| According to the latest global cancer statistics in 2020,colorectal cancer is the third most common malignant tumor in the world,and the second most deadly malignant tumor.Fluorouracil(FU)and oxaliplatin(OXA)are the main chemotherapy drugs in the treatment of colorectal cancer.The combination of the two drugs brings survival benefits to advanced patients,but there are still some patients with drug resistance resulting in poor prognosis.To explore the molecular mechanism of colorectal cancer chemotherapy is helpful to improve the efficacy of chemotherapy.As an endogenous,small,non-coding RNA,miRNA is an important post-transcriptional regulator that plays a key role in cell development,cell differentiation,cell proliferation and cell type-specific functions,and is involved in the pathogenesis of a variety of human diseases,including tumorigenesis,cell differentiation,distant metastasis and chemotherapy resistance.Therefore,studying mirnas and their functions has become an important strategy to find new cancer predictors and therapeutic targets.In the early stage of this study,non-coding RNA chip and bioinformatics analysis found that the combined application of FU and OXA significantly changed the expression of miRNA in colorectal cancer cells,and the expression of miR-183-5p was significantly reduced.Crucially,bioinformatics prediction results showed that SOCS3 was the target gene of miR-183-5p.Suppressor of Cytokine Signaling(SOCS)family is the negative regulator of JAK/STAT signaling pathway,which plays a crucial role in maintaining the stability of intracellular environment,inducing apoptosis and inhibiting overgrowth of cells.SOCS3 inhibits tumor progression by inhibiting JAK kinase and STAT signal transduction path ways.Current studies have found that JAK/STAT signaling pathway is involved in regulating PD-L1 expression in colorectal cancer,and activation of JAK-STAT3 pathway can drive PD-L1 expression.In vitro experiments have also shown that activation of JAK-STAT3 induces PD-L1 expression and induces tumor progression.Based on the above preliminary results and existing research progress,we speculated that FU combined with OX A affects the expression of PD-L1 in colorectal cancer through miR-183-5p/SOCS3 axis,thus promoting the chemotherapy effect.In this study,the role of miR-183-5p,SOCS3 and PD-L1 in the development of CRC was verified by evaluating the correlation between the expressions of miR-183-5p,SOCS3 and PD-L1 in the peripheral blood and tissues of CRC patients and clinicopathological characteristics(Part Ⅰ).Then,the expression of proliferation,apoptosis,metastasis,chemokines,immune escape related proteins and PD-L1 of CRC cells was further detected at the cellular level to verify the molecular mechanism of FU and OXA combined treatment affecting CRC cell growth and PD-L1 expression through miR-183-5p targeting SOCS3(Part Ⅱ).Finally,a mouse xenotransplantation model was constructed to verify the mechanism of FU and OXA combined application affecting the growth and PD-L1 expression of CRC transplanted tumor through miR-183-5p/SOCS3 axis in vivo(Part Ⅲ).Part Ⅰ:The correlation between the expressions of miR-183-5p,SOCS3 and PD-L1 in peripheral blood and tissues of patients with colorectal cancer and clinicopathological characteristicsObjective:To analyze the expression and clinical significance of miR-183-5p,SOCS3 and PD-L1 in peripheral blood and tissues of patients with colorectal cancer.Methods:Blood samples,cancer and paracancer tissue samples and paraffin sections were collected from patients and healthy controls.The expression of miR-183-5p in peripheral blood and tissues of patients was quantified by RT-qPCR.Immunohistochemistry was used to detect the expression of SOCS3 and PD-L1 proteins in cancer tissue samples.The relationship between the expression of miR-183-5p,SOCS3,PD-L1 and clinicopathological indicators of colon cancer patients was statistically analyzed.Results:1.miR-183-5p was significantly overexpressed in peripheral blood and cancer tissues of CRC patients,while SOCS3 was down-regulated and PD-L1 was overexpressed