| Objective: Several studies have shown that miRNAs play an important regulatory role in the development of colorectal cancer.The previous group tested colorectal cancer clinical specimens and cell lines,and found that miR-581 was differentially expressed in colorectal cell lines,and the expression in the highly metastatic cell line(SW620)was significantly lower than that of carcinoma in situ Cell line(SW480).In this study,a colorectal cancer cell line stably expressing luciferase was constructed and screened from the animal level.A nude mouse model of colorectal cancer subcutaneous ectopic transplantation tumor and a tail vein injection metastasis model were established.The effect of cancer cell proliferation ability and indirectly reflect the ability of miR-581 to affect the mechanism of colorectal cancer cell lines in vivo through the in vivo imaging system.Using reference genome transcriptome sequencing to predict and experimentally verify that miR-581 may be involved in regulating the signaling pathway of colorectal cancer cells.Method: 1.Construction of luciferase-labeled stable colorectal cancer transformants SW480(miR-581-NC-Luc,miR-581-KD-Luc)and SW620(miR-581-NC-Luc,miR-581-OE-Luc): Luciferase-labeled lentivirus infected SW480 and SW620 colorectal cancer cells in logarithmic growth phase;trypsinized,complete medium was made into 5 × 104 cells / m L cell suspension,and the corresponding cells were seeded according to the instructions Count into 6-well culture plates and continue to culture to ensure that the plating volume reaches about 15-30% when infected.According to the pre-experiment results,the infection medium was changed at 12 h,and the optimal virus amount was added for infection according to the MOI value of the virus.Observe the cell state and infection efficiency after infection.The cells are in good condition without a large number of deaths.The infection efficiency is guaranteed to reach about 80%-90%.q PCR/WB was tested to verify the interference of transcriptome level and proteomics level gene miR-581.2.Establishment of subcutaneous xenograft model of colorectal cancer in nude mice:Puromycin was added to the medium according to the grouping of SW480(NC,miR-581-KD)and SW620(NC,miR-581-OE)The stable transfection strains were selected and cultured to a cell concentration of 2E + 7/m L.The cell suspension was aspirated with a disposable 1ml sterile syringe,and slowly inoculated subcutaneously into the axillary skin of nude mice according to the grouping situation.Each inoculated 0.1ml.Five control nude mice were set,and the tumor cycle and tumor tissue growth of each component were observed at the same time.Every other week,the length and diameter of the subcutaneous tumor were measured with a vernier caliper,and the growth curve of the tumor was plotted.After five weeks,nude mice were sacrificed by vertebrae to exfoliate the subcutaneous tumor.Calculate the tumor volume according to the formula V =(a2b)/2(a represents the shortest diameter of the tumor,b represents the longest diameter of the tumor).The dissected tumor tissues were grouped for comparison and photographed.They were fixed in 4%paraformaldehyde and removed from the pathology section,and then verified by HE staining/IHC and other experiments.3.Construction of tail vein injection metastasis model of colorectal cancer: Twenty4-week-old female nude mice were randomly divided into 4 groups OE-Luc),5 nude mice were set in each of the experimental group and the control group.Culture the stable transgenic plants in each group to a cell concentration of 2E + 7/m L,and inject them into the tail vein of nude mice,each 0.1ml.The real-time fluorescence imaging system KODAK Iinage Station 2000 MM was used to observe the size and metastasis of tumors in vivo.Nude mice in each group began to observe the dynamic imaging in vivo from the first week after tail vein injection,and then observed weekly.The experiment was terminated after 5 consecutive weeks of observation,and the mice were subjected to the last live imaging before the end.After the imaging,the experimental animals were euthanized with 2% sodium pentobarbital(0.5 m L)by injection.After they were completely comatose,cervical dislocation was performed to confirm death.The animals were dissected with medical scissors and medical forceps,and the lungs,livers,and other organs were removed.Some of the specimens were stored in paraformaldehyde at room temperature and stored at-80 ° after freezing in liquid nitrogen for subsequent HE staining and immunofluorescence experiments Detection.4.Detection of EMT-related molecules: Western blot tests were performed to detect the expression of EMT-associated factors E-cadherin,Vimentin,Snail,and MMP-9 in the protein samples extracted from the above-mentioned groups of transplanted tumors.The process of EMT in cancer cells.5.MiR-581 participates in related signaling pathways in the development of colorectal cancer: using reference genome transcriptome sequencing,SW620-NC and SW620-miR-581-OE two groups of cells were subjected to RNA extraction,purification,and library construction.Next-Generation Sequencing(NGS),based on the Illumina sequencing platform,performs paired-end(PE)sequencing of these libraries.The original raw data was filtered,and the filtered high-quality sequence(Clean Data)was aligned to the reference genome of the species.Based on the comparison results,the expression level of each gene was calculated.On this basis,the samples were further analyzed for expression difference analysis,signal pathway enrichment analysis(KEGG),and function enrichment analysis(GO).Screening of miR-581-related signaling pathways was performed based on sequencing results and verified by experiments such as Western blot.Result: 1.Detection of miR-581 transfection efficiency and verification of interference effects: 72 hours after