| BackgroundColon cancer is one of the most frequently diagnosed gastrointestinal malignancies worldwide.The morbidity of colon cancer ranks second among all tumor types and mortality ranks third,which brings a heavy burden to patients,families and society.At present,the main treatments for colon cancer are surgery,radiotherapy,chemotherapy and molecular-targeted therapy.Although these treatments prolong the survival of the patients and improve the quality of life of the patients,most of the colon cancer patients have developed to the middle and advanced stages at the time of diagnosis,and even have distant organ metastasis,resulting in a poor prognosis.As an important part of tumor microenvironment,tumor angiogenesis plays an important role in tumor growth,immune cell infiltration and tumor metastasis.Aeveral studies have proved that anti-angiogenesis therapy is a very promising cancer treatment strategy.Therefore,exploring the molecular mechanisms of tumorigenesis,development and angiogenesis,identifying suitable targets and prognostic-related biomarkers of colon cancer is the main direction of scientific research in colon cancer.Long non-coding RNAs(lncRNAs)refers to a type of RNA with a transcription length>200 nt,lacking protein coding ability.Research has shown that lncRNA can regulate gene expression through many aspects,such as epigenetic regulation,transcriptional regulation and post transcriptional regulation,and it can participate in many biological processes such as cell proliferation,differentiation and apoptosis.DNA methylation is an epigenetic modification,which can inhibit gene expression.It plays an important role in maintaining chromosome structure,X chromosome inactivation,gene imprinting and tumorigenesis.LINC01666 is located on chromosome 2q31.1,with a total length of 1058 bp.Research has found that LINC01666 expression is up-regulated in a variety of malignant tumors,promoting tumor proliferation,migration and angiogenesis,such as epithelial ovarian cancer,bladder cancer,glioma,osteosarcoma and so on.In addition,LINC01116 targets miR-520a-3p and affects IL6R to promote the proliferation and migration of osteosarcoma cells through the Jak-stat signaling pathway.In glioma cells,LINC01116 recruits the transcription factor DDX5 to the promoter region of IL-1 to promote its expression and induces tumor cell proliferation.However,there are few reports about the expression and biological functions of LINC01116 in colon cancer.More data are needed to explore the regulatory mechanism of LINC01116 in colon cancer.ObjectiveThis study intends to detect the expression of LINC01116 in colon cancer tissue,analyze its prognostic relevance,and explore its impact on the biological function of colon cancer cells;predict and analyze the molecules related to the interaction of linc01116 through biological information,and explore its interaction mechanism;finally,further confirm the effect of LINC01116 on the proliferation and angiogenesis of colon cancer cells in vivo experiments.Part Ⅰ The expression,clinical significance and biological function of LINC01116 in colon cancerMethod:1.RT-qPCR was used to detect the RNA expression of LINC01116 in 80 cases of colon cancer tissues and adjacent normal tissues.To analyze the relationship between the expression of LINC01116 and clinicopathological conditions and postoperative survival time.2.The expression of LINC01116 in colon cancer cell lines and human normal colonic epithelial cells were detected by RT-qPCR.3.After transfection of pcDNA3.1-LINC01116 and sh-LINC01116 in colon cancer cells SW480 or HCT116,RT-qPCR or western blot was used to detect the expression of LINC01116.CCK-8 and clone formation assay were used to detect cell proliferation.The scratch assay was used to detect the migration ability of human umbilical vein endothelial cells(HUVEC)co-cultured with SW480 or HCT116 cells after transfection,and the tubule formation assay was used to detect the angiogenesis of HUVEC.Results:1.The RT-qPCR results showed that the expression of LINC01116 in colon cancer tissues was significantly higher than that in adjacent normal tissues,and the expression level of LINC01116 in colon cancer tissues was significantly correlated with the degree of tissue differentiation and tumor size(P<0.05).Kaplan-Meier survival analysis showed that the 5-year survival rate of