Study Of Anti-tumor Effects And Mechanisms Of Tumor Vaccine Induced By Down Regulating The Expression Of MUC1 In Melanoma Cells

Malignant melanoma is the most invasive and highly metastatic skin cancer.Studies have shown that its natural apoptosis rate is lower than other types of tumors.Melanoma only accounts for 5%of all skin cancers,but about 80%of all skin cancer-related deaths are attributed to melanoma,posing a serious threat to human health.The incidence of melanoma is increasing worldwide in despite of early detection and intervention,and the number of patients who die of metastatic disease continues to increase.The prognosis of advanced melanoma is poor,with a median survival of 6 to 9 months.In the past 30 years,extensive clinical research has been conducted,but the treatment options for metastatic disease are still limited,and melanoma is still considered one of the most difficult to treat malignancies.At present,the treatment methods for melanoma include surgery,radiotherapy,chemotherapy,targeted therapy,etc.Although surgery is still the current gold standard of treatment, immunotherapy and targeted molecular therapy have also shown good therapeutic effects in metastatic melanoma.Tumor immunotherapy,such as immune checkpoint blocking,adoptive immunization,chimeric antigen receptor(CAR-T),tumor vaccines,etc.,has shown good therapeutic effects in scientific experiments and clinical studies.Among them,the research of melanoma tumor vaccine has been under discussion and development for many years.Vaccines can activate the body’s specific cytotoxic T lymphocyte(CTL)effects and specific antibodies to inhibit and kill tumor cells.However,due to the weak immunogenicity of tumor cells and the complicated immune escape mechanism of tumors,there is a big gap between the actual effect of the vaccine and the expected result.In order to improve the immune effect of melanoma tumor vaccines and screen effective target antigens,researchers are also constantly working to solve this key scientific problem.Hypoglycosylated MUC1 is overexpressed in most human epithelial cancers and has attracted widespread attention as a carcinogenic molecule.A project carried out by the National Cancer Institute to prioritize the treatment of cancer antigens ranked MUC1 as the second candidate for 75cancer vaccine antigens,which emphasized the importance of MUC1 as a therapeutic target among other tumor-related cancer antigen potential.Studies have proved that MUC1 expressed by mouse cancer cells has a dominant epitope, which can induce mice to produce MUC1 antibodies,which can be used as a candidate antigen for vaccines.If its expression is regulated,it will affect the immunogenicity of the vaccine,and then affect the vaccine’s anti-tumor effect.In normal cells,a large number of MUC1 sugar chains prevent the presentation of antigen peptides and prevent the access of CTLs.MUC1 subunit andβ-catenin are jointly transported to the nucleus,inhibiting the expression of E-cadherin,and up- regulating the expression of EMT-inducing factors Snail,Slug,Vimentin and Twist.But if new glycopeptide epitopes appear in cancer cells due to its configuration changes,its steric hindrance will be lost and MUC1 itself is an immune target,which provides an opportunity to target these new glycopeptide targets.Our laboratory found in colon cancer vaccine research that MUC1 is an important target antigen,and the immunogenicity of tumor cell vaccines is significantly related to the expression of MUC1.Objective:The purpose of this study is to explore the effect of regulating the MUC1 molecule on the biological characteristics of the melanoma cell line B16F10,prepare melanoma vaccines, investigate its anti-tumor effect and its mechanism,and provide experimental evidence for verifying MUC1 as an effective candidate target antigen.Methods and contents:1.We used small RNA interference technique,construct a lentiviral recombinant vector containing pHBLV-U6-shMUC1-ZSGreen and pHBLV-U6-shNC-ZSGreen,and co-transfect 293T cells with the helper plasmids psPAX2 and PMD2.G,respectively.We collected the 293T cells culture supernatant and filter,concentrate the lentivirus by high-speed centrifugation,infect murine melanoma B16F10 cells with the packaged pLV-shMUC1 and pLV-shNC viruses,and then screen out the stable down-regulated MUC1 with the limiting dilution method as molecule-positive monoclonal cells.After that,real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)and western blotting(Western