| Nowadays,lung cancer has become the leading cause of male and female deaths worldwide,and its incidence is far ahead of many other tumor types.According to histopathological classification,lung cancer can be divided into two main categories: non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).Among them,LUAD is one of the most common types of lung cancer,and the incidence rate accounts for 20%-30% of all primary lung cancers.It can invade blood vessels and lymphatic vessels in its early stage,and metastasis often occurs before the primary tumor causes symptoms.LUAD is a huge threat to the health of human life.In recent years,follow-up studies have shown that the use of low-dose computed tomography(LDCT)to screen people with high lung cancer risk can effectively reduce lung cancer mortality,so many countries are gradually promoting similar screening methods.However,even if it relies on multiple measures for early screening and early treatment,highly preventable cancers such as lung cancer still have huge differences in survival and mortality in terms of ethnicity and geographical conditions.In addition,the incidence of cancer has become gradually younger.It is difficult to expand the screening of lung cancer from the long-term heavy smoking population to the general population in a short time.There is no effective method for lung cancer screening.Therefore,there is an urgent need for research on the most common pathological classification of lung cancer(LUAD)and the development of new targeted therapies.At present,there are more than 2,500 proteins that can be combined with chromatin and participate in human biological processes.A large part of these proteins that can interact with DNA are considered transcription factors.Due to its unique regulation mechanism,transcription factors can be widely involved in various physiological and pathological processes of the human body,and the most common one is malignant tumors.The HOX family genes is one of the most common transcription factor families,which can be divided into HOXA,HOXB,HOXC,HOXD four subfamilies,a total of 39 genes.As a member of the four subfamilies of human HOX genes,the HOXA gene cluster also plays a regulatory role in the pathological process of malignant tumors.Its 11 family members not only have abnormal expression in a variety of cancer tissues,but also have a certain impact on the prognosis and treatment of cancer.In the preliminary study of the research group,by analyzing the expression profiles of lung adenocarcinoma tissues contained in the TCGA database,we found that seven HOXA genes expressed differently in lung adenocarcinoma tumor tissues and control non-cancer lung tissues.Among them,HOXA1,HOXA10,HOXA11 are highly expressed in tumor tissues,and HOXA3,HOXA4,HOXA5,HOXA7 genes are low expressed.Through analysis and literature research,we found that the genes that show low expression in lung adenocarcinoma tissues are located at the 3 ’end of the HOXA gene cluster,especially HOXA3,HOXA4 and HOXA5,this three genes are located in similar positions,and are all expressed in the early stage of embryonic development.Their expression trends in various cancers are similar,and they have certain function complementary effects.However,as transcription factors,the molecular mechanism of their joint regulatory function in the pathological process of lung adenocarcinoma remains to be elucidated by our research.Therefore,combined with these early bioinformatics and literature tips,we propose the following hypothesis: HOXA3,HOXA4,HOXA5,as transcription factor proteins,can transcriptionally regulate downstream target genes to the occurrence and development of lung adenocarcinoma,especially to the effects of malignant biological processes,such as proliferation,migration and invasion abilities.Based on this hypothesis,our research group intends to verify the expression and clinical pathological significance of HOXA3,HOXA4,HOXA5 in lung adenocarcinoma through paraffin tissue samples and computational biology techniques.Furthermore,cell biology techniques were used to verify the effects of HOXA3,HOXA4 and HOXA5 on the proliferation,migration and invasion abilities of lung adenocarcinoma cells through in vitro experiments.Finally,through Ch IP-seq analysis technology,we obtained the downstream target genes of HOXA3,HOXA4,HOXA5 transcription regulation,and discussed the potential possibility of its regulatory axis and the potential of its target gene as a target for lung adenocarcinoma targeted therapy.Part 1.HOXA family gene expression significance in LUADMaterials and Methods1.Immunohistochemical method was used to detect the expression of HOXA3,HOXA4,HOXA5 on protein level in 100 cases of LUAD tissue sections and their adjacent lung tissue sections.2.Screening of TCGA(The cancer genome atlas)database,GTEx(The Genotype-Tissue Expression)database and GEO(NCBI Gene Expression Omnibus)database for sequencing and chip data which contain the LUAD tissue samples and their control non-LUAD lung tissue samples.Using meta-analysis technology to analyze the expression of HOXA3,HOXA4,HOXA5 at the m RNA level and the relationship between their expression trends and clinical