Expression Of Tumor Suppressor Gene PTEN Indifferent Tumor Cell Lines And Its Effect On CIK Killing Tumor Cell Function

ObjectiveAnalyse different tumor cell lines anti-oncogene PTEN mutation status, and study the PTEN gene mutation frequency in different tumor cells by high-resolution melting curve( HRM) method.Detect expression level difference of PTEN gene in the tumor cell lines using fluorescence quantitative PCR method,transfection change expression of PTEN in tumor cells in order to study the effect of PTEN on cell proliferation, cell cycle, migration the relative expression of immune related molecules,measure healthy CIK cells killing efficiency on different tumor cell by calcein method, analyse effects of PTEN relative expression level on CIK cell killing efficiency, so as to provide a theoretical basis and reference for tumor gene therapy based on PTEN and CIK immunotherapy in the future.Methods 1.Study on mutation of tumor suppressor gene PTEN in different tumor cell lines.According to ATCC corresponding cell culture methods, train and collect different tumor cells,extract the genomic DNA according to the kit instructor, design a pieces of specific screening mutation primers, screen PTEN gene mutation status of different tumor cells by HRM method, and further verify HRM screening results by sequencing to analyse the feasibility and accuracy of HRM method on screening PTEN gene mutationin tumor cells, and to explore cancer inhibition gene PTEN mutation frequency in different tumor cells. 2.Study on expression level difference of PTEN in different tumor cells by fluorescence quantitative PCR.Develop and collect different tumor cells, extract total RNA from the tumor cells, RT reverse transcript synthesize c DNA first strand, detect the tumor cell PTEN expression levels by using SYBGreen dye fluorescence quantitative PCR method and comparatively analyse differences of PTEN expression level in different tumor cells. 3.Effect of PTEN on the physiological characteristics of tumor cells and the effect on the expression level of immune related molecules.Change the expression level of PTEN in tumor cells by means of transfection and investigate the influence of PTEN expression changes on tumor cells proliferation, migration,and cycle;analyze the relation between PTEN expression level and the expression of p38 MAPK, costimulatory molecules CD80/86, B7-H1/H4 and chemokine receptor CCR6 and CCR7 in tumor cells and tumor cells with PTEN before and after transfection by the use of agarose electrophoresis and RT-PCR semi quantitative method. 4.Study on Difference of healthy human CIK cells killing efficiency on different tumor cells and relationship between killing effciency and PTEN different expression levels in different tumor cells.Measure healthy human CIK cells killing efficiency on different tumor target cells by calcein method, compare the differences of CIK cells killing efficiency on different tumor target cells, analyse the correlation between CIK cells killing efficiency and tumor target cells PTEN expression level,construct the pc DNA3.1-PTEN eukaryotic expression plasmid and transfect into Hep G2 and He La cells, compare the change of CIK cells killing efficiency on tumor cells with PTEN before and after transfection, and analyse the influence of tumor target cells PTEN expression changes on CIK cells killing sensitivity. Results 1.The tumor suppressor gene PTEN sequence of different tumor cells was basically normal and there are less mutations.Screen PTEN gene mutation frequency in a variety of different sources of tumor cells by use of HRM, and Sanger sequencing validate the accuracy of HRM screening results. Results show that almost all tumor cells’ PTEN gene are wild-type expression, gene nucleotide sequence are the same as NCBI published sequence, HRM screening only found that the melting curve of Jurkat cell line are different from other cells’ melting curve, TA cloning and sequencing confirm that the PTEN gene of Jurkat cell line is deletion mutation and sequencing also proved that the feasibility and accuracy of the screening of the mutation of PTEN gene in tumor cells by HRM. 2.There exists difference in the expression level of PTEN in different tumor cell lines.Extract the tumor cellstotal RNA,oligo(d T) reverse transcription synthesize c DNA, detect the different samples‘ PTEN m RNA transcription level by fluorescence quantitative PCR. The results showed that expression of PTEN in different tumor cells are different and some tumorcells are significant different,which provide basis for study on PTEN influencing tumor cell physiological activity and CIK immune reaction to tumor cells. 3.Changing the PTEN expression level of tumor cells by transfection has a certain impact on the cell proliferation,cell cycle and migration of tumor cells, and can change the expression of immune related molecules in tumor cells.Increase the expression of PTEN by transfection in Hep G2 and He La cells, MTT method was used to measure cells proliferation activity in tumor cells with PTEN before and after transfection, and the results prove that increasing expression of PTEN may reduce tumor cell proliferation activity and inhibittumor cells proliferation;PI staining method was used to determine the changes of cell cycle in tumor cells with PTEN before and after transfection,and the results show that PTEN can make tumor cells arrest in G0 / G1 to S phase in cell cycle;scratch migration repair experiment determine the influence of the changes of expression of PTEN on the ability of migration and repair of tumor cells and the results prove that improving the expression of PTEN may weaken the migration and repair ability of tumor cells. Analyse the relation between PTEN expression level and p38, CD80/86, B7H1, B7H4, CCR6 and CCR7 expression level by semi quantitative RT-PCR method Hep G2 and He La cells with PTEN before and after transfection,and the results showed that the difference of expression of PTEN truly can affect the expression of p38 and CD80 / 86, B7H1, B7H4, CCR6 and CCR7.PTEN affect the physiological characteristics of tumor cells and the expression of related immune molecules,which can provide theoretical basis for further studying the relation between the expression of PTEN in tumor cells and CIK killing efficiency. 4.Healthy human CIK’s killing efficiency on different tumor target cells are different, and there has a negatively correlation between killing efficiency and the level of PTEN expression in tumor target cells.Evaluate the killing efficiency of the healthy human CIK cells on different tumor target cells by calcein method.The results showed that the killing efficiency of healthy human CIK cells on different tumor cells exists obvious difference,CIK cell killing efficiency and tumor target cells PTEN expression level showed a significant negative correlation, further study construct the recombinant pc DNA3.1-PTEN expression system, transfect the tumor cells and found that changing the tumor target cells’ PTEN expression level can decrease the killing efficiency of CIK on tumor cells.ConclusionHave successfully established HRM method for screening the mutation of PTEN gene in tumor cells, have identified the tumor cells PTEN gene mutation frequency and the expression level. Have proved that PTEN gene can inhibit the proliferation,cycle operating,and migration repairing of tumor cells,and illustrates that PTEN expression level difference can influence tumor cell immune related molecule expression.It is proved that CIK cells killing efficiency on different tumor cells are obviously different, and found that CIK cells immune killing efficiency correlate significantly with tumor target cells PTEN relative expression level.