| Object: Gastrointestinal stromal tumors(stromal tumor Gastrointestinal,GIST)are the most common mesenchymal tumors of the digestive tract.Patients face problem of metastasis occur at an early stage,postoperative recurrence and poor clinical prognosis;At present,imatinib(imatinib mesylate,IM)is the most commonly used targeted drug to treated primary or metastatic GIST in patients.IM can be significantly prolonged the survival time and median survival period,but in the period of IM therapy,IM resistance become the main problem to cure the GIST.Mitochondrial translation extension factor(mitochondrial TU translation elongation factor Tu,TUFM)is found highly expressed in GIST and other kinds of tumor tissues,and associated with the tumorigenesis,progression,drug resistance formation process and bad prognosis.In this study,silencing the expression of TUFM on GIST-T1 cell and IM induced secondary resistant cell lines GIST-IR cells.To explore the functiom of TUFM in gastrointestinal stromal tumor and the process of drug resistance mechanism and influence to the related characteristics of cell biology.In order to found out the new clues to treated GIST and reversal of IM resistant of GIST.Method: 1.Construction of TUFM silencing plasmid and transfected to gastrointestinal stromal tumor cells GIST-T1 and its resistant cells GIST-IR in vitro.The relative expression of TUFM was detected by RT-PCR then evaluate the effect of interference.2.Use electroporation method TUFM silencing plasmid was transfected into GIST-T1 and GIST-IR in vitro.The transfection efficiency and assess the morphological changes of cells were observed by inverted fluorescence microscope.3.Use cell counting method and CCK-8 method to observe the effect of TUFM silencing GIST-T1 and GIST-IR cell proliferation.4.Cell scratch assay and Transwell migration assay were used to observe the effects of TUFM silencing migration on the GIST-T1 and GIST-IR cells;5.CCK-8 method was used to calculate the cell inhibition concentration and draw the IC50 curve,IM effect on drug sensitivity of GIST-IR cells was observed after TUFM silencing; 6.USE FCM method to observe the effects of GIST-T1 and GIST-IR in cell cycle and late apoptosis after TUFM silencing.Result: TUFM expression of GIST-T1 and GIST-IR were detected by RT-PCR.The results of RT-PCR showed that the expression of TUFM in GIST-T1 group was lower than that in negative control group and blank control group(P<0.05).2.Inverted fluorescence microscope to observe electrical efficiency analysis in the GIST-T1 and GIST-IR cell showed: GIST-T1 and GIST-IR under voltage 590 m V,30 ms shocking time,20 ug plasmid,was transfected into cells.After electroporation,cells without the apparently dead,plasmid GFP expression efficiency is up to 75%.Fluorescent cell morphology was observed by inverted fluorescence microscope 24 hours after transfection,between TUFM group,negative control group and blank group there no significant difference was found.3.Cell counting method and CCK-8 method were used to detect the proliferation and viability of GIST-T1 and GIST-IR after TUFM silencing.Cell counting method continuous cell plate silent counting GIST-T1 and GIST-IR TUFM group,negative control group and blank control group cells 6 days showed that TUFM silent group of GIST-T1 proliferation of cell count is about 24×104 and the negative control group for 30×104.The cell count of TUFM GIST-IR silence group was about 24×104,negative control group is 17×104.By transfection after 48-72 hours reached maximum,the inhibition rate was(26.2±2.0),GIST-IR silent group of proliferation inhibition in transfection after 48-72 hours to reach maximum value,GIST-T1 cell silence group proliferation is suppressed the inhibition rate was(25.0± 3.0).4.The effect of and GIST-IR cell migration on the GIST-T1 and TUFM cells was observed in vitro.Scratch test results showed that: GIST-T1 and GIST-IR silent group cell lateral migration ability decreased,48 hours after scratching,have no significant difference between negative control group with blank control.5.Transwell cell migration assay of GIST-T1 and GIST-IR in vitro vertical migration.Transwell migration assay results: TUFM silence after GIST-T1 cell silence group through the dorsal cell membrane cell number obvious reduction,longitudinal cell migration ability decreased significantly.Transwell back film,GIST-T1 cell eosin staining,statistical chamber on the back of the cell migration,silent small chamber groups on the back of each field cell number(4.7 ±2.8)and negative control,the number of every vision cells for(84.8±4.8)in the control group,each view cell number(88.2±5.7).GIST-IR TUFM silencing group through the cell membrane back decreased,cell longitudinal migration decreased significantly.Transwell and eosin staining,statistical chamber on the back of the cell migration,GIST-IR silence small chamber groups on the back that the number of cells in each field(30.8±3.8),negative control group,the number of every vision cells for(66.7±7.9)in the control group,each view cell number(66.3±5.8).The number of cells in GIST-T1 and GIST-IR group was less than that in the blank group and negative control group,and the difference was statistically significant(P<0.05).Detection of IM drug sensitivity of GIST-IR cells by 4.CCK-8 method.The GIST-IR cell viability was detected by in 0,10,25,50 and 75 μmol/L IM,and the results showed that the IC50 curve was calculated and plotted.There was no significant difference between the control group and the negative control group(P>0.05).6.Cell apoptosis and cell cycle of GIST-IR and GIST-T1 were detected by FCM.Experimental results show that GIST-T1 cell silencing group G0/G1 ratio for(781±0.15)compared with the negative control group G0/G1 cells(4.3±0.53)increased obviously,the difference has statistical significance(P<0.05).The phase of G1,S,G2/M phase silent groups of cell number and control cells between the differences had no statistical significance(P>0.05)in GIST-T1 cell.About GIST-IR silence group,the ratio of G0/G1 cells for(16.3±1.2)compared with the negative control group G0/G1 cell(7.2±0.0.45)increased,difference has statistical significance(P<0.05),the G1,S and G2/M phase silent groups of cell number and no statistical significance compared with differences between cells(P> 0.05).Conclusion: 1.The expression of TUFM significantly decreased after transfection TUFM silencing plasmid(P<0.05).TUFM expression silencing efficiency 70.8%,and TUFM expression cells in GIST-T1 cell and GIST-IR no significant difference;2.Electroporation can be efficient in vitro transfection plasmid to cell lines,and less cell damage,transfer efficiency was 75%.3.GIST-T1 and GIST-IR cells decreased the proliferation capacity,migration ability and advanced apoptosis,but had no significant changes in the IM sensitivity and cell cycle of GIST-IR. |
