| The incidence and mortality of Colorectal Cancer(CRC)are in the forefront of all tumors.The abnormality of epigenetic modification has been found to be an important cause of colorectal cancer.In recent years,it has been found that long noncoding RNA(long noncoding RNA,lncRNA)is of great significance in the pathological process of tumors.We compared lncRNA expression profile data of colon cancer tissues and normal tissues,and found a new lncRNA LNRRIL6(ak024522)expression increased significantly in tumor tissues.LNRRIL6 was located in chromosome 12p13.1,with 2017 nucleotides in length.Part ⅠOBJECTIVE:We would like to identify lncRNAs with biomarker function by analyzing the chip data,detect the expression level of lncRNA in clinical patients,and explore the relationship between lncRNA expression and CRC clinical phenotype,diagnosis,disease staging and prognosis.Methods:66 cases of colorectal cancer were enrolled and the tumor tissue and para-cancer tissues were collected.The expression of LNRRIL6 was detected by the fluorescence quantitative PCR..Statistical clinical data,including gender,age,tumor location,size,differentiation,staging and tumor metastasis were compared between the high LNRRIL6 expression group and the low expression group.The 5-year survival of 66 patients was followed up,and the difference in the survival time between the high expression group and the low expression group was analyzed by Kaplan-Meier.The expression of LNRRIL6 in human normal colonic epithelial cells NCM460 and colon cancer cell lines HCT-116,LoVo,SW480,SW620 and HT-29 cells was detected by fluorescence quantitative PCR.Results:The expression of LNRRIL6 in colorectal cancers was significantly higher than that of adjacent normal tissues(p<0.01).Compared with the low group,high group had larger tumor sizes(p=0.049),poorer differentiation(p=0.026),and more lymph node metastasis(p=0.037).Kaplan Meier survival analysis showed that the 5-year Overall survival rate(OS)of high group was significantly lower than that of the LNRRIL6 low expression group(p=0.0343).Real-time fluorescence quantitative PCR showed that the expression of LNRRIL6 in colon cancer cell lines was significantly higher than that of normal colonic epithelial cells.Part ⅡObjective:To detect the effect of LNRRIL6 on tumor size,proliferation and apoptosis,and to clarify the biological effects of LNRRIL6 on colon cancer cells.Methods:LNRRIL6 full-length lentiviral vector and oligonucleotide shRNA expression vector specific for LNRRIL6 cDNA were constructed;LoVo and HCT-116 cells were transfected.Cell proliferation and apoptosis were examined.A subcutaneous xenograft model was established to measure the volume of the transplanted tumor.Immunohistochemical staining was used to detect the positive expression of Ki67 and caspase-3 in the tumor.Results:The full-length sequence of LNRRIL6 has a composition of 2017 nucleotides.A cell line stably overexpressing LNRRIL6 and a lentiviral LNRRIL6-silent colon cancer cell line with different target sequences were constructed.Proliferation and apoptosis assay showed that overexpression of LNRRIL6 significantly increased the activity of HCT-116 and LoVo cells(p<0.01),and increased the positive rate of EdU and decreased the number of apoptotic cells(p<0.01).The number of cells in silencing LNRRIL6 cells decreased.p<0.01),and the number of cells positive for EdU was significantly decreased,and the number of apoptotic cells was significantly increased(p<0.01).In vivo xenograft models found that compared with the control group,the overexpression group accelerated the tumor proliferation rate after the 21st day(p<0.01).The tumor volume of the silent group was significantly smaller than that of the control group(p<0.01).On day 28,the tumor weight of the control group(LV-NC)was less than that of the overexpression group(LV-LNRRIL6)(p<0.01);the tumor weight of the control group(LV-shNC)was greater than that of the silent group(p<0.01);the number of Ki67 positive cells was The overexpression group was significantly more than the control