in cancer tissues.Specifically,RT-qPCR results showed that miR-183-5p expression in cancer tissues was higher than that in adjacent tissues in CRC patients(P<0.001).miR-183-5p expression was further increased in cancer tissues of patients with tumor invasion compared with patients without invasion(P<0.001).The expression of miR-183-5p in the blood of CRC patients was higher than that of healthy controls(P<0.001),and its elevated expression was correlated with lymph node metastasis,vascular invasion,and tumor T stage.2.Immunohistochemical analysis showed that SOCS3 protein expression was down-regulated in cancer tissues(P<0.05),and PD-L1 expression was significantly increased(P<0.05).The expression levels of SOCS3 and PD-L1 were closely correlated with lymph node metastasis,vascular invasion and late T stage(P<0.05).There was a significant correlation between SOCS3 and PD-L1 expression in CRC(P<0.05).The expressions of SOCS3 and PD-L1 in CRC cancer tissues were independent predictors of OS(P<0.001).Conclusions:1.MiR-183-5p expression was significantly increased in cancer tissues and peripheral blood of patients with colorectal cancer.2.SOCS3 expression was significantly decreased in cancer tissues,and PD-L1 was highly expressed in colorectal cancer tissues.The expression of SOCS3 and PD-L1 in colorectal cancer tissues has good prognostic value in different pT stages,and SOCS3 and PD-L1 are independent prognostic factors of OS.Part Ⅱ:Fluorouracil combined with oxaliplatin regulates SOCS3 through miR-183-5p and affects apoptosis,proliferation,invasion and PD-L1 expression of colorectal cancer cell line HCT-116.Objective:To explore the effect and molecular mechanism of FU and OXA combined treatment on biological behavior of colorectal cancer cells in vitro.Methods:1.MTT experiment was used to detect the IC50 value of oxaliplatin and fluorouracil on HCT-116 cells.2.miRNA-seq was used to detect the effect of fluorouracil and oxaliplatin on the expression of miRNA in HCT-116 cells.MiR-183-5p imhibitor,mimics transfection are used for knockdown and overexpression of miR-183-5p.pcDNA3.1-SOCS3 and siRNA-SOCS3 are used for SOCS3 overexpression and knockdown transfection.3.CCK-8 and clonogenesis assay were used to detect the proliferation of colorectal cancer cell line HCT-116.Transwell assay was used to detect the invasion and migration of cells.Apoptosis was determined by Annexin V-FITC/PI staining and Flow cytometry.The expression of miR-183-5p in cancer cells was detected by RT-qPCR.Western blot was used to detect the expression levels of SOCS3 and PD-L1 in colon cancer cells.Western blot was used to detect the expression of chemokines(CCL1,CCL4,CCL7)and immune escape related proteins(EGFR,STARD1,STARD3).The targeting relationship between miR-183-5p and its target gene SOCS3 was detected by dual luciferase reporter system.Results:1.The combination of FU and OXA significantly inhibits the proliferation,invasion and migration of HCT-116 cells,promotes apoptosis,and significantly down-regulates the expression of immunosuppressive factors PD-L1,CCL1,CCL4,CCL7,EGFR,STARD1,and STARD3.2.miRNA-seq results showed that the combination of oxaliplatin and fluorouracil significantly reduced the expression of miR-183-5p in HCT-116 cells.3.Further cell function experiments showed that knockdown miR-183-5p significantly inhibited the proliferation and apoptosis of colorectal cancer cells,and down-regulated the expressions of PD-L1,CCL1,CCL4,CCL7,EGFR,STARD1 and STARD3.However,overexpression of miR-183-5p had opposite effects.The dual fluorescence reporter assay combined with western blot showed that SOCS3 was a target gene of miR-183-5p.Functional exploration of miR-183-5p/SOCS3 axis showed that compared with the control group,overexpression of SOCS3 significantly reduced the expression of PD-L1,while simultaneous overexpression of miR-183-5p and SOCS3 significantly eliminated the inhibition of SOCS3 on PD-L1 expression.miR-183-5p relieved the inhibitory effect of SOCS3 on PD-L1 expression by negatively regulating SOCS3 expression.4.In the rescue experiment,SOCS3 