| Background:Colorectal cancer is a common malignant tumor of the digestive tract,which occurs at the junction of the rectum and the sigmoid colon.Surgery and chemotherapy have been the first choice for patients with colorectal cancer,however,their therapeutic effect is suboptimal,especially for patients with metastatic disease,with poor prognosis.In the treatment of colorectal cancer,targeted therapy is a new option that has successfully extended the overall survival of colorectal cancer patients.With the successful application of anti-epidermal growth factor receptor（EGFR）drugs and anti-angiogenic（VEGF）drugs,new drugs that block different key pathways are emerging at an unprecedented rate,among which immunotherapy targeting PD-1/PD-L1 benefits more tumor patients.In 2017,the U.S.Food and Drug Administration（FDA）first approved the PD-1 inhibitor pembrolizumab（commonly known as K drug）for the treatment of metastatic colorectal cancer patients with microsatellite high instability（MSI-H）/mismatch repair deficient（d MMR）.Programmed death receptor 1（PD-1）,an important immunosuppressive molecule,is a member of the immunoglobulin superfamily and widely exists on the surface of immune cell membranes.PD-L1 is the ligand of PD-1 and exists on the surface of tumor cells.The combination of PD-1 and PD-L1 initiates the programmed death of T cells,induces immune escape of tumor cells,and targets PD-1/PD-L1 for immune regulation of anti-tumors,anti-infection,anti-autoimmune diseases and organs transplantation and so on,with great significance.PD-1/PD-L1 antibody drugs have drawn more and more attention.At present,6 anti-PD1/PDL1 antibody drugs have been approved by the US FDA for marketing,achieved remarkable curative effect in the treatment of various types of tumors.However,there are also complications such as poor targeting and various adverse reactions,e.g.immune myocarditis during the treatment process,making the mortality rate as high as 46%,which poses a challenge for the widespread use of this type of therapy.Signal transducer and activator of transcription 3（STAT3）is a member of the signal transducer and activator protein family,known for its role in tumor cell proliferation,survival,invasion and immunosuppression,as a transcription factor involved in Cyclin D1,Bcl-2.The expression regulation of proteins such as is one of the most promising targets for cancer therapy.It has been reported in the literature that STAT3 can directly bind to the promoter region of PD-L1,thereby promoting the transcription and expression of PD-L1,suggesting that STAT3 may be involved in tumor cell immune escape through PD-L1;studies have also shown that silencing STAT3 can inhibit the proliferation of colorectal cancer cells induces apoptosis,but the problem of limited silencing efficiency of RNAi technology is also prominent.In the previous work of our research group,the attenuated Salmonella targeting vector was used to carry the sh STAT3 plasmid,which achieved good results in the treatment of prostate cancer,melanoma,breast cancer,laryngeal cancer and other transplanted tumors,effectively improving the problem of poor targeting.This study aims to use the attenuated Salmonella carrying the double-interfering sh STAT3/sh PD-L1 plasmid to precisely target tumor tissue,and combined with RNA interference technology,it can simultaneously prevent proliferation,promote apoptosis and enhance self-anti-tumor immune function.The anti-cancer function of the mechanism plays a role,has obvious tumor targeting,stability and safety,and has clinical application prospects and practical value in the targeted therapy of colorectal cancer.Objective:To investigate the anti-colorectal cancer effect of attenuated Salmonella co-expressing sh STAT3 and sh PD-L1 plasmids.Method:（1）Co-expressing sh STAT3 and sh PD-L1 plasmids were constructed by gene recombination technology;mouse colon cancer CT26 cell line was transfected by eukaryotic cell transfection technology.（2）The sh STAT3/sh PD-L1 recombinant plasmid was transformed into the attenuated Salmonella pho P/pho Q mutant strain by electroporation technology.