| PART ? Predictive value of blood lipid association with response to neoadjuvant chemoradiotherapy in rectal cancerBackground:At present,neoadjuvant therapy(nCRT)for colorectal cancer(CRC)in stage ?/? was generally accepted,but the treatment effects of different patients were very different.Some patients have obvious tumor regression after nCRT,but there were still many patients with no significant regression,or even progress.Therefore,it was very important to predict the patient's neoadjuvant treatment effect,and it was necessary to guide the individualized treatment of patients.However,there were currently no effective predictors for the effects of CRC nCRT.Through long-term clinical work,we found that the effect of nCRT in patients with hyperlipidemia was often worse than that of patients with normal blood lipids.Therefore,considering that the nCRT effect of CRC may be related to blood lipid levels,the clinical data collected by this study was retrospectively analyzed.To study the relationship between blood lipids and nCRT for CRC,and to compare with existing predictive indicators to determine whether blood lipid levels have a predictive effect on their therapeutic effects.Materials and methods:1.Patient data collection:Data of CRC patients who underwent nCRT were collected.The criteria for enrollment were as follows:(1)Proctoscopy confirmed that primary colorectal adenocarcinoma was located 3-12 cm above the edge of the anus(KPS>80);(2)According to the AJCC,the tumor stage was determined to be T3-T4 or lymph node metastasis positive;(3)No distant metastasis was confirmed by various auxiliary examinations.Exclusion criteria were as follows:(1)patients discontinued NT due to serious adverse reactions,(2)history of familial hereditary CRC,(3)patients receiving simple neoadjuvant chemotherapy(4)suffered from previous malignant tumors,or have received anti-tumor treatment.2.Treatment plan:combined with radiotherapy and chemotherapy,the target area of radiotherapy was three-dimensional conformal radiotherapy with pelvis as radiation field,the dose was 45Gy/25 times.Chemotherapy was a combination chemotherapy with capecitabine + oxaliplatin or FOLFOX6 regimen.3.Evaluation criteria for treatment response:Tumor regression(TRG)according to postoperative pathology,TRG was determined according to the degree of tumor activity,fibrosis and inflammation,TRG 0,no significant regression of tumor;TRG 1,tumor regressed(tumor)Fibrosis or vascular lesions ? 25%);TRG 2,moderate regression(26%? tumor fibrosis range ? 50%);TRG 3,significant regression(51%? tumor fibrosis range<100%);TRG 4,completely subside,no visible tumor cells,only fibrotic mass.TRG 0-2 were considered as non-responders,while the tumors that were TRG 3 or 4 were considered as responders.4.Statistical analysis:TC,TG,LDL,HDL and ?CEA were expressed as the mean± standard error of the mean.The ?2 test was used for univariate analysis.The t-test was used to analyze the hematological analysis values to assess the correlation with TRG.Logistic regression analysis showed whether there was a causal relationship between various independent factors and TRG.The ROC curve determines the diagnostic value of independent factors,and p<0.05 was regarded as statistically significant.Results:Between June 2011 and January 2015,the records of 176 patients with primary colorectal adenocarcinoma treated with nCRT followed by radical surgery were reviewed retrospectively.Total cholesterol(TC),triglyceride(TG),low-density lipoprotein(LDL),high-density lipoprotein(HDL),and pre-CEA were measured before nCRT,and post-CEA was measured before surgery.A total of 129(73.3%)responders(TRG 3-4)and 47(26.7%)non-responders(TRG 0-2)were assessed after the nCRT.TC,LDL,HDL,and ?CEA were 6.56±0.95,3.08±0.72,and 1.43±0.25 mmol/L and-0.69±8.33 ?g/mL in non-responders compared with 5.15±1.29,2.39±0.5,and 1.37±0.32 mmol/L and 16.67±30.18 ?g/mL in responders,respectively(p<0.05).TG,pre-CEA,and post-CEA were not significantly different.Multivariate logistic regression analysis revealed TC and ?CEA as independent factors in predicting TRG;TC showed a sensitivity of 62.79%,a specificity of 91.49%,Youden index was 0.543,a cut-off value was 5.52,and AUC was 0.800 compared with ?CEA(sensitivity 76.74%,specificity 65.96%,Youden index was 0.427,and AUC was 0.761).Conclusion:The results of the study indicated that serum TC levels were closely related to tumor response to nCRT.Statistical analysis showed significant differences in TC,LDL,and HDL levels between non-responder group(TRG 0-2)and the responder group(TRG 3-4);patients in non-responder group had higher TC and LDL values than responder group.And the HDL level was low.Logistic regression results showed that serum