| Background:As one of the most common malignancies,colorectal cancer(CRC)ranked third in the incidence and second in mortality.Although in recent decades,due to advances in diagnosis and treatment,the survival benefits of CRC patients worldwide have been significantly improved,but the morbidity and mortality of CRC still showed upward trend in China.No obvious symptoms were observed in the early stage of CRC,resulting in large numbers of patients were advanced stage or even late when diagnosed.Thus,early detection and effective non-invasive biomarker research are essential to promote early diagnosis and reduce CRC mortality.Micro RNAs(miRNAs)are a class of mature non-coding RNAs(typically 21–25nucleotides).They mainly regulate the expression of cancer-related genes in cells and tissues by directly silencing target mRNA or inhibiting protein biosynthesis.As potential biomarkers,miRNAs can be detected in human body fluids and tumor tissues.Due to their influence on oncogenesis and progression of cancer,miRNAs have been widely used in the field of diagnosis and prognosis of diverse malignant solid tumors.As they are mainly packaged in various extracellular vesicles(especially exosomes)or combined with protein complexes,circulating miRNAs are very stable,which makes them advantageous as non-invasive biomarkers in the early detection of tumors.miRNAs are involved in the transmission and inhibition of many signal pathways,and have great potential in the field of diagnosis,prognosis,and personalized targeted therapy in CRC.Although the diagnostic value of a few miRNAs has been confirmed in previous studies,there still need to explore more miRNAs to be used as potential biomarkers in CRC.Multi-dimensionally comprehensive and systematic evaluation of the biological functions of miRNAs in CRC will help to explore the rationale and mechanism of CRC and also provide a theoretical basis for prevention,early diagnosis as well as treatment of CRC.Purposes:1.Multi-dimensional screening of miRNAs related to the risk of CRC.2.Multi-dimensional identification of miRNAs related to the risk of CRC and their correlation with clinicopathological parameters.3.To explore the effects of these miRNAs on the biological behavior of colon cancer cells and related mechanism.Methods:Part 1: Multi-dimensional screening of miRNAs related to the risk of CRC1.Public database microarray analysisSearch and retrieve the relevant miRNA microarray information and analyze the results by GEO2 R through the GEO database.Collect the miRNA microarray information of tissues,serum,and exosomes,respectively.Sort each item according to sample numbers from high to low and exclude treatments such as radiotherapy and chemotherapy.Top 3 microarrays are chosen to analyze the results by GEO2 R.After taken intersection of all these results,multi-dimensional analysis of differentially expressed miRNAs related to the risk of CRC were performed.2.Self-prepared microarray analysis(1)A total of 9 cases of serum were collected in this part of study.6 CRC patients were enrolled who were admitted to Department of Anorectal Surgery,the First Hospital of China Medical University from June 2018 to December 2018.The TNM stage of all patients were at stage III and diagnosed as adenocarcinoma,including 3 cases of left-sided CRC and 3 cases of right-sided CRC.In addition,3 cases of non-cancer healthy controls were also enrolled.This study was approved by the Ethics Committee of the First Hospital of China Medical University,each subject signed informed consent forms.(2)After extracting serum exosomes and identifying them by transmission electron microscopy,RNA was extracted and then tested by Agilent miRNA microarray to analyze the difference of miRNAs among three groups of left-sided CRC,right-sided CRC,and non-cancer control.