| Objective: Colorectal cancer(CRC)is still a malignant tumor with the third highest incidence rate and the second highest fatality rate worldwide,which not only seriously affects human life and health,but also brings a great economic burden to society.Although multiple clinical methods of treatment for colorectal cancer,including classic cytotoxic drug chemotherapy,targeted therapy,and immunotherapy,etc,still parts of patients who cannot effectively benefit from them.In addition to genetic factors,the reason for the poor clinical treatment benefit results from its complex pathogenesis,the development is a long,multi-factor,multi-step cumulative process.In recent years,some non-coding RNAs with a length greater than 200 nucleotides,such as long non-coding RNAs(LncRNAs)which were initially thought to be "noise" during genome transcription without biological function.However,they are now considered as novel molecular markers to mediate tumorigenesis and development as well as drug resistance which have drawn widespread attention.Therefore,excavating LncRNA molecular markers as well as explaining the role of key proteins and intrinsic regulation or interaction mechanisms will provide new targets and diagnosis-treatment strategies for tumor early warning,prognosis evaluation and targeted anti-tumor drug development.Studies have shown that LncRNA is a class of RNA without coding ability,relying on its secondary structure to directly bind to proteins,mi RNAs and m RNA,etc.,as a regulator of tumor initiation,growth,invasion and metastasis to participate in multiple biological functions such as tumor proliferation and cycle,apoptosis and differentiation,thus becomes a star molecule chased by the academic community,especially in the field of tumor characteristics and function research.The precise and complex regulation of LncRNA at the level of development and gene expression has largely revealed the key points of tumor occurrence and development and drug resistance,but its intrinsic regulatory mode and molecular mechanisms in mediating tumor pathological characteristics are still in the initial stage of research,and still need to be deeply analyzed and explored.The latest research has found that small nucleoli RNA host genes(SNHGs)play a key role as LncRNAs in breast cancer,colorectal cancer,lung cancer,liver cancer and other tumors,and are involved in manipulating biological characteristics such as tumor cell proliferation,invasion and metastasis.The LncRNA SNHGs family has been found to contain at least 22 family members,including SNHG1 to SNHG22,most of whom have become hot molecules in the field of epimolecular biology.Among them,SNHG 8(small nucleolar RNA host gene 8)as a key member of the LncRNA SNHGs family,has the role of regulating a variety of changes in tumor biological behavior and function.Although current studies have confirmed that SNHG8 is involved in regulating pathological processes such as breast cancer,colorectal cancer,and gastric cancer,previous studies have focused on the simple verification of classical endogenous competitive RNA mechanisms(ce RNA),so in addition to the classical ce RNA mechanism,the endogenous molecular targets specifically regulated by LncRNA SNHG8 in colorectal cancer and their regulation and intrinsic molecular mechanisms are warranted to be clarified.Accmulting studies demonstrated that LncRNA can participate in the regulation of various biological functions in protein translation level,while during the entire process of protein translation,protein translation initiation is a speed-limiting step of protein synthesis,during which multiple protein translation initiation factors are involved in.Dysregulation of Protein translation significantly influence the tumorigenesis and progresssion,and translational initiation plays pivotal roles in cell proliferation,differentiation,and apoptosis,etc.Eukaryotic translation initiation factor 4A3(eIF4A3)is a major member of the DEAD cassette RNA helicase family and it is crucial during protein translation initiation.eIF4A3 plays an important role in the process of tumorigenesis and development,however,whether LncRNA SNHG8 can specific target the regulation of eIF4A3 to mediate tumor malignant phenotype and pathological process?Is the eIF4A3 protein regulated directly or indirectly? What are the critical factors that regulate downstream? Those above issues are worth studying and explaination.This research was conducted based on the expression and regulation of LncRNA,SNHG8 and eIF4A3 in colorectal cancer.The main contents and results were divided into three parts,which are described as follows:Part I: Investigating the expression of LncRNA SNHG8 and eIF4A3 in CRC cells and tissues and their correlation with clinicopathologic parameters and prognosis Objective: to investigate the expression of LncRNA SNHG8 and eIF4A3 in CRC tissues and cells,the correlation between the expression of LncRNA SNHG8 and eIF4A3 and the value of clinical early warning and prognosis evaluation.Methods:(1)The differential expression profiles of LncRNA SNHG8 and eIF4A3co-expressed in colorectal cancer and adjacent tissues were analyzed by differential expression profiling of LncRNA chip.