Apoptosis Of Human Keloid Fibroblast Induced By Small Interfering RNA-mediated CyclinD1 Gene Silencing

Ⅰ.Background and aimHypertrophic scar(HS) and keloid(K) are common pathological scars.Usually believed to be caused with wounds,they are proliferative diseases of dermatofibroma characterized in abnormal proliferation of fibroblast,overproduction and deposit of such extracellular matrix(ECM) as collagen with high incidence.Keloid,in particular,has such characteristics as producing too much,proceeding beyond the boundary of wounds to encroach on tissues nearby,never degenerating and being easy to recur after pure surgery.It not only can cause dysmorphia and functional disturbance,but also has influence on the outlook.What is more,it often goes with evident pruritus and pains.Therefore,it brings great harzards to the physical and mental health of the patient both in functions and outlook.However,we have not found out completely the pathogensy of this pathologican scar,or effective methods for its treatment,though a great deal of research has done on its cause.Scholars at home and abroad have conducted some series of research on the causes of pathological scars from the point of view of histology,pathology, physiology,immunology and genetics,and great achievements have been made.The real causes of the disease still remain unclear.There is one point,however,that lots of experts have come into conformity,that is,the factors in vivo and vitro all contribute to the too much proliferation of fibroblast of the pathological scars,and the over deposit of collagan,which is typical of all pathological scars,including keloid.It is the abnormal proliferation of fibroblast that brings about the series of manifestations of the scars clinically.Thus,phologican scars may be treated if proliferation of their fibroblast can be inhibited with whatever means during the process of wounds healing,or something is done to enhance the apoptosis of reduplicated cells to reduce the over-deposit of collogan.It is therefore the precondition of this research to find out the mechanism to regulate the apoptosis of fibroblast proliferation of pathological scars,particularly the key factors regulating abnormal proliferation of cells at early stage,as the regulation of proliferation and differentiation is closely related with cell cycles,and the regulatory core in cyclnes determines the final terminal of cell growth.That the establishment of the concept of cell cycle and the breakthrough in the research on it in recent years have laid down a foundation for the study of cell proliferation,differentiation and apoptosis in modern times.The discovery of Cyclin, CDK and CDI has certainly revealed the mechanism of such movement.The organic combination of the three brings about normal division of cells,becoming the core controlling cell cycle.The outcome:from the research on the core regulating cell cycle provides a new theoratical base for people to explore the mechanism of the abnormal cell proliferation and differentiation.Studies show that division of cell in vivo is affected by such factors as time and environment.Dissociation of normal cells is restricted by one another that coordinates closely to construct normal cell groups in order.Division would cause pathological change if it proceeds continuously without control or constraint.Therefore,if we find the CDK,Cyclin and CKI in the process of cell cycle,we would get the the way to control continuity of abnormal cell division.Priming at G1 is a critical step in the cell cycle.It is particularly important, therefore,to regulate cyclins at G1/S.As one of normal major regulating factors, CyclinD1 mainly regulates cyclin at G1 to enter Phase S.It is the key factor for regulation at early stage to accelerate cell cycle.Its over expression,however,will bring about abnormal cell proliferation.Therefore,high expression of cyclinD1 due to various causes is one of the main reasons that makes cells grow too fast.Research on many a tumors has found that the occurrence and development of tumors are closely related to abnormality of the composition of G1/S at the regulating point.The unique growth characteristics and clinical manifestation of keloid shows have been taken by many scholars as a kind of innocent tumor developing after a wound heals.Fibroblasts are functional cells for keloid to develop,and their disequilibrium inin proliferation and apoptosis is the cytological base for keloids to grow continuously and be hard to become vestigial.There was research showing that cyclinD1 wass in a state of high expression in fibroblasts.This implies that keloids may be caused by the regulating factors working on cell cycles,for they increase synthesis of cyclinD1 and speed up cell cycles to cause confusion of functional cells in proliferation and apoptosis.Therefore,we have collected typical samples of hyperplastic scars and keloids to observe how cyclinD1 was expressed in fibroblasts.The differences in expression at the edge of keloids and in the center