Investigation On The Expression Of Galectin-3 And Its Proliferation Signaling Pathway In Colorectal Cancer LoVo

Background and objectivesColorectal cancer(CRC)was the third most common malignant tumor in theworld, the incidence rate of its was increasing year after year, and mortality ratewas ascended the fastest in all alimentary cancer. There was about 1.5 years thatcolorectal cancer encircled one wrap of bowels, which was the one main reason ofsurvival rate depressed. To inhibit the proliferation of cancer and to promote theapoptosis could achieve the aim at depressing mortality rate. At present, the researchconsider that the development of cancer has the relation to the failure toanti-oncogene,activation of oncogene because of deletion and mutation. To knockdown the expression of gene effectively could depress the occur rate of cancer. RNAiinterfere technology as one method oppositely researching gene and its biologyfunction, was already used in side of signaling conduction of gene. Mitogen activatedprotein kinase (MAPK) has main three signaling pathway including PI3Ksignalingpathway,P38signaling pathway and ERK1/2signaling pathway, MAPK/ERKsignaling pathway was the most actively researched at present, which participated inmany physiological and pathology process including development,growth,proliferation,differentiation,malignant transform of cells. GAL-3 was one of fourteen of animal hemagglutinin (lectins) family which was considered by researcherat present to express in many cells including epithelia cells,lymphocyte,Fibroblastcells and cancer cells, also could express different position of cell includingmembrane, cytoplast, matrix, nucleolus etc, which indicated many biology function ofGAL-3 including adhesion of cell,proliferation,apoptosis,Pre-RNA montage cellinner according to the expression position of GAL-3. These function turn tocarbo-hydrogene recognition site of GAL-3 witch binds to ligand of it to precipitateconformation transform. The all research summaried indicated that:(1) the expressionof GAL-3 in colirectal cancer was related to the differention degree of cancer, thehigher the differention degree was, the lower GAL-3 expressed.on the contray, thelower the differention degree was, the higher GAL-3 expressed. (2) the expression ofGAL-3 in cancer was related to metaptosis of cancer.(3) the location of GAL-3 incolorectal cancer was main cellnucleus, but in the develop process from normal tissue,heavy grade atypical hyperplasia to canceration, GAL-3 gradually excreted tocytolymph, so GAL-3 was regarded as the gene which mostly related to developmentof colorectal cancer. (4) Reports were considered that GAL-3 because of higherexpression in colorectal cancer serum was regarded as a significance marker incolorectal cancer census. At present, the research on GAL-3 was mainly on stromaadhesion, metastasis and apoptosis, the few about proliferation and differention. Inaddition, there was few about research of GAL-3 in colorectal cancer, existed dispute,and the mechanism of action of GAL-3in colorectal cancer was not clear.So it wasnecessary to further verification the expression of GAL-3 in colorectal cancer and theresearch on proliferation mechanism, so we first researched different expression ofGAL-3 by RT-PCR and western blot on the levels both mRNA and protein andlocation in cell by immunocytechemistry in different Ducks stages colorectal cancer,screened out the colorectal cancer cell which express highest GAL-3. GAL-3siRNA was synthesized by chemic methods and transferred into LoVo, detected theproliferation by MTT, the apoptosis and cell cycle by FCM ofGAL-3 before and aftertransferred, observed the apoptosis cell by fluorescence microscope, screened out theeffective GAL-3siRNA by western blot. We synthesizedpGPU6/GFP/Neo/GAL-3shRNA vector, built the steady cell that depressed theexpression of GAL-3, detected the expression of ERK1/2, phospho-ERK1/2, MEK1,phospho-MEK1 and their upper signaling protein K-Ras, PKC and there downnucleus transfer gene c-Myc, We discussed the possible signaling pathway thatGAL-3 participated in the proliferation of colorectal cancer. In the end, hairlessmouse were inoculated with LoVo transferred pGPU6/GAL-3shRNA and LoVo cells.After tumor was formed, we observed tumor weigh and infiltrate depth, furthervalidated the relation between the expression of GAL-3 and proliferation of colorectalcancer.Material and methods一,The difference expression and cellular