| Background:Colorectal cancer(CRC)is one of the most common malignant tumors endangering human health worldwide.The incidence of CRC is increasing gradually in China.The occurrence and development of CRC involve a large number of genomic,transcriptomic,and proteomic interaction networks and regulatory pathways.A thorough elucidation of the molecular mechanisms behind CRC biology is critical for the early diagnosis and the development of personalized treatment for CRC.As key molecules of the EGFR signaling pathway,KRAS,NRAS,and BRAF gene mutations have been proved to play an important role in the onset and development of CRC.The mutation status of these three genes is an important reference value for choosing the most suitable personalized treatment for CRC patients.Most of the present studies focus on the frequency and prognostic value of KRAS,NRAS,and BRAF mutations.However,the relationship between these mutations and clinicopathological characteristics of CRC patients and the expression of other genes is still poorly understood.Therefore,in this study we further analyzed the relationship between these gene mutations and clinicopathological parameters in CRC patients.Circular RNA(circRNA)is a new family of noncoding RNA composed of covalently closed RNA molecules.CircRNA has become a research hotspot in recent years because,thanks to its closed-loop,it is characterized by high stability,strong specificity,and easy detection.It is expected that circRNA could become the most promising molecular marker for tumor diagnosis and prognosis evaluation.KRAS gene mutation has been proved to play an important role in the onset and development of CRC.However,we wondered whether KRAS was involved in CRC progression through other mechanisms besides mutation.We also wondered whether the KRAS gene was regulated by circRNA in the context of CRC.To the best of our knowledge,no similar studies have yet been reported.Objective:In this study,we detected common mutations in KRAS,NRAS,and BRAF genes by amplification-resistance mutation system polymerase chain reaction(ARMS-PCR)in 216 CRC tissues.In parallel,we used immunohistochemical staining to detect the expression level of CRC-related genes in protein level.The relationship between gene mutation,clinicopathological characteristics,and the gene expression level was analyzed with a variety of statistical methods,to generate more robust data for developing a personalized treatment of CRC patients.In addition,based on KRAS,NRAS,and BRAF mutations,we selected the most important candidate genes for further research.qRT-PCR was used to detect the expression of KRAS in CRC and adjacent tissues.To elucidate the involvement of noncoding RNA in CRC regulation,we used a bioinformatics method to predict the key circRNA/miRNA/KRAS regulatory axis in the development of CRC.We confirmed the existence of this axis at the molecular,protein,and cell function level.Thus,we demonstrated that noncoding RNAs have a role in regulating KRAS gene expression in the onset and development of CRC.This discovery set the foundation for the identification of new potential candidate biomarkers and molecular targets for the diagnosis and therapy of CRC.Method:1.A total of 216 CRC tissues were collected from patients.The mutation status of KRAS,NRAS,and BRAF in the tissues was detected by ARMS-PCR.?~2 test,Mann Whitney rank sum test,Student t-test were used to analyze the relationship between the mutations and clinical characteristics such as gender,age,tumor location,TNM stage,depth of invasion,lymph node metastasis,pathological type,and differentiation degree.2.The expression of p53,EGFR,CDX2,PMS2,MLH1,MSH6,MSH2,and Ki67 was detected by immunohistochemistry.?~2 test,Mann Whitney rank-sum test,and multiple correspondence analysis were used to investigate the correlation between the expression of these proteins and KRAS,NRAS,and BRAF mutations.3.A total of 40 pairs of CRC and the adjacent non-tumor tissues were collected from patients,and the expression of KRAS was detected by qRT-PCR.Using a bioinformatics pipeline,we identified the circRNA and miRNA molecules involved in the proliferation and invasion of CRC and with the potential to be upstream regulators of KRAS gene expression.Thus,we established a theoretical circRNA/miRNA/KRAS regulation axis.We identified two candidates:the circRNAs circGLG1 and the miRNA miR-622.The circGLG1 and miR-622 expression levels were detected by qRT-PCR in 40 pairs of clinical samples.Then the relationship between the expression levels of these two noncoding RNAs and KRAS was analyzed by the Pearson linear correlation method.4.We detected the expression levels of circGLG1 in three colon cancer cell lines(HCT8,HCT116,DLD1)and a normal human intestinal epithelial cell line(NCM460).siRNAs against circGLG1 were designed and transfected into DLD1 cells using Lipofectamine 2000 along with control siRNAs.After knockdown of circGLG1in DLD1 cell line,we performed the following tests:1)CCK-8 