| Background and objectivesPeutz-Jeghers syndrome(PJS)is an autosomal dominantly-inherited disorder characterized by multiple hamartoma polyps in gastrointestinal tract,mucocutaneous pigmentation and an high risk for development of gastrointestinal(GI)malignancies.Clinical manifestation are features of Intussusception,intestinal obstruction,GI bleeding which caused by a large number of polyps in digestive tract,and high recurrence rate of those polyps.Reported estimates of the clinical incidence of PJS is between 1/50000 to 1/200000.Risk of benign or malignant tumors in patients with PJS is significantly higher than the general population(HR 8.96),and most of the patients died of cancer.Its familial incidence of malignant tumor is 15 times higher than the general.LKB1(Liver Kinase B1)gene hase been identified as the major relative gene of PJS.As a tumor suppressor gene,LKB1 plays an important role on cell growth,cell proliferation,protein synthesis,cell apoptosis,autophagy by cell signaling pathway.The mutations in LKB1(including frame shift,nonsense,splice site mutation,in frame insertion or deletion and missense mutations)could reduce LKB1 kinase activity,even loss of the activity of the kinase,and then cause various disease including PJS and lung cancer by means of changing a series of signaling pathways.With the progress of examination technology,the popularity of high-throughput sequencing and MLPA(multiplex ligation-dependent probe amplification),the new mutations of LKB1 having been reported in worldwide.At present nearly 300 kinds of LKB1 mutations have detected in patients of PJS and other diseases.Despite LKB1 has identified as the disease-causing genes of PJS currently,its pathogenic mechanism is too complex to be fully elucidated.Mutations of LKB1 in different loci would affect different change in cell function.Our research team has collected biological samples and clinical data from 52 Chinese families with 116 PJS patients in the early research work.By gene sequencing,we found LKB1 mutation rate was 67.3%in Chinese PJS patients,and the mutation rate consistent with foreign reports(50-90%).We found 14 novel mutations,and 37%of these mutations on the end of extra 6 and entire extra 7(coding protein kinase domain XI in LKB1).We highly suspected that the area may be mutation hotspots of LKB1 in China PJS patients.After retrospective analysis of the clinical phenotype,we found mutations in domain XI of LKB1 associated with atypical hyperplasia in PJS hamartomatous polyps.But there is no studies on functional role of point mutations of domain ?,and its pathogenic mechanism is unclear.We selected p.M289R mutation as the representative,according to the mutation hazard rating by PolyPhen2 software.And we constructed the p.M289R mutant plasmid and its wild type plasmid to research whether the mutation plays any role in cell growth,cell proliferation,protein synthesis,cell cycle,autophagy,and the effect on LKB1-AMPK-mTOR pathway.Material and Methods1.Primary MaterialCell lines of SW480,SW1116,HT29,Hela,SW-620,293T,DLD1,HCT-116,LoVo.Lipofectamine(?)3000 Transfection Reagent,Trizol,RIPA Buffer,CCK-8 Kit,Propidium iodide,low metling agarose.Antibody of LKB1,p-AMPK(Thr172),p-p70S6K(Thr389),p70S6K,p-mTOR(Ser2448),mTOR,4E-BP1,p-4E-BP1(Thr37/46),S6K,p-S6K(Ser235/236),p-S6K(Ser240/244),cyclin Dl,beclinl,LC-3B,p53.2.PCR primer design12 pairs of PCR primers were designed to amplify the LKB1 gene exons and intron-exon boundaries using the primer sequences in the reported literatures or the software program Primer 3.0(http://frodo.wi.mit.edu/primer3/)3.Construction of LKB1 plasmids.pGCMV-GFP-Vecor empty vector,pGCM V-GFP-LKB1(WT)and pGCMV-GFP-LKB 1(p.M289R)plasmid were synthesized by GenePharma Company in Shanghai,China.And these plasmids were checked again by Sangon Biotech in Shanghai.4.Screening LKB1 gene none expression,or low expression of cell linesSW480?SW1116?HT29?Hela?SW-620?293T?DLD1?HCT-116?LoVo cell lines were maintained in DMEM(Gibco)supplemented with 10%FBS.The cells were incubated in a humidified incubator at 37 ? with an atmosphere of 5%CO2.Cells were lysed with RIPA buffer(added Protease Inhibitor Cocktails)or Trizol,and the basic LKB1 expression was evaluated by Western Blot and RT-PCR(Reverse