| Background and objectivesThere is always high incidences of colorectal cancer(CRC) in western countries,As was described in a statistic data made by Aemal et.al that new cases and mortality of colorectal cancer rank the third every year,separately 10% and 9%,there were no abvious differences between males and females.In recent years,there were more and more paients of colorectal cancer due to improved life levels and changing environment,including our country,and the morbidity rank the third in digestive carcinomas,especially in city there was higher moibidity.Three ways could be used to treat colorectal cancers at present,respectively surgery,chemotherapy and radiotherapy.For those patients discovered early and no metastas,surgery is the only way that can cure colorectal cancers,at the same time,survival rate of five years can be increased adjoint with chemotherapy or radiotherapy,but for those patients with metastas cancers,no radical ways can we adopt,the methods of chemotherapy or radiotherapy can only control the progression of colorectal cancer,and they may cause severe side effects,which limit their use clinically.therefore if we can early discover and know the mechanism of progression of colorectal cancer by various incidators, it is very possible that we could cure colorectal cancer one day.Phosphatidylinositol-3-kinase (PI3K) is a lipid kinase and is responsible for the phosphorylation of 3 position of the inositol ring of PI(4,5)P2, to generate PI(3,4,5) P3, a potent second-messenger required for fundamental cellular functions such as transcription, translation, proliferation, growth, and survival. There are three members in PI3K family. Class-IA PI3K received more and more attention due to its roles in the progression of various cancers. PI3Kp110βis one of subtypes of Class-IA PI3K,as is the same with other Class-IA PI3K, PI3Kp110βsimultaneously has lipid kinases and protein kinases.As a lipid kinases, PI3Kp110βcan classically converts phosphatidy-linositol-4,5-trisphosphate(PIP2) to phosphatidy-linositol-3,4,5-trisphosphate(PIP3) in thecytoplasm, thereby directly activating the activity of PI3 kinase (PI3K) and Akt,by this way PI3Kp110βrealizes its roles in regulating cells probliferation,growth,apoptosis,survival and so on Reasearches show that depression of PI3Kp110βcould downregulate the activion of PI3K/Akt signaling pathway, then regulate the progression of endometraial cancer,prostate cancer,maglignant glioma and breast cancer.But there were no reports that PI3Kp110βregulate the proliferation and apoptosis of colorectal cancer.FoxO transcription factors belong to the Forkhead family of proteins, a family characterized by a conserved DNA binding domain termed the Forkhead box (Fox). The family is primarily regulated by PI3K pathway. Direct phosphorylation by PKB/Akt inhibits transcriptional activation by FoxO factors, causing their displacement from the nucleus into the cytoplasm. Deregulation of cell cycle and cell apoptosis are colosely related with tumor progression. FoxO transcription factors may be involved and play an important role in regulating cell cycle and cell apoptosis:①Inactivation of FoxO transcription factors leads to cell cycle progression which contributes to the development of tumor.②Inactivation of FoxO transcription factors leads to impaired ability to repair DNA damage which results in genomic instability.③Depletion of FoxO transcription proteins leads to inhibition of apoptosis which may contributes to tumor progression.④FoxO transcription proteins are found in human translocation mutational tumors. At present, the relationship between biological changes resulted from the changes of PI3Kp110βexpression levels and the changes of FoxO transcription factors is not reported.In this study, we investigate the expression and significance of PI3Kp110βin the progression of colorectal cancer, including normal colorectal tissue, hyperplastic polyps,colorectal adenoma and primary colorectal carcinoma. Then we design and synthesis PIK3CB-siRNA expressional sequences to transfect HCT116 cells. After transfection, we observed the effect of PI3Kp110βexpression on colorectal cancer cell proliferation and cell apoptosis. We also investigated the expression changes of PI3K signaling pathway proteins including Akt, FoxO transcription proteins, associated cell cycle proteins and cell apoptosis proteins. We aim not only to investigate the molecular mechanism of tumor proliferation and apoptosis, but also to search new effective target for gene therapy of CRC.Materials and methodsThe expression and significance of PI3Kp110βin progression of colorectal cancerWestern blotting was used to detect the expression of PI3Kp110βin the progression of colorectal cancer, including normal colorectal mucosa, colorectal hyperplastic plopy,colorectal adenoma and primary colorectal carcinoma,and then we analyzed the significance of PI3Kp110βprotein in the progression of colorectal cancer.The effect of PI3Kp110βexpression on proliferation and apoptosis in colorectal cancer cells1.Screening of colorectal cancer cell lines The resuscitation of colorectal cancer cell lines SW620,SW480 and HCT116 was finished,all of them were cultured in 1640 medium containing fetal bovine serum,cells total RNAs and proteins were not extracted until cells stabilization,and then we conducted real-time PCR and western blotting to detect the expression of PIK3CB(PI3Kp110β) in order to screen out cells that have the most expression of PIK3CB(PI3Kp110β). 