patients with high expression of LINC01116 was lower than that of patients with low expression of LINC01116(P<0.05).2.LINC01116 was significantly up-regulated in colon cancer cells.3.RT-qPCR results showed that the expression level of LINC01116 in pcDNA3.1-LINC01116 group was higher than that in pcDNA3.1 group(P<0.05),and the expression level of LINC01116 in sh-LINC01116 group was lower than that in sh-NC group(P<0.05),but there was no significant difference between the expression level of LINC01116 in pcDNA3.1 group and sh-NC group and blank group.CCK-8 and clone formation assay showed that the proliferation activity and clone number of pcDNA3.1-LINC01116 group were higher than those of pcDNA3.1 group(P<0.05),and the proliferation activity and clone number of sh-LINC01116 group were lower than those of sh-NC group(P<0.05).The results of scratch assay and tubule formation assay showed that the migration rate and total tubule length of HUVEC cocultured in pcDNA3.1-LINC01116 group were significantly higher than those in pcDNA3.1 group,and the migration rate and total tubule length of HUVEC cocultured in sh-LINC01116 group were significantly lower than those in sh-NC group(P<0.05).Summary:LINC01116 was highly expressed in the tumor tissues of colon cancer patients and was significantly correlated with tumor size and tumor differentiation.The survival time of patients with high expression of LINC01116 was significantly lower than that of patients with low expression of LINC01116.Cell experiments showed that LINC01116 promoted the proliferation of colon cancer cells and angiogenesis in vitro.These results indicate that LINC01116 plays a tumor promoting role in colon cancer and can be used as a potential prognostic biomarker.Part Ⅱ LINC01116 promotes the proliferation and angiogenesis of colon cancer cells through EZH2/TPM1 signaling pathwayMethod:1.TCGA database analysis was used to evaluate the expression of TPM1 in colon cancer tissues and normal tissues.2.RT-qPCR and western blot were used to detect the expression of TPM 1 in colon cancer cell lines and human normal colonic epithelial cells.3.After pcDNA3.1-LINC01116 and sh-LINC01116 were transfected into SW480 and HCT116 cells respectively,the expression of TPM 1 was detected by qRT-PCR and Western blot.After sh-TPM1 was transfected into SW480 and HCT116 cells,the expression of TPM1 was detected by RT-qPCR and Western blot.After sh-LINC01116 and shLINC01116+ sh-tpml were transfected into SW480 and HCT116 cells respectively,CCK-8 and clone formation assays were used to detect cell proliferation.The scratch assay was used to detect the migration ability of human umbilical vein endothelial cells(HUVEC)co-cultured with SW480 or HCT116 cells after transfection,and the tubule formation assay was used to detect the angiogenesis of HUVEC.4.RNAInter(http://www.rnainter.org)was used to predict the interaction between E2H2 and LINC01116.UCSC(http://genome.ucsc.edu)online tool was used to predict TPM1-related transcription factors.TCGA database showed that EZH2 was highly expressed in colon cancer tissues and negatively regulated TPM1.The expression of EZH2 was detected by RT-qPCR and Western blot.RNA immunoprecipitation(RIP)assay and RNA pull down assay were used to detect the interaction of LINC01116 with EZH2.chromatin immunoprecipitation assay(ChIP assay)was performed to confirm the binding of EZH2 to TPM1 promoter.5.After pcDNA3.1-EZH2,sh-EZH2 and sh-EZH2+sh-TPM1 were transfected into SW480 and HCT116 cells respectively,the expression levels of EZH2 and TPM1 were detected by RT-qPCR and Western blot.CCK-8 assay and clone formation assay were used to detect cell proliferation,scratch assay was used to detect migration ability of HUVEC co cultured with SW480 or HCT116 cells,and tubule formation assay was used to detect angiogenesis of HUVEC.Results:1.TCGA database showed that the expression of TPM1 in colon cancer tissues was significantly lower than that in normal tissues.2.RT-qPCR and western blot showed that the expression of TPM1 in colon cancer cells was significantly lower than that in normal colon cells(P<0.05).3.In SW480 and HCT116 cells,the expression of TPM1 was down regulated after LINC01116 overexpression,and increased after LINC01116 silence(P<0.05).The proliferation and clone formation sh-LINC01116+sh-TPM1 group were significantly more than that of