Discovery Of Exosome-Delivered MiR-185-5p In The Plasma Of Patients As An Indicator For Colorectal Tumors And Mechanism Study

Background and objectiveThe overall global incidence of colorectal cancer(CRC)has risen to the third highest and is the second leading cause of cancer death in 2020.Based on projections of population age,growth base,and human development,the number of new CRC cases is expected to reach 3.2 million globally in 2040.In clinical practice,most CRC develops from adenomas.The disease progression process includes early-stage adenoma,advanced adenoma(AA),early-mid stage CRC and advanced CRC.Research shows that effective screening and early intervention of tumor patients in the early stage of disease can significantly improve the survival rate of patients.Therefore,effective screening of colorectal AA and early diagnosis of CRC in high-risk population can reduce the incidence rate and mortality of CRC every year.The "liquid biopsy" technique based on circulating blood to detect specific tumor markers for the diagnosis of malignant tumors is a relatively non-invasive,simple and accurate method with good promise for early diagnosis of tumors.Although the commonly used clinical tumor markers CEA and CA199 have shown high value in the diagnosis of advanced CRC,they have limitations in the diagnosis of colorectal AA and early-mid stage CRC.Therefore,there is an urgent need to explore potential circulating specific markers that can reflect the progression of colorectal tumors and play a continuous tracing diagnostic role for colorectal tumors to enable screening of colorectal AA and early-mid stage CRC in high-risk populations.Meanwhile,an indepth exploration of its related molecular mechanisms affecting the development of CRC will provide an important theoretical basis for molecularly targeted therapies for CRC.Exosomes are phospholipid bilayer structured vesicles released by cells that act as carriers for intercellular communication.Exosomes derived from tumor cells can transport the informative material they carry,such as micro RNAs(mi RNAs),from donor cells to recipient cells to perform biological functions.In various types of tumors,the expression of mi RNAs in circulating exosomes of patients has been reported to differ from that of healthy volunteers and is highly effective for early-stage tumor diagnosis.Therefore,circulating exosome-mi RNA expression has been used as a new class of biological markers for the diagnosis of tumors.Investigating the mechanisms of exosome-delivered mi RNAs that regulate tumor development as biological markers may also provide ideas for the discovery of new molecular targeted therapeutics.The purpose of the study is to explore a biological marker with high diagnostic efficacy for both colorectal AA and CRC,and to continuously tracing the diagnosis of the disease via plasma exosome RNA deep sequencing screening and cohort validation.Furthermore,we will further explore the biological functions and molecular mechanisms of target mi RNAs affecting CRC progression,and provide new clues for CRC molecular precision therapy.Part I Screening and validation of biological markers for plasma exosome-delivered mi RNAs in colorectal AA and CRCMethods1.Plasma samples were collected from clinical patients and divided into healthy volunteers(Healthy donor,HD),AA and,CRC groups based on colonoscopy and histopathological results.2.Plasma exosomes were extracted by precipitation method,and exosome morphology,size concentration and exosome surface specific protein markers were identified by transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA)and western blotting assay(WB)from screening stage(including 6 HD,6 AA,and 6 stage I-II CRC patients plasma samples).3.Candidate exosome-mi RNAs were screened by deep RNA sequencing after identification of exosomes.4.Based on the sequencing results,the expressions of candidate mi RNAs were screened for continuous increase or decrease in plasma exosomes from HD to AA,and further to CRC group(screening criteria: fold change>1.5 or <0.67 between the two groups,P<0.05).5.Taq Man mi RNA assays were used to validate the expressions of candidate mi RNAs in two independent cohort samples(Cohort I: 22 HD,24 AA and 29 stage III CRC patients plasma samples;Cohort II: 22 HD,41 AA and 35 stage III-IV CRC patients plasma samples).6.The receiver operating characteristic(ROC)curve was constructed and the efficacy of candidate mi RNAs for the diagnosis of AA and CRC was quantified using the area under curve(AUC);the AUC values were compared with biological markers CEA and CA199 in clinical.Results1.According to the results of exosome RNA deep sequencing,mi R-185-5p,mi R-126-3p,mi R-30d-5p and mi R-4433b-3p expressions showed a continuous increase or decrease in plasma exosomes from HD to AA,and further to CRC group.2.Validation by cohort samples,mi R-185-5p expression showed a significant continuous increase in plasma exosomes from HD to AA,and further to CRC group.3.The AUC values for mi R-185-5p diagnosis of AA(AA vs.HD)were 0.737 and0.720 in Cohort I and Cohort II,respectively.4.The AUC values for mi R-185-5p diagnosis of stage I-II CRC and stage III-IV CRC were 0.887 and 0.803 in Cohort I and Cohort