TC and ?CEA levels were independent factors in the effect of tumor nCRT,while LDL and HDL had no significant significance.The possible reason for this phenomenon was that the levels of LDL and HDL were themselves affected by serum cholesterol and participate in cholesterol metabolism,so LDL and HDL were not considered independent factors.From logistic regression analysis,TC can be used as a predictor of the sensitivity of nCRT in CRC,and the sensitivity,specificity and area under the curve of TC were better than ACEA,and since TC can be obtained before nCRT,and the sensitivity and specificity were significantly better than those predicted in other studies.Therefore,TC can be regarded as a predictive factor for the accuracy of CRC neoadjuvant therapy.PART ? Cholesterol regulates cell proliferation and apoptosis of colorectal cancer by modulating miR-33a-PIM kinase pathwayBackground:Colorectal cancer(CRC)was the third most common malignancy in the world and the fourth leading cause of cancer-related deaths.About 21%of patients have reached the stage of progression.For a long time,some studies have found that the development of CRC is closely related to high fat intake in the diet,especially cholesterol levels.In the first part of this study,the preliminary analysis of the new adjuvant treatment effect of CRC was related to the serum cholesterol level of patients,but the mechanism of is still unclear.Therefore,this part focused on the mechanism of cholesterol affecting the chemotherapy effect of CRC tumor.miRNAs were non-coding RNAs that regulated gene expression at the post-transcriptional level and participate in various physiological activities of the organism.It has been confirmed that a variety of miRNAs are abnormally expressed in tumors and were closely related to tumorigenesis.Therefore,our study hypothesized that cholesterol may regulate the growth and apoptosis of CRC cells by regulating the expression of tumor-associated miRNAs,and then analyze the relationship between cholesterol and CRC from the perspective of miRNAs.miR-33a played an important role in cholesterol metabolism,can regulate the synthesis and extracellular transport of cholesterol in cells,and bioinformatics and literature analysis found that miR-33a was abnormally expressed in many tumors,including colorectal cancer The expression of miR-33a was significantly lower than that of peritumoral tissues,thus our consideration of cholesterol may affect the proliferation and apoptosis of CRC cells by regulating the expression of miR-33a.Materials and methods:1.Cell culture:HT-29 and SW480 cells were cultured in MyCoy'5A and high glucose DMEM,respectively,and water-soluble cholesterol was used to verify the effect of cholesterol.Cells were induced to apoptosis by treatment with 200 mg/L oxaliplatin(OXA).2.CCK8 experiment and flow cytometry(FCM):(1)CCK8 experiment:cells were seeded in 96-well plates at 3000 per well,and after starvation overnight,different concentrations of cholesterol were employed(0,5,10,20 and 50 ?M).CCK8 detected cell proliferation at 24h-96h.OXA was used to induce apoptosis after cholesterol culture,and CCK8 was used to detect the effect of different concentrations of cholesterol on apoptosis.(2)FCM assay:Cells were seeded into 6-well plates at 3-5 × 105 per well.After starvation overnight,the cells were treated with 5 ?M and 10 ?M cholesterol for 24 h,and parts of cells were collected,stained by PI/RNase staining according to the instructions,and analyzed for cell cycle changes by FCM.Then,the remaining parts of cells were induced to apoptosis by OXA for 12 h,and the cells were collected and stained by Annexin V-FITC/PI,and the apoptosis was measured by FCM.3.Cell transfection and real-time quantitative reverse transcription PCR(RT-qPCR):cell transfection:cells were seeded in 6-well plates and transfected at a density of 30-50%.miR-33a mimics and inhibitor were diluted to 30 nM and 50 nM in serum-free medium,transfected with INTERFERin reagent.Transfection efficiency was detected by RT-qPCR.4.Westem bolt(WB):Total protein was extracted by protein extraction kit,separated by SDS-PAGE gel and transferred to PVDF membrane.After blocking,the primary antibody was incubated overnight.After the primary antibody was incubated,the secondary antibody was incubated for 2h.5.Dual luciferase reporter assay:HT-29 cells were seeded in 96-well plates until cell transfection reached 70%.HT-29 cells were transfected with the firefly luciferase vector(Firefly):Renilla luciferase vector(Renilla)and miR-33a mimics.After culture for 48h,firefly and Renilla luciferase OD values were measured.The ratio of firefly/sea kidney fluorescein(luc/R-luc)was recorded.6.Statistical analysis:GraphPad Prism and SPSS