miR-581-Luc transfected colorectal cancer cells SW480 and SW620 cells,q PCR was used to detect the miR-581 transcription in the two groups of cells;under the premise that the previous group’s dual luciferase report has verified that miR-581 targets negatively regulated PRDX1,Western blot and cellular immunofluorescence assays are used to detect the expression of PRDX1 and indirectly detect the interference effect of miR-581.From the q PCR results of miR-581,the Western blot results of PRDX1,and immunofluorescence results,it can be concluded that the miR-581 of the SW480 experimental group was successfully knocked down compared to the control group;the overexpression of miR-581 in the SW620 experimental group was more successful than that in the control group;and it was confirmed that the growth characteristics and cell activity of colorectal cells in the negative control group transfected with luciferase-free virus were not different from those in the blank control group..2.The results of nude mice bearing colorectal cancer cells in different groups:(1)SW480 cell line: the control group(miR-581-NC)and the experimental group(miR-581-KD)were each inoculated with 4-week-old female nude mice.The inoculation volume was 0.1ml x 2E + 7 / m L,the tumor formation period was 21 days,the tumor formation rate was 100%,and the observation period was 5 weeks.The results showed that,compared with the control group,the tumor volume and mass of the nude mice in the miR-581 knockout group were significantly reduced.(2)SW620 cell line: the control group(miR-581-NC)and the experimental group(miR-581-OE)were also inoculated with 5 4-week-old female nude mice,the cell inoculation amount was also 0.1ml x 2E + 7 m L.The tumor period was 21 days,and the tumor formation rate was 100%.The observation period was also 5 weeks.Results: compared with the control group,the tumor volume and tumor mass of nude mice in the mir-581 overexpression group increased significantly.The expression of PRDX1 in the miR-581 knockdown group was significantly higher than that in the miR-581 overexpression group.The expression of ki-67 in the miR-581 overexpression group was significantly higher than that in the mir-581 knockout group.3.Result of distant metastasis of colorectal cancer cells by tail vein injection: The bioluminescence signal of tumor cells can be detected in the chest position by in vivo imaging system in nude mice in the second week after inoculation,but not all groups can detect it.With the increase of time,the fluorescence signal gradually increased,and the fluorescence signal reached the strongest at 5 weeks after the inoculation.The experiment lasted for 6 weeks from vaccination to final execution.The statistics of metastasis results were as follows:(1)SW480 group: NC group had 5 tail vein inoculations,3 lung metastases;miR-581 knockdown group had 5 tail vein inoculations,4 lung metastases.No liver metastases were found in the NC group and miR-581 knockout group.The average expression of fluorescence in each nude mouse was divided into T-TEST test according to the control group and experimental group.The analysis showed that the average fluorescence expression of each nude mouse in the experimental group was higher than that in the control group(P <0.05);(2)SW620 group: NC group inoculated 5 tail veins,4 lung metastases;miR-581 was up-regulated In the group,5 mice were inoculated by tail vein,and 3 lung metastases.Neither the NC group nor the miR-581 up-regulation group found liver metastases.The average expression of fluorescence in each nude mouse was divided into T-TEST test according to the control group and experimental group.After analysis,the average fluorescence expression of each nude mouse in the experimental group was lower than that in the control group(P <0.05).From the analysis of the total metastasis rate,the number of metastases,and the fluorescent expression in the metastatic area of??two colorectal cancer cells SW480 and SW620,it was initially concluded that compared with the control group,overexpression of miR-581 may be in nude mice.Inhibition of lung metastasis in colorectal cancer.4.Western blot results of EMT-related molecules and signal pathway verification results: Tissue tumor tissue proteins of the experimental group and control group of SW480 and SW620 were extracted,respectively,and the expression of EMT-related molecules was detected by Western blot.SW480 group: Compared with the negative control group and the blank control group,the expression of E-cadherin in the miR-581 knockdown group decreased,and the expressions of Vimentin,Snail,and MMP9 increased.E-cadherin expression was increased in the miR-581 overexpression group,while Vimentin,Snail and MMP9 expression was decreased.Based on the KEGG signal enrichment analysis in the sequencing results,a signal pathway with a P-value <0.05 and related to tumorigenesis and development was selected for verification.It was confirmed by Western blot that miR-581 is mainly involved in ECM/ITGA/PI3K/AKT signaling pathways in colorectal cells to participate in the survival of colorectal cells,and miR-581 overexpression can promote CSF3,ITGA2 B,etc.Molecular expression is upregulated.Conclusion: 1.miR-581 overexpression promotes tumorigenicity of colorectal cancer cells in vivo and significantly promotes its proliferation ability,but inhibits the lung metastasis ability of colorectal cancer cells in vivo.2.The expression of PRDX1 in tumor tissues was negatively correlated with the expression of miR-581,which was consistent with the conclusion that miR-581 targeted negative regulation of PRDX1 at the cell level.3.Overexpression of miR-581 can inhibit the EMT process of colorectal cancer.4.miR-581 regulates colorectal cancer cell metastasis mainly through ECM/ITGA/PI3K/AKT signaling pathway. |
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