and clone formation tests were used to detect cell proliferation activity;2)Transwell and scratch healing tests were used to detect cell invasion and migration activity;3)miR-622 expression was detected,along with KRAS expression,the latter both at the mRNA and protein levels.Furthermore,a miR-622 inhibitor was co-transfected into DLD1 cells knocked down for circGLG1.Then,cell proliferation,invasion,migration activity,and expression of KRAS were tested.5.We designed and cloned the wild type and the mutant double luciferase reporter plasmids of circGLG1 and KRAS mRNA.The binding of miR-622 with circGLG1 and KRAS mRNA was further verified by the double luciferase reporter test.Result:1.In CRC patients,the mutation rate of the KRAS gene was 39.8%.The mutations detected were the following:G12D(16.2%),G12V(8.8%),G13D(8.3%),G12S(1.9%),G12C(1.9%),G12A(1.4%),G12R(0.9%),and a double mutation(G12D+G12S)(0.5%).The mutation rate of NRAS was 2.7%,and the mutations detected were G12D,G13D,and Q61R(0.9%each).The mutation rate of BRAF was1.4%,and the only mutation detected was V600E.The mutation rate of KRAS in female patients was higher than that in males(53.3%vs 32.6%,P=0.003),whereas NRAS and BRAF mutations were not significantly different among the two sexes.The mutations in KRAS,NRAS,and BRAF had no significant correlation with patients'age,TNM stage,depth of invasion,lymph node metastasis,histopathological type,and differentiation degree of the adenocarcinoma.2.The expression of the EGFR protein was correlated to NRAS mutation.The mutation rate of NRAS in the group positively expressing EGFR was higher than that in both the weak expression and the negative expression groups(5.3%vs 1.4%vs 0%,P=0.047).The expression of PMS2,MLH1,and MSH2 was correlated to BRAF mutation.The mutation rate of BRAF in the PMS2-negative expression group was higher than that in the positive expression group(16.7%vs 1%,P=0.010).The mutation rate of BRAF in the MLH1-negative group was higher than that in both the weak expression group and the positive expression group(16.7%vs 7.7%vs 0.5%,P=0.001).The mutation rate of BRAF was higher in the MSH2-negative group than that in the MSH2-positive group(33.3%vs 1.0%,P=0.005).3.The average expression level of KRAS in CRC tissues was significantly higher than that in the adjacent non-tumor tissues(P<0.001).Using bioinformatics analysis,we hypothesized that circGLG1 may regulate the proliferation and invasion of CRC through a circGLG1/miR-622/KRAS axis.We found that indeed the expression of circGLG1 was up-regulated(P<0.01)and positively correlated with the expression of KRAS in CRC tissues(R~2=0.586,P<0.001).In parallel,we found that the expression of miR-622 was significantly down-regulated(P<0.01)and negatively correlated with the expression of KRAS in CRC(R~2=-0.339,P<0.05).4.We found that the expression of circGLG1 in CRC cells was higher than that in normal intestinal epithelial cells,and it was especially higher in DLD1 cells(P<0.001).Thus,we designed two siRNAs against circGLG1 and knocked down its expression in DLD1 cells and tested the following:1)CCK-8 and clonal formation tests confirmed that the proliferation of DLD1 cells was inhibited;2)Transwell and scratch healing tests confirmed that the invasion and migration of DLD1 cells were inhibited;3)miR-622 expression increased and KRAS expression decreased,both at the mRNA and the protein level.Then,we co-transfected a miR-622 inhibitor to downregulate miR-622 activity in DLD1 cells.In these cells,the effect of circGLG1on cell proliferation and invasion and the expression of miR-622 and KRAS were lost.5.Double luciferase reporter assays showed that when expression vectors for wild type circGLG1/KRAS and a miR-622 mimic were co-transfected,the luciferase activity in DLD1 cells was significantly reduced.However,when a miR-622 mimic and vectors containing mutant forms of circGLG1/KRAS were co-transfected,we observed no significant difference in luciferase activity.These results suggest that miR-622 physically interacts with circGLG1 and KRAS mRNA.In conclusion,we detected KRAS,NRAS,and BRAF gene mutations in CRC patients and analyzed the relationship between different clinicopathological characteristics and gene mutations.We found that the EGFR expression level correlates to NRAS mutation.To our knowledge,this is the first time that this correlation has been reported.We also confirmed that PMS2,MLH1,and MSH2expression levels were correlated to BRAF mutations.In addition,we confirmed that KRAS is differentially expressed in CRC tissues and the adjacent non-tumor tissues.We found a KRAS regulatory mechanism affecting cell proliferation and invasion involving the noncoding RNAs circGLG1 and miR-622.Our study provides a new theoretical and experimental basis for the molecular mechanism of CRC regulation,providing new potential biomarkers and key molecular targets for the diagnosis and treatment of CRC. |
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