（3）FITC-Annexin V/PI flow cytometry and Western blot were used to detect the effects of recombinant plasmids on CT26 cell apoptosis and cycle.（4）The effect of sh STAT3/sh PD-L1 recombinant plasmid on the cell viability and proliferation ability of mouse colon cancer CT26 cell line was detected by CCK-8 assay and clone formation assay.（5）Transwell chamber invasion assay and cell scratch assay were used to detect the effect of recombinant plasmids on the migration ability of CT26 cell.（6）Treatment of subcutaneous tumor-bearing model mice with attenuated Salmonella carrying sh STAT3/sh PD-L1 recombinant plasmid:intraperitoneal injection,statistical analysis of tumor volume and mouse body weight;mouse tumor tissue and its various organ tissues sections were stained with H＆E to detect the cell morphology of tumor tissue and main organs;Western blot experiment and immunohistochemical staining were performed on mouse tumor tissue to detect the expression of apoptosis and cycle-related proteins;The changes of serum cytokines IL-6,IFN-γand TNF-αwere detected by ELISA.（7）Treatment of orthotopic colorectal cancer model mice with attenuated Salmonella carrying sh STAT3/sh PD-L1 recombinant plasmid:intraperitoneal injection,statistical analysis of mouse body weight,tumor number,to observe the therapeutic effect.Result:（1）The sh STAT3/sh PD-L1 recombinant plasmid was successfully constructed;the sh STAT3/sh PD-L1 recombinant plasmid was successfully introduced into the attenuated Salmonella pho P/pho Q mutant strain.（2）The results of flow cytometry and Western blot showed that the recombinant plasmid could induce G1 arrest in CT26 cells（P<0.001）,and the expression of G1arrest-related protein Cyclin D1 was significantly down-regulated（P<0.01）.The proportion of apoptotic cells was significantly increased（P<0.001）,the Cleaved Caspase-3 protein was significantly increased（P<0.001）,the expression level of Bcl-2was significantly decreased（P<0.001）,and the ratio of Bcl-2 to Bax protein was decreased（P<0.001）.0.01).It is suggested that the recombinant plasmid inhibits cell proliferation by promoting apoptosis and cycle arrest;（3）The results of CCK-8 experiment and clone formation experiment showed that the recombinant plasmid could significantly inhibit the growth and clone formation ability of CT26 cells（P<0.001）,and the difference was statistically significant.（4）The results of Transwell chamber invasion assay and cell scratch assay showed that the number of migrating cells in the recombinant plasmid transfection group was the least,and the wound healing distance was the shortest,and the difference was statistically significant（P<0.001）,indicating that the recombinant plasmid could significantly inhibit colon cancer CT26 cell migration ability.（5）In vivo experiments,it showed that attenuated Salmonella carrying the sh STAT3/sh PD-L1 recombinant plasmid could target tumor sites,significantly inhibit tumor growth（P<0.001）,and had no toxic and side effects on the main organs of mice;influenced apoptosis and expression of cycle-related proteins;activated immune system function,increased the infiltration of CD4+and CD8+T cells in tumor tissue of tumor-bearing mice;regulated the synthesis and secretion of cytokines,increased the content of IFN-γin serum of tumor-bearing mice,IL-6,TNF-αcontent is reduced.Conclusion:（1）An attenuated Salmonella vector carrying the sh STAT3/sh PD-L1 recombinant plasmid was successfully constructed;（2）The sh STAT3/sh PD-L1 recombinant plasmid can simultaneously silence the expression of STAT3 and PD-L1 in colorectal cancer cells,thereby causing cell cycle arrest,inhibiting cancer cell proliferation and migration,and promoting cell apoptosis;（3）The attenuated Salmonella carrying the sh STAT3/sh PD-L1 recombinant plasmid can specifically aggregate in tumor tissue and has good tumor targeting;（4）The sh STAT3/sh PD-L1 recombinant plasmid carried by attenuated Salmonella can significantly inhibit the growth of colorectal cancer in C57BL/6N mice by inhibiting cell proliferation,promoting apoptosis and increasing immune cell infiltration,and has a good anti-tumor effect.become an effective new strategy for the treatment of colorectal cancer. |
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