Effects And Mechanisms Of LncRNA MIR4713HG On UBE2C Expression On Invasion And Metastasis Of Colorectal Cancer

BackgroundColorectal cancer(CRC)is one of the most common gastrointestinal malignancies.Although the early diagnosis and early treatment of colorectal cancer have made certain progress with early diagnosis and early treatment of colorectal cancer,the prognosis of colorectal cancer patients did not improve significantly.Therefore,it is important to identify new molecular biomarkers and therapeutic targets for the early detection and treatment of colorectal cancer.Also,these novel markers could improve the early diagnosis rate and the survival rate of colorectal cancer patients.In recent years,long noncoding RNAs is one of the hotspots in oncology research.LncRNAs play important roles in epigenetics,transcriptional levels,and post-transcriptional levels.Also,lncRNAs are widely involved in the key process related to tumor biological behavior.Therefore,lncRNAs are expected to become new biomarkers for diagnosis and prognostic assessments of colorectal cancer.Therefore,studying the roles of the biological function and molecular mechanism of lncRNAs in the progression of colorectal cancer is of great significance to elucidate the progress of colorectal cancer.This study aimed to screen differentially expressed lncRNAs between colorectal cancer and normal colorectal tissues based on data downloaded from The Cancer Genome Atlas(TCGA).The screened lncRNAs were then used for performing the univariate Cox regression analysis to identify prognosis-related lncRNAs.Finally,a total of 10 candidate lncRNAs were included in the construction of the risk prognosis model by using the multivariate Cox regression analysis.LncRNA MIR4713 HG was then selected and studied.Subsequently,the role of LncRNA MIR4713 HG on the proliferation,migration,and invasion abilities was validated in vitro and in vivo.The Western blot,RT-q PCR,RNA pull-down,and RIP assays were used to validate the relationship between LncRNA MIR4713 HG and UBE2 C protein.The results demonstrated that LncRNA MIR4713 HG was directly combined with the UBE2 C to regulate the stability of UBE2 C protein and the malignant biological behavior of colorectal cancer cells.In summary,this study aimed to screen prognostic related lncRNAs in colorectal cancer based on data downloaded from the TCGA database.Finally,the LncRNA MIR4713 HG was selected and studied.The effect of LncRNA MIR4713 HG on the proliferation,migration,and invasion of colorectal cancer was studied in this study.This study aimed to provide theoretical supplements in studying the progression of the mechanism of colorectal cancer.Objective:To detect the expression level of LncRNA MIR4713 HG in colorectal cancer tissues and normal tissues,and evaluate its correlation with the prognosis of colorectal cancer patients.Methods:1.We obtained the RNA expression profiles from the TCGA database.Data related to colorectal cancer and normal tissues were downloaded.The “edge R” package was used to screen differentially expressed lncRNAs.2.Univariate and multivariate Cox regression analyses were used to construct the risk score model.Kaplan-Meier analysis was used to evaluate the different expression levels of lncRNAs on the prognosis of colorectal cancer.3.FISH assay was performed to determine the location and expression of LncRNA MIR4713 HG in cells.Its relationship correlated to clinicopathological characteristics of colorectal cancer patients was analyzed.Results:1.We obtained the RNA expression profiles from the TCGA database,including 44 cases of normal tissues and 568 cases of colorectal cancer tissues.541 lncRNAs were screened.2.The screened lncRNAs were selected for univariate Cox regression analysis,and a total of 48 lncRNAs related to the prognosis of colorectal cancer patients were obtained(P<0.05).16 lncRNAs(P<0.01)were selected for multivariate Cox regression analysis.Finally,10 lncRNAs were used to construct the prognostic lncRNAs model.The high expression level of LncRNA MIR4713HG was closely related to the poor overall survival time of colorectal cancer.3.The results of FISH and Rt-q PCR demonstrated a high expression level of LncRNA MIR4713 HG in colorectal cancer.4.Patients with a high expression level of LncRNA MIR4713 HG showed a larger tumor volume,higher TNM stage,higher lymph node metastasis,higher incidence rate of lymph node metastasis,higher incidence rate of vascular tumor thrombus and nerve invasion.Conclusion:LncRNA MIR4713 HG is highly upregulated in colorectal cancer tissues.The expression of LncRNA MIR4713 HG is closed related to the poor overall survival rate and clinicopathological characteristics of colorectal cancer patients.Part Two.Effects of LncRNA