sh-LINC01116 group(P<0.05).HUVEC cell migration rate and the total length of tubules formed after co-culture in sh-LINC01116+sh-TPM1 group were significantly greated than that in sh-LINC01116 group(P<0.05).4.Bioinformatics analysis showed that LINC01116 and EZH2 have strong binding ability,and EZH2 is a potential transcription factor of TPM1.The TCGA database showed that EZH2 expression was up-regulated in colon cancer tissues and negatively correlated with that of TPM1(P<0.05).The results of RT-qPCR and western blot showed that EZH2 was highly expressed in colon cancer cell lines,SW480 and HCT116(P<0.05).RIP results showed that LINC01116 was significantly enriched in the protein complex pulled down by the EZH2 antibody(P<0.05),indicating that LINC01116 could bind to EZH2.Meanwhile,ChIP assay showed that EZH2 bind to the promoter of TPM1.After overexpression of LINC01116,EZH2 antibody-enriched TPM1 promoter significantly increased(P<0.05).RNA pull down results showed that H3K27me3 protein level was significantly increased in the complex enriched by LINC01116(P<0.05).5.After pcDNA3.1-EZH2,sh-EZH2 and sh-EZH2+sh-TPM1 were transfected into SW480 and HCT116 cells respectively,the expression of EZH2 in pcDNA3.1-EZH2 group was stronger than that in pcDNA3.1 group,while the expression of TPM1 was lower.Compared with sh-NC group,the expression of EZH2 in sh-EZH2 group was inhibited,but the expression of TPM1 was increased(P<0.05).CCK-8 and clone formation assay showed that the proliferation activity and clone number of pcDNA3.1-EZH2 group were higher than that of pcDNA3.1 group(P<0.05).Compared with sh-EZH2+sh-TPM1 group,sh-EZH2 group had lower proliferation activity and clone number(P<0.05).Compared with pcDNA3.1 group,scratch assay and tubule formation assay showed that the migration rate of HUVEC cells and the total length of tubules were increased in pcDNA3.1-ezh2 group(P<0.05).Compared with sh-EZH2+sh-TPM1 group,the migration rate of HUVEC cells and the total length of formed tubules in sh-EZH2 group decreased(P<0.05).Summary:The expression of TPM1 in colon cancer tissues and cell lines was significantly down-regulated,while EZH2 was significantly increased.LINC01116 promoted the proliferation and angiogenesis of colon cancer cells by recruiting EZH2 to the promoter region of TPM 1,thereby reducing the expression of TPM 1.Part Ⅲ LINC01116 promotes tumor growth and angiogenesis of colon cancer in vivoMethod:sh-LINC01116 or sh-NC was transfected into SW480 colon cancer cells.A nude mouse xenograft model was established by using SW480 cancer cells.Tumor volume and mice weight were detected.RT-qPCR or western blot was used to detect the expression of LINC01116,E2H2 and TPM1 in tumor tissues.HE staining was performed to detect tumor tissue pathology.Immunohistochemistry of CD31 and Ki-67 was performed to observe tumor cell proliferation and angiogenesis.Result:Two weeks after injection of sh-LINC01116 or sh-NC transfected SW480 cells,tumor formation rate was 100%.The expression of LINC01116 and E2H2 in sh-LINC01116 group was significantly lower than that in sh-NC group,while TPM1 expression was significantly increased(P<0.05).The results of HE staining showed that tumor cell density in sh-LINC01116 group was significantly lower than that in sh-NC group.The necrotic tumor cells in sh-LINC01116 group were significantly up-regulated than that in sh-NC group.Two weeks later,tumor size of sh-LINC01116 group was significantly smaller than that in sh-NC group(P<0.05).The results of immunohistochemistry showed that the expression of CD31 and Ki-67 in tumor tissues of sh-LINC01116 group was significantly lower than that of sh-NC group.Summary:LINC01116 promotes the progression of colon cancer in vivo.In vivo results show that LINC01116 promoted the expression of EZH2 and reduced the expression of TP M1,which was consistented with the results at cellular level.Conclusion:LINC01116 was highly expressed in tumor tissues of colon cancer patients and associated with a poor,suggesting that LINC01116 was a potential molecular marker for patients’ prognosis.By recruiting EZH2,LINC01116 promoted the methylation level of TPM 1 promoter region and then inhibited its expression,thus promoting colon cancer cell proliferation and angiogenesis.In vivo experiments also verified the"oncogene" function of LINC01116. |
PDF Full Text Request