Construction Of ELANE、ELA1、HPPE Gene Overexpression Vector And Its Effect On Human Cancer Cells

Background and objectives:Neutrophils,as one of the most abundant cells in human myeloid leukocytes,are the main responders against a variety of pathogens including bacteria and viruses.Mature neutrophils are retained in the bone marrow or released into the blood influenced by regulatory factors.The neutrophils released into the blood,passing through rolling and migrating from blood vessels to local tissues under the action of various integrins and chemokines.Neutrophils play an important role in activating and regulating innate and adaptive immunity.Recent studies have found that neutrophils,known as tumor-associated neutrophils(TANs),are not only found in anti-infection process,but also in tumors tissue.TANs are important component of tumor microenvironment(TME).As an important inflammatory cell in TME,TANs are diverse and heterogeneous,showing both anti-tumor and pro-tumor effects.In 2009,Fridlender,an American scholar,proposed naming,and called TANs mediating anti-tumor effect type N1 and mediating pro-tumor effect type N2.In the complex environment of TME,TANs can be polarized,and it was found that the two types of TANs can be transformed into each other,and this process can be induced and regulated by cytokines and epigenetic signals.Most previous studies focused on the tumor promotion of N2-TANs,while studies on N1-TANs were few and limited,ignoring its anti-tumor effect and application prospect.Previous studies on antitumor mechanism have shown that N1-TANs mediated cytotoxicity can be mediated by direct cytotoxic effects,mainly refers to antibody dependent cell mediated cytotoxicity(ADCC).Meanwhile,various chemokines,growth factors and cytokines in TME can affect the mutual transformation of the two types of TANs,which is a specific inflammatory cell death mode of TANs.Neutrophil extracellular traps(NETs),which consist of DNA and proteins,may become a research topic in recent years.Elastase neutrophil expressed(ELANE),as a subfamily of serine protease,can hydrolyze many proteins in addition to elastase,which has been regarded as a tumor promoting factor in previous studies.Until recently,different research results have been exposed by American scholar Cui et al.In vitro experiments,ELANE was observed to be the main antitumor protein in neutrophil conditioned medium,selectively killing tumor cells and inhibiting tumor formation,while having no killing effect on non-tumor cells.Subsequently,in vivo validation experiments in animal models,it was found that the antitumor effect of ELANE was impaired by serine protease inhibitors in TME.Porcine pancreatic elastase(PPE),with 32% amino acid homology,is a serine protease from porcine pancreas.It has similar protein structure and substrate specificity to ELANE,it is not easily inhibited by serine protease,therefore increasing the killing efficiency on the tumor.At the same time,ELANE inhibited tumor growth and induced non-specific effects mediated by CD8+T cells,and the effects were mutual,when CD8+T cell depletion would weaken the anti-tumor effects of TANs.Based on the above research status and the objective of the experiment,the human PPE(human porcine pancreatic elastase)is constructed.Previous investigations have found that human pancreatic elastase 1(chymochypsin like elastase 1,ELA1)is also an elastase gene,which has a similar coding sequence to ELANE.In summary,this study identified key genes,aiming to explore the anti-tumor effects of ELANE,ELA1 and HPPE protein.In order to provide new ideas for tumor treatment.Methods:1.The target gene plasmid was obtained according to the gene sequence information,and the expression vector was determined according to the needs of subsequent experiments.The vector should be equipped with fluorescent labels and screening labels to facilitate the efficiency of cell transfection subsequently.Appropriate restrict enzyme sites were selected on the target plasmid and vector,cleaved both of them through double-restrict enzyme sites,then performing T4 binding.The binding product is transformed and expanded for culture,so as to construct recombinant expression vectors and empty vectors.The vectors were respectively named as follows: empty vector Lentip EF1-IFNlp-cop GFP-Puro(abbreviated as PEF),empty vector without fluorescence Lenti-p EF1-IFNlp-Puro(abbreviated