| Part one The expression of miR-106 a in human colorectal cancer tissuesObjective: To detect the concentrations of miR-106 a in colorectal cancer tissues and non-tumor adjacent tissues by Real-time polymerase chain reaction,and analyze the relationship between the expression of miR-106 a and clinicopathological factors of all patients.And evaluate the diagnostic value of miR-106 a in colorectal cancer tissues.Methods:1 A total of 42 colorectal cancer patients that underwent radical resection were obtained between Oct 2013 and Mar 2014 at the First Hospital of Hebei Medical University.All cases did not receive any form of preoperative radiotherapy or chemotherapy and have complete clinical data.All pathological diagnosis were given by two experienced pathology doctors.The fresh tumor tissues were obtained during the operation,and the corresponding normal tissues were obtained from a section of the resected specimen at least 5cm from the edge of tumor.2 Total RNA was extracted from the frozen tissues using TRIzol reagent.Then the concentration and purity of all RNA samples were detected and reverse transcription was carried out by the One Step Prime Script? miRNA c DNA Synthesis Kit.Finally the the expression of miR-106 a was detected by Real-time polymerase chain reaction.3 Differences were tested for significance with Mann Whitney test by statistical package for social sciences(SPSS)PC version 19.0.Differences were statistically significant with P<0.05.All values were reported as the P50(P25,P75)or mean±SD.Results:1 The expression level of miR-106 a was significantly up-regulated in tumor tissues compared to adjacent tissues [26.2882(14.5358,49.3492)vs.8.7187(5.4891,13.6726)].There was significantly statistical difference(P<0.0001).2 The expression of miR-106 a in colorectal cancer tissues has relationship with the clinicopathological characteristics of colorectal cancer patients.The expression level of miR-106 a in colorectal cancer tissues was significantly higher in adenocarcinoma patients than in mucinous carcinoma patients(100.96±262.20 vs.18.14±18.22,P=0.0348).miR-106 a expression in colorectal cancer tissues was increased in moderate-well differentiation patients compared with that in worse differentiation patients(108.87±272.79 vs.18.12±19.20,P=0.0343).And miR-106 a expression in CRC tissues was increased in T1+T2 patients compared with that in T3+T4 patients(271.91±525.60 vs.47.24±74.53,P=0.0431).There was no significant association between miR-106 a expression and other clinicopathological characteristics,such as gender,age,distant metastasis,Dukes staging,tumor location,and lymph node metastasis.Conclusions:The expression level of miR-106 a was significantly up-regulated in tumor tissues compared to adjacent tissues,and has relationship with the clinicopathological characteristics of colorectal cancer patients such as differentiation,pathological type of tumor and the depth of invasion.miR-106 a may be a potential biomarker of colorectal cancer.Part two The expression of miR-106 a in human colorectal cancer plasmObjective: The aim of this study was to evaluate the diagnostic value of miR-106 a in plasm of colorectal cancer patients.We investigate miR-106 a expression in plasm of colorectal cancer patients compared with neoplasm free healthy individuals,and analyzed the relationship between the expression of miR-106 a and clinicopathological factors of all patients.And examine the miR-106 a expression in plasma of colorectal cancer patients pre-and post-operation.Methods:1 A total of 40 colorectal cancer patients that underwent radical resection were obtained between Oct 2013 and Mar 2014 at the First Hospital of Hebei Medical University.Plasma samples were collected before surgical resection and seven days after the operation respectively.Meanwhile,collect 42 plasma samples from 42 cases of age-matched healthy physical examination people as the control who do not have tumor history.All cases did not receive any form of preoperative radiotherapy or chemotherapy and have complete clinical data.All pathological diagnosis were given by two experienced pathology doctors.2 Total RNA was extracted from plasma using the miRNeasy Serum/Plasma Kit.Then the concentration and purity of all RNA samples were detected and reverse transcription was carried out by the One Step Prime Script? miRNA c DNA Synthesis Kit.Finally the the expression of miR-106 a was detected by Real-time polymerase chain reaction.3 All values were reported as the P50(P25,P75)or mean±SD.Differences were tested for significance with Mann Whitney test by statistical package for social sciences(SPSS)PC version 19.0.Differences were