(3)Analysis of microarray results: After removing repetitive and non-specific probes,the adjusted P value<0.05,|log FC|≥1 and hsa-miRNA as screening conditions.Those which met the screening conditions are identified as differently expressed miRNAs in CRC.Part 2: Multi-dimensional identification of miRNAs related to the risk of CRC1.This study enrolled 56 CRC patients who took surgical operations at Department of Anorectal Surgery,the First Hospital of China Medical University from June 2018 to June 2019.All CRC tissue and paired normal mucosal tissue(distal margin)were removed and collected.A total of 100 cases of serum were also collected,including 51 CRC patients and 49 healthy non-cancer populations.This study was approved by the Ethics Committee of the First Hospital of China Medical University,each subject signed informed consent forms.2.The RNA from tissue,serum and exosomes were harvested and reverse transcribed into c DNA,respectively.2-ΔCt method was applied to calculate and access the relative expression levels of hsa-miR-1539,hsa-miR-6751-3p,hsa-miR-631,hsa-miR-4443,hsa-miR-6073,hsa-miR-1185-1-3p,hsa-miR-765,hsa-miR-150-5p,and hsa-miR-6086.Analyze their correlation with the risk of CRC,ΔCT=CT target miRNA-CT U6.The relationship between these target miRNAs and clinicopathological parameters were also analyzed in a multi-dimensional manner based on the patient’s pathological data and hospital admission records.Part 3: Effects and potential mechanism of hsa-miR-1539 on biological function of colon cancer cells1.This part of the research included 32 pairs of cancerous tissues and matched normal mucosal tissues of CRC patients who underwent surgical treatment at Department of Anorectal Surgery,the First Hospital of China Medical University from June 2018 to June 2019.HCT116 cell lines were purchased and obtained from Cell Bank of Chinese Academy of Medical Sciences.2.The effect of hsa-miR-1539 on the biological behavior of colon cancer cells We constructed three kinds of plasmids,including overexpression,knockdown,and negative control of hsa-miR-1539.All these plasmids were extracted and transfected into HCT116 cells.Universal microscope was used to evaluate the transfection efficiency of the plasmids.And the relative expression of hsa-miR-1539 was calculated to access the expression efficiency of the plasmids.Then these transfected cells were placed in 96 wells after transfection 24 h.The cell viability was observed by CCK-8 assay at 24 h,48h,72 h to evaluate the proliferation activity of HCT116 cells.In addition,the cell proliferation was also detected by EdU assay.Cell apoptosis was analyzed by detecting the mRNA levels of the pro-apoptotic indicator Caspase 3 and the anti-apoptotic indicator Bcl-2,as well as flow cytometry.3.Prediction,verification,and functional analysis downstream target genes of hsa-miR-1539The prediction of potential target genes of hsa-miR-1539 were described by bioinformatics.Besides,analysis of GO and KEGG pathway enrichment were also performed.These underlying target genes of hsa-miR-1539 were verified by RT-qPCR in tissue and dual luciferase reporter experiments.Furthermore,by constructing overexpression,knock-down and negative control plasmids,HCT116 cells were transfected with these plasmids to detect the mRNA expression of target gene to explore and confirm downstream target genes of hsa-miR-1539.Results:Part 1: Multi-dimensional screening of miRNAs related to the risk of CRC1.At tissue level,we analyzed the miRNA microarray information related to CRC tissues and normal mucosa tissue in the current public databases from different study populations.We chose the top three microarrays based on the number of samples(GSE115513,GSE18392,GSE48267).Taken the intersection of the results and we finally got 14 differently expressed miRNAs,including hsa-miR-30a*,hsa-miR-183,hsa-miR-1,hsa-miR-552,hsa-miR-31,hsa-miR-182,hsa-miR-503,hsa-miR-139-5p,hsa-miR-551 b,hsa-miR-133 a,hsa-miR-137,hsa-miR-224,hsa-miR-135 b,hsa-miR-378*.2.At serum level,we analyzed the miRNA microarray information related to serum from CRC patients and non-cancer populations.We chose the top three microarrays based on the number of samples in the current public databases(GSE106817 、GSE113740 、 