| [Objective]Colorectal cancer is one of the three common malignant tumors,and its morbidity and mortality are high.Colonoscopy is currently a common method for monitoring colorectal cancer,but it is an invasive and invasive test,and it is difficult to find tiny flat lesions in colonoscopy,its role in monitoring colorectal cancer still exists.Limitations are urgently needed for the discovery of a non-invasive biomarker.MicroRNAs are differentially expressed in colorectal cancer tumor tissues and adjacent normal tissues,and play an important regulatory role in the development of tumors.Earlier in this study,high-throughput gene chip analysis of colorectal cancer tumor tissues and adjacent normal tissues revealed that miR-7-5p expression was significantly down-regulated in colorectal cancer tumor tissues,however,its specific mechanism of action in colorectal cancer remains unclear.In order to further confirm the results of the chip,this study will detect the expression of miR-7-5p in tumor tissues and adjacent normal tissues of 10 patients with colorectal cancer.[Methods](1)Collecting 10 groups colorectal cancer tissue specimens and normal intestinal mucosa tissue from patients with a colorectal cancer whose pathological diagnosis of colorectal cancer was performed at the digestive endoscope center of the First Affiliated Hospital of Kunming Medical University from January 2018 to October 2018.All patients included in the study have signed the informed consent form,and there is no history of tumor and surgery,and no history of chemoradiotherapy and other immunobiological treatments.Clinicopathological data were collected on patient age,gender,tumor location,degree of differentiation,depth of invasion,and tissue type.RT-PCR technology was used to compare and analyze the difference between miR-7-5p expression in colorectal cancer tissues and adjacent normal tissues,and analyze the relationship between miR-7-5p and clinicopathological data.(2)The target genes of miR-7-5p were predicted by the bioinformatics database TargetScan,miRPathDB,miRDB,miRWalk,and the target genes with higher predicted value and high intersection were selected as the targets of miR-7-5p.Western blot was used to detect the expression of miR-7-5p target genes in colorectal cancer tissues and adjacent normal tissues.The miR-7-5p inhibitor and miR-7-5p mimic were transfected into CaCo2 cell lines,and the targeting relationship between the two was verified by Western blot technology.(3)The miR-7-5p mimic was transfected into CaCo2 cell line,and the proliferation of cells after overexpression of miR-7-5p was detected by the CCK-8 method,the TUNEL method combined with flow cytometry was used to detect the apoptosis of cells after overexpression of miR-7-5p,detecting the mRNA levels of proliferation and apoptosis related molecules(Bax,Bcl-2,Caspase-3,MMP2)by RT-PCR,by regulating the expression of miR-7-5p in colon cancer cell line CaCo2 to observe its effect on colon cancer cell proliferation and apoptosis,and to explore its internal mechanism.[Results](1)The results of RT-PCR technology showed that miR-7-5p was lower expressed in colorectal cancer tissue(0.409±0.095)compared with normal tissue adjacent to cancer(1.000±0.014),and the difference was statistically significant(P<0.01).MiR-7-5p expression in colorectal cancer tissues and patient age(t=1.06,p=0.694),gender(t=1.345,p=0.068),tumor location(t=4.142,p=0.757),Depth of infiltration(t=0.698,p=0.318),degree of differentiation(t=-0.592,p=0.757),and lymphatic metastasis(t=0.081,p=0.072)were not related.(2)The target genes of miR-7-5p were predicted through the bioinformatics database TargetScan,miRPathDB,miRDB,miRWalk,and the target gene TFF3 with higher predicted value and high intersection was selected as the target of miR-7-5p.Western blot results It showed that TFF3 was highly expressed in colorectal cancer tissue(1.575±0.012)compared with normal tissue adjacent to it(1.000±0.020),and the difference was statistically significant(P<0.01).And Western blot results showed that the expression of TFF3 protein was negatively correlated with the expression level of miR-7-5p.After overexpressing miR-7-5p in colon cancer cell CaCo2,the overexpressing miR-7-5p control group(0.729±0.041),the blank group(normally cultured colon cancer CaCo2 cells)(1.000±0.066),and the blank control group(Mimic control transfected with colon cancer cells)(0.847±0.042)could inhibit the expression of intestinal trefoil factor(TFF3),and the difference was statistically significant(P<0.001).After inhibiting miR-7-5p in colon cancer cell CaCo2,the blank group(normally cultured colon cancer CaCo2 cells)(1.000±0.022),the blank control group(colon cancer cell transfected mimic control)(0.984±0.066),comparing with the miR-7-5p inhibitor control group(1.000±0.041),the miR-7-5p inhibitor control group significantly increased the TFF3 protein level,the difference was statistically significant(P<0.05).(3)After miR-7-5p mimic was transfected into CaCo2 cell line,RT-PCR was used to verify that the transfected miR-7-5p control group(914.490±86.630)could successfully overexpress miR-7-5p compared to the corresponding blank group(1.000±0.200)and blank control group(1.720±0.060)(P<0.01).CCK8 cell viability test found that the blank group had almost no effect on cell viability(1±0.007),while the overexpressing miR-7-5p control group(0.930±0.007)significantly reduced the relative survival rate of the cells compared with the blank control group(0.990±0.005)(P<0.001).Observation under the microscope showed that compared with the blank group and the blank control group,the miR-7-5p overexpression control group could significantly reduce the number of cells.The effect of miR-7-5p overexpression on the apoptosis of CaCo2 cells was detected by flow cytometry.It was found that the overexpression miR-7-5p control group can significantly increase the proportion of early apoptotic cells(50.700±0.989)and late apoptotic cells(40.525±0.515),and reduce the proportion of living cells(0.455±0.422),and the effect of dead cells(1.295±0.074)is smaller.Fluorescence detection of the effect of miR-7-5p overexpression on CaCo2 cell apoptosis revealed that after miR-7-5p was overexpressed in colon cancer cell CaCo2,transfection with mimic control had no effect on apoptosis,and overexpression of miR-7-5p can significantly increase Annexin V-FITC(green fluorescence)and propidium iodide(red fluorescence).RT-PCR detection found that overexpression of miR-7-5p in colon cancer cell CaCo2 can significantly increase mRNA expression levels of Bax,Bcl-2,Caspase-3,and MMP2.[Conclusion](1)miR-7-5p is lowly expressed in colorectal cancer tissues,and its expression level in colorectal cancer is not related to patient age,gender,tumor location,depth of invasion,degree of differentiation,lymphatic metastasis,and so on.(2)miR-7-5p can participate in regulating the occurrence and development of colorectal cancer by targeting the TFF3 gene.(3)miR-7-5p can inhibit the proliferation of colorectal cancer and promote apoptosis by inhibiting its target gene TFF3,thereby exerting its tumor suppressive effect. |
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