are of particular importance in the study of the clinical manifestation of keloids and in guidance in the treatment.Presently a lot of research on pathological scars focus mainly on the cause,and treatment is limited to injection of medicine and surgical removal of focus of infection,which cannot thoroughly cure the diease,which may appear again later, particularly the scar fibroblast proliferation.There is an urgent need clinically for one method to inhibit the proliferation after the heeling of wounds but before the formation of scars.The mechanism to regulate the apoptosis of proliferation at cyclin provides a new way for us to inhibit proliferation at the level when scar fibroblasts break.With the emergence and rapid development of genetic engineering,gene therapy will become an ideal way to treat pathological scars.Some scholars have tried employing antisensenecleic acid technology to inhibit the gene expression of factors bringing about the formation of scars with sequence specificness,or transferring inhibiting genes into the fibroblasts of hyperplastic scars to build up collagenous degradation.This,however,does not seem to work well.The appearance of RNA interferring(RNAi) further provides vast space for treating genetically pathological scars.RNAi refers the phenomenon of degradation of the specific gene mRNA brought about by introducing exogenous double-stranded RNA.They are the congenerous in sequence,a sort of post-transcriptional gene silencing(PTGS),first put forward by Fire in I the research on the gene functions of edlworms,and then was confirmed that RNA interferring was the natural mechanism protecting gene groups, found commonly inside animals and plants.After a great deal of research,some scholars proposed a mode for RNAi to work in mammalian cells:exogenous or endoneous dsRNA was cut into small interferring RNAs(siRNA) with two basic group protrutions at 3' under the effect of Dicer after entering cells.The siRNA is combined with the specific enzyme of RNAi to develop RNA-induced silencing complex(RISC) which is further combined with RNA the exogenous target of siRNA under the instruction of siRNA antisense strand.They are cut at the near center to inhibit the expression of target genes.Through polymerase chain reaction the cut materials will develop into new dsRNA to work continuouly on mRNA until finally degrading target mRNA completely.Therefore,a small amount of dsRNA can complete the effect of remarkable interference.However,experiments have found that dsRNA introduced would initiate the virus protective mechanism in cells when it was larger than 30bp,which increased the synthesis of interferon in cells and activate simultaneouly the protein kinase K(PKR) and 2'5' adenyl acid synzyme(-AS).The activated PKR phosphorylation translates the initial factors eIF2αto decrease its activity so that the synthesis of protein in cells is closed completely.The activated 2'3'(-AS) is can activate a ribonuclease(RNase L) to degrade the non-specific mRNA,resulting in the non-specific and entire inhibition the expression of genes that causes apoptosis.This factor limits the development of RNAi in a short time.Now when it is easy to get it withouth dsRNA and employ it in and out of body,siRNA can specifically and effectively stop or inhibit the expression of target genes.RNAi will certainly become a hot point of research in medical field.Surprising development has been made in the research on RNAi in extensive fields including medicine and it has been applied in widely in treating various diseases,particularly tumors.Although some scholars in plastics have-started to inhibit the expression of genes related to the formation of scars with the specifiness of RNAi technology,but there was no report about Apoptosis of human keloid fibroblast induced by small interfering RNA-mediated cyclinD1 gene silencing.We thus suppose that if the high expression of cyclinD1 in the fibroblasts of pathological scars causes the abnormal cell proliferation at early stage,we may knock out its synthesis of mRNA with RNAi technology to cut down the synthesis of CyclinD1 so as to inhibit the proliferation of fibroblasts at the early state of cell cycle.This may help us treat scar proliferation,which will provide new theoractical evidence for further research on the treatment of pathological scars.