II,respectively(CRC vs.HD).By significance analysis,the AUC value for mi R-185-5p diagnosis of stage I-II CRC was significantly higher than that of CA199(p=0.0334);the diagnostic specificity and sensitivity of mi R-185-5p were higher than that of CEA and CA199.The AUC value of mi R-185-5p for the diagnosis of stage III-IV CRC were not significantly different from that of CEA and CA199.5.The AUC values for mi R-185-5p diagnosis of stage I-II CRC and stage III-IV CRC were 0.70 and 0.631 in Cohort I and Cohort II,respectively(CRC vs.AA),and the results of significance analysis showed no significant difference with both CA199 and CEA.6.Univariate and multivariate logistic regression analysis showed that exosomedelivered mi R-185-5p expression was an independent factor for the diagnosis of colorectal AA,stage I-II and stage III-IV CRC.ConclusionExosome-delivered mi R-185-5p could serve as a biomarker for continuously tracing the transition from precancerous lesions(AA)to tumorigenesis(CRC stages I–II)and further to tumor progression(CRC stages III–IV).Part II Exploring the effect of mi R-185-5p on the malignancy of CRCMethods1.A total of 28 pairs of tumor tissues and adjacent normal tissues were collected from clinical CRC patients for the detection of mi R-185-5p expression by RT-q PCR.2.Culture of colonic normal epithelial cell line FHC and CRC-derived cell lines(SW480,Caco-2,HCT116,RKO)and detection of mi R-185-5p expression in the cell lines by using RT-q PCR assay.Supernatant exosomes were extracted from FHC cell lines and CRC-derived cell lines by precipitation method,and mi R-185-5p expression in supernatant exosomes was detected by Taq Man mi RNA assay.Comparison of the proliferation capacity of FHC and four CRC-derived cell lines by 48-h cell proliferation assay.3.Increasing mi R-185-5p expression in cell lines(with NC mimics as negative control)by transfection of human(RKO and SW480)and mouse(MC38)CRC-derived cell lines with mi R-185-5p mimics.The effect of increased mi R-185-5p expression on cell line proliferation and migration ability was tested by CCK-8 assay,cell wound healing assay and cell transwell migration assay.4.Down-regulation of mi R-185-5p expression in cell lines(with NC inhibitors as negative control)by transfection of human(RKO and SW480)and mouse(MC38)CRC-derived cell lines with mi R-185-5p inhibitors(inhibitors).The effect of inhibited mi R-185-5p expression on cell line proliferation and migration ability was tested by CCK-8 assay,cell wound healing assay and cell transwell migration assay.5.Overexpression of mi R-185-5p(RKO-Lv-185)and control(RKO-Ctrl)RKO stable cell lines were constructed for subcutaneous tumor bearing mice.The growth volume and mass of subcutaneous tumors in the two groups were compared by measurement and weighing.6.Overexpression of mi R-185-5p(MC38-Lv-185)and control(MC38-Ctrl)MC38stable cell lines were constructed for subcutaneous tumor bearing mice.The growth volume and mass of subcutaneous tumors in the two groups were compared by measurement and weighing.Detection of mi R-185-5p expression in subcutaneous tumors of two groups by Taq Man mi RNA assay.The expression of Ki67 and tunelpositive cells in subcutaneous tumors of two groups of mice was detected by immunohistochemical methods in paraffin sections.7.Stable overexpression of mi R-185-5p(Lv-185-Exo)and control(Ctrl-Exo)RKO cell line supernatant exosomes were extracted by ultracentrifugation.Exosomes morphology,size concentration and exosome surface-specific protein markers were tested by TEM,NTA and WB methods,respectively.Detection of mi R-185-5p expression in Lv-185-Exo and Ctrl-Exo by Taqman mi RNA assay.8.After identification of exosomes,co-incubation culture was performed with SW480 cell line.Labeling of exosome surface-specific molecules PKH26 and SW480 cell nuclear DAPI,respectively,and detection of the binding of supernatant exosomes to SW480 cell line using fusion immunofluorescence technique.9.After 24 h co-culture of exosomes with SW480 cell line,the effects of exosomedelivered mi R-185-5p on cell proliferation and migration ability were tested by cell function assay.Results1.The expression of mi R-185-5p in 19 CRC tumor tissues was higher than that in adjacent normal tissues in 28 pairs of tissues.2.The mi R-185-5p expression in both CRC-derived cell lines and supernatant exosomes was significantly higher than that in the colonic normal epithelial cell line FHC.Mi R-185-5p expression may be associated with the proliferation capacity of CRC-derived cell lines.3.Increased mi R-185-5p expression promoted the proliferation and migration of RKO,SW480 and MC38 cell lines compared to the negative control.4.Down-regulation of mi R-185-5p expression inhibited the proliferation and migration of RKO,SW480 and MC38 cell lines compared to the negative control.5.The subcutaneous tumor volume and mass of RKO-Lv-185 tumor-bearing mice were significantly larger than that of the RKO-Ctrl group.6.The subcutaneous tumor volume and mass of MC38-Lv-185 tumor-bearing mice were significantly larger than that of the MC38-Ctrl group.The expression of mi R-185-5p in subcutaneous tumors of MC38-Lv-185 tumor-bearing mice was higher than that of MC38-Ctrl group,Ki67-positive