| Background and ObjectiveAnaplastic thyroid carcinoma(ATC)is one of the most aggressive solid tumors in humans and has a median post-diagnosis survival of 3 to 5 months.The 1 and 10 years overall survival rates were estimated at 10-20% and less than 5%,respectively.Although there are some reports of long-term survival,the reliability of its diagnosis is questioned.Undifferentiated thyroid carcinoma accounts for only 1-3% of thyroid malignancies,but its contribution is as high as 14-50% annual mortality in all of thyroid cancer.Moreover ATC has limited therapeutic options and is generally resistant to standard radiotherapy and chemotherapy.Therefore,understanding the metabolism and molecular pathogenesis of ATC will provide new hope for the treatment of this disease.The character of metabolism in cancer and non-cancer cells are very different,Many cancer cells are reprogrammed in metabolism.We know that normal cells mainly use the mitochondrial to metabolism of glucose under the adequate oxygen condition.Only in the case of hypoxia,it will use the way of glycolysis metabolism,the pyruvate converts into lactic acid and rapid supply of energy.Then,once oxygen is abundant,lactic acid is converted back to pyruvate,redirected into the mitochondria,redox,and eventually generate of carbon dioxide,water and ATP.However cancer prefer to using glycolytic rather than mitochondrial oxidative phosphorylation as the glucose metabolism for cell growth and proliferation,even in the presence of oxygen condition(The effect is called the Warburg Effect).lactate dehydrogenase(LDH)is a key enzyme in nicotinamide adenine dinucleotide(NAD+)-dependent cellular glycolytic metabolic pathways.The expression of LDH in cancer cells does have a clear effect on the maintenance and malignant progression of tumor cells metabolism.LDH is a tetrameric enzyme composed of two distinct subunits(LDHa and LDH-b)encoded by isolated genes.LDH catalyzes the interconversion of pyruvate and reduced nicotinamide adenine dinucleotide(NADH)to lactic acid and NAD+.The catalytic direction is dependent on the ratio of LDHa to LDH-b in the LDH complex;LDHa promotes the reduction of pyruvate to lactate to regenerate NAD+ from NADH,whereas LDH-b favors reverse direction catalysis.In this study,we focused on the pathway of glucose glycolysis and metabolism of cancer cells,by Oncomine database analysis to determine the differential expression of LDH in different types of thyroid cancer pathology.Subsequently,a stable gene silencing cell line was established by constructing a recombinant lentiviral vector whose expression was controlled by Doxycycline(DOX)in Hth83 cell line of thyroid undifferentiated carcinoma.Then,the lentivirus-infected cells were subjected to a monoclonal screening,then we will find a monoclonal cell line in which LDHa and LDHb gene expression can be sustained and stably silenced.The effects of shLDHa and shLDHb on the tumor phenotype of ATC cell,enzyme activity and lactate production were also studied.The orthotopic transplantation tumor models were established in nude mice,and the tumorigenicity was observed when the expression of LDH is suppressed by shRNA in ATC cells at nude mice thyroid;This study not only helps to elucidate the biological function and metabolism of LDH enzyme in ATC cells,but also may provide new ideas and intervention targets for the treatment of ATC.The first chapter :The preparation and biological function study of ecombinant lentivirus lenti-shLDHa and lenti-shLDHb in ATC Hth83 cell linePart I: Preparation,identification and monoclonal screening of recombinant lentivirus lenti-shLDHa and lenti-shLDHb in Hth83 cell line Methods1.Amplify a linearized vector p-inducer-EmGFP-luciferase that induces gene silencing.2.LDHa and LDHb gene fragments were designed to prepare linearized interfering vectors.The lentiviral vectors Lenti-shLDHb and Lenti-shLDHa were constructed by annealing,ligation,transformation,identification and construction of LDH genes.3.Lentivirus packaging,purification,concentration,the use of gradient dilution method for the determination of virus titer,with the infection multiplicity(Multiplicity of Infection,MOI)= 10?25?50?75?100?200?500 infection of Hth83 cells,The ratio of green fluorescent cells to the total number of cells was determined by fluorescence observation.4.The recombinant lentivirus Lenti-LDHa and negative control virus infected Hth83 cells were observed morphological changes,MTT test to detect cell proliferation,Western-blot detection of protein expression.5.The density of 0.1?g / mL DOX is used to induce shLDHa expression in Hth83 cell line,which is