as PEF without GFP),The recombinant expression vectors named following,Lenti-p EF1-IFNlp-ELANE-cop GFP-Puro(ELANE+PEF),Lenti-p EF1-IFNlp-ELANEPuro(ELANE+PEF without GFP),Lenti-p EF1-IFNlp-ELA1-cop GFP-Puro(ELA1+PEF),Lentip EF1-IFNlp-ELA1-Puro(ELA1+PEF without GFP),Lenti-p EF1-IFNlp-HPPE-cop GFP-puro(abbreviated as HPPE+PEF),Lenti-p EF1-IFNlp-HPPE-cop GFP-puro(abbreviated as HPPE+PEF without GFP).2.The constructed recombinant expression vector and empty vector were verified,including restrict enzyme cleaving verification and E.coli colony PCR verification.The product was compared with the target band position by agarose gel electrophoresis,then the recombinant vector was sent to sequencing,and the successful construction of the recombinant expression vector and empty vector was judged according to the comparison results of gene map.The constructed recombinant expression vector was transferred to the E.coli for long-term preservation and expanded culture,it is conceivable that the extracted plasmid was convenient for subsequent experiments.3.The constructed recombinant expression vector and empty vector were transfected into 293 T cells through lentivirus-mediated transfection.According to the expression of the recombinant vector,the fluorescence expression of the recombinant vector with fluorescence protein expression was observed during transfection,and the drug screening was conducted directly for the recombinant vector without fluorescence protein expression.The screened cells were cultured until they were in good condition.The stable transfected cell lines were frozen and expanded for subsequent experiments.4.The stable transfected cells were verified through following methods,RNA was extracted for q PCR experiment as the transcription level verification,protein was extracted for western blot experiment as the translation level verification,so as to verify the expression of the target protein.5.Functional verification of the stable transfected cell lines was carried out.A variety of human lung cancer cell lines and human breast cancer cell lines were selected to conduct experiments on the effect of target proteins on tumor cell proliferation and the killing efficiency on tumor cells.Results:1.The constructed recombinant expression vectors were identified by double restrict enzyme cleaving verification and E.coli colony PCR respectively.Target bands were visible in the products after agarose gel development.Combined with sequencing results,it indicates that the recombinant vector is successfully constructed.2.Lentivirus transfection was performed to construct transfected cell lines.According to the recombinant vector,fluorescence expression of transfected cell lines could be seen under inverted fluorescence microscope.The non-fluorescent transfected cell lines were co-cultured for puromycin,then the expression of transfected cell lines was detected.The constructed transfected cell lines were named as follows,PEF-293 T,PEF without GFP-293 T,ELANE+ PEF-293 T,ELANE+PEF without GFP-293 T,ELA1+ PEF-293 T,ELA1+PEF without GFP-293 T,HPPE+ PEF-293 T,HPPE+PEF without GFP-293 T.3.The constructed transfected cell lines were verified,and the results of q PCR showed that the recombinant expression vectors had significantly high amplification of target genes compared with empty vectors(P<0.0001),western blot results showed high expression of the target protein,the stable transfected cell lines were successfully constructed(P<0.01).4.The results of functional experiment indicated that the proliferation of lung cancer cells and breast cancer cells in experimental group significantly slowed down compared with negative control group(P<0.05),the killing efficiency of experimental group of lung cancer and breast cancer cells was significantly higher than that of negative control group(P<0.05).Conclusions:1.Recombinant overexpression vectors of ELANE,ELA1 and HPPE were successfully constructed.2.Stable transfected cell lines expressing ELANE,ELA1 and HPPE protein were successfully constructed.3.Through cell proliferation experiments,it was proved that the up-regulation of ELANE,ELA1 and HPPE protein could inhibit the proliferation of human lung cancer cells and human breast cancer cells.The killing efficiency of ELANE,ELA1 and HPPE protein on human lung cancer cells and human breast cancer cells were demonstrated by the Calcein-AM experiments.