| Objective:Globally,colorectal cancer(CRC)is the third largest cancer and the second largest cause of cancer death.Incidence rate and mortality incidence of colorectal cancer are high in China.The incidence rate and mortality rate are high.The early diagnosis rate is low.Recurrence and distant metastasis after diagnosis are the main causes of poor prognosis and low survival rate.Surgery,radiotherapy and chemotherapy,molecular targeted therapy and nutritional immunotherapy are the main methods for colorectal cancer treatment.However,the total survival time of patients affected by many factors still has a great room for improvement.Therefore,it is urgent to explore new anti-tumor drugs and their anti-tumor mechanisms to provide new ideas for the prevention and treatment of colorectal cancer.Previous researchers have shown that PUFAs is the main means for colorectal cancer treatment.Previous researchers have shown that PUFAs is the key to the treatment of colorectal cancer Colorectal cancer has antitumor effect,and the imbalance of intake ratio of co-6 andω-3 polyunsaturated fatty acids(PUFAs)plays a key role.However,the molecular mechanism of the effect of polyunsaturated fatty acids on local microenvironment of colorectal cancer needs further study.In order to further explore its anti-tumor molecular mechanism,this study will analyze the effects of AA,DHA and EPA on the biological function of colorectal cancer cells and detect AA and DHA,The changes of gene expression profiles related to the development of colorectal cancer with EPA provide theoretical basis for adjuvant chemotherapy of colorectal cancer.Methods:(1)The effects of AA,DHA,and EPA on cell proliferation,apoptosis,cycle,invasion and migration and chemosensitivity were analyzed.The cells HT29 and RKO were selected as the study objects.AA,EPA,DHA,5-FU and oxa were added to the medium as the observation group,PBS solution was treated as the negative control group.The proliferation,apoptosis,cycle,invasion and migration of cells were analyzed.The cells of oxa+AA,oxa+DHA,oxa+EPA were combined with each other.Oxa+PBS were used as control.PUFAs was added to the culture medium The changes of chemosensitivity of post tumor cells.(2)The changes of gene expression profile were analyzed.After AA,DHA and EPA treated cells,mRNA chip analysis was performed to screen the difference genes.The GA/KEGG clustering analysis and signal path analysis were carried out by using bioinformatics software.(3)Verification of chip results:the main pathway genes screened after AA,DHA and EPA were verified by QRT PCR at RNA level,and the differences between the two PUFA treatments and PBS treatment group were analyzed.The expression of AA,DHA,and EPA related genes and the downstream products of related genes were analyzed by Western blot method at protein level.The expression of target gene was knocked out by Si RNA method.The expression of target genes downstream of AA,DHA and EPA were analyzed to verify whether PUFA affected the signal pathway.Results:(1)In CCK-8 cell viability test,different doses of AA,DHA,EPA,5-FU and oxa inhibited the growth of HT29 and RKO cells in varying degrees.As shown in figures 1-1 to 1-5,the survival rate of HT29 cells in control group(100 ±8.52%),AA(125um)group(73.92 ±3.56%),DHA(100um)group(54.06 ±2.06%)and EPA(100um)group(68.34 ± 3.06%)was 100 ± 8.52%,73.92±3.56%,68.34 ±3.06%respectively,The 48 hour cell survival rate of 5-FU(4um)group was 49.71 ±1.16%,and that of oxa(4um)group was 25.34±1.68%.Therefore,the 48 hour cell survival rate of HT29 treated with AA,DHA,EPA,5-FU and oxa was significantly lower than that of the control group(P<0.05).As shown in Figure 1-6,the survival rate of HT29 cells in oxa(0.8um)group(94.66 ± 7.69%)was significantly higher than that in oxa(0.8um)+AA group(87.76±6.58%),oxa(0.8um)+DHA group(84.56 ± 5.36%),oxa(0.8um)+EPA group(73.12±4.96%)(P<0.05).The survival rate of RKO cells in control group(100 ± 9.02%),AA(125um)group(53.06 ±2.53%),DHA(100um)group(76.19±3.66%),EPA(100um)group(59.52±2.53%),The 48 hour cell survival rate of 5-FU(4um)group was 77.41±4.53%,and that of oxa(4um)group was 66.95±3.72%.Therefore,the 48 hour cell survival rate of RKO cells treated with AA,DHA,EPA,5-FU and oxa was significantly lower than that of the control group(P<0.05).As shown in Figure 1-6,the survival rate of RKO cells in single drug oxa(0.8um)group(69.05± 4.32%)was significantly higher than that in oxa(0.8um)+AA group(54.97 ± 3.65%),oxa(0.8um)+DHA group(48.82 ±3.89%),oxa(0.8um)+EPA group(68.09±4.37%),the difference was statistically significant(P<0.05).