| Colorectal cancer(CRC)is one of the most common malignancies in the gastrointestinal tract.The most noticeable problem is that CRC is the third most common diagnosed cancer,and it has been estimated that more than 1 million people were diagnosed with CRC each year.In recent years,the morbidity and mortality rates of CRC have been increasing annually.Especially in China,the rates have reached even exceeded the level of Western countries.CRC peaks at age 50-60 years in China,10 years earlier than the Western countries[1].Surprising advances have been made in diagnosis and treatment of CRC in recent years,but the pathological disease mechanisms are still unclear.Thus,it is necessary to further elucidate the molecular and cellular mechanisms and provide a theoretical basis for early diagnosis and targeted therapy of CRC.Glucose phosphate isomerase(GPI)plays an important role in glycolysis and gluconeogenesis,it catalyzes the intercoversion of glucose-6-phosphate and fructose-6-phosphate.GPI could be secreted to extracellular,which has been identified as a motility factor: autocrine motility factor(AMF).The proliferation,apoptosis and migration of tumor cells were affected due to the combination of AMF and autocrine motility factor receptor(AMFR).Studies have documented that GPI/AMF was overexpressed in various cancers,including breast,kidney,lung,endometrial,osteosarcoma,gastrointestinal[2-6].And increased GPI/AMF levels in urine and serum of tumor patients was associated with CRC[7].Moreover,GPI not only plays an important role in the development and progression of cancer,but also affects apoptosis,metastasis and invasion of tumor cells[8-10].Aquaporins are found in tumor cells and are associated with the occurrence and development of cancer.Chulso Moon presumed that AQP1,AQP3,AQP5 were expressed in all the seven colorectal cancer cell lines including HCT-116,HT-20 et al.[11].AQP3 was expressed in the respiratory system(airway,lung,etc.),digestive system(stomach,colon),urinary system,sensory organs(eye,lacrimal gland).The activation of AQP3 enhances the migration of some tumor cells(eg,pancreatic cancer cell line MPC-83).The present study found that AQP3 expression in CRC tissues was significantly higher than that in adjacent tissues,and the expression of AQP3 was associated with lymph node metastasis,differentiation,and distant metastasis of CRC.PI3K/AKT signaling pathway is associated with the development and progression of various malignancies and exists widely in all kinds of cells.PI3K/AKT signaling pathway is abnormally activated in CRC[12],change the expression levels of downstream genes such as Matrix Metalloproteinases(MMPs)resulting in increased cancer cells migration,invasion and metastasis[13-14].The studies also reported that PI3K/AKT signaling pathway played an important role in h EGF-induced AQP3 expression[15].Therefore,we assume that whether downregulation the expression of GPI/AMF can affect the apoptosis and migration of CRC cells HCT-116? Is the migration ability of CRC cells HCT-116 related to AQP3? Does the PI3K/AKT signaling pathway play a crucial role in this process?ObjectiveIn this study,we screened out GPI high expression CRC cell line at first,and then explored the effects of GPI on apoptosis,migration,invasion and the expression of AQP3 in colon cancer cells,and the possible mechanisms.It can provide clues and experimental basis for targeting GPI in the treatment of colorectal cancer.MethodsHuman colorectal cancer cell lines(HT-29,HCT-116,SW480,caco2)were cultured in vitro.Western-blot and RT-PCR were used to detect the expression levels of GPI in different human colorectal cancer cell lines,and to screen out GPI highly expressed CRC cell line.CCK-8 was employed to determine the appropriate concentration of puromycin for screening of GPI stable transfected HCT-116 cell line;HCT-116 cells was infected by si GPI lentiviral plasmid,and the transfection efficiency was observed under fluorescence microscope;After HCT-116 cells were stably transfected,the expression level of GPI was detected by Western-blot and RT-PCR.The cell cycle and apoptosis of HCT-116 cells were detected by Flow cytometry.The effects of GPI on the migration of HCT-116 cells were detected by wound healing assay;Transwell was used to detect the migration and invasion of HCT-116 cells;Cell cycle related protein