| Colorectal cancer(CRC)is one of the most common cancers in the world,with morbidity and mortality ranking in the top five.The incidence of colorectal cancer in China has steadily increased over the past 30 years,creating a huge burden on families and society.Postoperative chemoradiotherapy is the main treatment for patients with advanced colorectal cancer.while,due to primary or secondary chemotherapy resistance,especially multidrug resistance(MDR),the effect of chemotherapy is poor and the prognosis of patients is poor.This phenomenon is the main obstacle in clinical treatment.MDR is defined as resistance to a drug.They also resist a range of anti-cancer drugs that are not related to the structure or mechanism.The occurrence and development of MDR involves many gene changes and effects.Among them,MDR1(multidrug resistance protein 1)in the ABC transport protein family was studied mostly.MDR1 coding protein P-gp(P-glycoprotein)output pump is a kind of ATP energy dependence drugs,drugs in the cell can be pumped to extracellular,thereby reducing drug in the accumulation of intracellular,led to the decrease of the effect of chemotherapy 。 In recent years,microRNAs(microRNAs)have received widespread attention.miRNA is a kind of non-coding small molecule RNA which is widely existed in many kinds of organisms and has about 20 nucleotide sequences,and has the function of regulating gene expression.Mature microRNAs combine with RNA-induced silent complexes(RISC)and form RISC complexes,which can specificity recognition target mRNA 3 ’noncoding region,thus cause the degradation of the target mRNA,translation,after the transcription level control of one or more of the gene expression.MiRNA,as an endogenous small molecule RNA,with abnormal expression in almost all tumor individuals.miR-143 is located on human chromosome 5 and has a precursors of the stem ring structure.It plays an anti-cancer role in a variety of tumors.Some studies have found that miR-143 abnormal expression can regulate cell proliferation,cell growth,clone formation,apoptosis,cell cycle,etc.It has been reported that miR-143 can inhibit the expression of cyclin D1 in prostate cancer cells.However,the relationship between miR-143-3p and multidrug resistance in colorectal cancer is rarely reported.ObjectiveThis experiment in the level of organization through the detection of miR-143-3p,the NF-kappa B p65 and P-gp expression in colorectal cancer tissue and normal mucosa tissues,analysis of miR-143-3p,the NF-kappaB p65,P-gp’s relationship with clinical pathology characteristic and the correlation of the three.The level of cell was expressed by transfection of miR-143-3p mimics、 miR-143-3p inhibitor 、 miR-143-3p mimics NC and inhibitor detection NF-kappaBp65,P-gp mRNA expression,and the correlation was analyzed.Method1.30 cases who received the first treatment underwent surgical resection and postoperative pathology confirmed is colorectal cancer From June 2016 to march,2017 in a number of hospitals in chengde city,hebei province.。Among the 110 cases of paraffin specimens,69 were colorectal cancer tissues,and 41 cases of normal intestinal mucosal tissue(over ten centimeters from the edge of the lesion).There were 80 fresh tissues,including 50 cases of colorectal cancer and 30 cases of normal intestinal mucosal tissue(over 30 centimeters from the edge of the lesion).2.Using immunohistochemical SP method to detect the NF-kappa B and P-gp p65 protein in colorectal cancer tissues and normal mucosa protein expression levels in the organization,and analyzes the relationship of NF-kappa B p65 protein and P-gp the between the expression and clinical pathological features and the correlation of expression in colorectal cancer tissues.3.Western blot was used to detect the expression of NF-kappa B p65 protein and P-gp in colorectal tissue and normal intestinal mucosal tissues.4.The real-time fluorescent quantitative PCR technology(qRT-PCR)to detect the expression of NF-kappa B p65 mRNA and P-gp mRNA in large intestinal carcinoma tissues and normal mucosa tissues,further analysis the NF-kappaB p65 mRNA in colorectal cancer tissue and P-gp mRNA expression and the relationship between the clinical pathological features.5.The expression of NF-κB p65 and MDR1 mRNA was observed by transfection of miR-143-3p mimics miR-143-3p miR-143-3p miR-143-3p inhibitor NCand miR-143-3p mimics NC negative control.6.Statistical analysis was performed using SPSS19.0 statistical software,and the experimental results were obtained.Result1 Immunohistochemical