GSE124158).Taking the intersection of results,we finally got 915 differently expressed miRNAs.3.At exosomal level,we analyzed the existing serum-based exosomes and cellular exosomes microarray information in public databases(GSE39833,GSE40247).The results of miRNA microarray analysis showed that there were 97 differentially expressed miRNAs in serum exosomes of CRC patients and 14 differentially expressed miRNAs were found in CRC cellular exosomes.Self-prepared serum exosomes miRNA microarray results showed that there were 15 differentially expressed miRNAs(10up-regulated miRNAs and 5 down regulated miRNAs)in LCRC,and a total of 14 differentially expressed miRNAs(8 up-regulated and 6 down regulated miRNAs)in RCRC,compared with non-cancer control group.There were three different miRNAs between LCRC and RCRC.4.Comprehensive analysis of self-provided exosomal miRNA microarray and GEO serum miRNA microarrays found that there were 18 differentially expressed miRNAs,namely hsa-miR-1539,hsa-miR-150-5p and hsa-miR-6751-3p,hsa-miR-4443,hsa-miR-631,hsa-miR-6073,hsa-miR-1185-1-3p,hsa-miR-6086,hsa-miR-5100,hsa-miR-4463,hsa-miR-4530,hsa-miR-6756-3p,hsa-miR-3663-3p,hsa-miR-630,hsa-miR-1246,hsa-miR-4310,hsa-miR-3180-5p,hsa-miR-1296-3p.Combined with the results of self-prepared exosomal miRNA microarray and GEO exosome miRNA microarray,hsa-miR-1539 was observed overexpression in serum-based exosomes from CRC patients,besides,hsa-miR-150 was also different in both GSE39833 and RCRC patients.Part 2: Multi-dimensional identification of miRNAs related to the risk of CRC1.Tissue level: RNA was harvested from 56 pairs of CRC cancerous tissue and matched normal mucosal tissue from distal margin,and then reversely transcribed them into c DNA.Based on the differentially expressed miRNAs of first part study,we selected9 hsa-miRNAs as target miRNAs,namely hsa-miR-1539,hsa-miR-6751-3p,hsa-miR-631,hsa-miR-4443,hsa-miR-6073,hsa-miR-765,hsa-miR-1185-1-3p,hsa-miR-150-5p and hsa-miR-6086.The expression levels of these 9 miRNAs were also detected and accessed by RT-qPCR in CRC tissue and matched normal mucosal tissue.The expression level of hsa-miR-1539(P value <0.0001),hsa-miR-6751-3p(P value<0.0001),hsa-miR-631(P value <0.0001),hsa-miR-4443(P value <0.0001),hsa-miR-6073(P value <0.001),hsa-miR-1185-1-3p(P value = 0.028),and hsa-miR-765(P value <0.001)were significantly up-regulated in CRC tissues.The expression level of hsa-miR-150-5p was significantly down-regulated in CRC tissues(P value=0.002),whilst no significant difference of hsa-miR-6086 expression level was shown(P value=0.186).The overexpression of hsa-miR-1539 in CRC tissue was significantly correlated with the increase of Ki67 index(P value=0.035).Up-regulation of hsa-miR-150-5p was significantly correlated with lymphatic metastasis(P value=0.014)as well as advanced TNM stages(P value=0.008).There was a trend of difference between hsa-miR-6751-3p expression level and tumor-infiltrating lymphocytes degree(P value=0.056).Hsa-miR-1185-1-3p was significantly related to lymphatic metastasis(P value<0.001),advanced TNM stages(P value=0.003),besides,its overexpression is associated with perineural invasion(P value=0.017)and poor pathologic differentiation(P value=0.041).Hsa-miR-4443 was associated with lymphatic metastasis(P value=0.004),advanced TNM stages(P value = 0.003)significantly,and its down-regulation was observed in the older group(P value = 0.04).Hsa-miR-6073 was positively correlated with high Ki67index(P value = 0.02).Hsa-miR-765 was also related to lymphatic metastasis(P value =0.05)and angiolymphatic invasion(P value= 0.037)significantly.Hsa-miR-6086 was related to lymphatic metastasis(P value = 0.004)and advanced TNM stage(P value =0.011).2.Serum level: SerumRNA was extracted from 51 CRC patients and 49 healthy controls,and reversely transcribed them into c DNA.And the correlation between the risk of CRC and relative expression levels of hsa-miR-1539,hsa-miR-150-5p and hsa-miR-6751-3p were detected and accessed by RT-qPCR.No significant