Research On Screening Of Mycoplasma Hyorhinis-targeting Aptamer And Its Activity In Anti-esophageal Squamous Cell Carcinoma

Esophageal cancer is one of the most common malignant tumors in digestive tract worldwide with high incidence and mortality rate.Although the strategies of diagnosis and treatment for esophageal cancer is improving in recent years,such as the replacement of open surgery with minimal invasive surgery and the targeted therapy and immunotherapy are also widely used at the same time,but the 5-year survival rate was only 15-25%.So it is urgent to study the occurrence and development mechanism of esophageal cancer and exploit new diagnostic methods and therapeutic strategies.Researches have shown that microbial infection is an important factor in the occurrence and development of tumors.Mycoplasma is the smallest prokaryote in nature,and it is widely parasitic in humans and animals.Mycoplasma is a conditional pathogenic pathogen,which could cause a variety of infection and inflammation.In recent years,multiple studies have shown that mycoplasma infection is closely related to human malignant tumors.Among them,Mycoplasma hyorhinis(M.hyorhinis)has been reported to be detected in various malignant tumors.Through interacting with host cells,M.hyorhinis can lead to chromosome abnormalities,induce malignant transformation of normal cells,or activate the signal pathway of tumor cells to promote the migration and invasion of tumor cells and increase the malignancy.The current research shows that about 50.9% of esophageal cancer patients have M.hyorhinis infection in tumor tissue.In addition,the clinical detection of mycoplasma mainly depends on separated cultivation method or PCR method at present,and there is a lack of more rapid and in situ detection methods.Aptamer is an oligonucleotide molecule composed of RNA or ss DNA that screened by Systematic Evolution of Ligands by Exponential Enrichment(SELEX).Aptamer could bind to proteins,sugars,small molecules,ions and other target molecules with high affinity and specificity.Besides,aptamer also has the advantages of small molecular weight,low cost,ease of chemical synthesis and modification and no batch difference.As an excellent molecular ligand,aptamer has been widely used in molecular imaging,biosensor,disease detection and diagnosis,targeted therapy and other biomedical fields.According to the above background,in order to further clarify the relationship between M.hyorhinis and ESCC,revealing the role and mechanism of mycoplasma infection in the development of ESCC,and developing a novel mycoplasma detection method and anti-tumor strategy based on SELEX,our paper has carried out the following experiments.The details are as follows:(1)In order to clarify the infection of M.hyorhinis in ESCC,we extracted the DNA of tumor tissues from ESCC patients and identified the infection rate by real-time quantitative PCR.Secondly,ESCC cell line Eca-109 was co-cultured with M.hyorhinis to infect the cells.We found that the proliferation ability of Eca-109 cells did not change after infection,but the infection significant enhanced the migration and invasion ability.In addition,we constructed the tumor metastasis model of nude mice by injecting cells through tail vein and found that mycoplasma infection could significant promote the lung metastasis of ESCC in mice.Thus,we proved that ESCC patients have M.hyorhinis infection,and the infection could induce the migration,invasion in vitro and metastasis in vivo.(2)In order to obtain aptamer for recognizing M.hyorhinis,we screened and identified aptamers targeting the infected cells by cell-SELEX.By truncating the aptamer,we obtained the aptamer ZY3 A with higher affinity,and the chemical modification improved its serum stability.Then,we constructed a xenograft tumor model in nude mice with mycoplasma infection,we found that aptamer ZY3 A could recognize M.hyorhinis at the site of xenograft tumor and perform in situ imaging in vivo.This experiment proved that aptamer ZY3 A is a potential molecular tool for M.hyorhinis imaging in vivo.(3)In order to clarify the target of aptamer ZY3 A and clarify the combination mechanism of ZY3 A to its target,we used the Aptamer pull-down assay and LC-MS/MS and identified that the target of ZY3 A is high affinity transport system protein p37.p37 is a highly conserved and specific membrane mycoplasmal protein for M.hyorhinis,and it is also a key protein for infection.Using anti-p37 antibody,we proved that the aptamer ZY3 A and anti-p37 antibody have similar binding ability and highly co-localization in cellular immunofluorescence.Then we proved the direct binding ability of ZY3 A to recombinant p37 protein through enzyme linked oligonucleotide analysis(ELONA),and clarified the specific interaction sites between ZY3 A and p37 by molecular docking simulation,In this part,we made it clear that the target of aptamer ZY3 A is p37.(4)The present studies have shown that M.hyorhinis infection depends on the interaction of p37 with host receptor proteins.Our experiment found that the aptamer ZY3 A could neutralize the infection of M.hyorhinis on ESCC cells with a concentration dependent manner.We found that TLR4 protein was the receptor protein of p37 on ESCC cells by detection of host receptors.Through GST pull-down and immunoprecipitation experiments,We proved that the aptamer ZY3 A could block the interaction between TLR4 on cell surface and p37 on mycoplasma,inhibit the adhesion of M.hyorhinis to the cell surface and neutralize the infection of M.hyorhinis.Under this effect,ZY3 A inhibited the cell migration and invasion caused by mycoplasma infection.By further analyzing the activity or content of NF-κB and MMP2,which related to cell migration and invasion,we found that mycoplasma infection could activate the phosphorylation of NF-κB P65 and increase the expression of MMP2,while the neutralization of aptamer ZY3 A could counteract this activation.The above results show that M.hyorhinis infects ESCC cells through the interaction between p37 and TLR4,and activates relevant downstream pathways to induce cell migration and invasion.Aptamer ZY3 A could block this process and reverse the migration and invasion ability of cells.(5)Finally,we investigated the ability of aptamer ZY3 A to neutralize mycoplasma and its anti-tumor effect in nude mice.By injecting M.hyorhinis infected ESCC cells into nude mice from tail vein to build a tumor metastasis model,and then injecting aptamer ZY3 A for treatment,we found that ZY3 A can effectively inhibit the tumor metastasis,reduce the number of tumor metastasis nodules and the content of M.hyorhinis in lung tissue,which implying that nucleic acid aptamer ZY3 A can be used as a potential anti-mycoplasma agent in the treatment of ESCC.