Ⅱ.Materials and methods1.Subjects The Samples came from patients eceiving plastic surgery in Nanfang Hospital.These included 77 cases of hyperplastic scar,with 56 males and 21 females aged between 4 to 45 years old and the periods of scar hyperplasy ranging 40d～5a; there were 10 samples of keloid,among which 6 were males and 4 females aged between 18～46 years old.There were 24 cases of normal skin from the patients above.These were skin residues left after the surgery.The patients did not have a history of taking medication to prevent scars for long,nor did they suffer from tumors or other severe diseases.The patients were told about the purpose before the sampling and they agreed to it.The diseased parts with keloid sampled included the ear lobe, the deltoid region and the protothorax.The lower orthophoria part was divided into central and ambitus areas,both confirmed clinically and with pathologic diagnosis.The normal skins,taken from the skin-supply areas of the patients above, were residuces left after the surgery.The samples of hyperplastic scars were divided into five groups according to the time hyperplasy occurred:1.Those occurred at Months 1 to 3;2.those at Months 4 to 6;3.those at Months 7 to 9;4.those at month 10 to 12 and 5.those appeared 12 months after the surgery.Samples of keloid were divided into two groups:the central group with 2/3 of the center and the edge group with 1/3 of the edge.Those in the NS group were taken as the control group,and PBS as negative the control group.2.Methods(1) Detecting cyclinD 1 with immunohistochemistryDetect with SP method of immunohistochemistry the expression difference of CyclinD1 in different pathological phase of hyperplastic scar and fibroblast in different area of keloid.(2) siRNA design and synthesis for CyclinD1With siRNA target finder,the softwear online by Ambion,we designed s ome siRNA-cyclinD1 molecues whose positive-sense strand being 5'-CAAACA GAUCAUCCGCAAAtt and antisense strand 5'-UUUGCGGAUGAUCUGUUUGt t.The siRNA-cyclinD1 molecues interfered nucleotides of Cyclin D1 genes pos itioning at No.664-684,and the target serial was AACAAACAGATCATCCG CAAA.Then with chemical methods,we had the positive-sense and anti-sense s-trands of siRNA-cyclinD1 molecues synthesized respectively(entrusted to Sh anghai Jikai Genes Co.).They became double-stranded siRNA-cyclinD1 mole cues after being treated with denaturalized anneal.(3) laboratory groups and transfected siRNA-cyclinD1 molecuesThere were two groups for the transfection laboratory:blank control group in which no transfection was related to siRNA,and siRNA group spectific to Cyclin in which transfected Smad2 specific to siRNA.Tests were repeated three times in each group.(4) Detecting CyclinD1 gene expression with RT-PCRGross RNA of cells were taken when the fibroblasts were transfected with siRNA-cyclinD1 for 24,48 and 72 hours and reverse transcription reaction was conducted for the observation of the change of the relative expression amount in mRNA in CyclinD1.(5) Analyzing apoptosis with double tagged flow cytometry10~6 cells after transfected siRNA-cyclinD1 molecues were collected to be detected with flow cytometry and the percent of cells in four areas(UL,UR,LL and LR) were analyzed with Cellquest softwear.The percent of cells in UR and LR standed for the percentage of apoptosed cells.(6) Detecting DNA fragment of apoptosis10~6 cells were collected respectively from the transfection siRNA-CyclinD1 group(the experimental group) and the siRNA group without transfection(the control group),and 10μl of DNA were colbcted from each for 1.2%agarose gel electophoresis.(7) Detecting the activity of fiborblast with MTTThe activity of fibroblasts was detected with MTT when siRNA-cyclinD1 was transfected into fibroblasts for 24,48 and 72 hours,with the cells in siRNA without transfection in the control group.The rate of cell proliferation inhibited at different time point was worked out.The formulas was the rate of cell proliferation inhibited= (1—A570 value after transfected siPNA-cyclinD1/A750 value of cells in the control group)×100%.The tests were repeated three times at each time point.(8) Data processingAnalysis on the data was done with SPSS12.0 for Kruskal-Wallis H test),and comparisons among groups were made for mono-factor variance with LSD method.Ⅲ.Results(1) The expression of CyclinD1 in fibroblast at different stage of hyperplastic scarAt the early stage of hyperplastic scar,more fibroblasts showed positive expression with CyclinD1,while fewer fibroblasts showed positive expression at middle and later stages,and in some samples the CyclinD1 had negative expression in the fibroblasts.The expression turned from being strong to weak with the development of the scars.Statistical analysis showed great difference in the expression of CyclinD1 protein between Group 1 and Group NS;between Group 2 and Group NS,and between Groups 1 and 5(X~2=68.53,p＜0.001);while comparisons in pairs in other groups showed no statistic significance.(P＞0.05)(2) The expression of CyclinD1 in fibroblast at different parts of keloidAt the ambitus of a keloid,CyclinD1 showed high positive expression,while at the center,positive expression dropped greatly.Statistical treatment showed marked difference in the expression of Cyclin D1 protein between the ambitus and Group NS (P＜0.001),between the ambitus and the center(P＜0.001),and between the center and Group NS(P＜0.001).(3) The change in form for fibroblast after being transfectedWhen siRNAwas transfected for 24 hours,some fibroblasts turned from fusiform into spherical or oval shape,and with time proceeding,the percentage of round or oval cells increased gradually.The cells in the untreated group and the hollow transfected bangosome group remained basically in the same shapes.