| Colon cancer is one of the most common gastrointestinal cancer,and also the main reason that caused death among kinds of magligent cancers worldwide.The mechnism of colon cancer occurrence is still unclear.Mount evidences indicate that colon cancer is the result of many genes interaction together.As a solid cancer,hypoxia is a typical feature during ocolon cancer occurrence and progression.Many studies indicate that HIF1-α play very important roles under hypoxic conditions through regulate one hundred of genes.As a member of PLD family,PLD2 play key roles in many solid cancers.Our research group previous reports showed that PLD2 is one of very important proteins that regulate the apoptosis of cells through proteomic analysis after interference HIF1-αgene.We speculate that PLD2 may be regulated by HIF1-α under hypoxic conditions and maintain the growth of colon cancer.There are no articles about the correlation of PLD2 and HIF1-α in colon cancer so far.Therefore,there has very important research value about this issue.In order to illuminate this issue,the report was divided into following parts: Part ⅠExpression of PLD2 and HIF1-α in colon cancer tissue and its clinical significance Objective:To investigate the expression and correlation of PLD2 and HIF1-α in colon cancer Methods:The expression of PLD2 and HIF1-α in thirty human colon cancer specimens and their corresponding normal colon mucosa tissue were detected by using immunohistochemistry staining.The relationships between their expression and clinical pathological characteristics of colon cancer patient were analyzed by λ2-test.The correlations between PLD2 expression and HIF1-α expression was assessed using Spearman rank correlation;RT-PCR and Western blot were used to detected the mRNA and protein expression levels of PLD2 and HIF1-α in thirty human colon cancer specimens and their corresponding normal colon mucosa tissue respectively. Results:The positive expression of HIF1-α and PLD2 in colon cancer tissues were 76.67% and 73.33% respectively,there was significant difference compare with normal colon tissues respectively(p<0.05),and co-expression of them associated with the volume of tumor(p<0.05).The Spearman rank correlation analysis showed that there present positive relationship between PLD2 expression and HIF1-α expression in colon cancer tissues(r=0.56,p<0.05).The mRNA and protein expression level of HIF1-α and PLD2 were high in colon cancer tissues,but weakly detected in normal colon mucosa tissue.The mRNA and protein expression levels of HIF1-α and PLD2 significantly elevated compare to corresponding normal colon mucosa tissue respectively(p<0.05). Conclusion:The expression of HIF1-α and PLD2 were high in colon cancer,and co-expression of them associated with the volume of colon tumor;There was a significant positive correlation between the HIF1-α expression and PLD2 expression in colon cancer. Part Ⅱ HIF1-α up-regulates the expression of PLD2 in colon cancer cell under hypoxic conditions Objective:To investigate HIF1-α regulates the expression of PLD2 in colon cancer cells in vitro and vivo under hypoxic conditions Methods:Western Blot and RT-PCR were used to detect the protein and mRNA expression levels of HIF1-α and PLD2 in colon cancer cell lines(SW620,SW480,Lovo and HCT116) and FHC respectively. The effect of different hypoxia time(0h,12 h,24h and 48h)on the protein and mRNA expression levels of HIF1-α and PLD2 in SW480 and SW620 were detected by Western Blot and Real-Time PCR respectively. Then adenovirus that carry human-specific HIF1-α interference RNA(SiRNA)was designed and constructed successfully.The effect of it on the expression of HIF1-α was assessed by Fluorescence microscpe,Western Blot and Real-Time PCR.The protein and mRNA expression levels of PLD2 in SW620 and SW480 after interference HIF1-α gene under hypoxic conditons or cultured under different concentration of CoCl2 were detected by using Western Blot and RT-PCR respectively. Establishment of colon cancer cells in nude mice transplantation tumor model to investigate effect of HIF1-α SiRNAon the expression of PLD2 in vivo. Results: HIF1-α and PLD2 protein and mRNA expression levels were high in colon cancer cells(SW620,SW480,Lovo,HCT116),but weakly detected in FHC,there was a significant difference between colon cancer cells(SW620,SW480,Lovo,HCT116)and FHC(P<0.05);Hypoxia induced the protein and mRNA expression levels of PLD2 in SW620 and SW480 increased compare to normoxia group(P<0.05);HIF1-α protein and mRNA expression levels were significant decreased by Ad-HIF1-α-SiRNA under both hypoxic conditions or normoxia conditions,there have significant difference compare to control group(P< 0.05);PLD2 high protein and mRNA expression levels in colon cancer cells induced by hypoxia was significantly decreased after HIF1-α gene expression was interferenced compare to