sorted by flow cytometry in 96-well plates,cultured 10-14 days and then take a monoclonal amplification.Western-blot was used to verify the expression of LDHa.Monoclonal cell Hth83-shLDHa-4 and Hth83-shLDHa-31 were selected and verified.6.The density of 0.1?g / mL DOX is used to induce shLDHb expression in Hth83 cell line,which is sorted by flow cytometry in 96-well plates,cultured 10-14 days and then take a monoclonal amplification.Western-blot was used to verify the expression of LDHa and detected the Luciferase signal.Monoclonal cell Hth83-shLDHa-6 and Hth83-shLDHa-18 were selected and verified.Result1.Based on the successful transfection of Luciferase protein in the original Hth83 cell line,we successfully constructed the lentivirus-infected cell lines Hth83-shLDHa and Hth83-shLDHb which will suppress the expression of LDH by DOX induction.The fluorescence showed that the expression efficiency was up to 75%,satisfied the next experiment requirements.2.The results of western-blot showed that the expressions of LDHa and LDHb were significantly inhibited by DOX induction at different concentrations and times.The growth inhibition,cell dispersion,visible cell debris and cell contracture were observed at high concentrations of DOX group in Hth83 cells.3.Used the different concentration of DOX and time,MTT test showed that there was statistical difference in the decline of cell proliferation ability at the treatment group with DOX concentration of 10 ?g/mL.4.The monoclonal cell Hth83-shLDHa-4,Hth83-shLDHa-31,Hth83-shLDHb-6 and Hth83-shLDHb-18 were successfully selected,Western-blot proofed that LDHa?LDHb protein can be well inhibited in different time and dose,which will help us to find out the best concentration and time to inhibit the target gene and also help us to continue the further cell function work.Part II: Effect of inhibiting LDH expression on cell function,enzyme ctivity and lactic acid concentration in monoclonal Hth83 cell line Methods1.DOX induced the expression of shLDHa ? shLDHb genes in Hth83-shLDHa-4,Hth83-shLDHa-31,Hth83-shLDHb-6 and Hth83-shLDHb-18 cell lines,respectively.which were detected by lactic acid assay kit about the lactic acid concentration in vitro.2.DOX induced the expression of shLDHa ? shLDHb genes in Hth83-shLDHa-4,Hth83-shLDHa-31,Hth83-shLDHb-6 and Hth83-shLDHb-18 cell lines,respectively.Using Lactate dehydrogenase Kit detected the lactate dehydrogenase activity.3.DOX induced the expression of shLDHa ? shLDHb genes in Hth83-shLDHa-4,Hth83-shLDHa-31,Hth83-shLDHb-6 and Hth83-shLDHb-18 cell lines,respectively.MTT assay is used to detected the ability of cell proliferation.4.DOX induced the expression of shLDHa?shLDHb genes in Hth83-shLDHa-4,Hth83-shLDHa-31,Hth83-shLDHb-6 and Hth83-shLDHb-18 cell lines,respectively.Cell monoclonal formation activity assayed by plate assay.5.DOX induced the expression of shLDHa genes in Hth83-shLDHa-4,Hth83-shLDHa-31 cell lines respectively.Scratch experiment is adopted to evaluate the Cell migration ability.6.DOX induced the expression of shLDHa genes in Hth83-shLDHa-4,Hth83-shLDHa-31 cell lines,respectively.Flow cytometry(FCM)measured the changes in the number of apoptotic cells.Result1.Lactic acid production decreased by DOX induction in 24 hours at Hth83-shLDHa-4 and Hth83-shLDHa-31 experimental groups(p <0.05).After 96 hours,there was no significant difference in lactate production between the experimental and control groups.The tumor lactic acid production no significant difference(p> 0.05).Hth83-shLDHb-6,Hth83-shLDHb-18 experimental group compared with the control group,in 24 hour high concentration of DOX induction group,lactic acid production decreased slightly,low concentration DOX experimental group compared with the control group in Hth83-shLDHb-18,lactic acid production were not Significant difference(p> 0.05).Lactic acid production were no difference after 96 hours in Hth83-shLDHb-6,Hth83-shLDHb-18 cell.(p> 0.05).2.The activity of lactate dehydrogenase in Hth83-shLDHa-4 and Hth83-shLDHa-31 experimental groups were significantly lower than the control group by DOX induction(p <0.05)and the activity of lactate dehydrogenase was further decreased in the 96 H group compared with the 24-hour group(p <0.05).Hth83-shLDHb-18 experimental group compared with the control group in 24 hours,lactate dehydrogenase activity was slightly decreased(p <0.05).Meanwhile Hth83-shLDHb-6 experimental group compared with the control group,lactate dehydrogenase activity was no difference(p > 0.05).In 96 hours group Hth83-shLDHb-6,Hth83-shLDHb-18 lactate dehydrogenase activity was no significant difference compared with the control.