cells were significantly more than that in MC38-Ctrl group,and tunel apoptotic vesicles were significantly less than that in MC38-Ctrl group.7.The cell supernatant exosomes extracted by ultracentrifugation were qualified.Mi R-185-5p expression was significantly higher in Lv-185-Exo than in Ctrl-Exo.8.The result of fusion immunofluorescence suggested that exosomes can be internalized by SW480 cell line.9.Results of cellular function experiments suggested that SW480 cell line coincubated with Lv-185-Exo had significantly enhanced proliferation and migration capacity compared with the SW480 blank control group and Ctrl-Exo co-incubation group.ConclusionThe reason for elevated circulating exosome mi R-185-5p expression in CRC patients was likely to originate from the release of CRC tumor cells.Mi R-185-5p expression affected the proliferation and migration of CRC-derived cell lines(RKO,SW480,and MC38)in vitro;overexpression of mi R-185-5p promoted tumorigenesis and tumor development of CRC-derived cell lines(RKO and MC38)in vivo.Exosomedelivered mi R-185-5p promoted malignant proliferation and migration of SW480 cell line.Part III Mechanism of mi R-185-5p regulation of CRCMethods1.Two sets of RKO cell lines(2 replicate wells per set)were transfected with mi R-185-5p mimics and NC mimics,respectively,and RNA was extracted for RNA sequencing.2.According to RNA sequencing results,analysis was performed to screen significantly downregulated genes in the mi R-185-5p mimics group compared to NC mimics(screening principle: fold change<0.5,P<0.05).Bioinformatics analysis was then performed.Combined with the results of mi RNA target gene prediction database,candidate downstream genes were screened.3.The expression of candidate downstream genes was tested by RT-q PCR and WB assays after RKO and MC38 cell lines transfected with mi R-185-5p mimics and NC mimics,respectively.4.By constructing the 3’UTR wild-type and mutant reporter gene plasmid of the candidate downstream gene,the double-luciferase reporter method was performed to verify that mi R-185-5p and the candidate downstream gene could bind to each other.5.The TCGA database was searched to understand the expression of candidate downstream genes in CRC tumor tissues and para cancerous tissues.6.A total of 26 pairs of tumor tissues and adjacent normal tissues were collected from clinical CRC patients,and the expression of candidate downstream genes in CRC tumor tissues and adjacent normal tissues was detected by RT-q PCR method.The relationship between mi R-185-5p expression and candidate downstream gene expression was verified in the tumor tissues.7.Overexpression candidate downstream gene coding sequence(CDS)plasmids were constructed and RKO and MC38 cell lines were transfected;The effect of overexpression of candidate downstream genes on cell proliferation and migration capabilities was tested by CCK-8,wound healing and transwell cell migration assays.8.RKO-Lv-185 and MC38-Lv-185 cell lines and corresponding negative control cell lines were cultured and transfected with overexpression candidate downstream gene CDS plasmids,respectively.The effects of overexpression candidate downstream gene plasmids on cell proliferation and migration capabilities were tested by WB,CCK-8 assay,wound healing and transwell cell migration assays.Results1.Combining the results of RNA sequencing and mi RNA gene prediction database,AKR1C2 was screened as a possible candidate downstream gene for mi R-185-5p to promote CRC progression.KEGG results showed the highest enrichment score of pathways for downregulated differential genetic was steroid hormone biosynthesis pathway.2.Compared to the NC mimics group,m RNA and protein levels of AKR1C2 were significantly decreased in both mi R-185-5p mimics group of RKO and MC38 cell lines.3.The results of dual luciferase reporter assay suggested that luciferase activity was significantly downregulated after co-transfection with mi R-185-5p mimics and AKR1C2 3’UTR wild-type plasmids in HEK-293 cell lines compared to NC mimics.In contrast,co-transfection of mi R-185-5p mimics and AKR1C2 3’UTR mutant plasmids in HEK-293 cell lines did not significantly alter luciferase activity compared to NC mimics.4.AKR1C2 expression levels were significantly decreased in CRC tumor tissues compared with adjacent normal tissues;a negative correlation was found between mi R-185-5p expression and AKR1C2 expression in CRC tumor tissues.5.The results of cell function assay suggested that overexpression of AKR1C2 plasmid transfection could significantly inhibit the malignant proliferation and migration of RKO and MC38 cell lines.6.The WB results suggested that transfection of overexpressed AKR1C2 plasmid "rescued" the reduced AKR1C2 protein expression caused by overexpression of mi R-185-5p in RKO and MC38 cell lines.7.The results of cell function assay suggested that overexpression of AKR1C2 plasmid transfected with RKO-Lv-185 and MC38-Lv-185 cell lines could effectively inhibit the enhanced proliferation and migration caused by overexpression of mi R-185-5p.ConclusionMi R-185-5p interacted with AKR1C2 and down-regulated its expression,thus promoting the malignant proliferation and migration of CRC-derived cell lines.