Cyclin D1,apoptotic related proteins Bax,Bcl-2,p53,Cleaved-caspase3,PI3K/AKT signaling pathway proteins,AQP3 and migration related proteins were detected by Western-blot.Besides,in order to investigate the relationship between GPI/AMF,PI3K/AKT signaling pathway and MMP3,MMP9,AQP3,HCT-116 cells were treated with si GPI or/and LY294002.Results1.RT-PCR and Western-blot results identified that HCT-116 is the colorectal cancer cell line which showed higher expression level of GPI compared with HT-29,SW480 and caco2 cells.Therefore,HCT-116 was selected for followed experiment.2.CCK-8 showed that the inhibition rate of HCT-116 cells was nearly 95% when the concentration of puromycin was 0.8 ?g/m L.So puromycin(0.8 ?g/m L)was used for screening stable transfected HCT-116 cells.3.Fluorescence microscope showed that transfected cells emitted green fluorescence,which indicated that the HCT-116 cells were successfully infected by lentivirus plasmid.The RT-PCR and Western-blot results showed that GPI gene m RNA and protein levels were down-regulated.The inhibition rates of GPI m RNA and protein in si GPI HCT-116 cells were 30.0% and 55.0% respectively,compared with parental,there was a significant difference(P<0.05),but not in mock(P>0.05).4.Cell cycle analysis showed that the percentage of G0/G1 phase in si GPI cells was increased from(58.07 ? 1.69)% to(65.81? 2.16)% and the percentage of G2/M,S phase was significantly reduced compared with the parental group(P<0.05).There were no significant differences between mock and parental group(P>0.05).5.Apoptosis analysis showed that the percentages of early apoptosis were increased from(8.48? 1.97)% to(24.74? 4.75)%(P<0.05)after stably infected by lentivirus which silenced the expression GPI.There were no significant differences between mock and parental group(P>0.05).6.The Western-blot results showed that the expression of Cyclin D1,and Bcl-2 proteins decreased in si GPI cells compared with parental,while the expression of p53,Bax,Cleaved-caspase-3 increased(P<0.05).There were no significant differences between mock and parental group(P>0.05).7.Wound healing assay suggested that the migration rate of si GPI group was slowed down.There was no significant difference between mock and parental group.Moreover,HCT-116 cells were treated with si GPI or/and LY294002 could cause cell migration to slow down.Especially when combined si GPI with LY294002,the cell migration rate of HCT-116 cells became more slowly.8.Transwell assay revealed that the number of transmembrane cells in the si GPI group was significantly decreased,and the number of cells in the mock group was not significantly changed compared with the parental group.Silencing GPI or/and LY294002 could reduce the number of cells passed through the membrane,especially when CRC cells were treated with both GPI si RNA and LY294002,this phenomenon was more obvious.9.The expression of migration related proteins and PI3K/AKT signaling pathway proteins were detected by Western-blot.The results showed that MMP3,MMP9,AQP3,PI3 K,p-AKT proteins were significantly decreased in si GPI cells compared with parental(P<0.05),but total AKT expression did not differ between si GPI group and parental group.10.Western-blot results showed that LY294002 could inhibit PI3K/AKT signaling pathway,and inhibit the expression of MMP3,MMP9,AQP3,but the expression of GPI did not change.When CRC cells were treated with GPI si RNA and LY294002,the expression of GPI,MMP3,MMP9,AQP3,PI3 K,p-AKT decreased significantly(P<0.05),but total AKT expression did not change significantly compared with LY294002 alone inducing HCT-116 cells;and the expression of MMP3,MMP9,AQP3,PI3 K,p-AKT proteins decreased significantly(P<0.05),but the expression of GPI,AKT did not observed significantly change compared with si GPI HCT-116 cells.Conclusion1.Down-regulation of GPI can arrest the cell cycle of HCT-116 in G0/G1 period and induce apoptosis.Its possible mechanism is that silencing GPI can activate the p53 signaling pathway and inhibit the expression of Cyclin D1 while disrupting Bcl-2/Bax proportions and activating caspase3.2.The inhibitory effect of GPI on migration of human colon cancer cells may be achieved by inhibiting the activation of PI3 K / AKT signaling pathway and reducing the expression of MMP3,MMP9 and AQP3. |
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