2.Screening of effective interference sequences Cells transfection was conducted according to the manufacturer's protocol of Lipofectamine 2000 (Invitrogen).48h after transfection cells total RNAs and proteins were extracted,and we respectively conducted real-time PCR and Western blot analysis to detect the expression of PIK3CB(PI3Kp110β),at the last we selected a piece of sequence of the best interference effect to finish the rest research.3. Cell proliferation assays Cells were cultured for 72h after siRNA transfection, and cells proliferation was determined at 24h,48h and 72h after transfection respectively using Cell Counting Kit-8 (CCK-8) solution.4. Apoptosis Assays we respectively conducted FCM and JC-1 staining analysis to detect the effect of depression of PI3Kp110βprotein on HCT116 cells apoptosis.Research of cell proliferation and apoptosis signaling pathway PI3Kp110βinvolved in colorectal cancer cellsWestern blot was used to analyzed expression changes of signaling pathway proteins after depression of PI3Kp110βprotein. The cell proliferation and apoptosis signaling pathway proteins include Akt,p-Akt, cytoplasmic proteins p-FoxO1a (FKHR, Ser256),p-FoxO3a (FKHRL1, Ser253),,nuclear proteins FoxOla (FKHR),FoxO3a (FKHRL1), cell cycle associated proteins cyclinD1,cdk4,cdk6 and cell apoptosis proteins bcl-2,bcl-6 and bax..Statistical AnalysisAll experiments results were from at least three separate experiments. For western blot results, one-way analysis of variance (ANOVA) and Student's t test were used in group comparison, repeatedly measured data analysis of variance were cell proliferation, LSD analysis method was used in group comparison. Dates are expressed as the mean±SD. A value of P<0.05 was considered statistically significant.ResultsThe expression and significance of PI3Kp110βin progression of colorectal cancer The difference for the expression of PI3Kp110βprotein in normal colorectal tissue, colorectal hyperplastic plopy,colorectal adenoma and primary colorectal carcinoma was significant (P<0.05),the expression of PI3Kp110βin colorectal cancer was four folds than that of normal colorectal tissue.The effect of down-regulating PI3Kp110βexpression on proliferation and apoptosis in colorectal cancer cells1. Human PIK3CB-siRNA expression sequences were synthesized successfully, which were identified PI3Kp110βmRNA by BLAST analysis.2.48h after transfection of HCT116 cells, we detected the changes of PI3Kp110βmRNA and protein by western blot and real-time PCR.Compared to the control groups,the target group had the least expression of PI3Kp110βmRNA and protein.3. To determine the effect of PI3Kp110βexpression on proliferation of colorectal cancer cells, CCK-8 assay was performed at Oh,24h,48h and 72h, respectively. Compared with the control cells, PIK3CB-siRNA cells grew much slowly at 24h,48h and 72h,and the change at 24h and 48h was significant (P<0.05). The results indicate down-regulating PI3Kp110βexpression could inhibit colorectal cells proliferation.4. In order to evaluate the effect of down-regulating PI3Kp110βexpression on the induction of apoptosis, we conducted JC-1 staining analysis 48h after transfection. Compared with negative control group, the motochondria membrane potential of target group was significantly decreased, cells presented green fluorescence by confocal microscopy, while the negative control cells presented red fluorescence, the change of motochondria membrane potential between groups was significant.5. To further determine the effect of PI3Kp110βprotein on colorectal cancer cells apoptosis, we conducted FCM analysis.Compared with negative control group, the target group had higher apoptosis rates 48h after transfection(P<0.05).The results further indicated that down-regulating PI3Kp110βexpression could increase the apoptosis rates of colorectal cancer cells. Research of related signaling pathway of PI3Kp110βexpression in colorectal cancer cellsTo further explore the mechanism underlying PI3Kp110βdepression on cells proliferation and apoptosis, we examined expression levels of Akt, phospho-Akt, FoxO transcription factors in nucleus and phosphorylated FoxO transcription factors in cytoplasm. The results showed that down-regulating PI3Kp110βexpression led to substantial reduction in the levels of phospho-Akt (P<0.05). Consistent with this reduction in the phospho-Akt level, Western blot analysis showed significantly decreased expression of phospho-FoxO1 (FKHR) in cytoplasm of PI3Kp110βdepression cells (P<0.05), the prominent accumulation of FoxO1 (FKHR) in nucleus was simultaneously observed (P<0.05). As potential downstream targets of FoxO transcription factors, the expression levels of cell cycle-associated proteins and cell apoptosis-related proteins were also determined. Results showed that the expression levels of cyclin D1, cdk4,bcl-2 and bax were significantly decreased in down-regulating PI3Kp110βexpression cells (P<0.05), while the expression level of cdk-6 and bcl-6 was not significantly changed(P>0.05).Conclusions1. Western blot results show that the expression levels of PI3Kp110βgradually increased from normal colorectal mucosa, colorectal hyperplastic plopy,adenoma to primary colorectal adenocarcinoma, indicating that PI3Kp110βplayed an important role in the progression of colorectal cancer.2. Down-regulating PI3Kp110βexpression could inhibit HCT116 cells proliferation.3. Down-regulating PI3Kp110βexpression could decrease the motochondria membrane potential of HCT116 cells,and induce cell apoptosis.4. Down-regulating PI3Kp110βexpression could increase apoptosis rates of HCT116 cells.5. Down-regulating PI3Kp110βexpression could inhibit the activity of Akt and activate FoxO1 transcription factors, which, in turn, activated transcription of target genes such as those involved in cell cycle regulation and apoptosis. Thus, the colorectal cancer cells proliferation arrest and enhanced apoptosis were closely related with activation of FoxO transcription factors. |
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