pathological parameters.In addition,through the online public database,verify the impact of HOXA3,HOXA4,HOXA5 on the prognosis of LUAD patients.Results1.At the protein level,through immunohistochemical techniques,we found that HOXA3,HOXA4,HOXA5 showed a low expression trend in cancer tissues.2.At the m RNA level,through a joint analysis of the online database,it was found that compared with normal lung tissue,HOXA3,HOXA4,HOXA5 showed a significant low expression trend in lung adenocarcinoma tissue.And the expression of HOXA3 is related to some clinicopathological parameters.In the histological grade,the expression level of HOXA3 in patients with higher LUAD grade(stage Ⅲ ～ Ⅳ)is higher than that in patients with lower cancer grade(stage I ～ Ⅱ).However,in the regional lymph node status(N)stage in TNM stage,compared with patients with N0 stage(ie,no regional lymph node metastasis),HOXA3 expression in tumor tissues of patients with N1 ～ N3(ie,different degrees of regional lymph node metastasis)is significantly higher(p<0.05).In terms of prognosis,the survival analysis data provided by the KMplot database shows that patients with higher expression of HOXA3 and HOXA5 have a higher overall survival rate.Materials and Methods1.Using lentiviral transfection technology,screen out two LUAD cell lines(A549,HCC827)with over-expressed HOXA3,HOXA4,and HOXA5.2.Use untreated cell lines,control cell lines transfected with empty virus,and experimental group cell lines transfected with target genes for in vitro experiments such as CCK-8 experiment,scratch experiment,and invasion experiment.Results1.Fluorescence microscope observation and real-time fluorescence quantitative reverse transcription polymerase chain reaction(RT-q PCR)technology verified that HOXA3,HOXA4,HOXA5 were significantly over-expressed in the screened cell lines.2.In vitro experiments verified that the proliferation,migration and invasion abilities were significantly inhibited in HOXA3,HOXA4,HOXA5over-expressed cell lines.Part 3.The possible targeted regulation of HOXA family genes and oncogeneCHEK1 in LUADMaterials and Methods1.Through the collation and analysis of Ch IP-Seq data,annotate the genome of HOXA4 and HOXA5 transcription factor binding sites,and intersect these target genes with the differentially expressed genes in LUAD to find the potential target genes of HOXA4 and HOXA5 in LUAD.2.Analyze the signal pathways of these target genes,collect the genes most enriched in the ten most significant KEGG signaling pathways involved in the transcriptional regulation of HOXA4 and HOXA5 in lung adenocarcinoma,andPart 2.The potential functional mechanism of HOXA family genes in LUAD perform PPI network analysis.3.The hub genes in the PPI network are verified in LUAD expression and prognostic function,and their potential targeting sequences with HOXA3 are predicted.Results1.A total of 16972 downstream target genes that can be regulated by HOXA4 and 1791 downstream target genes that can be regulated by HOXA5 were found.These target genes and the differentially expressed genes in LUAD tissues were intersected to obtain 1100 potential target genes of HOXA4 in lung adenocarcinoma and 126 potential target genes of HOXA5 in lung adenocarcinoma.2.Signal pathway analysis revealed that HOXA4 target genes in LUAD were significantly enriched in the "cell cycle" pathway,while HOXA5 target genes in LUAD were significantly enriched in the "p53 signaling pathway".Through further PPI analysis,we found the most significant hub genes in the KEGG signaling pathway involved in HOXA4 and HOXA5.3.According to the key gene analysis of the previous step,we found that the oncogene CHEK1 plays an important role in the transcriptional regulation of HOXA4 and HOXA5.We verified CHEK1’s m RNA expression significant over-expression in LUAD through the sequencing data of the TCGA database.There is a negative correlation between CHEK1 expression and HOXA4 and HOXA5 expression.In terms of prognosis,patients with low expression of CHEK1 have higher overall survival and disease-free survival than patients with high expression of CHEK1.Moreover,because of the lack of experimental evidence of HOXA3’s transcriptional regulation of CHEK1,we predicted the potential binding site of HOXA3 on the CHEK1 promoter sequence through an online database,suggesting that HOXA3 has the potential ability to transcriptionally regulate CHEK1.Conclusions1.In LUAD tissues,the expressions of HOXA3,HOXA4,and HOXA5 are significantly down-regulated,and the expression of HOXA3 is related to the clinicopathological parameters of LUAD development.HOXA3 and HOXA5 have a certain prognostic prompt.2.The over-expression of HOXA3,HOXA4,HOXA5 inhibits the proliferation,migration and invasion ability of lung adenocarcinoma cells in vitro.3.HOXA3,HOXA4,HOXA5 all have potential transcriptional regulation effect on oncogene CHEK1,and the abnormal expression of CHEK1 will also affect the occurrence and development of lung adenocarcinoma.In summary,HOXA3,HOXA4,HOXA5 may affect the proliferation,migration and invasion ability of lung adenocarcinoma cells by transcriptionally regulating the oncogene CHEK1. |
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