location of GAL-3 indifferent Ducks stage eoloreetal cancer cells.To research the expression of GAL-3 in eoloreetal cancer by RT-PCR andwestern-blot methods.Human fiber cell NIH3T3 and gastric cancer SGL7901cell were regarded aspositive groups, human embryo kidney T lymphocyte as negative groups, accordingto the NM-002306sequence on NCBI, GAL-3 primer was synthesized, b-actin wasregarded as refer, the results of RT-PCR and Western-blot of relatively ratio everygroups cell were analyzed by gel analysis instrument, compared the difference inevery groups. From the RT-PCR and protein level we screened out the highestexpressing GAL-3. Immunocytechemisity was used to observe GAL-3 expression location in cell colorectal cancer.二,The expression of GAL-3in colorectal cancer and its relationshipwith proliferation and apoptosis.1,To analysis and screen out the effective GAL-3RNAiAccording to on line Ambio company software design we synthesized fourGAL-3 siRNAs (small interference RNAs) and one RNAi which was no relationshipwith GAL-3 sequence and was regarded asGAL-3-NC and regarded as negativegroup, they were instantaneous transferred LoVo, distilled the plasm protein after 72h,the result of western blot was analysised by gray and screened the most effectivelysiRNA which could significant induce GAL-3 gene silencing.2,To research the relationship both GAL-3 and proliferation of colorectalcancer by MTTTo observe the changes of cell growth we departed three groups incloudingLoVo,GAL-3NC and GAL-3siRNA, About 4000 LoVo cells in one hole wereinoculated in 96 walls, on the basis of screened siRNA, cell proliferation wasanalysed by MTT on three points 24h,48h and 72h after transferred GAL-3siRNAandGAL-3NC.3 To research the relationship by FCM between GAL-3 and apoptosis and cellcycle in colorectal cancerLoVo Cell apoptosis was detected using Annexin-V-EGFP reagent kit, cell cyclewas detected by PI, the apoptosis cell was observed by fluorescence microscope.三,To research the signaling pathway of gal-3 participating incolorectal cancer proliferation1,To construct and identify the p-GAL-3shRNA efficacious recombinantexpression vector According to the result of above and the structure of pGPU6/GFP/Neo vector,one pair of small interfering RNAs (siRNAs) of GAL3 mRNA (GenBank:NM002305) was designed, and two corresponding single-strand short hairpin RNAs(shRNAs) containing BbsⅠand BamH sites and 9nt hairpin structure, weresynthesized and annealed.The annealed products and the linear pGPU6/GFP/Neoplasmid, were ligated for 4h at 16℃using T4 ligase. The recombinded plasmids weredigested with BamHⅠ, HindⅢand PstⅠfollowed by electrophoresis with 1% agaroseand sequence to idetify. Cell transfection pGPU6/GFP/Neo and recombinant plasmidsp-GAL-3shRNA were transferred into LoVo using Lipofectamine 2000^TM.Thepositive cell colnes were selected by 0.8μg/ml puromycin and subsequentlyproliferated respectively. GAL3 exprssion detecting the expression changes of GAL3were detected using reverse transcription polymerase chain reaction (RT-PCR),western-blotting on mRNA and protein levels.2,Detected expression change of ERK1/2,phospho-ERK1/2,MEK1,phospho-MEK1,K-Ras,PKC and c-myc on GAL-3siRNA transferred LOVOPlasm protein ditill was as the same as above, we first detected the expression ofERK1/2. phospho-ERK1/2. MEK1 and phospho-MEK1 before and aider transferedGAL-3shRNA, K-ras and PKC were also detected, finally we detected theexpression of the nucleus gene c-myc. We could definitude whetherK-ras/MEK1/ERK1/2 signaling pathway participated in the proliferation functionmediated byGAL-3四,Tumorigenicity assayFifteen BALB/c hairless mouse(5-6 weeks) were immediately three groups,each group was five, colorectal cancer LoVo were hypodermic inoculation on back,weigh region invasion and distantly metaptosis, toumr, liver and spleen wereobserved with HE. 五Statistical assayThe data was demonstrated as (?)