group(p<0.01),while the control group was significantly more than the silent group(p<0.01);the overexpression group caspase-3 positive cells were less than the control group(p<0.01);The number of cells in this group was less than that in the silent group,and the difference was significant(p<0.01).Part IIIObjective:To predict the effect of LNRRIL6 on IL-6 based on the localization of LncRNA-LNRRIL6 and bioinformatics prediction of LNRRIL6 targeting.Methods:The cytoplasm and nucleus were separated,and the subcellular localization of LNRRIL6 in the nucleus and cytoplasm was detected by qRT-PCR.Chromatin RNA isolation and purification experiment(ChIRP).The IL-6 content in the supernatant was detected by enzyme-linked immunosorbent assay(ELISA),and the activation of STAT3 was detected by Western blot.Results:LNRRIL6 binds to the IL-6 promoter region,and more than 80%of LNRRIL6 is in the nucleus.The results of bioinformatics analysis showed that the 870bp-1180bp of LNRRIL6 has high homology with the IL-6 promoter region,and 83%of the sequences match each other.CHIRP detects that the LNRRIL6 antisense probes bind to each other and the IL-6 promoter region.LNRRIL6 can activate the IL-6/STAT3 pathway.The expression of IL-6 mRNA in HCT116 cells overexpressed by LNRRIL6 and IL-6 protein in cell culture supernatants were significantly increased by ELISA and qRT-PCR.In contrast,LNRRIL6 silenced HCT116 cells.Significant reduction within the system.Increased p-STAT3 phosphorylation activation in overexpressing cells.In the silence group,the p-STAT3 protein content was decreased.There was a good positive correlation between IL-6 expression level and LNRRIL6 RNA level in tumor tissues(r=0.6917,p<0.0001).After the IL-6 signaling pathway inhibitor was used,it was found that the effect of LNRRIL6 on tumor viability disappeared(p<0.01).Part IVObjective:To study the mechanism of inhibition of IL-6/STAT3 combined with PDL1 inhibition of colon cancer cell growth.Material method:The expression of STAT3 mRNA and PD-L1 were detected in CT26.WT cells and mouse intestinal mucosa cells.STAT3 silencing lentivirus were constructed and transfected into CT26.WT cells.The mouse xenograft model was constructed and grouped according to the transfected vector.The subcutaneous tumor volume was measured periodically.Tumor tissue’s HE staining,apoptosis detected by Tunel,detection of C-met,PD-L1,STAT3 protein immunohistochemical staining,gene expression,protein expression.Result:CT26.WT cells overexpressed STAT3 mRNA and PD-L1.The mice transfected with STAT3 silencing lentivirus have relatively small tumors,and the mice in the combined silent group have slower tumor growth and the smallest volume.HE staining showed that compared with the control group,The cells in the STAT3 alone group were relatively loose,and a small amount of vacuoles appeared,while the combined silent group showed a large number of vacuoles.The pattern is extremely irregular and there is a more serious inflammation.The apoptosis of the silencing STAT3 alone group was enhanced,and the apoptosis rate was significantly increased in the combined silencing group.Immunohistochemistry,real-time PCR and Western blot showed that the expression of C-met,STAT3 and PD-L1 in the combined silencing group decreased significantly.The difference in results was statistically significant(P<0.05).Conclusion1.LNRRIL6 is highly expressed in colorectal cancer tissue and colorectal tumor epithelial cells;LNRRIL6 promotes the proliferation of colorectal cancer cells and inhibits apoptosis;The expression of LNRRIL6 in tumor is positively correlated with tumor size,tumor differentiation and tumor metastasis,and is negatively correlated with overall survival;2.LNRRIL6 is expressed in the nucleus and interacted with the IL-6 promoter region,thus promoting the activation of STAT3/IL-6 signaling pathway;.LNRRIL6 promotes tumor progression via 11-6 signaling.3.Combined application of PDL1 and inhibition of IL-6/STAT3 signaling pathway can inhibit tumor proliferation. |
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