(2)The differential expression and correlation of LncRNA SNHG8 and eIF4A3 in colorectal cancer including colorectal cancer were analyzed based on the Tumor Genome Atlas(TCGA)data and the bioinformatics platform.(3)The expression of LncRNA SNHG8 and eIF4A3 in 7 colorectal cancer cell lines and 29 pairs of colorectal cancer tissues and their matched para-cancerous tissues was detected by real-time quantitative PCR(q RT-PCR).(4)The expression of LncRNA SNHG8 was detected in 125 pairs of colorectal carcinoma and adjacent tissues by ISH assays.The expression of eIF4A3 was detected in125 pairs of CRC and adjacent tissues by IHC assays.The correlation between high/low expression(cut-off)and regression analysis expression of LncRNA SNHG8 and eIF4A3 was analyzed by ROC curve method.(5)Correlation between high/low expression of LncRNA SNHG8 and eIF4A3 and clinicopathologic parameters by Logistic regression analysis.Correlation between high/low expression of LncRNA SNHG8 and eIF4A3 and prognositic values by Log-rank test and multivariate Cox prognostic analysis.Results:(1)Based on the data set of GEO chip GSE137511,the dysregulation profiles of 4pairs of lncRNAs-m RNAs microarrays in CRC tissues were analyzed.The results showed that both LNCRNA SNHG8 and eIF4A3 were upregulated(3.45 and 4.22 in log2 FC,respectively).Further analysis of TCGA data showed that LncRNA SNHG8 and eIF4A3 were significantly upregulated in CRC,and there was a significant positive correlation(P < 0.0001).A positive correlation was confirmed between SNHG8 and eIF4A3 in 30 pairs of CRC patients(R = 0.5375,P = 0.0022).(2)The expression and correlation of LncRNA SNHG8 and eIF4A3 in CRC pateints:compared with adjacent-tissues,the positive rate of LncRNA SNHG8 and eIF4A3 in CRC was significantly higher(P < 0.0001),the expression of LncRNA SNHG8 and eIF4A3 was positively correlated(P = 0.009).(3)The correlation between the expression of LncRNA SNHG8 and eIF4A3 and the clinicopathologic parameters in CRC patietns: the high/low expression of cut-off of LncRNA SNHG8 or eIF4A3 was determined by ROC curve.The results showed that the high/low expression of SNHG8 and eIF4A3 were significantly associated with the primary site,perienteric ratio,DFS and OS status of the CRC patients.(4)LncRNA SNHG8 and eIF4A3 as independent prognostic risk markers: high/low expression of LncRNA SNHG8 and eIF4A3 were associated with DFS and OS by Kaplan-Meier Log-rank test and multivariate Cox regression risk model.That is,high expression of LncRNA SNHG8 and eIF4A3 was associated with shortened survival in patients with CRC,suggesting that it could be as independent prognostic biomarkers for survival evaluation.Conclusion:(1)Based on the expression profile of LncRNA chip,TCGA data and bioinformatics anaylis,the platform data set were evaluated,and the target gene co-expression regulatory network of LncRNA was analyzed and enriched.The translation initiation factor eIF4A3,which was enriched and co-expressed by LncRNA SNHG8,was found to be up-regulated in 7 colorectal cancer cell lines and 30 CRC tissues.There was a significant positive correlation between the high expression levels of the two candiators in CRC samples.(2)The clinical and pathological features of colorectal cancer were compared with those of adjacent colorectal cancer(n = 125)to explore the potential clinical value of LncRNA SNHG8 and eIF4A3.The data revealed that LncRNA SNHG8 and eIF4A3 was upregualted in CRC tissues,correlated with clinicopathologic parameters such as perienteral and primary position.More importantly,the high expression of LncRNA SNHG8 and eIF4A3 was associated with poor prognosis in patients with CRC(DFS and OS),suggesting that these indicators may be candidate markers for prognostic risk assessment for predicting survival in CRC.Part II: Biological Characteristics and role of colorectal cancer cells mediated by targeting regulation of eIF4A3 expression by LncRNA SNHG8Objective: This part is to analyze the relationship between regulation of eIF4A3 expression by interfering S LncRNA NHG8 expression and biological characteristics and