The Effect And The Mechanism Of Midkine On Gastric Cancer Cell Proliferation

ObjectiveGastric cancer is one of the most common malignancies in the world,particularly in Eastern Asian countries such as China,Korea,and Japan.Despite the advance in diagnosis and treatment,the prognosis for advanced gastric cancer is poor,with a 5-year survival rate about 20%～30%.Over the past years,many evidences have clearly demonstrated that multiple genetic alterations are responsible for the development and progression of gastric cancer.Changes of specific genes in gastric cancer play important roles in cell adhesion,signal transduction,cell differentiation, development and metastasis.Better understanding of the molecule mechanism of gastric cancer will profit the diagnosis,treatment and precaution of the disease.Numerous growth factors and their downstream signaling systems are involved in the development,progression,and dissemination of cancer.Midkine(MDK,MK) is a member of heparin-binding growth factors,and is the only members of a family distinct from other heparin-binding growth factor families.MK was found to be the product of a retinoic acid-responsive gene discovered by screening for induced genes during the differentiation of embryonal carcinoma cells.MK is a secreted protein,which distributes extensively in the intermediate stage of pregnancy and expresses lower after birth.In normal human reference tissues,MK was highly expressed in the mucosa tissue of the small intestine,moderately in the thyroid,weakly in the tissues of the lung, colon,stomach,kidney,and spleen,and not at all in the liver.Midkine expressed generally low or undetectable in adults,whereas high in various human cancers,including esophageal,gastric,urinary bladder,pancreas, colorectal,breast,lung carcinomas,neuroblastomas and Wilms' tumors.The biological roles of MK is diverse.It not only promots the neurite extension and neuronal survival, but also affects the proliferation,differentiation and apoptosis of tumor cells through its biological activities that include pmitogenesis,transformation,anti-apoptosis, angiogenesis and migration.Since not much is known about the relationship between midkine and gastric cancer,the purpose of this study is to investigate the exprssion and the biological activities of midkine in gastric cancer and illustrate the possible signal transduction pathways by which MK acts in human gastric cancer.Mehtods1,The expression of MK in gastric cancer specimens(1)Immunohistochemical analysis was used to detect the expression of MK in cancer specimens and non-cancerous tissues.(2)Real-time quantitative RT-PCR was used to detect the expression of MK in cancer specimens and matched normal mucosa.2,The effects of MK on gastric cancer cell proliferation(1)Real-Time RT PCR was used to investigate the expression of MK in four gastric cell lines.(2)Molecular cloning technology was used to constructe the recombinant retrovirus vector of human MK gene.The vector was identified with enzyme digestion and sequencing.(3)Liposome was used to transfect the retrovirus expression vector of MK and the corresponding empty vector into gastric cancer SGC7901 cells and then screened by G418.The stable clones are further identified by Real-Time PCR and Western blot.(4)MK siRNA was used to transiently knockdown the MK expression in BGC823 cells.(5)MTT assay and growth curve were used to investigate the effect of MK on the proliferation of SGC7901 cells. (6)MTT assay was used to investigate the effect of MK knockdown on the proliferation of BGC823 cells.(7)Soft agar assay was used to investigate the effect of MK on the clonality of SGC7901 cells.(8)Nude mice assay was used to investigate the effect of MK on the cell growth of SGC7901 cells in vivo.3,The mechanism of MK on gastric cancer cell proliferation(1)Western blot assay was used to determin the effects of different expression of MK on two major survival pathways:PI3K/AKT,ERK1/2.(2)Western blot assay was used to investigate the effects of different expression of MK on cell cycle protein:cyclinA,cyclinB1,cyclinD1,Cdk2,Cdk4,Cdk6.Results1,The expression of MK in gastric cancer specimens(1)The expression of MK was found significantly higher in gastritic t issues(54%),compared with that in adjacent non-cancerous tissues(28%)(P<0.05).(2)In 11 of 19 patients(57.9%),MK expression was upregulated in the tumor tissue two fold higher than the expression in the matched normal mucosa.Howerever, in 2 of 19 patients(10.5%),MK expression was upregulated in the matched normal mucosa two fold higher than the expression in tumor tissue(P<0.05).2,The effects of MK on gastric cancer cell proliferation(1)Among the four gastric cancer cell lines,the highest expression of MK was BGC823,then was MGC803 and MKN1,and the lowest was SGC7901.(2)By the identification of sequencing,the retrovirus vector of MK was successfully established.(3)As identified by Real-time quantitative RT-PCR and Western blot,the gastric cancer cell models with increased MK expression was successfully established. (4)The two target interference groups all had a significant efficiency in gene silencing.The mRNA level was decreased by 46.9%in 10nM siRNA group and 74.8% in 20nM siRNA group compared to control(non-target siRNA group),respectively.(5)As the results of MTT assay and growth curve show,the proliferation speed of clone 7 and clone 11 which express MK highly are much faster than the vector.(6)As the results of MTT assay show,the proliferation speed of 10nM and 20nM MK RNA interference groups which express MK lowly are much slower than non-target RNA interference group.(7)As the results of soft agar assay show the clonality of clone 7 and clone 11 which express MK highly are much stronger than the vector..(8)As the results of nude mice assay show up-regulation of MK promote the growth of gastric cancer cells in vivo3,The mechanism of MK on gastric cancer cell proliferation(1)As identified by Western blot,ERK and Akt phosphorylation was clearly increased in MK over-expressed SGC7901 cells,and decreased in MK hypo-expressed BGC823 cells.(2)As identified by Western blot,MK up-regulated the expression of cyclinA, cyclinD1,Cdk2,Cdk4,Cdk6,while transiently knocking down of MK could down-regulate the expression of these protein.Conclusions1,MK is found to express highly in gastric cancer tissues,compared with normal gastric tussues.2,MK can promote to the proliferation of SGC7901 cells in vitro and in vivo.3,MK contributes to the proliferation of SGC7901 cells through activating the ERK1/2 and Akt pathways.4,MK contributes to the proliferation of SGC7901 cells through upregulating the expression of some cell-cycle regulatory proteins,such as cyclinA,cyclinD1,Cdk2, Cdk4,Cdk6.