early keloid.