silencing significantly promoted the malignant biological behavior of HCT-116 cells,and significantly upregulated the expressions of CCL4,CCL4,CCL7,EGFR,STARD1,STARD3 and PD-Ll.The malignant biological behavior of colorectal cancer cells was significantly recovered in the si-SOCS3+miR-183-5p inhibitor group,si-SOCS3+ si-PD-L1 group and si-SOCS3+FU+OXA group.Conclusions:FU combined with OXA down-regulated PD-L1 through miR-183-5p/SOCS3 axis and inhibited the malignant biological behavior of HCT-116 cells.Part Ⅲ:Fluorouracil combined with oxaliplatin regulates SOCS3 and affects tumor proliferation and PD-L1 expression through miR-183-5p in the HCT-116 cell line Balb/C nude mouse transplanted tumor model.Objective:In order to construct xenograft tumor model of HCT-116 cell,to verify the molecular mechanism of the combined application of FU and OXA through the miR-183-5p/SOCS3 axis to reduce the growth and immunosuppression of colon cancer transplantation tumor in vivo.Methods:1.Balb/c was injected subcutaneously into nude mice to construct a colon cancer xenograft model.Intratumoral injection of miR-183-5p agomir and antagomir serves as an in vivo intervention for the overexpression and knockdown of miR-183-5p.2.Lentiviral transfection to construct SOCS3 knockdown HCT-116 cells.3.FU and OXA combined intratumoral injection to simulate chemotherapy;The growth curve of transplanted tumor was plotted by volume measurement.TUNEL staining was used to detect the apoptosis of the transplanted tumor tissue.Immunohistochemistry was used to detect the expression of SOCS3 and PD-L1.The expression of miR-183-5p in transplanted tumor tissues was detected by RT-qPCR.Western blot was used to detect the expression levels of SOCS3 and PD-L1 in transplanted tumor tissues.Results:1.Compared with the control group,miR-183-5p agomir significantly promoted the tumor-forming ability and tumor growth of the transplanted tumor,while the injection of miR-183-5p antagomir resulted in the growth inhibition of the transplanted tumor.2.The in vivo function study of SOCS3 on xenograft tumors showed that knocking down SOCS3 significantly enhanced the ability of HCT-116 cells to form tumors under the skin and tumor growth;among them,the growth and tumor weight of transplanted tumors in nude mice in the SOCS3 silence group significantly increased,and cell apoptosis The rate was significantly lower than that of the control group.Further experiments showed that the combined application of FU and OXA significantly reduced the growth of transplanted tumors and promoted the apoptosis of transplanted tumor cells;while knocking down SOCS3 significantly weakened the inhibitory effect of combined chemotherapy on the volume and growth of subcutaneous tumors in nude mice.In addition,the combination of FU and OXA reduced the expression levels of miR-183-5p and PD-L1 in the transplanted tumor tissues.However,the expression of PD-L1 was not significantly affected in the transplanted tumor group formed by HCT-116 cells knocking down SOCS3.Conclusions:The expression of miR-183-5p in colorectal cancer tissues and peripheral blood was significantly increased,while the expression of SOCS3 was significantly decreased in colorectal cancer tissues,and PD-L1 was highly expressed in colorectal cancer.The combined detection of SOCS3 and PD-L1 in colon cancer tissues had better prognostic value than a single marker at different pT stages.Uni variate and multivariate analyses showed that SOCS3 and PD-L1 were independent prognostic factors for OS.In vitro and in vivo experiments confirmed that the combination of FU and OXA significantly reduced the expression of miR-183-5p in colorectal cancer cells.Down-regulation of miR-183-5p reduced its negative regulation of SOCS3 and inhibited the expression of PD-L1.miR-183-5p,SOCS3 and PD-L1 are closely related to the efficacy and prognosis of colorectal cancer chemotherapy,and joint intervention of these three key factors is a potential effective strategy to promote the efficacy of colorectal cancer chemotherapy. |
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