function of colorectal cancer cells,and regulation of downstream effector eIF4A3.The aim of this study is to reveal the effect of S LncRNA NHG8 on the phenotype and function of colorectal cancer cells during the development of colorectal cancer by regulating eIF4A3 expression,and to provide key evidence for further elucidating the roles and mechanism of LncRNA SNHG8 in regulating the phenotype of colorectal cancer cells.Methods:(1)The expression levels of LncRNA SNHG8 and eIF4A3 in colorectal cancer cell lines and tissues were detected by q RT-PCR assays and the expression levels of LncRNA SNHG8 and eIF4A3 Mrna in HCT116 and SW620 cells were detected before and after the treatment of HCT116 and SW620 cells.The MRNA recovery of LncRNA SNHG8 and eIF4A3 was determined after the SNHG8 silencing combined with the double intervention of eIF4A3 overexpression.(2)Western Blot assays was performed to detect the expression of eIF4A3 protein in colorectal cancer cells after the HCT116 and SW620 cells were silenced or over-expressed LncRNA SNHG8.The expression of eIF4A3 protein in HCT116 and SW620 cells was detected by silencing LncRNA SNHG8 combined with overexpression of eIF4A3.(3)The growth,survival and proliferation activities of HCT116 and SW620 cells were examined by MTT assays after interfering LncRNA SNHG8 expression.The proliferation and survival ability of HCT116 and SW620 cells were measured after double intervention of LncRNA SNHG8 silence combined with eIF4A3 overexpression.(4)Cell cycle detection: After silent or overexpression of LncRNA SNHG8,HCT116 and SW620 cell cycle were detected by Flow cytometry.The cell cycle recovery of HCT116 and SW620 cells was detected by LncRNA SNHG8 silencing combined with dual intervention of eIF4A3 overexpression.(5)Apoptosis anaysis: after silencing or overexpressing of LncRNA SNHG8 in HCT116 and SW620 cells,the percentage rates of cellular apoptosis were measured by Flow cytometry.The recovery of apoptosis of HCT116 and SW620 cells was detected by SNHG8 silencing combined with the double intervention of eIF4A3 overexpression.(6)The ability of invasion and migration: the ability of HCT116 and SW620 cells to invade and migrate was conducted by Trans-well assay after over-expression of LncRNA SNHG8.The abilities of invasion and the migration ability were analyzed in HCT116 and SW620 cells.Results:(1)The expression of EIF4A3 was significantly associated with LncRNA SNHG8 expression in CRC cells: The enhanced expression of eIF4A3 was detected in HCT116 and SW620 cells by q RT-PCR,whereas the expression of eIF4A3 m RNA was significantly inhibited by silencing LncRNA SNHG8.Western blot and immunofluorescence assays showed that overexpression of LncRNA SNHG8 could significantly increase the expression of eIF4A3 protein in HCT116 and SW620 cells.Silencing of LncRNA SNHG8 could significantly inhibit the expression of eIF4A3 protein in HCT116 and SW620 cells.The expression level of LncRNA SNHG8 and EIF4A3 was not significantly different in the resuce experiments.(2)Intervention of LncRNA SNHG8 expression mediated alterations of cell proliferation and cell cycle in CRC cells: The proliferation of HCT116 and SW620 cells was significantly increased at 0,1,2,3,4 days by MTT Assay(P < 0.0001).The proliferation of CRC cells was dramatically inhibited after silencing LncRNA SNHG8(P< 0.0001).The results showed that over expression of SNHG8 could significantly increase the cell cycle related indexes including proliferation index(Pi)and s phase percentage(SPF)of HCT116 and SW620 cells(P < 0.0001).Silencing of LncRNA SNHG8 could significantly inhibit the cell cycle,and the PI and SPF value decreased significantly(P < 0.0001).MTT Assay showed that no significant difference in proliferation ability and activity was confirmed in HCT116 and SW620 cells(P < 0.0001)in the treatment of overexpressing of eIF4A3 combined with silencing of LncRNA SNHG8 expression.Similarly,no significant difference in PI and SPF values of HCT116 and SW620 by cell cycle proliferation activity(P < 0.0001).