differences were shown between hsa-miR-1539,hsa-miR-6751-3p and the risk of CRC(P values were 0.104 and 0.617,respectively).However,the expression level of hsa-miR-150-5p had a downward trend(P value = 0.079).In addition,subgroup analysis showed that the expression of serum-based hsa-miR-1539 was decreased in left-sided CRC(P value =0.031),besides,serum-based hsa-miR-150-5p was also downregulated in right-sided CRC significantly(P value = 0.010).Hsa-miR-1539 expressed differentially with diverse tumor location(P value=0.047),and at the same time,there was a trend of different expression of hsa-miR-150-5p(P value=0.064).The high level of VEGF was related to significant increased expression of hsa-miR-1539(P value=0.028)and hsa-miR-6751-3p(P value=0.002),whereas significantly decrease in the expression level of hsa-miR-150-5p(P value=0.018).Down-regulation of hsa-miR-150-5p was also associated with high EGFR expression(P value = 0.042).Moreover,overexpression of hsa-miR-6751-3p was associated with poor differentiation of tumor cells(P = 0.016).3.The exosomal level: After extracting serum exosomes by reagent kit assay,three classical and common approaches were adopted to identify exosomes,for instance,transmission electron microscopy(TEM),nanoparticle tracing analysis(NTA)and western blot.After proving success of this method,we extracted a total of 100 serum exosomal RNA from 51 CRC patients and 49 healthy controls,and then reversely transcribed them into c DNA.The relationship between hsa-miR-1539,hsa-miR-150-5p,hsa-miR-6751-3p and the risk of CRC were detected and identified by RT-qPCR approach.Hsa-miR-1539 expression level was elevated in CRC patients(P value=0.003),and the expression of hsa-miR-150-5p showed a downward trend(P value=0.063),and there was no difference in expression level of hsa-miR-6751-3p(P value =0.644).There was no significant difference between clinicopathological parameters and the overall expression levels of hsa-miR-1539,hsa-miR-150-5p and hsa-miR-6751-3p.However,the expression of hsa-miR-150-5p showed a downward trend(P value= 0.068)in poor differentiation group.Part 3: Effects and mechanism of hsa-miR-1539 on biological function of colon cancer cells1.Effect of hsa-miR-1539 on the biological behavior of colon cancer cellsCell proliferation effect: After constructing and transfecting overexpression,knockdown,and negative control plasmids of hsa-miR-1539 into HCT116 cells for 24 hours,the transfection efficiency is higher than 50%,accessed by universal microscope.The overexpression efficiency is 75.1 times that of the negative control(P value<0.01),and the knockdown efficiency is 52.9% that of the negative control(P value<0.01).The cell viability of HCT116 cells treated with different transfection plasmids was detected by the CCK-8 assay at 24 h,48h,and 72 h.The results displayed that OD value of knockdown group dropped significantly at 48 h,in contrast to negative control group(P value<0.05).Moreover,at 72 h,the OD value among three groups was significantly different(P value<0.01),in which the overexpression group augmented(P value<0.05),and the knockdown group was declined(P value<0.01).The proliferation effect of HCT116 cells transfected with three different plasmids was also detected by EdU assay.The results showed that the percentage of EdU positive cells was increased in overexpression group(P value < 0.01),whilst the percentage of EdU positive cells was decreased in knockdown group(P value < 0.01).Apoptosis effect: After transfection of HCT116 cells with overexpression,knockdown,and negative control plasmids of hsa-miR-1539 for 24 hours,mRNA level of Caspase 3(pro-apoptotic indicator)and Bcl-2(anti-apoptotic indicator)were assessed and calculated among these three groups.The mRNA level of Caspase 3 and Bcl-2 were significantly different(P value<0.05).The expression level of Caspase 3 was0.597±0.201 in the overexpression group and 1.522±0.233 in the knockdown group.The expression level of Bcl-2 in the overexpression group was 1.557±0.557,and the