CELF5 Promoting The Development Of Lung Cancer And Its Molecular Mechanism

Background:Lung cancer is the tumor with the highest morbidity and mortality in the worldwild.Lung cancer is divided into small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).of which non-small cell lung cancer(NSCLC)accounts for the highest proportion,about 85%,and its pathological types are mainly adenocarcinoma and squamous cell carcinoma.In the worldwide,the incidence and mortality of lung cancer among men rank first,while among women,the incidence of lung cancer is second only to breast cancer,but the mortality rate still ranks first.According to a report from the National Cancer Registry of China in 2015,due to the continuous increase in life expectancy and the continuous increase in the number of elderly people,the incidence and mortality of lung cancer have shown a continuous upward trend.The total number of new cases of lung cancer in China is The number of deaths was 730,000 and 600,000 respectively.At present,it has become the first malignant tumor in my country in terms of morbidity and mortality.Due to the insidious symptoms of lung cancer,most lung cancer patients are already in the middle and advanced stages when they are diagnosed,and the prognosis is poor,with a five-year survival rate of 13%17%.In addition to traditional surgical treatment,radiotherapy and chemotherapy,the current treatment for lung cancer,although many emerging treatment methods,such as targeted therapy and immunotherapy,have also begun to be applied in clinical practice.However,because lung cancer,like other tumors,has the characteristics of recurrence and metastasis,the prognosis of lung cancer patients is still not ideal.The 5-year survival rate of patients with non-small cell lung cancer is only 15%-20%.In the past decade,with the development of molecular sequencing technology,lung adenocarcinomas have identified epidermal growth factor receptor gene mutations and echinoderms microtubule-associated protein 4-anaplastic lymphoma kinase fusion gene and other lung cancer drivers.Gene mutations,and molecular targeted therapies for these specific driver genes have been developed.Compared with standard chemotherapy,the new molecular targeted therapy can significantly improve the progression-free survival of lung cancer patients and has relatively low side effects.Although molecular targeted therapy has changed the clinical outcome of certain subgroups of lung cancer patients,the 5-year survival rate has increased by 5%,but it is still less than 20%.In response to this situation,the specific molecular mechanism of the occurrence and development of lung cancer and the new therapeutic targets in the treatment process are rigorously to be resolved.Researching out more molecular mechanisms that can be used in the diagnosis and treatment of lung cancer patients is of great value for the diagnosis and treatment of lung cancer patients.In the era of precision medicine,these studies on the molecular biology of lung cancer can be used to screen lung cancer patients more accurately and promote the development of individualized treatment for lung cancer patients.During the progression of NSCLC,some proto-oncogenes are overexpressed,while the expression of other genes is suppressed.For example,during the occurrence and development of NSCLC,the expression level of calnexin(calnexin,CANX)is significantly upregulated,and some researchers believe that the detection of serum CANX level can be used clinically as a molecular marker for the detection of early lung cancer.In addition,intercellular adhesion molecules(ICAM)and other molecular markers related to the diagnosis and prognosis of non-small cell lung cancer can also be used as potential new targets for molecular targeted therapy.RNA binding protein is a protein that binds to single-stranded or double-stranded RNA and can regulate the expression of transcripts.At present,thousands of RNA binding proteins have been discovered,which are widely expressed in various organisms.However.their specific biological functions and mechanisms are still unclear.CELF5 is a member of the CELF/Bruno-like protein family,which is an RNAbinding protein widely found in mammalian cells.CELF5 can regulate mRNA expression through alternative splicing,transcription inhibition,transcription activation and deacetylation.CELF5 protein is mainly distributed in the cytoplasm.It is composed of three conserved RNA recognition sequences,RRM1,RRM2 and RRM3.RRM2 is located at the N-terminus of the protein and RRM3 is located at the C-terminus of the protein.CELFs can participate in the editing,splicing and translation process of target mRNA.Previous studies have found that members of the CELF protein family can participate in the occurrence and development of malignant tumors by activating related downstream signaling pathways or regulating the expression of downstream target genes.For example,the study by WangHuaiming et al.found that in colorectal cancer,CELF1 can bind and regulate the expression of ETS2 to promote cell invasion and chemotherapy resistance.YiuTo Yeung et al.found that CELF2 can inhibit the growth of non-small cell lung cancer by inhibiting the PREX2/PTEN axis.A study of ovarian cancer found that CELF2 can inhibit the progression of ovarian cancer by stabilizing FAM198B.Another study found that CELF2 can promote the occurrence of liver cancer by activating the PI3K/Akt signaling pathway.The study also found that in colorectal cancer,CELF6,as a potential tumor suppressor,inhibits cell proliferation by upregulating the expression of p21.In the human body,the expression of CELF5 is mainly distributed in the forebrain,midbrain and hindbrain,and the expression is low in the corpus callosum and pons.Previous studies on CELF5 have mainly focused on the central nervous system.Studies have found that the abnormal expression of CELF5 is closely related to the occurrence of central nervous system diseases.In recent years,studies have found that CELF5 can participate in the regulation of malignant tumors by interacting with human cytomegalovirus and regulating its genomic DNA synthesis,suggesting that CELF5 may be closely related to the occurrence of malignant tumors.There are no reports about the function and mechanism of CELF5 in human