(3)Intervention of LncRNA SNHG8 expression to regulate the invasiveness and apoptosis of colorectal cancer cells: Trans-well assays showed that overexpressing of SNHG8 significantly increased the invasiveness of HCT116 and SW620 cells(P <0.0001).Nevertheless,knockdown of SNHG8 inhibited the invasion and migration abilities of HCT116 and SW620 cells(P < 0.0001).Similarly,the apoptosis rate was significantly inhibited after overexpressing of SNHG8(P < 0.0001).Conversely,the apoptosis rate was remarkably enhanced by silencing of LncRNA SNHG8(P < 0.0001).After overexpressing of eIF4A3 combined with silencing of LncRNA SNHG8 expression,no significant difference was detected in the invasiveness and apoptosis rates in the HCT116 and SW620 cells(P < 0.05).Conclusion: LncRNA SNHG8 may play a role of oncogene in colorectal cancer through single intervention or combined intervention of eIF4A3,which can regulate the expression of eIF4A3,it can promote cell proliferation,invasion,migration,cell cycle and inhibit cell apoptosis.These phenotypic alterations were recovered in the double-intervention experiment,indicating that LncRNA SNHG8 play a critical role in promoting cell proliferation via regulating of eIF4A3 protein expression,which provide the basis for further revealing how LncRNA SNHG8 targets the regulatory mode and the functional position of eIF4A3.The findings also provides data support for the involvement of the transcriptional and posttranscriptional levels of LncRNAs in turmorigenesis,and therapeutic strategies.Part III: the key sequence and mode of action of targeting eIF4A3 protein in LncRNA SNHG8 to regulate the proliferation of colorectal cancer cells Objective: Part III mainly focuses on the mode of action and specific binding sites of eIF4A3 protein in LncRNA SNHG8 targeting recognition and specific recruitment.The aim of this study is to reveal the regulatory mechanism or mode of action between the LncRNA SNHG8 and eIF4A3.These findings provide a new idea for the individualized diagnosis and treatment of colorectal cancer and the exploration of new targets and new regulatory approaches.Methods:(1)Bioinformatics prediction and evaluation: the second order sequence,conformational folding and free energy of LncRNA SNHG8’s first order nucleotide sequence in FASTA sequence was evaluated using the RNAfolder searching the key protein domains of eIF4A3 protein by using the online platform of STRING.CATRIPIAD online platform was utilized to predict the strength of the direct interaction and the interaction elements.(2)In situ hybridization combined with immunofluorescence was performed to detect the expression,distribution and co-localization of SN8 and eIF4A3 proteins in HCT116 colorectal cancer cells.(3)Inhibition of cell translation activity: The inhibition of protein translation was carried out,that is,the cells were treated with CHX(200 g/ml),and the proteins were collected based on the time courses.Western Blot was used to detect the effect of SNHG8 on the efficiency of translation and the expression of eIF4A3 protein.(4)Western blot assays were utilized to investigate the effect of eIF4A3 expression on the expression of m TOR and EGFR in SW620 and HCT116 cells.(5)The proliferation ability of colorectal cancer cells was measured by CFA assay:After silencing or overexpressing of eIF4A3 in SW620 and HCT116 cells,the effect of eIF4A3 on the proliferation and cloning ability was analyzed.(6)Edu(5-Ethynyl-2’-Deoxyuridine,thymidine Nucleoside analogue)assay to detecting the cell proliferation ability: the replication and proliferation activity of DNA in colorectal cancer cells was compared after treated by silencing or overexpressing of eIF4A3 in SW620 and HCT116 cells.(7)Scratch repair assays to investigate the changes of cell migration ability: After silencing or overexpression of eIF4A3 in SW620 and HCT116 cells,the effect of eIF4A3 on the migration rate was analyzed in CRC cells.(8)RNA pull down assay: RNA pull-down assay was performed to identify the function of SNHG8 directly and specifically binding eIF4A3 protein,and to find the critical sequence and action animo acids of eIF4A3 protein.(9)Indirect regulation of SNHG8 and EIF4A3 m RNA by Ce RNA mechanism evaluation: Using Encori: possible micro RNAs interaction between enriched SNHG8 and eIF4A3 m RNA,we simultaneously analyzed whether SNHG8 was specifically regulated by dual luciferase reporter to oberve the alterations of transcriptional activity.(10)To establish subcutaneous transplanted tumor model in nude mice,track the growth of tumor,draw the growth curve of transplanted tumor,and observe the survival time of tumor-bearing mice.Detection of SNHG8 and eIF4A3 m RNA expression in tumor tissues by q RT-PCR.(11)Data analysis,statistical methods using SPSS19.0 statistical analysis.In the analysis of continuous variables,t test or One-Way Anova(One-Way Anova)was used,Mean ± SD(Mean ± SD)was used,and chi-square test was used in the analysis of discontinuous variables.When P < 0.05,there was a significant statistical difference.Results:(1)Based on the hypothesis that LncRNA SNHG8 targets eIF4A3 protein,we evaluated the secondary sequence and conformational folding free energy of LncRNA SNHG8 by using the RNAfolder bioinformatics online platform.The results showed that the minimum free energy of LncRNA SNHG8 was-179.30 kcal/mol.The key domains of eIF4A3 were confirmed by the on-line platform of STRING,including DEXDC and HELICC domains,and further using Catrapid to predict the strength of direct interaction between the two proteins and the interacting element GAUGA,it is suggested that they have the sequence and structural basis of direct combination and action.