Blot)were used to detect the expression of MUC1 molecule at the mRNA and protein levels.2.To explore the effect of down-regulating MUC1 molecules on several biological characteristics of B16F10 cells:plate cloning experiments and CCK8 experiments to detect the cloning and proliferation of B16F10 cells after down-regulating MUC1 expression;Transwell experiments and scratch experiments to detect the migration ability of B16F10 cells after down-regulating MUC1 expression;qRT-PCR detects the expression levels of mRNAs of E-cadherin,N-cadherin and vimentin related molecules of EMT.3.Take 1mL of B16F10 cells,Scramble cells and Lv-shMUC1-B16F10 cells at a concentration of 5×10~6/mL,freeze and thaw them three times,prepare them into tumor vaccines and then immunize C57BL/6 mice three times,every two immunizations at an interval of 10 days,on the10th day after the last immunization,the mice were challenged with 1×10~5 WT B16F10 cells subcutaneously in the abdomen.Real-time observation of the survival status,tumor emergence time and tumor volume of different immunized mice to evaluate the anti-tumor effects of different groups of tumor vaccines;flow cytometry(FCM)to analyze the CTL activity and NK activity of spleen cells of immunized mice Cell killing ability;ELISA was used to detect serum anti-MUC1antibody titers and levels of IFN-γand TGF-βcytokines in mice immunized with tumor vaccines.qRT-PCR was used to detect the mRNAs expression level of related molecules in tumor tissues.Results:1.Infected B16F10 cells with the packaged lentivirus.After the stable infection strain was selected,the qRT-PCR and Western Blot test results showed after pLV-shMUC1 infected B16F10cells,the expression of MUC1 molecule was significantly reduced,which was statistically significant compared with the control group(P<0.05).After pLV-shNC was infected with B16F10cells,the expression of MUC1 molecule was not significantly different from the uninfected group(P>0.05).2.In vitro results showed that:Compared with wild-type B16F10 cells and Scramble cells,Lv-shMUC1-B16F10 cells have reduced clonal formation,proliferation and migration capabilities; qRT-PCR detection of EMT related the molecular results showed that the expression of E-cadherin mRNA in Lv-shMUC1-B16F10 cell was increased,while the expression of N-cadherin and vimentin mRNA was decreased.The difference was statistically significant(P<0.05).3.In vivo the tumor vaccine experiment results in mice showed that:mice immunized three times with wild-type B16F10 cells,Scramble cells and Lv-shMUC1-B16F10 cell vaccines,after challenged wild-type B16F10 cells,the B16F10 cell and Scramble vaccine groups,the anti-tumor growth effect was good,which was reflected in the tumor-free survival period is the longest,the mouse tumor growth was the slowest,and the size was small,while the Lv-shMUC1-B16F10 cell vaccine group had the worst anti-tumor effect,and the difference was statistically significant (P<0.05).According to FCM analysis,the spleen cells of the Lv-shMUC1-B16F10 cell vaccine group had the weakest CTL activity and NK ability;the ELISA test results showed that the Lv-shMUC1-B16F10 cell vaccine group had the lowest IFN-γlevel,and TGF-βlevel was the highest among all groups,and the differences were statistically significant(P<0.05).qRT-PCR was used to detect the mRNAs expression of EMT-related molecules and granzyme and perforin in tumor tissues.The results showed that the expression of E-cadherin mRNA in the Lv-shMUC1-B16F10tumor vaccine immune group was decreased,and the mRNA of N-cadherin and vimentin was increased.The expression of granzyme and perforin mRNAs were lower than those of B16F10 and Scramble tumor vaccine immunized group,the difference was statistically significant(P<0.05).Conclusions:1.The MUC1 molecule is related to the ability to promote the proliferation,migration and invasion of B16F10 cells.After down-regulating the MUC1 expression,the above biological properties are all weakened.2.The wild-type B16F10 cell vaccine and Scramble cell vaccine can induce a strong anti-tumor effect in immunized mice and antagonize the growth of melanoma.The mechanism of its effect is related to its high expression of MUC1 antigen to induce a strong immune response.After down-regulating the MUC1 molecular expression,it can affect the immune effect and anti-melanoma effect of the vaccine.This study proved for the first time that the MUC1 molecule may be an effective candidate target antigen for melanoma vaccine development.