Adenine Monophosphate-activated Protein Kinase Regulates Mitochondria Function And Warburg Effect Via Nuclear Transcription And Direct Mitochondrial Binding

Chapter 1 IntroductionTumor has been reported to rank second of the most fatal diseases.The data from world health organization(WHO)showed that there would be 50% increase in the cancer morbidity before 2020.Cancer is related to various factors,including environment and genetics.There are still no certain mechanisms about the pathogenesis of cancer,not to mention effective therapies.Therefore,study of the mechanism about cancer is of enormous importance.Now,it is reported that energy metabolism disorder is very important in cancer tumorigenesis,correcting it may be the new strategy.AMPK is the most important energy sensor for cells.Also,it has been recognized as the most important and effective tumor suppressor in recent 10 years.Even in aerobic condition,glycolysis provide energy for cancer cells,this is called as Warburg effect.In recent studies,we found LKB1/AMPK could regulate Warburg effect,but we do not know the mechanism.mitochondria is the power plant inside cells,which could supply most of the energy necessary for cell functions,so it has closed relationship with AMPK.It's said when the aerobic respiration is increased,or glycolysis is decreased,the cancer then was inhibited.There has been a lot of reports about the relationship between AMPK and mitochondria from different perspectives.Our group has focused on the study of AMPK for a long time.In one experiment before,we need extract mitochondria protein from A549-LKB1 and A549-WT cells,and found that there is more pellet from A549-LKB1.Therefore,we concluded that AMPK could affect the function of mitochondria and the quantity of mitochondria.We,then,constructed different plasmids,and obtained LKB1 over-expressed cells,AMPK? knock-out cells,and mitochondria AMPK? over-expressed cells through transfection.Then,we performed counting of the mitochondria.After verification of our hypothesis,we studied the AMPK effect on mitochondria by analysis of mitochondria biosynthesis,mitochondria fission and fusion,which could affect the quantity and performance of mitochondria.We measured the aeroblic respiration and glycolysis function in these cells to reflect the metabolism of these cell using seahorse metabolism instrument.Moreover,we found that the expression of AMPK? decreased after knock-out of AMPK?.Also,we studied the instability mechanism of AMPK? after knock out of AMPK?.At last,we performed nude mice assay to find the function of AMPK in cancer inhibition,the promotion of mitochondria number,fission and fusion.Chapter 2 The AMPK distribution and function in cellObjective: To examine the distribution of AMPK? and the cancer inhibited function of mitochondria AMPK?.Methods: First of all,we established A549-LKB1-AMPK?-Null cell line using Crispr-Cas9 transfection system.Then we used Western blot assay to detect if the stable cell line is established successfully.Next,AMPK? distribution was detected by Western blot.We examined the functions of AMPK? and mitochondria AMPK? in cell proliferation by counting the cell numbers and MTT.Wound healing assay were performed to detect the functions of AMPK? and mitochondria AMPK? in cell migration;we used plate clone formation assay to ensure the clone formation ability of AMPK? and AMPK?.Results: Firstly,we constructed A549-LKB1-AMPK?-Null cells and A549-mito-AMPK/RFP cells by Crispr/Cas9 system and Lenti-virus system.Then we ensured the stable cell lines are constructed successfully by Western blot assay.We almost could not found AMPK? expression in A549-LKB1-AMPK?-Null cells.When we detected AMPK? expression in A549-mito-AMPK/RFP cell,we could find two lanes.When we detected the AMPK? distribution,we found in A549-LKB1 cells,we could found AMPK? expression in both mitochondria and cytoplasm.But in A549-LKB1-AMPK?-Null cells,we could not found it.When we compared the proliferation,migration and plate clone formation ability in A549-LKB1-AMPK?-Null cells and A549-LKB1,we found when we knock-outed AMPK?,cells get faster proliferation,migration and plate clone formation ability,But A549-mito-AMPK? cells have lower proliferation and plate clone formation compared with A549-mito-RFP.Conclusion:(1)We constructed AMPK? knock-out cell A549-LKB1-AMPK?-Null cells and mitochondria special over-expressed cells A549-mito-RFP/AMPK? cells successfully.(2)By MTT,wound healing and clone formation assay,we found no matter AMPK? and AMPK? could inhibit cancer cells proliferation,migration and clone formation ability.Chapter 3 The effect of AMPK? and AMPK? in mitochondria1.The effect of AMPK? and AMPK? in mitochondria mass.Objective: To investigate the role of AMPK? and mitocondrial special AMPK? in mitochondria number.Methods: q PCR was used to detect the mitochondria DNA number and nuclear DNA number in A549-LKB1-AMPK?