software were used for statistical analysis.The experiment was repeated triple to avoid errors.Differences in t-test or one-way ANOVA.In the PCR analysis,the p value<0.05 and the fold difference>2 were statistically significant differences.In the other results analysis,the p value<0.05 was defined as the difference.Results:1.Cell viability was increased after cholesterol treatment:from 24h to 96h,the cell viability of HT-29 was increased 9.17 times after cholesterol treatment.The viability of SW480 cells increased to a maximum of 2.64 times.After induction of apoptosis,apoptosis was significantly inhibited after cholesterol treatment.The inhibition rate of apoptosis in HT-29 cells was up to 3.45 times,and that of SW480 cells was up to 1.88 times.2.Cholesterol promoted cell cycle and inhibited apoptosis;After treatment with cholesterol,the G1 phase of HT-29 cell cycle decreased by 27.62%,and S phase and G2 phase increased by 18.96%and 8.98%,respectively.SW480 cells showed the same trend as HT-29.In the apoptosis assay,OXA-induced apoptosis decreased by 12%and 15.3%in HT-29 and SW480,respectively,after cholesterol treatment.3.miR-33a mediated the effect of cholesterol on CRC cells:The relative expression of miR-33a in cholesterol-treated HT-29 cells was reduced by 5.7-fold.SREBP2 mRNA expression was reduced by 7.17-fold.The expression of miR-33a and SREBP2 mRNA in SW480 cells was the same as that of HT-29 cells.After miR-33a mimics transfected cells,the expression of miR-33a was up-regulated 10 and 4 times in HT-29 and SW480 cells,respectively,but the expression of miR-33a was not significantly decreased after transfection with miR-33a inhibitor.The cell viability of the HT-29 and SW480 cells in the miR-33a inhibitor group was significantly higher than that in the NC group,but there was no significant difference in cell viability between the cholesterol-treated inhibitor group and the inhibitor NC cholesterol-treated group.In the transfected cells of HT-29 and SW480 cells,the S+G2 phase of the mimics group was significantly lower than that of the NC group and the cholesterol group.In the inhibitor group,the S+G2 phase was similar to its cholesterol-treated group but higher than its NC group.The percentage of apoptosis in HT-29 and SW480 transfected cells was 8.7%higher in the mimics group than in the NC group,and the apoptosis rate in the inhibitor group was 10.6%lower than that in the inhibitor NC group.When these groups were given cholesterol pre-treatment before apoptosis,the apoptosis of the mimics group was reduced by nearly 10%compared with the cholesterol group and the NC group,while the apoptosis of the inhibitor group was reduced by 10.6%compared with the NC group.The same phenomenon was found in SW480 cells.4.PIM1/PIM3 was involved in the role of cholesterol in CRC cells as a target gene for miR-33a:expression of PIM1 and PIM3 protein in the cholesterol group were significantly enhanced in HT-29 cells.Moreover,the expression of PIM1/PIM3 protein was significantly inhibited in the miR-33a mimics group.Dual luciferase reporter assays analyzed specific target relationships between miR-33a and PIM kinase.The results showed that the expression inhibition rate of miR-33a-PIM-3'UTR(WT)luciferase was about 60%.After the binding site mutation,the inhibition of the 3'UTR(MUT)group was reduced to about 25%.5.PIM kinase inhibited CRC apoptosis by phosphorylating C-Myc,p21,p27 and Bad proteins:expression of p-Myc,C-Myc,p-p21 and p-p27 in cholesterol-treated groups were up-regulation,the expression of p21 and p27 was suppressed.Moreover,the expression of the cell cycle-associated proteins Cyclin E,CDK2,CDK4 and Cyclin B was elevated.In apoptotic cells,the expression of p-Bad protein was significantly increased,and the expression of downstream apoptotic proteins Bcl-xL,Bcl-2 and Caspase 3 was increased.The expression of PIM kinase protein was increased after miR-33a inhibition,and the expression of p-p21,p-p27,p-Myc and p-Bad was increased.Conclusion:From CCK8 and FCM results,cholesterol can stimulate CRC cell proliferation,promote cell cycle progression and inhibit apoptosis.Cholesterol inhibited miR-33a and SREBP2 mRNA expression,and when transfected with miR-33a mimics or inhibitor,there was a significant difference in the effect of cholesterol.In addition,PIM protein expression was up-regulated in the cholesterol-treated group,and it was identified as a target gene of miR-33a by dual luciferase reporter gene assay.PIM kinase regulates CRC cell proliferation by phosphorylating p27,p21 and Bad proteins.Therefore,it is concluded that cholesterol can regulate the development of CRC through the miR-33a-PIM kinase pathway. |
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