±s, SPSS10.0 stastistic soft was used to analysestatistically(one factor analysis of variance) these data, repeatedly measured dataanalysis of variance were cell proliferation and apoptosis, random unit group designdata analysis of variance were used to analysis the weigh of tumor. Bonferrorianalysis method was used in group comparison. There was significant difference asP≤0.05Results1,GAL-3 was mainly expressed in plasm of colorectal cancer. In NIH3T3, HT293,SGL7901and four different Duck s colorectal cancer SW1116 A stage, SW480 Bstage, and D stage SW620and lovo, the expression level on mRNA and protein ofgal-3 were respectively0.969±0.05,0.504±0.02,0.986±0.01,0.589±0.09,0.624±0.06, 0.843±0.01, 1.258±0.01and 1.634±0.017,0.616±0.01,1.435±0.01,0.978±0.01, 0.999±0.06,,1.228±0.06, 2.051±0.01, there were significantdifference on the both level mRNA and protein(F=268138.2, P=0.000,F=942152.0,P=0.000) amony every cell. From above we found that the expression ofGAL-3 was the weakest in SW1116 cells, but the strongest in LoVos.2,By the methods of instantaneous transfer we successful transferred fourGAL-3siRNA—676,GAL-3siRNA—737,GAL-3siRNA—208 and GAL-3siRNA—433 into colorectal cancer and detected the protein result of above siRNA bywestern blot, the ratio of GAL-3siRNA andβ-actin were 0.883±0.02,0.597±0.10,0.07±0.02,0.09±0.03respectively, there were significant difference(F=977.544, P=0.000).According to the above result, the GAL-3siRNA—433 waselected as the effective interfere RNAi. We respectively detected the proliferation ofLoVo on three points 24h, 48h and 72h after transferred GAL-3siRNA-433,the result was that LoVo cell continue grown, but slower, there was significantdifference(F=149.710, p=0.000),there was significant difference on different pointbetween GAL-3siRNA and LoVo and GAL-3NC (P＜0.05),but there was nosignificant difference between LoVo and GAL-3NC groups. The result of cell cycleand cell apoptosis were found that the growth of LoVo was blocked on G1 stage aftertransferred GAL-3siRNA,the apoptosis happened every cell cycle, but there washigher on nonage than metaphase and afternoon, but there was no significantdifference between above two groups(P＞0.05),there was significant difference bothon two stage above respectively between GAL-3siRNA and LoVo and GAL-3NCgroups(P＜0.05).Above result indicated that GAL-3 played a role in promotingproliferation and restraining apoptosis of colorectal cancer.3 Through enzyme excision,sequence checked and BLAST, pGUP6/GAL-3shRNAwas successful constructed, the expression of GAL-3 was significantly checked ingroups transferred p-GAL-3shRNA((P＜0.05). Two aspects confirmed the transferwas successful, at the same time it was confirmed that pGUP6/GAL-3shRNA couldsignificantly result to GAL-3 gene silence. Fluorescence microscope was used todetected the efficiency of transfer, the result of green fluorescence indicated thatpGUP6/GAL-3shRNA was successful transferred into LoVo cell. ERK1/2 and MEKwere both expression in LoVo,there was significant difference amonypGUP6/GAL-3shRNA and P-LoVo and LoVo (P＜0.05), but there was no significantdifference between P-LoVo and LoVo (P＞0.05). we also received the same resultwhen phospho-ERK1/2 and phosphor-MEK1 were detected in LoVo transferredpGUP6/GAL-3shRNA groups, which indicated phospho-ERK1/2 andphospho-MEK1 were activation in LoVo of transferred pGUP6/GAL-3shRNA, Todetected the expression of PKC, K-Ras and c-myc in three groups includingpGUP6/GAL-3shRNA,LoVo and P-LoVo, we found that above three protein were expressed the weakest in pGUP6/GAL-3shRNA than in both LoVo andP-LoVo,there was significant difference when the expression of PKC, K-Ras andc-myc were compared in above three groups (p＜0.05).4,Tumorigenicity assay Experiment of Hairless mouse hypodermic inoculation was found that theweigh of three goups (LoVo. p-LoVo,p-GAL3-LoVo) were respective 0.550±0.022,0.524±0.022,0.0887±0.022, There was significant difference amony among three groups(P=0.001). Tissue pathology confirmed poorly differentiated adenocarcinoma, partial nodulartransfer was not found, there was not tumor infiltrated in liver and spleen on the basis of the resultby HE method.Conclutions1,The expression location of GAL-3 in four colorectal cancer was main plasm.Infour different Ducks staging from A-D includingSW1116,SW480,SW620,LoVo, theexpression of GAL-3 was lower inSW1116,but the highest in LoVo.2,On the basis of principle of interfere design and the results of RT-PCR andwestern-blot, GAL-3siRNA-433 was filtered as the efficacious RNAi, GAL-3mediated the biology function of colorectal cancer by promoting its proliferationand restraining its apoptosis on nonage of cell cycle.3,PKC/Ras/ERK1/2 signaling pathway participated in the proliferation of colorectalcancer by mediated GAL-3,and activated c-myc nucleus gene.4,Through the experiment of Hairless mouse hypodermic inoculation withp-GAL-3shRNA, LoVo and p-LoVo, it was confirmed that the weigh of interferencegroups was far lighter than control groups.