-Null,A549-LKB1,A549-WT,A549-mito-RFP and A549-mito-AMPK?cells.We extracted cytoplasma protein and mitochndria protein of these cells,then quantitied the protein.Next,we used Western blot to detect the special mitochondria protein marker ATPase sythesis ? expression in total protein.Mitochondria special dye Mito Tracker-Red was used to stain live cells mitochondria,then used confocal microscope to get images to find out the red fluorescence intensity and quantity.At last,we used adenovirus And-AMPK-CA and control And-GFP to infect A549-LKB1-AMPK?-Null and A549-LKB1 cells for 48 hours,then using q PCR to detect mitochondria number just like the method above.Results: It is shown that when LKB1 was over-expressed,the cells express higher AMPK phosphorylation level,then,the mitochondria number,the mitochondria protein mass,ATPase sythesis ? expression are higer than A549-WT cells,.But if we knockout AMPK? gene,they are decreased.Withing this results,we hypothesized AMPK? and AMPK? in cytoplasm could both have positive effect on mitochondria number.To confirm our hypothesis,we used two adenovirus named as Adn-AMPK-CA and Adn-GFP to infect A549-LKB1-AMPK?-Null and A549-LKB1 cells.We found after infected by adenovirus,the two cells have much more mitochondria DNA number,and the number of mitochondria DNA increases more obviously in A549-LKB1 cells.At last,we stained the three live cells with Mito-Tracker-Red.We were easy to see that in A549-LKB1 cells,the red fluorescence intensity is stronger and the number is bigger.By the way,we found the size of the cell in A549-LKB1-AMPK?-Null is smaller than A549-LKB1.We did the staining experiments in A549-mito-RFP and A549-mito-AMPK cells.Quite unexpectedly,we did not get the same results in the two cells.There is no significant difference in mitochondria mass.And the sizes of the two cells have no significant difference.Conclusion:(1)AMPK could increase the mitochondria mass.(2)mitochindria AMPK has nothing to do with the mitochondria mass.2.The effect of AMPK? and AMPK? in mitochondria synthesis.Objective: To detect the role of AMPK?,AMPK? and mitochondria AMPK? in mitochindria synthesis.Methods: We detected the m RNA and protein expression of mitochondria synthesis related gene like PGC1 ?,Nrf-1 m RNA using q PCR and Western blot assays in A549-LKB1-AMPK?-Null,A549-LKB1,A549-WT,A549-mito-RFP and A549-mito-AMPK cells.Also,we treated cells with AMPK activated drugs like metformin and berberine for 24 hours,treated A549-mito-RFP and A549-mito-AMPK cells with DOX for 24 hours to shut off the mitochondria AMPK expression,then extracted m RNA and total protein,used q PCR asssay to detect Nrf-1 m RNA expression;used Western blot to detect AMPK phosphoralation level,PGC1? and Nrf-1 protein expression.At last,adenovious Adn-AMPK-CA and Adn-GFP was used to infect A549-LKB1-AMPK?-Null,A549-LKB1 and B16 cells for 48 hours,then measured the Nrf-1 protein expression.Results: By comparing the basic PGC1? and Nrf-1 m RNA expression in A549-LKB1 and A549-LKB1-AMPK?-Null cells,we found both of the two gene m RNA expressions in A549-LKB1 cells are higher.After infected by adenovirus or treated with AMPK activated drugs,the two cells have much more PGC1? and Nrf-1 m RNA expression,and it is much more obviously in A549-LKB1 cells.And the Western blot assay got the same results even in B16 cells.We performed the Western blot experiment when treated the cells with DOX in A549-mito-RFP and A549-mito-AMPK,we did not find difference in Nrf-1 m RNA expression.Conclusion:(1)AMPK?,? could increase mitochondria synthesis.(2)Mitochondria AMPK has nothing to do with mitochondria synthesis.3.The effect of AMPK? and AMPK? in mitochondria fission and fusion.Objective: To examine the roles of AMPK? and mitocondrial AMPK? in mitochondria fission and fusion.Methods: We detected the m RNA and protein expression of mitochondria fusion related gene MFN-1,MNF-2 m RNA using q PCR and Western blot assays in A549-LKB1-AMPK?-Null,A549-LKB1 and A549-WT cells.Also,we treated A549-LKB1 and A549-WT cells with AMPK activated drug metformin(10m M)for different hours,treated A549-mito-RFP and A549-mito-AMPK cells with AICAR(1m M)for different hours to active AMPK expression,then extracted total protein,used Western blot to detect AMPK phosphoralation level and Drp-1 phosphoralation level.At last,adenovious Adn-AMPK-CA and Adn-GFP was used to infect A549-WT,A549-LKB1 and B16 cells for 48 hours,then measured the Drp-1 phosphoralation level.Dox was used to treat A549-mito-RFP and A549-mito-AMPK cells and used Western blot to detect Drp-1 phosphoralation level.Results: By comparing the basic MFN-1 and MFN-2 m RNA expression in A549-WT,A549-LKB1 and A549-LKB1-AMPK?