(2)In the cell cycle assay,HT29 cells were treated with 50 100,After 150 umol/L AA,DHA and EPA intervention,the proportion of G1 phase cells in control group(without drug intervention)was 63.86± 3.86%,in AA group(58.51 ±2.89%),in DHA group(54.77±3.12%)and in EPA group(51.52±1.68%),in DHA group(65.38±2.45%),in DHA group(62.97±3.26%)and in EPA group(56.27 ±2.58%),in AA group(60.86 ± 3.89%)and in EPA group(60.86± 3.89%),There was no significant change in cell cycle(P<0.01).After intervention with 50100150umol/L AA,DHA and EPA,the percentage of RKO cells in G1 phase(42.49±.56%)in control group(without drug intervention)was lower than that in AA group(46.38±2.36%),(50.82 ± 3.78%),(52.92 ± 2.56%);lower than that in DHA group(44.69 ±3.46%),(49.27 ±3.68%),(78.29±5.79%);lower than that in EPA group(44.59 ±3.78%),(49.87±3.69%),(61.03 ±4.56%),The difference was statistically significant(P<0.01).(3)In the apoptosis detection experiment,the proportion of living cells in RKO cell control group(without drug intervention)was 94.34±6.97%.When AA,DHA,EPA(50,100,150 umol/L)were treated for 48 h,the proportion of apoptosis in the experimental group gradually increased with the increase of concentration,among which The proportion of early withering in AA group was(2.95±0.95%),(4.66 ± 1.11%),(21.57 ± 3.56%);the proportion of late withering was(2.52±0.45%),(10.43 ± 0.98%),(7.07 ±0.78%);the proportion of early withering in DHA group was(3.32±0.53%),(4.1 ± 0.26%),(4.86±0.35%);the proportion of late withering was(3.69± 0.79%),(3.59 ± 0.69%),(3.91 ± 0.49%);the proportion of early withering in EPA group was(3.32 ±0.53%),(4.1±0.26%),(4.86± 0.35%)The proportion of late withering was(11.89±2.45%),(26.64 ± 3.25%)and(10.98 ± 2.16%),the difference was statistically significant(P<0.01).(4)The wound healing rate of HT29 and RKO cells treated with PUFAs was significantly slower than that of the control group(P<0.05).(5)When the differential gene expression was verified by QRT PCR,the relative expression of tollip gene in HT29 cells increased by 8.33 times,decreased by 0.87 times,and increased by 2.09 times after treated with AA,DHA,and EPA for 48 hours,respectively,with statistical significance(P<0.01).The results of other genes are shown in table 4-1.In RKO cells,the relative expression of ACVR1B gene after treated with AA,DHA,and EPA for 48 hours,respectively 2.37 times,0.60 times and 1.28 times respectively,with significant difference(P<0.01).The results of other genes are shown in Table 4-2;Conclusion:1.After different concentrations of PUFAs(AA,DHA,EPA)intervened HT29 and RKO cells,the cell survival rate decreased with the increase of concentration.PUFAs can inhibit the proliferation of HT29 and RKO cells,and PUFAs can enhance the chemosensitivity of anti-tumor drug oxa.2.The apoptosis rate of HT29 and RKO cells treated with low,medium and high concentrations of PUFAs(AA,DHA,EPA)increased gradually with the increase of PUFAs concentration after 48 h culture.PUFAs can promote the apoptosis of HT29 and RKO cells.3.Low,medium and high concentrations of PUFAs could prevent RKO cells from cell cycle arrest in G1 phase,but had no significant effect on HT29 cell cycle.4.PUFAs can delay wound healing and inhibit invasion and migration of colorectal cancer cells HT29 and RKO.5.HT29 and RKO cells treated with polyunsaturated fatty acids AA,DHA and EPA could increase or decrease the relative expression of TLIP.AA had the most obvious effect on the relative expression of TLIP in HT29 cells,which was 8.33 times of the normal group,and EPA had the most obvious effect on the relative expression of sema6a,which was 0.46 times of the normal group.In RKO cells,AA had the most obvious effect on the relative expression of ACVR1B,which was 2.37 times of that in the normal group,while EPA had the most obvious effect on the relative expression of mapkl,which was 0.27 times of that in the normal group. |
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