| Background and Objects:The incidence and mortality rate of colorectal cancer(CRC)are increasing in recent years in China,ranking 2nd and 5th,respectively.CRC is one of the most common malignant tumors worldwide,and the incidence rate of CRC has become younger.Due to the occult incidence,about one-third of patients present with metastatic disease at their first diagnosis,recurrence and metastasis have become the main causes of death.Therefore,it is urgent to clarify the molecular mechanism of CRC metastasis and explore new biomarkers for the diagnosis and treatment of high-risk groups.Diet,air pollution,lifestyle and other factors are considered to be environmental risk factors for the development of CRC,and epigenetic regulation is an important mechanism for environmental risk factors to participate in the development of CRC.Non-coding RNA regulation is one of the epigenetic regulatory mechanisms,and long non-coding RNA(Lnc RNA)is widely involved in the occurrence and development of CRC due to its special spatial structure and expression specificity.In this study,through microarray analysis and weighted gene co-expression network analysis(WGCNA),the Lnc RNA HIF1?-AS2,which with high expression specificity in CRC,was selected.The aims of the study were to clarify the molecular mechanism of CRC metastasis,and to evaluate the practical application value of HIF1?-AS2 as prognosis and targeted treatment of CRC.Methods:1.Identification and analysis of differentially expressed Lnc RNAs in CRC tissues1.1 96 CRC patients and 96 adenoma(ADE)patients were recruited,who had not received any radiotherapy or chemotherapy,and were diagnosed by Clinicopathology.CRC and matchedAdjacent tissues of CRC patients,adenoma tissues of ADE patients were collected.6 pairs of patients were randomly selected for microarray analysis,combined with WGCNA,Gene Ontology(GO)and Kyoto Encyclopedia of genes and genomes(KEGG)to analyze the differential expression of Lnc RNA and messenger RNA(m RNA)in Adjacent,ADE and CRC tissues.HIF1?-AS2,which involved in the regulation of CRC development and significantly related to environmental risk factors,was selected for further study.1.2 The expression of HIF1?-AS2 in fresh tissues was detected by Quantitative real time polymerase chain reaction(q RT-PCR),and verified by In situ hybridization(ISH)in tissue microarray(TMA).The correlation between HIF1?-AS2,expression level and the risk of CRC was analyzed by proportional hazards model(Cox model).2.HIF1?-AS2 participates in the regulation of CRC metastasis2.1 The HIF1?-AS2 knockout CRC cell line was constructed by CRISPR/Cas9,and the overexpression of HIF1?-AS2 was constructed by plasmid transfection.The effects of HIF1?-AS2 on the function of CRC cells were analyzed by Colony Formation,Transwell experiment and flow cytometry.2.2 A nude mouse tumor-bearing model was constructed,and the effect of HIF1?-AS2 on the metastasis of CRC was evaluated through IVIS Spectrum in Vivo Imaging System(IVIS)and microplate reader.3.HIF1?-AS2 regulates the effect of HIF1?/RMRP/IGF2 axis on CRC metastasis3.1 In-depth analysis of the structure of HIF1?-AS2 through the University of California Santa Cruz(UCSC)genome browser database,to clarify the structural relationship between HIF1?-AS2 and HIF1?,and to verify the regulatory relationship in CRC cells.Bioinformatics prediction database and dual luciferase reporter gene experiment were used to explore the molecular mechanism of HIF1?-AS2 competitive adsorption of mi R-18a/b-5p and mi R-20a-5p to regulate HIF1?expression.3.2 The next generation sequencing technology was used to analyze the transcriptome of HIF1?-AS2 control and knockout CRC cell lines.Combined with literature search,bioinformatics prediction and Chromatin Immunoprecipitation(Ch IP),the downstream molecule RMRP regulated by HIF1?-AS2 was screened.The expression level of RMRP in CRC was detected by q RT-PCR.The effect of RMRP on CRC metastasis was evaluated both in vitro and in vivo.3.3 Co-Immunoprecipitation(Co-IP)combined with Mass Spectrometry(MS)analysis was used to screen the downstream genes of RMRP involved in the development of CRC,IGF2R,was selected.Through Co-IP,enzyme-linked immunosorbent assay(ELISA),q RT-PCR and Western blot(WB)experiments to verify the effect of RMRP on promoting IGF2 expression through competitive adsorption of IGF2R.Detected the expression level of IGF2 by q RT-PCR,and to explore the effect of IGF2 on CRC metastasis.3.4 Through Patient-Derived tumor Xenograft(PDX)mouse models to evaluate the effects of HIF1?-AS2 on the tumorigenesis and metastasis of CRC both in vitro and in vivo.The molecular mechanism of HIF1?-AS2 regulating CRC metastasis through HIF1?/RMRP/IGF2axis was verified by q RT-PCR and WB assay.4.Preliminary exploration of liposome shuttle HIF1?-AS2 small molecule inhibitor for the treatment of CRC4.1 The specific cationic lipid was prepared by a thin film hydration method for constructing the liposome shuttle HIF1?