knockdown group was 0.779±0.058.The apoptosis effect of HCT116 cells transfected with three different plasmids was also detected by flow cytometry.The results showed that the proportion of early apoptotic cells was decreased in overexpression group(P value < 0.01),and the proportion of late apoptotic cells was increased in knockdown group(P value < 0.01).2.Prediction and function analysis of downstream target genes of hsa-miR-1539Potential target genes of hsa-miR-1539 were identified using an integrated analysis of miRWalk and Target Scan datasets.In total,155 potential genes were identified using synthesis analysis of public bioinformatics algorithms.GO analyses demonstrated that the majority of target genes were enriched in biological processes such as transcription,development and growth,and cell proliferation.At the same time,they were also enriched in molecular functions such as RNA polymerase II transcription factor activity and growth factor activity.Enrichment analysis of the KEGG pathway revealed that the target genes of hsa-miR-1539 may be involved in multiple cancer signaling pathways such as PI3K-Akt,MAPK,Ras,Rap-1,AMPK,Jak-STAT,HIF,p53,etc.3.Verification of downstream target genes of hsa-miR-1539 by RT-qPCR and dual luciferase experimentsThe potential target genes were identified by RT-qPCR at tissue level.The results showed that GJA4(P value = 0.003),ARFGAP(P value <0.001),ZHX3(P value =0.008),FLT1(P value = 0.004)were highly expressed in CRC tissues,whilst GPIHBP1(P value <0.001)and IGF1(P value <0.001)were downregulated in CRC tissues.There were no significant differences of NFIA,MMP19 and TNFRSF14(P values were 0.29,0.866,0.224,respectively)in CRC tissue versus normal tissue.The results of the dual luciferase experiment report indicated that compared two group of the hsa-miR-1539 vector plasmid + 3’UTR negative control plasmid and the hsa-miR-1539 vector plasmid + 3’UTR IGF1 wild type plasmid,the expression of luciferase differed by >20%(P value<0.001).Compared two groups of the hsa-miR-1539 vector plasmid+3’UTR IGF1 wild type plasmid group and hsa-miR-1539 vector plasmid+3’UTR IGF1 mutant plasmid group,the expression level of luciferase differed by >20%(P value<0.001).Therefore,hsa-miR-1539 can bind to the 3’UTR region of IGF1 and inhibit its luciferase activity.In contrast to hsa-miR-1539 vector plasmid +3’UTR negative control plasmid,the expression of luciferase of hsa-miR-1539 vector plasmid + 3’UTR GPIHBP1 wild type plasmid differed by <20%.Compared two groups of the hsa-miR-1539 vector plasmid + 3’UTR GPIHBP1 wild type plasmid and the hsa-miR-1539 vector plasmid + 3’UTR GPIHBP1 mutant plasmid,the expression of luciferase differed by <20%.Hsa-miR-1539 cannot suppress luciferase activity of GPIHBP1 due to unable to bind to 3’UTR region.4.Overexpression or knockdown of hsa-miR-1539 affect the expression of its downstream target gene IGF-1After overexpressing hsa-miR-1539 in HCT116 cell,the expression level of IGF-1was declined obviously(0.59±0.17,P value <0.05),in contrast to negative control group.However,with hsa-miR-1539 knocking down,the expression level of IGF-1 elevated dramatically(2.04±0.35,P value<0.05).Conclusions:1.This study screened out and identified a new panel of miRNAs related to the risk of CRC from multi-dimensional analysis of tissue,serum and exosomes.Among them,exosomal hsa-miR-1539 had high sensitivity in terms of diagnostic efficiency and serum-based hsa-miR-1539 had high specificity for warning the risk of left-sided CRC.Serum-based hsa-miR-150-5p may sign the risk of right-sided CRC.A panel of tissue-based miRNAs(hsa-miR-1539,hsa-miR-6751-3p,hsa-miR-631,hsa-miR-4443,hsa-miR-6073,hsa-miR-1185-1-3p,hsa-miR-765,hsa-miR-150-5p)may warn the risk of CRC and were related to clinicopathological parameters.2.Hsa-miR-1539 is capable of promoting cell proliferation and suppressing apoptosis in CRC.Hsa-miR-1539 may perform biological functions by regulating its downstream target gene IGF-1. |
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