| [Objective](1)The differential expression of miRNAs in colorectal cancer tissues and adjacent non-cancerous normal mucosa tissues was analyzed by high-throughput Miseq sequencing.(2)The expression of miRNA452-5p and miRNA215-5p in colorectal cancer tissues and adjacent normal mucosa tissues was verified by quantitative real time PCR(RT-qPCR)technique.The relationship between miRNA 452-5p and miRNA215-5p may play an important role in the biological function of colorectal cancer and its regulation of downstream target genes.(3)To evaluate the diagnostic value of miRNA452-5p,miRNA215-5p and 2-miRNA panel by using ROC and Area under the ROC curve,and to explore the potential colorectal Molecular markers and therapeutic targets for the diagnosis of cancer.[Methods](1)3 cases of colorectal cancer were collected and the normal intestinal mucosa adjacent to the paracancerous tissues was sent to Shanghai Kang Cheng bio Co.,Ltd.to screen the differential expression of miRNA by high throughput Miseq sequencing.(2)To collect 30 specimens of patients diagnosed as colorectal cancer by enteroscopy at the Digestive Endoscopy Center of the First Affiliated Hospital of Kunming Medical University from July 2017 to October 2017.Patients signed informed consent and determined that the patient had not done chemoradiation and immunotherapy.The clinical and pathological data including age,gender,tumor location,degree of differentiation,infiltration range,Ducks stage and tissue type were collected.The experiment group was colorectal cancer tissue and adjacent normal mucosa tissue.(3)The expression of miRNA452-5p and miRNA215-5p in colorectal cancer tissues and adjacent normal mucosa tissues of all patients was detected by RT-qPCR and the relationship between them and clinical pathological data was analyzed,(4)Draw the ROC curve and the area under the curve AUC to evaluate the diagnostic value of miRNA452-5p.miRNA215-5p and 2-miRNA panel.[Result](1)Gene high-throughput Miseq sequencing showed that miRNAs were up-regulated in miRNAs and down-regulated in 48 miRNAs in colorectal cancer tissues and adjacent normal mucosa.Using Mirdbv5 and Targetscan7.1 two target gene prediction software to detect a total of 412 miRNA target genes upregulated,down-regulated miRNA target genes 372.The results of GO analysis show that in these miRNAs that are significantly regulated,GO processes include:biosynthesis,cellular metabolism,active intracellular processing,cell cycle regulation,intracellular signal transduction,intracellular trafficking and the like.The results of KEGG pathway show that in colorectal cancer tissues,KEGG pathways that are significantly regulated mainly involve pathways such as metabolism,genetic information processing,environmental information processing and cellular processes.(2)The results of real-time PCR showed that:(1)The expression of miRNA452-5P was 205.19 ± 123.27 in colorectal cancer tissue,576.32 ± 182.4 in normal intestinal mucosa tissue,and the expression of miRNA452-5P in colon cancer tissues(T =6.078,p<0.001).?miRNA215-5p expression in colorectal cancer tissues was 3.46 ±1.27,and in normal intestinal mucosa tissues was 2.07 ± 1.22,The expression of miRNA215-5p in colon cancer tissues was significantly up-regulated(t =-2.836,p<0.001).(3)The relationship between the expression of miRNA452-5p and clinicopathological characteristics of patients with colorectal cancer:(1)The expression of miRNA452-5p was not related to age,the relative expression of miRNA452-5p was 236.68(95.32,372.06),and the relative expression of miRNA452-5p Was 222.63(116.07,498.36).There was no significant difference between the two groups(Z =-0.525,P = 0.601).The expression of miRNA452-5p was not related to sex,the relative expression level in male group was 251.27(105.06,436.77),the relative expression level in female group was 210.24(104.24.459.65).there was no significant difference between the two groups(Z =-0.426,P = 0.634).? The expression of miRNA452-5p was not related to the degree of differentiation.The relative expression of miRNA452-5p was 234.43(124.53,459.65),and the relative expression in well differentiated group was 210.84(88.90.406.53).There was no significant difference between the two groups(Z =-0.283,P = 0.777).The expression of miRNA452-5p was not related to tumor location,the relative expression of rectal group was 124.53(85.38.251.27),and the relative expression of colonic group was 276.79(186.84.488.81).There was no significant difference between the two groups(Z ?