-AS2 small molecule inhibitor.The basic physical and chemical properties pf liposome,such as average diameter,polydispersity index(PDI),Zeta potential and stability was detected by transmission electron microscope.The concentration of liposome was detected by particle tracking analyzer.Meanwhile,the RNA integrity,complexity,and safety was measured by Gel retardation assay and cytotoxic experiments.4.2 The"inflammatory cancer transformation"mouse model of CRC was constructed using an azoxymethane-dextran sodium sulfate(AOM-DSS),to detect the inhibitory effect of liposome shuttle HIF1?-AS2 small molecule inhibitor in the progression of CRC,and to evaluate the potential therapeutic value of HIF1?-AS2 in metastatic CRC.Results:1.Identification and analysis of differentially expressed Lnc RNAs in CRC tissues1.1 WGCNA analysis of Lnc RNA and m RNA expression profiles in CRC tissuesTo assess Lnc RNA and m RNA co-expression patterns,WGCNA was used to identify genes that played pivotal roles in CRC development.Module 1(M1),Module 14(M14)and Module 15(M15),were gradually increased or decreased from Adjacent,ADE to CRC tissues,and DEGs in both M1 and M14 were enriched in the regulatory network related to the development of CRC.Further analysis found that HIF1?-AS2 and its corresponding linear RNA-HIF1?were enriched in immune related regulatory network,and the expression level was highly positively correlated,suggesting that HIF1?-AS2 might play an important regulatory role in the progression of CRC.Therefore,HIF1?-AS2(NCBIIDNR?045406)was selected for subsequent research.1.2 The association between the expression level of HIF1?-AS2 and the prognosis of CRC patientsThe expression level of HIF1?-AS2 was markedly increased from Adjacent,ADE to CRC tissues as detected by q RT-PCR in fresh tissues(P<0.001).Furthermore,the results of ISH in TMA showed that the expression of HIF1?-AS2 in CRC was significantly higher than that in Adjacent tissues.Combined with the analysis of 10-year follow-up information of patients,the results showed that the higher expression of HIF1?-AS2 the less survival time of patients.Cox model analysis showed that HIF1?-AS2 might be an independent negative prognostic factor for CRC.2.HIF1?-AS2 participates in the regulation of CRC metastasis2.1 Functional study of HIF1?-AS2 in CRC cellsCoding Potential Assessment Tool(CAPT)database confirmed the non-coding protein properties of HIF1?-AS2.RNAscope and q RT-PCR experiments showed that HIF1?-AS2 was mainly located in the cytoplasm in CRC cells.To determine whether HIF1?-AS2 regulated the metastasis progression and its potential functional mechanisms,first of all,7 CRC cell lines were divided into two groups(high or low HIF1?-AS2 levels)based on expression levels using q RT-PCR analysis.RKO,DLD-1 and SW620 cells were ascribed to the high-level group and SW480,HCT15,HT29 and HCT116 were ascribed to the low-level group.Next,HIF1?-AS2was knock out with CRISPR/Cas9 technology in the high-HIF1?-AS2 group,with overexpression in the low-HIF1?-AS2 group.Data showed that the proliferation,migration and invasion capacities of RKO cells were markedly impaired by HIF1?-AS2 knockout and enhanced with HIF1?-AS2 overexpression in SW480 cells(P<0.001).The results of flow cytometry showed that HIF1?-AS2 knockout led to a significant G1 phase arrest and S/G2phase reduction in cell cycle,in addition to the induced higher proportion of apoptosis(P<0.001).2.2 HIF1?-AS2 regulates the metastasis of CRC in a xenograft mouse modelTo further evaluate the function of HIF1?-AS2 in vivo,HIF1?-AS2 knockout and control RKO cells with stably expressing luciferase protein were constructed,followed with subcutaneously nude mice xenograft assays.The results showed that HIF1?-AS2 knockout reduced tumor growth and hepatic spreading after injection.Furthermore,HIF1?-AS2overexpression enhanced tumor growth and hepatic spreading of SW480 cells(P<0.001).3.HIF1?-AS2 regulates the effect of HIF1?/RMRP/IGF2 axis on CRC metastasis3.1 HIF1?-AS2 promotes the expression of HIF1?by competitively adsorbing mi R-18a/b-5p and mi R-20a-5pThe results of UCSC confirmed that HIF1?-AS2 is an antisense Lnc RNA corresponding to the HIF1?.Microarray analysis showed that 7 Lnc RNAs,which was gradually elevated from Adjacent,ADE to CRC tissues,including HIF1?.Therefore HIF1?was selected for further study.Notably,HIF1?level was decreased after HIF1?-AS2 knockout(P<0.01)and increased after HIF1?-AS2 overexpression(P<0.001).The results of Target Scan,RNA22,mi RWalk,mi RDB,and mi RBase databases showed that mi R-18a-5p,mi R-18b-5p and mi R-20a-5p could potentially bound to the promoter region of HIF1?,and this prediction was confirmed by Luciferase reporter assays.The results of q RT-PCR showed that HIF1?-AS2 overexpression promoted HIF1?expression(P<0.01),the addition of mi RNA overexpression inhibited the expression of HIF1?(P<0.01).3.2 HIF1?