(2)RNA pull-down test showed that the SNHG8-WT probe could target drag and accumulate eIF4A3 protein,but the mutant SNHG8-Mut could hardly accumulate eIF4A3 protein as the negative control,it is suggested that the Gauga binding element of SNHG8 is the direct sequence for the target recognition of eIF4A3.(3)Inhibition of protein translation,after CHX treatment,silence LncRNA SNHG8 on eIF4A3 protein translation speed and efficiency results: the expression of eIF4A3 protein decreased significantly after 2,3 and 4 hours of CHX treatment,which suggested that SNHG8 could significantly increase and accelerate the level of eIF4A3 protein,and silence hgsn8 could significantly inhibit its translation efficiency.(4)When eIF4A3 was silenced in SW620 and HCT116 cell lines,the expression levels of downstream associated factors m TOR and EGFR decreased significantly(P <0.0001).In contrast,overexpression of eIF4A3 in the above cells significantly increased the expression of m TOR and EGFR proteins in the two types of cell Lines(P < 0.0001).(5)By silencing eIF4A3 expression in SW620 and HCT116 cells,the proliferation activity and colony-forming ability significantly decreased(P < 0.0001),overexpression of eIF4A3 could significantly enhance the proliferation and clonogenic ability of SW620 and HCT116 cells(P < 0.0001).(6)When eIF4A3 was silenced in SW620 and HCT116 cells,the proliferation activity of the cells significantly reduced.Correspondingly,when eIF4A3 was overexpressed in the above cells,EDU in SW620 and HCT116 cells significantly increased,and the proliferation activity of the DNA was enhanced.(7)The results of scratch repair showed that the migration ability of SW620 and HCT116 cells was decreased after the expression of eIF4A3 was silenced,whereas,the migration ability of SW620 and HCT116 cells was significantly enhanced after the overexpression of eIF4A3 in these cells.(8)Based on ce RNA mechanism,the interaction of mi R411-5p between LncRNA SNHG8 and eIF4A3 m RNA was predicted and enriched by ENCORI.We did not find any significant difference between wild type and mutant type of LncRNA SNGH8 and control group(P < 0.05)based on the double luciferase reporter gene analysis.The above results did not find that mi R411-5p can be specifically regulated by LncRNA SNHG8,and the underlying mechanism still needs to be verified.(9)Xenografts in nude mice showed that LncRNA SNHG8 promoted the proliferation of colorectal cancer via eIF4A3: After subcutaneous injection of SW620 cells in nude mice,overexpression of eIF4A3 significantly increased the size and weight of the xenograft tumor.Importantly,a shortened survival time was observed in the overexpressiong of eIF4A3 group compared with the LncRNA SNHG8 group or LncRNA SNHG8-eIF4A3 mutation group.The results demonstrated that LncRNA SNHG8 could promote the proliferation of xenografts in nude mice by regulating eIF4A3 expression.Conclusion:(1)In the aspect of elucidating the characteristics and mechanism of mediating proliferation of colorectal cancer: We excavate and find that LncRNA SNHG8 has the key element GAUGA which specifically recognizes eIF4A3 protein,and the specific combination of the two can promote eIF4A3 protein translation and stable expression;Then upregulates the expression of downstream factors such as m TOR and EGFR.At the same time,the indirect regulatory mechanism of ce RNA was excluded by the experiment.Therefore,based on the post-transcriptional perspective,this study elucidates the new mechanism of targeting and stabilizing eIF4A3 protein translation in LncRNA SNHG8,which provides a new target for drug development and a new approach for clinical diagnosis and treatment.(2)At the level of in vivo model validation: We used the nude mouse model to demonstrate that LncRNA SNHG8 could promote the changes of tumor growth rate,tumor volume and tumor weight by regulating the expression of eIF4A3.More importantly,LncRNA SNHG8 and eIF4A3 combined intervention could significantly shorten the survival time of nude mice,which confirmed that the targeted regulation of eIF4A3 protein by LncRNA SNHG8 could promote the proliferation and prognosis of transplanted tumor. |
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