lung cancer.Toll-like receptors(Toll-like receptors,TLRs)are a kind of pattern recognition receptors(PRRs),which were first proposed by NussleinVolhard et al.in 1980.TLRs can be used to detect pathogen-associated molecular patterns(PAMPs)on invading pathogens.It can recognize a variety of PAMPs,such as bacterial lipopolysaccharide,bacterial flagellin,and viral nucleic acid.TLR3 is a member of the Toll-like receptor family,which is a nucleic acid sensitive receptor.Among various types of Toll-like receptors,TLR3 plays an important role in the regulation of virus-mediated innate immune response.It can recognize virus replication products(dsDNA)and synthetic ligand polyinosinepolycytidylic acid.TLR3 is widely expressed in a variety of cells and is involved in the activation of a variety of signaling pathways within the cell.TLR3 is involved in the occurrence and development of many diseases.In recent years,studies have also found that TLR3 plays an important role in the occurrence of malignant tumors.In a study on prostate cancer,it was found that Chuanpi glycosides can participate in the occurrence of prostate cancer by regulating the TLR3/IRF3 signaling pathway.A study on breast cancer found that TLR3 plays a role of tumor suppressor gene in the process of breast cancer.Songhuijuan et al.found that agonists of TLR3 can treat melanoma by regulating dendritic cells in the body.The study by BeatriceVanbervliet-Defrance et al.found that in malignant mesothelioma,cisplatin can promote apoptosis by down-regulating c-FLIP to release TLR3.TLR3 can also be used as an important marker for screening high-risk patients with early non-small cell lung cancer.In addition,studies have also found that human papillomavirus type 16 can promote the proliferation and migration of non-small cell lung cancer cells through the TLR3 signaling pathway.The above research results indicate that TLR3 is closely related to the occurrence of tumors.Propuse:Our group is committed to reaching new molecular mechanism in the development of lung cancer,the role of CELF5 in the development of lung cancer and its specific molecular mechanism,which will helps to provide new targets for the treatment of lung cancer in the future.Materials and Methods:1.Firstly,our group first used qRT-PCR to detect the expression level of CELF5 in human lung cancer cell lines NCL-H1229,A549,and H1975.In the following in vitro cell experiments,we constructed sh-Ctrl and sh-CELF5 stable lung cancer cell lines(A549 and H1975)through RNA transfection technology.Subsequently,we used BrdU experiments,cell flow cytometry,CCK-8 experiments,and clone formation experiments to explore the effects of knockdown of CELF5 on the phenotype of lung cancer cells in vitro.In the in vivo experimental study,we divided the selected BALB/c female nude mice into two groups.The two groups of mice were injected with sh-Ctrl and sh-CELF5 lung cancer cell lines subcutaneously,and cultured for 35 days and 7-35 days.During this period,the tumor size was measured every 7 days and the tumor growth curve was drawn.2.Secondly,in order to find the downstream target of CELF5,we used catRAPID omics software to predict the RNA that CELF5 might bind,and screened out the 10 genes with the highest matching degree,and tested whether CELF5 can directly bind to the above genes through the RNA-pulldown experiment.We use RNA-pulldown-qRTPCR technology to detect the mRNA that can be enriched by CELF5 in lung cancer cells.In further research,we used qRT-PCR method to verify the effect of knockdown of CELF5 on the expression of the above genes in lung cancer cell lines,and use Wetsernblot method to detect the changes of the above genes at the protein level.3.Finally,we constructed CELF5 single knockdown and CELF5 and TLR3 double knockdown lung cancer cell lines,and through flow cytometry,CCK-8 experiment and clone formation experiment,we explored CELF5 single knockdown and CELF5 and TLR3 double knockdown on lung cancer in vitro.The influence of cell phenotype.In in vivo animal experiments,we divided the selected BALB/c female nude mice into three groups.The three groups of mice were injected with sh-Ctrl,sh-CELF5 and shCELF5+sh-TLR3 lung cancer cell lines subcutaneously,and cultured for 35 days During the 7-35 days of culture,the tumor size is measured every 7 days and the tumor growth curve is drawn.Results:1.Firstly,our team finds that CELF5 has high abundance expression in different lung cancer cell lines.The low expression of CELF5 can inhibit the proliferation,growth and cloning ability of lung cancer cells,and promote the apoptosis of lung cancer cells.The tumor-forming ability of mice injects with the sh-CELF5 cell line subcutaneously is significantly reduced.2.Secondly,we find that TLR3,MTCYBP42,OR7E117P,GTF2IRD2P1 and PNP can effectively bind to CELF5 protein.After knocking down CELF5,the mRNA expression levels of TLR3,HERC2P5,OR7E117P and HSD17B12 increases significantly in these lung cancer cells.After knocking down CELF5,the expression of TLR3 gene increases significantly in the protein level.The above experimental results suggest that TLR3 is a downstream target gene of CELF5,and CELF5 can inhibit the expression of TLR3.3.Finally,after knocking down CELF5,the expression of TLR3 gene at the protein level increases significantly.Knockdown of CELF5 can inhibit the proliferation and cloning ability of lung cancer cells,but these effects can be reversed by co-transfection with sh-TLR3.The tumor formation ability of mice with sh-CELF5 cell line injected subcutaneously is significantly weakened,but the injection subcutaneously of shCELF5+sh-TLR3 cell line can enhance the tumor formation ability of mice.Conclusions:In summary,CELF5 is highly expressed in non-small cell lung cancer cell lines,and it plays the role as an oncogene in the development of lung cancer;TLR3 gene is the downstream binding target gene of CELF5,and CELF5 can inhibit the expression of TLR3;TLR3 can inhibit the oncogene effect of CELF5 in the process of lung cancer.Therefore,we infer that CELF5 may be an important oncogene for lung cancer.Inhibiting the expression of CELF5 may be a new strategy for the treatment of lung cancer in the future.CELF5 is expected to become an important target for the precise treatment of lung cancer in the future.