results.1.1 the expression of NF-kappaB p65 protein and P-gp in colorectal tissue and normal mucosal tissues.The positive expression rate(81.4%)was significantly higher in colorectal cancer tissues(81.4%)than normal mucosa(24.4%),and the difference was statistically significant(P<0.05).The positive expression rate of P-gp in colorectal cancer was significantly higher(89.9%)than that of normal mucosa(19.5%),and the difference was statistically significant(P<0.05).1.2 the relationship between NF-kappaB p65 protein and P-gp expression and clinical pathological features in colorectal cancer.In colorectal cancer,the expression level of NF-kappaB p65 was correlated with the degree of differentiation,infiltration depth and lymph node metastasis of colorectal cancer,and the difference was statistically significant(P < 0.05).The expression level of P-gp in colorectal cancer was correlated with the differentiation degree and infiltration depth of colorectal cancer,and the difference was statistically significant(P < 0.05).1.3 correlation between expression of NF-kappaB p65 protein expression in colorectal cancer and P-gp expression.In colorectal cancer,the expression of NF-kappaB p65 protein and P-gp was positively correlated,and the difference was statistically significant(rs=0.490,P < 0.05).2.Western Blot experiment results.In colorectal cancer tissues,the expression of NF-kappaB p65 protein was significantly higher than that of adjacent normal mucosal tissue,and the difference was statistically significant(P<0.05).The expression of P-gp in colorectal cancer tissues was significantly higher than that of the normal mucosal tissue adjacent to cancer,and the difference was statistically significant(P<0.05).3.Experimental results of real-time fluorescence quantitative PCR(qRT-PCR).3.1 the expression of P-gp mRNA and NF-kappaB p65 mRNA in colorectal cancer tissues was significantly higher than that of normal intestinal mucosa,and the differences were statistically significant(P<0.05).The expression of mir-143-3p in colorectal cancer tissues was significantly higher than that of normal intestinal mucosa,and the difference was statistically significant(P<0.05).3.2 Expression of P-gp mRNA and NF-kappa B p65 mRNA in colorectal cancer tissue and tissue differentiation degree,the Duke ’s stage,lymph node metastasis,the differences were statistically significant(P< 0.05),while with gender,age,tumor location,tumor shape,infiltration depth were unrelated,there were no statistically significant differences(P > 0.05).3.3 The expression of miR-143-3p in colorectal carcinoma was related to the differentiation of colorectal carcinoma and the stage of differentiation,lymph node metastasis and depth of invasion(P < 0.05),but not to sex,age,tumor location and tumor morphology.The difference was not statistically significant(P > 0.05).3.4 The expression of miR-143-3p expression in colorectal cancer tissues and MDR1 mRNA expression was shown to be negatively correlated with the expression of miR-143-3p in colorectal cancer tissues and the expression of P-gp mRNA,with statistical significance(r=-0.467,P<0.01).3.5 the expression of NF-κB p65 mRNA and MDR1 mRNA in colorectal cancer cells decreased 48 h after transfection with miR-143-3p mimics.3.6 the expression of NF-κB p65 mRNA and MDR1 mRNA in colorectal cancer cells was significantly higher than that in control group 48 h after transfection with miR-143-3p inhibitor.3.7 the expression of NF-κB p65 mRNA and MDR1 mRNA in colorectal cancer cells transfected with miR-143-3p mimics NC and miR-143-3p inhibitor NC for 24 hours was similar to that in control group.Conclusion1.The expression of NF-κB p65 protein and P-gp in colorectal carcinoma was significantly higher than that in adjacent normal mucosa,which was positively correlated with the expression of NF-κB p65.It was confirmed that both of them were involved in the carcinogenesis and development of colorectal cancer.2.The expression of NF-κB p65 mRNA and MDR1 mRNA in colorectal carcinoma was significantly higher than that in adjacent normal mucosa tissues,which was positively correlated with the expression of NF-κB p65 mRNA and MDR1 mRNA.3.The decrease of miR-143-3p expression in colorectal carcinoma was related to the stage of differentiation,depth of invasion and lymph node metastasis.There was a negative correlation with the expression of NF-κB p65 mRNA and MDR1 mRNA.4.MiR-143-3p regulates the expression of NF-κ B p65 mRNA in colorectal cancer cells. |
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