-Null cells,we found both of the two gene m RNA expression in A549-LKB1 cells are lower,after infected by adenovirus or treated with AMPK activated drugs,Drp-1 phosphoralation level have much more higher,especially in A549-LKB1 cells.And the Western blot assay got the same results even in B16 cells.When we performed the Western blot experiment when treated the cells with metformin different times in A549-mito-RFP and A549-mito-AMPK,we did not find difference in the ATPase sythesis ? protein expression,AMPK phosphoralation level and Drp-1 phosphoralation level.Conclusion:(1)AMPK?,? could increase mitochondria fission and inhibit mitochondria fusion.(2)Mitochondria AMPK has nothing to do with mitochondria fission and fusion.Chapter 4 The effect of AMPK?and AMPK?in energy metabolismObjective: To examine the roles of AMPK? and mitocondria AMPK? in energy metabolism.Methods: Lactate level was detected in the culture supernatant of A549-LKB1,A549-LKB1-AMPK?-Null,A549-mito-RFP and A549-mito-AMPK cells by lactate kit.Seahorse metabolism instrument were used to detect glycolysis ability with the XF glycolysis stress test kit and basic ECAR and OCAR.Results: From the lactate measured assay,we found the A549-mito-RFP culture supernatant has the higher lactate level than A549-mito-AMPK,but in A549-LKB1 and A549-LKB1-AMPK?-Null cell culture supernatant,A549-LKB1 cell has the lower lactate level.And the XF glycolysis stress test showed the same result: A549-LKB1-AMPK?-Null cell has lower basic glycolysis level and glycolytic capacity,when we tested the OCAR and ECAR values in A549-mito-RFP and A549-mito-AMPK cells,we found A549-mito-RFP cell has lower OCAR value and higher ECAR than A549-mito-AMPK cells.Conclusion:(1)Mitochondria AMPK could inhibit the glycolysis and promote the aerobic oxidation.(2)AMPK? could promote the glycolysis.Chapter 5 The effect of AMPK? in AMPK? stabilityObjective: To investigate the AMPK? function in AMPK? stability and the mechanism.Methods: Western blot was used to detect the AMPK? expression in A549-LKB1-AMPK?-Null,A549-LKB1 and A549-WT cells.Also,we detected total AMPK? and mitochondria AMPK? after infected with Adn-AMPK-CA adenovious.Cells were treated with drugs like 3-MA,MG132,leupeptin and CHX,then protein was extracted and AMPK? stability was measured by Western blot.Results: In A549-LKB1-AMPK?-Null cells,we knock-outed AMPK?,the AMPK? protein expression is also decreased.After infected with Adn-AMPK-CA adenovious in A549-LKB1-AMPK?-Null and A549-LKB1 cells,the AMPK? protein expression is increased in both of the two cells,but it is much more obviously in A549-LKB1 cells.Autophagy inhibitor 3-Methyladenine and proteasome inhibitor MG-132 were used to test the degradated signal pathway of AMPK? when we knockouted AMPK?.Only 3-MA could up-regulated AMPK?,as the same time,we detected autophagy marker LC3?/?,the ratio in A549-LKB1-AMPK?-Null cells is higher obviously.Also,after treated with CHX,we could detect exogenous AMPK?.Conclusion:(1)AMPK? play a vital role in AMPK? stability and when we knocked down AMPK?.(2)AMPK? could degradate by autophagy-lysosome pathway.Chapter 6 The tumor formation ability of AMPK? and mitochondriaAMPK? in miceObjective: To examine the tumor formation ability of AMPK? and mitochondria AMPK? in nude mice.Methods: Enough A549-mito-RFP ? A549-mito-AMPK cells were cultured,and then injected into six 4-week-old female nude mice separately,we measured the tumor sizes every 3 days,then sacrificed the mice in 39 days,weight the tumor tissues,detected the mitochondria synthesis,mitochondria fission and fusion in the tumor tissues by Western blot assays.We injected B16 cells into 12 4-week-old female nude mice,then separate the mice into two groups randomly.Intragastric administration was performed to treat with berberine or the same volume normal saline every day,then sacrificed the mice in 20 days,weight the tumor tissues,detected the mitochondria synthesis,mitochondria fission and fusion in the tumor tissues by Western blot assays.Results: We found A549-mito-RFP and B16 control tumor tissues are much bigger than A549-mito-AMPK and B16 treated with berberine tissues.As a result,the weight of A549-mito-RFP and B16 control tumor tissues are heavier.We then tested the p-Drp1 and PGC1? protein expression in these tumor tissues.Conclusion: AMPK? and mitochondria AMPK? could inhibit the tumor formation ability in nude mice.Chapter 7 ConclusionAs a tumor suppressor and energy sensor,AMPK has distribution in cytoplasm and mitochondria.Both AMPK could inhibit tumorigenesis in vivo and vitro.AMPK could increase mitochondria number,mitochondria biosynthesis,mitochondria fission,decrease mitochodria fusion,but mitochondria AMPK has nothing to do with these.In metabolism mitochondria AMPK could inhibit the glycolysis and promote the aerobic oxidation.In a word,AMPK and mitochondria AMPK may have different role in cells,but they could inhibit tumorigenesis using their own way.