transcription polymerase chain reaction)respectively.We chose cell lines in which LKB1 was almost not detectable to continue our study.5.Establish and verify a stable LKB1(Wide type and Mutation)cell line with SW1116/Hela cell3 different palsmids were transfected in SW1116/Hela cell line by Lipofectamine 3000,48 hours after transfection,adding G418 antibiotics The positive clones were selected by G418,and highly expressing clones were more selected by flow cytometry,and get stable highly expressing 3 different gene cell lines.Extracting the total mRNA,total protein of stable cell line.Comparing and detecting LKB1 expression on mRNA and protein levels,in different stable cell line.6.The impact of different stable cell lines' protein in the LKB1 AMPK mTOR signaling pathway and p53.Extracting the total protein from different stable cell lines.Comparing and detecting expression LKB1,p-AMPK(Thrl72),p-p70S6K(Thr389),p70S6K,p-mTOR(Ser2448),mTOR,4E-BP1,p-4E-BP1(Thr37/46),S6K,p-S6K(Ser23 5/236),p-S6K(Ser240/244),cyclin D1,beclin1,LC-3B,p53 in stable cell lines by Western Blot.7.The impact of proliferation activity in different stable cell lines.Counting different stable cell lines,1000 cells/well were plated in 96 well plates.CCK-8 reagent was added at 12h?24h?36h?48h?72h?96h after cell plated.And measuring the absorbance of each well at wavelength in 450nM,Detection time points were 0.5h?1.0h?1.5h?2.0h.Comparing activities of cell proliferative in different cell lines.8.The impact of cell colony formation capability in different stable cell lines.Counting different stable cell lines.2,000 cells/plate.Melt 1%Agar(DNA grade)in microwave,cool to 40? in a water bath.Warm 2×RPMI + 20%FCS to 40 ? in water bath.Allow at least 30 minutes for temperature to equilibrate.For plating add 3ml 2 × RPMI +10%or 20%FCS and 3ml 0.7%Agar to a tube,mix gently and add 1.5ml to each replicate plate.Incubate assay at 37 ? in humidified incubator for 10-14 days.Stain plates with 0.5ml of 0.005%Crystal Violet for>1 hour,count colonies using a dissecting microscope.9.The impact of cell cycle in different stable cell lines.Digesting cells with EDTA-free trypsin,centrifuged cells at 1100 rpm 5min;wash cells twice with PBS;Then fixed cell with cold 75%alcohol.After 12 hours,centrifuged cells at 900 rpm 5min;washing cells twice with PBS.Add mixture of PI and RNAase.15min at room temperature,to stain cells.Then performing cell cycle analysis by Flow Cytometr10.Statistical analysisResults obtained were analyzed using SPSS 16.0.The data were expressed as Mean±SD with at least 3 independent expriments.One way ANOVA was used to analyze the quantitive data with significant difference being considered if P values were less than 0.05.Result:1.SW1116 cell and Hela cell were detected by Western Blot and RT-PCR,both of them hardly express LKB1 protein and mRNA.And established stable expression cell lines of LKB1 wild-type,mutation(p.M289R)and empty based on SW1116 cell and Hela cell.2.By Western Blot,RT-PCR,the stable expression cell lines of LKB1 wild-type,mutation(p.M289R)were detected;compared with the control group and empty group,the stable expression LKB1 in mRNA and protein of LKB1 wild-type and mutation(p.M289R)was significantly increased(P<0.05).There are no significantly differences between LKB1 wild-type and LKB1 mutation(p.M289R)in expression of LKB1 mRNA and protein.3.CCK8(Cell Counting Kit8)assay showed that the LKB1 wild-type was significantly lower(P<0.001)at OD450 compared with control group,empty group and LKB1 mutation(p.M289R),.While there were no significant difference among control group,empty group and LKB1 mutation(p.M289R)at OD450(P>0.05).Suggests that the LKB1 wild-type inhibited cell proliferation,proliferative activity of LKB1 mutation(p.M289R)is stronger than the LKB1 wild-type.4.By soft agar assay,colony formation in stable expression cell lines showed:compared with the number of cell clones in the control group,empty group and LKB1 mutation(p.M289R),the LKB1 wild-type significantly decreased in the number of clones(P<0.001).In SW1116 cells,there were significant different between LKB1 mutation(p.M289R)and the control group,empty group in cell clones(P>0.05);In Hela cells,proliferation of LKB1 mutation(p.M289R)was less than