(p > 0.05).3.After 24 hours DOX induction,the cell viability of Hth83-shLDHa-4 and Hth83-shLDHa-31 cell lines was not significantly different from that of the control group.After 48,72 and 96 hours of DOX induction,the cell viability of the experimental group was significantly higher than that of the control group and with the increase of DOX concentration,cell viability was significantly reduced(p <0.05).Hth83-shLDHa-6 and Hth83-shLDHa-18 cell lines inducted by DOX after 24 h,48 h and 72 h,the cell viability was not significantly different from that of the control;but the activity of the experimental group was lower than that of the control group after 96 hours p <0.05)..4.When the expression of LDHa and LDHb gene were inhibited,the cell clones formation rates of monoclonal cell lines Hth83-shLDHa-4,Hth83-shLDHa-31,Hth83-shLDHb-6 and Hth83-shLDHb-18 were significant inhibited(P <0.01).With the increase of dose of DOX,the inhibition of clonogenicity also increased.5.When the LDHa gene expression was inhibited,there was no significant in the cell migration between the monoclonal cell lines Hth83-shLDHa-4,Hth83-shLDHa-31 and the control group.(p> 0.05).6.Inhibition of LDHa gene expression were not induced the change of cell cycle and ratio of apoptosis in the Hth83-shLDHa-4,Hth83-shLDHa-31 cell lines(p> 0.05).The second chapter: The effect of knockdown LDHa on the growth of ransplanted tumor in nude mice Methods1.Established the animal model of human xenografts in vivo implantation of Hth83-shLDHa-4,Hth83-shLDHa-31 cell lines at nude mice thyroid gland.2.The nude mice were given diets with DOX and without DOX respectively starting from the fifth day after surgery.The imaging system was used to monitor the changes of Fluorescence signal and tumor weight every two days.3.The experimental group and the control group were given a diet with or without DOX,respectively.After the xenograft model was established,three nude mice xenograft tissues were extracted,the expression of LDHa in the tissues was examined by Western blot.4.The experimental group and the control group were given a diet with or without DOX,respectively.After the xenograft model was established,three nude mice xenograft tissues were extracted,lactate dehydrogenase detection kit was used to detect the LDHa concentration in the tissue.5.To monitor the total survival of tumor transplant model nude mice,draw the survival curve.Result1.Successfully established an nude mouse xenograft model in situ companied with LDHa gene down regulation.2.The growth of tumor in experimental group was slower than that in control group.Fluorescence imaging system of live animal showed that the fluorescence intensity of experimental group was significantly lower than that of control group(P = 0.003).And the tumor volume and weight of nude mice in experiment group were less than those in control group(P = 0.0031 and P = 0.002).3.The expression of LDHa protein in the experimental group was lower than that in the control group(P = 0.286).4.Lactate dehydrogenase activity in the experimental group of tumor tissue was lower than the control group(P = 0.231).5.The overall survival of experiment group is longer than the control group in human thyroid tumor transplant model of nude mice.Conclusion1.Oncomine database indicated that LDHa is highly expressed in undifferentiated thyroid carcinomas,and LDH is a key molecule in cancer metabolic pathways,suggesting that LDH has a good research prospect in undifferentiated thyroid carcinoma.2.The inhibition of LDHa gene expression could effectively inhibit the proliferation and clone formation of Hth83 cells,but had no significant effect on cell migration,cell cycle and apoptosis.3.The inhibition of LDHa and LDHb gene expression can effectively inhibit the activity of lactate dehydrogenase in Hth83 cells and temporarily reduce the intracellular lactate concentration.4.The expression of shLDHa gene inhibited the activity of lactate dehydrogenase and the concentration of lactate in thyroid undifferentiated carcinoma Hth83 cells line by lentiviral transfection method.however,the effect of silencing LDHa was better than that of silencing LDHb gene.5.The expression of shLDHa gene can significantly inhibit the growth of human thyroid carcinoma xenografts in nude mice,and prolong the survival of nude mice in xenograft thyroid carcinoma.6.Down regulates the expression of LDHa gene inhibits the expression of LDHa protein and lactate dehydrogenase activity in xenografts nude mice.It is suggested that LDHa may be a potential therapeutic target for ATC. |
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