statistically significant with P<0.05.The sensitivity and specificity of miR-106 a were analyzed by receiver operating characteristic(ROC)curve in CRC diagnosis.Results:1 The expression level of miR-106 a in plasma was significantly higher in colorectal cancer patients than in healthy control group,and there was significantly statistical difference(P=0.0041).2 miR-106 a expression in plasma post-operation is lower than pre-operation,but there was no significantly statistical difference(0.16±0.18 vs.8.80±40.50,P=0.16).3 The area under the ROC curve is 0.858(95%Cl:0.772-0.944),The sensitivity and specificity are 73.8% and 87.1% respectively to distinguish colorectal cancer patients from healthy people.ROC curve analysis showed that the expression level of miR-106 a in plasma has a certain amount of diagnostic value to distinguish colorectal cancer patients from healthy people.4 No significant correlation was found between the miR-106 a expression in plasma and the clinicopathological parameters of the colorectal cancer patients,such as gender,age,distant metastasis,Dukes staging,tumor location,pathological type,differentiation,depth of invasion and lymph node metastasis(P >0.05).Conclusions:The expression level of miR-106 a in plasma was significantly higher in colorectal cancer patients than in healthy control group.Plasma expression level of miR-106 a is useful in the early diagnosis of colorectal cancer.No significant correlation was found between the miR-106 a expression in plasma and the clinicopathological parameters of the CRC patients.miR-106 a may be a potential biomarker of colorectal cancer.Part three The function of miR-106 a in human colorectal cancer cellsObjective: To investigate the role of miR-106 a in the development of colorectal cancer cells.We measure miR-106 a expression of HCT116 cells and its effects on cell apoptosis and proliferation.Methods:1 The HCT116 cells were transfected with miR-106 a mimic,mimic control,inhibitor or inhibitor control by lipofectamine 2000.The expression of miR-106 a was detected after forty-eight hours.2 HCT116 cells were transfected with miR-106 a mimic or mimic control,miR-106 a inhibitor or inhibitor control.Then,the proliferation of HCT116 cells were measured by CCK-8 assay.We determine the values of OD450 at the first day,the second day,the third day,the fourth day and the fifth day respectively,and draw the curves of cell proliferation.3 Fluorescein isothiocyanate(FITC)-conjugated annexin V and propidium iodide(PI)staining were performed by means of the Annexin V-FITC Apoptosis Detection kit.The apoptosis condition of HCT116 cells were detected by flow cytometric analysis.Results:1 The expression of miR-106 a was significantly up-regulated by miR-106 a mimic comparison with mimic control in HCT116 cells.Inversely,after transfection with miR-106 a inhibitor,the expression of miR-106 a was significantly decreased compared to inhibitor control transfected cells.There are significantly statistical differences respectively(P<0.05).2 The cell proliferation was significantly up-regulated by miR-106 a mimic comparison with mimic control in HCT116 cells.Inversely,after transfection with miR-106 a inhibitor,the proliferation of HCT116 cells was also reduced compared to inhibitor control transfected cells.There are significantly statistical differences respectively(P<0.05).3 After transfected with miR-106 a mimic or mimic control,cell status was drawed by flow cytometer,early apoptotic cells were located in the lower right quadrant.The HCT116 cells transfected with miR-106 a mimic have lower rate of early apoptosis than the control group.The average of early apoptosis rate were calculated from the flow cytometric analysis and presented as the mean±SD(miR-106 a mimic vs.mimic control,8.68%±0.75% vs.12.45%±1.16%,n=4).After transfected with miR-106 a inhibitor or inhibitor control,cell status was drawed by flow cytometer also.The HCT116 cells transfected with miR-106 a inhibitor have higher rate of early apoptosis than the control group.The average of early apoptosis rate were calculated from the flow cytometric analysis and presented as the mean±SD(miR-106 a inhibitor vs.inhibitor control,8.803%±0.27% vs.6.733%±0.60%,n=4).There are significantly statistical differences respectively(P<0.05).Conclusions:Up-regulation of miR-106 a can promote proliferation and decrease early-apoptosis in HCT116 cells.Down-regulation of miR-106 a can suppress proliferation and accelerate early-apoptosis in HCT116 cells.Our results suggested that miR-106 a promoted cell proliferation through inhibiting the early apoptosis in CRC cells. |
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