The Study Of The Impact About Cell Transfer When Targeting Interference The PPARγ MRNA Of Salivary Gland Adenoid Cystic Carcinoma

Objectives: The invasiveness and transitivity is the essentialcharacteristics of therioma. The transfer of therioma is composed of invadingsurrounding tissue of the tumor, moving along the blood and lymphatic system,proliferation in the target organs and so on, and the reason and the mechanismof it which is closely related to the change of its gene level is very complicated.If the therioma loses its ability of transfer, the level of malignancy will greatlydecrease. There is not a clear conclusion that can interpret the invasion and themetastasis of the malignant tumor roundly in the medical scientific research sofar, and there is no effective method to the treatment of the invasion andmetastasis of malignant tumor at present. The study which is aimed at the genethat related to the metastasis of the malignant tumor has become one of theways in which medical science workers try to conqur the tumor.Salivary adenoid cystic carcinoma(SACC), one of the most commonand the most aggressive salivary gland cancers, is in a second place in theincidence of the salivary gland malignant tumor. It grows slowly,is easy torelapse after treatment in local recurrence, and its ability of invadingsurrounding tissues is strong. One of its main biological characters is that it iseasy to have distant metastasis along bood, and the common position is lung,which has become one of the main reasons that lead to the patients’ death. Atpresent the primary therapeutic method is surgery, but it is difficult to make aone-time clean surgery because of its invasiveness. It is not sensitive tochemotherapy and radiotherapy, has a bad prognosis, and the treatment of therecurrence and metastasis it causes is quite difficult, so looking for a kind ofeffective method to treat the salivary gland adenoid cystic carcinoma hasbecome the focus of attention and a difficult problem of the stomatology medical workers. With the development of the genetic and molecular levelresearch on the causes, the development and the treatment process of variousdiseases, the research of gene therapy on the salivary glands of xianyangcystic cancer also gradually in-depths. high pulmonary-metastatic cell lineSACC-LM and low pulmonary-metastatic cell line SACC-83has the sameparents, the differences between the two kinds of cell focuses on the transferphenoty that the SACC-LM cell line’s transfer capacity is obviously higherthan that of the SACC-83cell line.In recent years, the research of RNAi (RNA interference) has madebreakthrough progress attributing to the experts from various fields, has beenjudged one of the ten major scientific progress in2001by 《Science》,andtaken the first place of the ten important scientific progress in2002. The RNAiphenomenon was discovered in1995, and aroused people’s concern andattention beginning from1998. Using the RNAi technology can eliminate orclose the expression of specific gene by restraining silent specific gene, so thistechnology has been widely used in the fields of exploring gene function,infectious diseases and the malignant tumor gene therapy. RNA interference(RNAi) belongs to the post-transcriptional gene silence, and it importsdsRNA which is compounded in vitro and homologous and complementarywith target genes mRNA into cells.PPAR (peroxisome proliferators activated receptors), one of the groupsof nuclear hormone receptor superfamily, is newly discovered that it belongsto the nuclear hormone receptor, as the same as thyroid hormone receptor,retinoids receptors, steroid hormone receptor, and vitamin D receptor, they arealso known as fatty acid receptor. The study found that the human’s PPAR hasthree subtypes, namely: PPAR alpha, PPAR delta also called PPAR beta) andPPAR γ. Among them, the PPAR γ is the most widely research one now. Atfirst, there has no PPAR natural ligand be found, while with the continuousimprovement of molecular biology and appraisal methods, there has beenhundreds of PPAR γ ligands were identified now, and be divided intosynthetic ligands and natural ligand. The PPAR γ participates in lots of diseases’ pathophysiological process, Such as: diabetes mellitus,atherosclerosis, inflammation, obesity, etcr, and plays an important role. ThePPAR γ mainly expresses in adipose tissue, and also expresses in the bodymacrophages and other fat storage cell, such as: liver, kidney, lung, etcr,butwe can’t fond its expresses in muscle tissue almost, and PPAR γ1is its majorpattern of manifestation, expressing widely. Recent studies found that, PPAR γis expressed in the malignant tumors in human oral and maxillofacial,respiratory system, digestive system and reproductive system, and theexpressed quantity is related to the malignant degree and the transfer or notof the tumor. The research shows that, dealing with the PPAR γ and its ligandthrough a variety of means can affect its growth and metastasis, and this hasbeen applied to the treatment of the malignant tumor,such as: lung cancer,breast cancer, etcr. The PPAR γ gene locates in the chromosome3p25,containing nine exon, and its genomic structure covers100KB.Among the oral and maxillofacial malignant tumor, SACC (salivarygland adenoid cystic carcinoma) can arise distant metastasis easily throughblood transport, and the most of the target place is the lungs. As one of theimportant features of malignant tumor, its malignant degree will be reducedinfinitely if the tumor loses the property of transfer. The research on therelativity of tumor metastasis is indispensable, important and basal inconquering the cancer treatment problem. The research shows that theexpression of PPAR γ which is studied as the most widely and maturehormone receptor is different in the high pulmonary-metastatic cell line andlow pulmonary-metastatic cell line of SACC, the expression quantity ofhigh pulmonary-metastatic cell line is higher than low pulmonary-metastaticcell line obviously. But there is few reports about the relationship betweenPPAR γ and SACC distant metastasis. This experiment uses the RNA I (RNAinterference) technology to silence the PPAR γ gene’ expression withspecificity, and researches the effect about the salivary gland adenoid cysticcarcinoma lung transfer after PPAR γ gene has been silenced throughestablishment Salivary gland adenoid cystic carcinoma lung transfer model in nude mouse’ body, to further enrich the researches about the SACC transfer.Methods:1Detect the differences of the expression of PPAR γ gene of these twokinds of cells above by PT–PCRExtracting the total RNA of these two kinds of cells, reversetranscribing it into cDNA, as template, detecting the difference of PPAR γexpression quantity by PT–PCR based on the cDNA, β-acton gene as acomparison，analysising the statistical data by the methods of single factoranalysis of variance (ANOVA) of SPSS13.0.2Construct the siRNA’ express carrier of the PPAR γThe Wuhan Genesil Biotechnology Co.Ltd designs and constructs thePPAR γ siRNA express carrier according to the design principles of the smallmolecule interference RNA (siRNA, small interfering). At the same time, theyselect the sequence which is lack of homology compare with PPAR γ toconstruct the negative control carrier. In the end, they have cloned the U6promoter sequences into PPAR γ siRNA express carrier. U6was compoundedaccording to the Genbank sequence (ACCession number X06980) in vitro,replacing original promoter, obtaining the siRNA express carrier which weneed finally.3The transfectionAccording to the product description of Invitrogen, we transfect SACC-LM and SACC-83cells with ationic liposomes Lipofectamine2000, positiveplasmid expression carrier, negative plasmid expression carrier and blankplasmid, naming these cells which have been transfected as:SACC-LM positive group,SACC-LM negative control group,SACC-LM blank control group;SACC-83positive group,SACC-83negative control group,SACC-83blank control group.4Observe plasmid transfect condition under the microscope Pick up six fields respectively in these holes where is culturing such cellsrandomly, under the microscope,48h later, observe the transfect efficiency,analysis statistical data by the method of single factor analysis of variance(ANOVA) of SPSS13.0.5Detect the restrain efficiency PPAR γ by PT–PCRExtract the total RNA of these cells those have been transfected and havenot been transfected, reverse transcribe them into cDNA, and detect thechange of PPAR γ expression quantity by PT–PCR on the base of the cDNA,β-acton gene as a comparison.6Detect the change of the migration ability of tumor cells in vitro byTranswell roomDetect the chang of adhesion migration ability of SACC-LM and SACC-83when PPAR γ has been silenced with the method of Matrigel matrixmembrane simulation extracellular matrix and the transwell room, comparedwith the cells which were transfected by negative plasmid and blank plasmid,analysis statistical data by the method of single factor analysis of variance(ANOVA) of SPSS13.0.7Test the change of transfer ability of tumor cell in Nude mouse bodyDivid a total of104BALB/C nu/nu female nude mouses which is4weeks old into eight groups randomly, use4＃syringe to inject cell suspensionat the place of caudal vein where is2-3cm distance from the tip,0.1ml eachmouse, inject once again1week later. Put mouses to death8weeks later.Anatomy in strict aseptic conditions, and take their lungs out. Use surgeryshear to strip all the metastasis that can be catched sight under the microscope,weigh the weight of the metastases which has been separated with theelectronic balance, record the data, analysis statistical data by the method ofsingle factor analysis of variance (ANOVA) of SPSS13.0.Results:1β-action for the internal reference, in these belts that we have got fromthe experimental results, the belt of SACC-LM group is significantly brighterthan SACC-83group, analysis the statistical data,P=0.00＜0.05. we can detect that the PPAR γ gene expression quantity of SACC-LM cell is obviouslyhigher than SACC-83cells.2There is bright green fluorescence can be seen after48hours in thesecells those have been transfected by plasmid. The result that we aculat thetransfection efficiency of SACC-LM positive group, SACC-LM negativecontrol group, SACC-LM blank control group; SACC-83positive group,SACC-83negative control group, SACC-83blank control group is87.80%,88.02%,86.51%，88.20%,85.31%,87.76%. There is no significantdiference among these different groups,P=0.609＞0.05.3After RNA interference, in these belts that we have got from theexperimental results, the belts of negative control group is significantly palerthan those control groups, it suggests that, after RNA interference, the PPAR γexpression quantity of SACC-LM and SACC-83cells reducessignificantly.4After RNA interference, the count of SACC–LM cells and SACC–83cells those get through the Matrigel artificial basement membrant reducessignificantly,P=0.00＜0.05. While there is no significant difference in theaverage number of the cell which get through the membrane in SACC-LMpositive group compared with SACC-83group, SACC-83negative controlgroup and SACC-83blank control group,P=0.444＞0.05.5. In the eight groups of the nude mouses, we have found no intumescentnodules and lymph nodes in their bodies except for their lungs. The group ofSACC-LM which is RNA interference has been found that the visible lungmetastases has significantly reduced, and the group of SACC-83, after theRNA interference, has no obvious visible metastases in the mouses’ lungs.The pulmonary metastases’ average weight of the SACC-LM positivegroup and the SACC-83positive group has significantly reducedcompared with those of the control groups, P=0.000＜0.05，has significantdifference. While there is no significant difference on the average weight ofpulmonary metastases of the SACC-LM positive group compared with theACC-83group, the SACC-83negative control group and the SACC-83blank control group, P=0.301＞0.05.Conclusion: The migration ability of the SACC cells in vitro and thelung metastasis will be reduced significantly after the RNA interference,Confirmed that the metastasis ability of SACC will be reduced if theexpression of PPARγ is inhibited. The PPARγ plays an important role in thetransfer process of SACC, PPARγ can be a new therapeutic target for SACC.