(4) The influence of transfected siRNA-cyclinD1 on the level of mRNA the CyclinD geneWhen the fibroblasts were transfected with 50nM siRNA-cyclinD1,the relative expression amount of the CyclinD gene in the fibroblasts gradually became less within 24～72 hours.(5) The influence oftransfected siRNA-cyclinD1 on the cycle of fibroblastsWhen the fibroblasts were transfetected with specific siRNA-cyclinD1 molecules for 24,48 and 72 hours,the experiment was repeated for three times before the results were analyzed with SPSS12.0 in mon-factor variance,showing that transfection of siRNA-cyclinD1(1) had influence on cells in the groups at G0/G1 (F=22.18,P＜0.001);(2) affected the cells at Phase S(F=29.88,P＜0.001); and(3) did not have any influence on the cells at Phase G2/M(F=2.77,p＜0.001).Comaprisons made between pairs of groups showed that the average percentage of cells at Phase G0/G1 were(60.38±1.77)%,(64.94±1.62)%and (65.18±2.20)%respectively,much higher than those in the control group without siRNA after 72 hours(54.45±1.77%)(P＜0.01,＜0.001,＜0.001);the average percentages of cells at Phase S were(18.15±1.28)%,(17.28±0.89)%and (11.54±1.56)%,much lower than that(22.17±1.68)%(P values being 0.019, 0.007 and 0.001 respectively) in the control group;and the average percentages at Phase G2/M were(21.47±3.04)%,(17.78±2.27)%and(23.28±1.83)% respectively,showing no difference with that(22.17±1.68)%(P values being 0.73, 0.09,1.0 respectively).(6) Apoptosis induced after siRNA-cyclinD1 was transfected into fibroblastsWhen specific siRNA-cyclinD1 molecues were transfected into fibroblasts for 24,48 and 72 hours,and the experiment was repeated for three times before the results of cyclines were analyzed in mono-factor vairance with LSD method among groups that showed that transfection with siRNA-cyclinD1 had influence on the apoptosis rate(F=458.77,P＜0.01).Comparisons between pairs of groups indicated that when siRNA-cyclinD1 was transfected into fibroblasts for 24,48 and 72 hours,the average apoptosis rates were(7.82±0.45)),(16.07±1.58)%and (18.49±0.82)%respectively,much higher than that(0.68±0.12)%in the control group,the differences were all of statistic significance(the P values being＜0.001).(7) Gragmentation of DNA when siRNA-cyclinD1 was transfectedWhen the fibroblasts were transfected with siRNA-cyclinD1,no fragmentswere found in 24 hours,but in 48～72 hours fragments as large as 100～400bp appeared; No fragments were found in the cells in the control group in 72 hours.(8) The proliferation of fibroblasts after siRNA-cyclinD1 was transfected24,48 and 72 hours after siRNA-cyclinD1 were transfected into fibroblasts,the growth of the cells were determined with MTT rules(A570).The experiment was repeated three times and analysis was made in multi-factor variance with SPSS12.0 with the results showing that transfection of siRNA-cyclinD1 had influence on the proliferation in each group(p＜0.001).Comparisons among groups with LSD method at the same time point made showed that(1) cell growth in the siRNA-cyclinD1 transfected group was inhibited in contrast with that in the group without siRNA transfection,the difference being statistically significant(p＜0.001); (2) cell growth in the siRNA-cyclinD1 transfected group was inhibited in contrast with that in the group without any treatment,the difference being statistically significant(p＜0.001);and(3) the cell growth in the group without siRNA transfection indicated no statistical significance(p=0.854).The cell proliferation inhibiting rates after siRNA was transfected was worked out in accordance with the formular:cell growth inhibiting rate=(1—transfected A570/untreated cells A570)×100%,and we got 18%,37%and 50%,tending to increase gradually,with the difference showing statistically significance(p＜0.001) compared with that in the group with siRNA transfection.Ⅳ.Conclusion1.CyclinD1 played an important role in the formation of pathological scars.Shortening proceeding of Phase G1 resulted in abnormal proliferation of fibroblasts that encouraged the formation of pathological scars.2.The high expression of CyclinD1 in fibroblasts at early stage of hyperplastic scars was closely related to the prosperous hyperplasy of scars at early stage.3.The high expression of CyclinD1 in fibroblasts at the edge of keloid not only promoted the their formation,but also probably one of the reasons for keloid to offent normal tissues locally and grow infiltratively.4.siRNA-cyclinD1 molecues could effectively inhibit CyclinD1,the target genes, and the amount of mRNA dropped greatly to reduce the expression of CyclinD1.The cell cycline of fibroblasts was restricted when it moved from G1 to Phase S and had to stop at G1,and cell tended to apoptose with time going and cell proliferation was inhibited.5.This research confirms that siRNA-cyclinD1 can interfere the cell expression of CyclinD1 and then inhibit the abnormal hyperplasy of keloid at early stage.