control group(P<0.05); PLD2 protein and mRNA expression levels was elevated as the concentration of CoCL2 increasing compare to control group(P<0.05);There was a statistical difference in tumor growth between HIF1-α interference group and blank control group after 21 days indicated by tumor growth curve(P<0.01),but the growth of xenograft tumor had no difference between HIF1-α interference group and blank control group in the early stage(p>0.05),the expression level of PLD2 in xenograft tumor in HIF1-α gene interference group was significantly lower than blank control group(P<0.05). Conclusion: HIF1-α could up-regulates the expression of PLD2 in colon cancer cells under hypoxic conditions, inhibited the expression of HIF1-α and PLD2 could suppress the growth of colon cancer cells subcutaneous xenotransplanted tumor. Part Ⅲ Effect of PLD2 activity inhibition on the apoptosis of colon cancer cells under hypoxic conditions and possible mechnism Objective:To investigate the effect of PLD2 on the apoptosis of colon cancer cells under hypoxic conditions through inhibiting PLD2 activity. Methods:Effect of hypoxia on the activity of PLD2 in colon cancer cells(SW620,SW480,Lovo and HCT116) and FHC was detected by using PLD activity assay;Effect of PLD2 activity inhibitor VU0364739 on the activity of PLD in colon cancer cells(SW620 and SW480) and FHC under hypoxic conditions was detected using PLD activity assay,and all cells were divided into following four groups::normoxia group,hypoxia group,hypoxia plus VU0364739(50nM) group and hypoxia plus VU0364739(100n M) group;Effect of PLD2 activity inhibitor VU0364739 on the apoptosis rate of SW620,SW480 and FHC under hypoxic conditions was detected by using flow cytometry,and on the protein expression level of Bcl2,survivin,p-PI3 K /PI3 K and p-AKT /AKT were detected by Western Blot; SW620 and SW480 were divided into following four groups: hypoxia control group, hypoxia plus PLD2 inhibitor group, hypoxia plus PI3 K inhibitor group, hypoxia plus PLD2 inhibitor and PI3 K inhibitor groups,flow cytometry was used to detect the apoptosis rate and Western Blot was used to detect the protein expression level of Bcl2, Survivin,p-PI3 K /PI3 K and p-AKT /AKT in those four group cells;The expression of PLD2,Survivin and Bcl2 in fifty human colon cancer specimens and their corresponding normal colon mucosa tissue were detected by using immunohistochemistry staining,correlation between PLD2 expression and Survivin expression and Bcl2 expression was assessed by using Spearman rank correlation. Results:The activity of PLD in colon cancer cells(SW620,SW480,Lovo and HCT116) and FHC was significantly high induced by hypoxia,there was a difference between hypoxia group and normoxia group(p<0.05);High PLD activity induced by hypoxia was significantly reduced by PLD2 inhibitor VU0364739 compare to hypoxia control group(p<0.05);Hypoxia induced the apoptosis rate and the protein expression level of Bcl2,Survivin,p-PI3 K /PI3 K and p-AKT/AKT in SW620 and SW480 increased compare to normoxia group(p<0.05),but PLD2 activity inhibitor promote the apoptosis rate of SW620 and SW480 significantly increased compare to hypoxia control group(p<0.05),and lead to the protein expression level of Bcl2,Survivin,p-PI3K/PI3 K and p-AKT/AKT decreased(p<0.05).However, athough hypoxia also induce the apoptosis rate and the protein expression level of Bcl2,Survivin,p-PI3 K /PI3 K and p-AKT /AKT increased in FHC compare to normoxia group(p<0.05),but those was not effected by the PLD2 activity was inhibited under hypoxic conditions,there have no difference between PLD2 activity inhibit group and hypoxia control group(p>0.05).Inhibition PI3K/AKT signaling pathway,inhibition PLD2 activity or both of them leaded to the apoptosis rate of SW620 and SW480 increased and the protein expression level of Survivin,Bcl2,p-PI3K/PI3 K and p-AKT/AKT in those cells decreased,there have difference comapare to hypoxia control group(p<0.05),but the apoptosis rate and Survivin,Bcl2,p-PI3K/PI3 K and p-AKT/AKT protein expression level in any two group of this three group all have no difference(p>0.05).The positive expression of PLD2,Survivin and Bcl2 in 50 colon cancer tissues was 64%,68% and 60% respectively, but weakly detected in normal colon mucosa tissues,the protein expression level of PLD2,Survivin and Bcl2 in colon cancer tissue were significantly higher than normal colon mucosa(p<0.05),there present positive relationship between PLD2 expression and Survivin expression(r= 0.379,P<0.05)and Bcl2 expression( r=0.405,P < 0.05) in colon cancer tissues through Spearman rank correlation analysis. Conclusion: PLD2 inhibited apoptosis of colon cancer cells under hypoxic conditions may through up-regulates the expression of survivin and Bcl2 via activating PI3K/AKT signaling pathway. |
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