-1.747,P = 0.081).The expression of miRNA452-5p was not related to the depth of invasion.The relative expression level of miR452-5p was 114.38(90.25,413.93)in T2 group and 242.85(166.40,453.24)in T3 + T4 group,but the difference was not statistically significant(Z =-1.091,P = 0.275).The expression of miRNA452-5p was not related to lymphatic metastasis.The relative expression of miR452-5p was 138.06(90.25,413.93)in lymphatic metastasis group,and 242.85(213.65.453.24)in lymphatic metastasis group.There was no statistical difference between the two groups Significance(Z =-0.945,P = 0.345).?miRNA452-5p expression was not related to Dukes stage,the relative expression of A + B group was 186.84(99.57,462.17),the expression of C + D group was 251.27(139.95,470.04),the difference between the two groups was not significant Statistical significance(Z =-0.367,P = 0.713).? miRNA452-5p expression and tissue typing is not related to the collection of colorectal cancer histopathology are adenocarcinoma,and did not make specific differentiation,it is not related.The relationship between miRNA215-5p expression and clinicopathological features of patients with colorectal cancer:(1)The expression of miRNA215-5p was not related to age,the relative expression of miRNA215-5p was 3.95(2.76,5.54)>60 years old,and the relative expression of miRNA215-5p was 3.22 2.65,7.11).There was no significant difference between the two groups(Z =-0.053,P = 0.958).The expression of miRNA215-5p was not related to sex.the relative expression level in male was 4.07(2.79,5.45).and the relative expression in female was 3.05(2.70,0.18).There was no significant difference between the two groups(Z =-0.370.P = 0.711).?miRNA215-5p expression was not related to the degree of differentiation,the relative expression of low-moderate differentiation group was 4.25(2.74.8.64),the relative expression of well-differentiated group 3.58(2.68,5.06),there was no significant difference between the two groups(Z =-0.542,P = 0.588).The expression of miRNA215-5p was not related to the tumor location.The relative expression level of rectal group was 2.65(1.86,4.22).while the relative expression level of colon group was 4.07(2.79,7.11).There was no significant difference between the two groups(Z ? 1.756,P = 0.079).?miRNA215-5p expression was not related to the depth of invasion,T2 group relative expression was 3.13(2.68,5.58),T3 + T4 group relative expression was 4.47(2.53,6.74),between the two groups,the difference was not statistically significant(Z=-1.091,P ? 0.275).?miRNA215-5p expression was not related to lymphatic metastasis,the relative expression of lymphatic metastasis group was 3.13(2.78,5.11),the expression of lymphatic metastasis group was 4.41(2.53,6.74),no significant difference between the two groups Significance(Z =-0.105,P = 0.916).?miRNA215-5p expression was not related to Dukes stage,the relative expression of A+ B group was 3.13(2.75,5.11),the expression of C + D group was 5.25(3.21,6.74),the difference between the two groups was no Statistical significance(Z =-0.849,P =0.396).?miRNA215-5p expression and tissue typing is not related to the collection of colorectal cancer histopathology are adenocarcinoma,and did not make specific differentiation,it is not related.(4)The AUC of plotting ROC curve and area under the curve showed that the area under the curve of miRNA452-5p curve was 0.859(P<0.001,95%CI 0.719-0.999),and the area under the miRNA 215-5p curve was AUC 0.879(P<0.001,95%CI = 0.757-1.0).The binary logistic regression method was used to introduce miRNA452-5p and miRNA215-5p into the regression equation to construct 2-miRNAs panel.The area under the 2-miRNAs panel curve was AUC =0.930(P<0.001,95%CI ? 0.833-1.0).[Conclusion](1)The expression of miRNA452-5p was significantly downregulated in colorectal cancer tissues,the expression of miRNA215-5p was significantly up-regulated in colorectal cancer tissues,and the expression of miRNA452-5p and miRNA215-5p in colorectal cancer correlated with the age,sex and differentiation,Tumor location,depth of invasion,lymphatic metastasis,Dukes stage and histological type were not correlated,and both of them were of diagnostic value for colorectal cancer.(2)Construction of 2-miRNAs panel,which has high diagnostic value for colorectal cancer and can be used as a potential molecular marker and therapeutic target for the diagnosis of colorectal cancer. |
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