MIR4713 HG on malignant biological behavior of colorectal cancerObjective:To evaluate the effects of LncRNA MIR4713 HG on the progression of colorectal cancer.Methods:1.The expression levels of LncRNA MIR4713 HG were determined in cells by RT-q PCR.2.Short hairpin RNA(sh RNA)was used to knock down LncRNA MIR4713 HG,and the expression of LncRNA MIR4713 HG was up-regulated by plasmid.The efficiency of knockdown and overexpression of LncRNA MIR4713 HG were verified by RT-qPCR.3.The effects of LncRNA MIR4713 HG were determined by CCK-8,wound healing,Transwell invasion assays.4.The effects of knockdown and overexpression of LncRNA MIR4713 HG on the proliferation of colorectal cancer in vivo were verified by subcutaneous tumorigenesis in nude mice.Results:1.The results of RT-q PCR showed that the expression level of LncRNA MIR4713 HG in colorectal cancer cell lines was significantly increased compared with normal human colorectal mucosal cells.The expression level of LncRNA MIR4713 HG in SW837 cells was relatively high,which was used for subsequent knock down experiments.The expression level of LncRNA MIR4713 HG in SW620 cells was relatively low,which was used for subsequent overexpression experiments.2.Knockdown and overexpression efficiency were verified by RT-q PCR,and the results showed that sh MIR4713HG-KD1 and sh MI4713HG-KD2 had better knockdown efficiency on LncRNA MIR4713 HG in SW837 cell line compared with sh MIR4713HG-KD3.Also,LncRNA MIR4713 HG lentiviral overexpression plasmid can significantly upregulate the expression level of LncRNA MIR4713 HG in SW620 cell line.3.Compared with the control group,knockdown LncRNA MIR4713 HG significantly inhibited the proliferation ability of SW837 cells.Also,would healing assay demonstrated that the migration ability of SW837 cells was attenuated after knocking down the expression of LncRNA MIR4713 HG.Besides,the invasion ability was decreased after LncRNA MIR4713 HG knockdown in SW837.On the contrary,the proliferation,migration and invasion abilities were increased after upregulating the expression of LncRNA MIR4713 HG in SW620 cells.4.Compared with the control group,tumor weight was significantly reduced in LncRNA MIR4713 HG knockdown group.On the contrary,tumor weight was significantly increased in overexpressed group.Conclusion:LncRNA MIR4713 HG,as a oncogene,can promote the proliferation,migration and invasion abilities of colorectal cancer cells.LncRNA MIR4713 HG could be regarded as a potential new target for the treatment of colorectal cancer.Part Third.Molecular mechanism of LncRNA MIR4713 HG in the invasion and metastasis of colorectal cancerObjective:To investigate the molecular mechanism of LncRNA MIR4713 HG promoting the progression of colorectal cancer.Methods:1.FISH assay was used to verify the localization of LncRNA MIR4713 HG in colorectal cancer cells.2.We used bioinformatics analysis to screen protein encoded m RNAs which co-expressed with LncRNA MIR4713 HG.The potential downstream regulatory targets of LncRNA MIR4713 HG were screened through GO and KEGG analyses.3.Western blot was used to verify the expression changes of UBE2 C protein after LncRNA MIR4713 HG was knocked down.4.RPISeq database was used to predict the possibility of binding between LncRNA MIR4713 HG and UBE2 C protein.5.The binding of LncRNA MIR4713 HG to UBE2 C protein was confirmed by RNA Binding Protein Immunoprecipitation(RIP)and RNA pull down assay.6.LncRNA MIR4713 HG in SW837 cells was knocked down by sh MIR4713HG-KD1.After treating with CHX,the stability of UBE2 C protein was detected.7.LncRNA MIR4713 HG in SW837 cells was knocked down by sh MIR4713HG-KD1.After treating with proteasome inhibitor MG132(20μM),Western blot was used to verify the regulation of LncRNA MIR4713 HG on UBE2 C protein degradation.8.LncRNA MIR4713 HG in SW837 cells was knocked down by sh MIR4713HG-KD1.The regulation of UBE2 C protein ubiquitination by LncRNA MIR4713 HG was confirmed by Immunoprecipitation(IP)assay.Results:1.The results of FISH assay show that LncRNA MIR4713 HG was expressed in both cytoplasm and nucleus of colorectal cancer cell lines.2.The predicted results of RPISeq database suggested the possibility of binding between LncRNA MIR4713 HG and UBE2 C protein.RNA pull down and RIP experiments confirmed the mutual binding between LncRNA MIR4713 HG and UBE2 C protein.3.After knockdown of LncRNA MIR4713 HG,the stability of UBE2 C protein was significantly decreased.4.Western blot results showed that LncRNA MIR4713 HG could not continue to affect the stability of UBE2 C protein after inhibiting proteasomal activity.Conclusion:LncRNA MIR4713 HG regulates the proliferation,migration and invasion of colorectal cancer cells by maintaining UBE2 C protein stability.