the control group,empty group in cell clones(P<0.03).Therefore,LKB1 wild-type inhibit cell proliferation,and LKB1 mutations p.M289R decrease the ability of inhibition of cell proliferation of LKB1 wild-type.5.Cell cycle distribution assessed by flow cytometry showed that there are no significantly different between LKB1 wild-type and LKB1 mutation(p.M289R)in the percentage of G1,G2 and S phase(p>0.3).6.By Western Blot showed:compared with the control group,empty group and LKB1 mutation(p.M289R),the LKB1 wild-type significantly enhanced expression of Thrl72 sites in AMPK protein phosphorylation(P<0.01),but no significant differences in the expression of AMPK total protein(P>0.05).In downstream signaling pathway of mTOR,compared with the control group,empty group and LKB1 mutation(p.M289R),the LKB1 wild-type reduced the expression of phosphorylation levels in p-mTOR(Ser2448 sites),P70S6K(Thr389 sites),p-S6K(Ser235/236sites),p-S6K(Ser240/244sites),p-4E-BP1(Thr37/46sites)(P<0.01).In contrast,LKB1 mutation(p.M289R)less expression in those phosphorylation sites than the control group and empty group,but more expression in those phosphorylation sites than the LKB1 wild type.It's suggested that LKB 1 wild type inhibit AMPK-mTOR signaling pathway,and LKB1 mutation p.M289R reduced the inhibition of LKB1 wild-type.7.Differences in cell autophagy:compared with the control group,empty group,the LKB1 wild-type and LKB1 mutation(p.M289R)significantly increased expression in LC-3B?Beclinl(P<0.05);But there were no significantly different between LKB1 wild-type and LKB1 mutation(p.M289R)in expression of LC-3B?Beclinl(P>0.05)8 Differences in expressing p53:compared with the control group,empty group,the LKB1 wild-type and LKB1 mutation(p.M289R)significantly increased expression in p53(P<0.05);But there were no significantly different between LKB1 wild-type and LKB1 mutation(p.M289R)in expression of p53(P>0.05).Conclusion1.It has been found that SW1116 cells didn't express LKB1 gene.And stable cell lines expressing LKB1 wild-type,LKB1 mutant and empty vector were established in SW1116 and Hela cell.There are no significantly different between LKB1 wild-type and LKB1 mutation(p.M289R)in expression of LKB 1 protein and mRNA.2.By the soft agar assay for colony formation and CCK-8 test the LKB1 mutation(p.M289R)could reduce the ability of to inhibition of cell proliferation in LKB1 wild-type.3.LKB1 mutation(p.M289R)lost or reduce the ability to activate p-AMPK(Thrl72 site)in LKB1 wild-type.4.LKB1 mutation(p.M289R)abnormally activated the phosphorylation of mTOR pathway.By increasing the phosphorylation in p-mTOR(Ser2448 site),P70S6K(Thr389 site),p-S6K(Ser235/236 sites),p-S6K(Ser240/244 sites),p-4E-BP1(Thr37/46sites)the promotion of protein synthesis and cell proliferation were achieved.There were no significantly differences between LKB1 wild-type and LKB1 mutation(p.M289R)in expression of mTOR,P70S6K,S6K,4E-BP1 protein.5.Compared with control group and Empty group,LKB1 mutation(p.M289R)and LKB1 wild-type enhanced the level of cell autophagy.And there were no significantly differences between LKBI wild-type and LKB1 mutation(p.M289R)in expression of autophagy.Therefore,cell autophagy may not directly affected on increasing cell proliferation caused by LKB1 mutation(p.M289R).6.There were no significantly differences between LKB1 wild-type and LKB1 mutation(p.M289R)in cell cycle,which confirming the ability of LKB1 mutations(p.M289R)to enhance cell proliferation without altering the cell cycle.7.Compared with control group and Empty group,LKB1 mutation(p.M289R)and LKB1 wild-type enhanced.expression of p53 in cells.But there were no significantly differences between LKB1 wild-type and LKB1 mutation(p.M289R)in expression of p53.Therefore p53 may not directly affect on increasing cell proliferation and abnormally activating mTOR pathway caused by LKB1 mutation(p.M289R).LKB1 point mutation p.M289R in domain XI may be one of pathogenic mutations in PJS.It reduced activity of LKB1 kinase and abnormally activated the LKB1-AMPK-mTOR signaling pathway.Finally enhanced the proliferation of cells.And the cell proliferation maybe no connection with cell autophagy,cell cycle and p53. |
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