-AS2 regulates the effect of HIF1?/RMRP axis on CRC metastasisRNA-seq was performed to analyze the transcriptome profile associated with HIF1?-AS2,and numerous genes which potentially associated with HIF1?-AS2 have been identified.RMRP,which plays an important role in tumor progression,was selected.The expression of RMRP decreased after HIF1?-AS2 knockout(P<0.001),and increased after HIF1?overexpression(P<0.001).The results of UCSC showed that HIF1?could potentially bound the promoter region of RMRP,in addition,Ch IP assays confirmed that.Therefore,there was a potential positive correlation between HIF1?-AS2,HIF1?and RMRP.The expression levels of RMRP in fresh tissues were significantly increased form Adjacent,ADE to CRC(P<0.05),which was consistent with the data retrieved from The Cancer Genome Atlas(TCGA)and the ISH results in TMA.Cox model analysis showed that RMRP was a potential prognostic factor in CRC.Moreover,RMRP was positively correlated with the metastasis capacity of CRC both in vitro and in vivo.3.3 The effect of RMRP/IGF2R/IGF2 axis on CRC metastasisThe results of Co-IP and MS showed that there was a interaction betwwen IGF2R and RMRP.ELISA and q RT-PCR experiments confirmed the degradation of IGF2 accelerated in the presence of IGF2R in CRC cells.The specific binding of IGF2R to RMRP was significantly decreased when IGF2 was overexpressed,indicating that there was a mutual regulatory relationship between RMRP,IGF2R and IGF2.Furthermore,WB experiments showed that the degradation of IGF2 was significantly inhibited when RMRP was overexpressed,and the opposite phenomenon was observed when RMRP or HIF1?-AS2 was down regulated.Briefly,RMRP promoted the expression of IGF2 by competitively binding IGF2R.3.4 HIF1?-AS2 regulates CRC metastasis in PDX mouse modelTumor masses from CRC patients with differential expression levels of HIF1?-AS2(HIF1?-AS2.high-high expression of HIF1?-AS2,HIF1?-AS2.low-low expression of HIF1?-AS2)were subjected for PDX model.After three passages in nude mice,single cell suspension was constructed,and the data showed that HIF1?-AS2 promoted proliferation,migration and invasion both in vitro and in vivo(P<0.01).Furthermore,the expression of functional molecules in tumor tissue in mice in PDX model was detected by q RT-PCR and WB analysis.As a result,the expression levels of HIF1?,RMRP and IGF2 were consistent with HIF1?-AS2in PDX model,suggesting that HIF1?-AS2 might mediate CRC metastasis by regulating HIF1?/RMRP/IGF2 axis.Additionally,PDX cells were injected into nude mice via the tail vein.The results of Magnetic Resonance Imaging(MRI)showed that HIF1?-AS2 knockdown significantly inhibited hepatic spreading in CRC metastasis PDX models.4.Preliminary exploration of liposome shuttle HIF1?-AS2 small molecule inhibitor for the treatment of CRC4.1 Construction and identification of liposome shuttle HIF1?-AS2 small molecule inhibitorPegylated cationic liposomes was constructed for the efficient entrapment of HIF1?-AS2si RNA(lipoplexes).Transmission electron microscope(TEM)imaging revealed that the cationic liposomes showed well-defined spherical morphologies,and there was no difference in physicochemical properties between liposome and lipoplexes.The concentration of liposomes was 2.26×1011 particles/m L.In addition,the long-term stability of cationic liposomes was good,and there was no degradation of the HIF1?-AS2.si RNA in the presence of Rnase A.The complexation between HIF1?-AS2 si RNA and cationic liposomes was higher than 99.14%.In addition,the results of the CCK-8 showed that liposomes have low cytotoxicity for cells.4.2 Lipoplexes decreases tumorigenesis in the AOM/DSS mouse model of CRCNext,the role of lipoplexes in CRC progression was detected using an AOM-DSS model.Lipoplexes developed a markedly lower number of tumors and longer overall survival time when compared with PBS and liposome control group,or with Cy5-si-HIF1?-AS2 group(P<0.05).In addition,liposome shuttle HIF1?-AS2 small molecule inhibitor was better enriched in the liver and lung tissues of mice,and the targeting ability was significantly improved.Conclusions:1.The expression level of HIF1?-AS2 from Adjacent,ADE to CRC tissues was increased gradually,and was significantly related to the survival time of CRC patients.2.HIF1?-AS2 can promote the migration and invasion of CRC cells,as well as the occurrence of metastasis of CRC.3.HIF1?-AS2 promotes the expression of HIF1?by competitively adsorbing mi R-18a/b-5p and mi R-20a-5p;HIF1?specifically bind the promoter region of RMRP to regulate its expression;RMRP promotes the expression of IGF2 by competitively binding IGF2R.HIF1?-AS2 mediates CRC metastasis by regulating HIF1?/RMRP/IGF2 axis.4.Liposome shuttle HIF1?-AS2 small molecule inhibitor inhibit the tumor metastasis in the AOM/DSS mouse model of CRC.HIF1?-AS2 is a potential therapeutic target for CRC. |
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