Prognostic Model Established By A Four-gene Signature And Preliminary Study On The Mechanism Of C1QTNF6 Acting As A Tumor Promoter In Lung Adenocarcinoma

Purpose: The latest cancer epidemiological data shows that lung cancer is the most common malignant tumor in the world in terms of morbidity and mortality,among which more than 80% is non-small cell lung cancer(NSCLC).Lung adenocarcinoma(LAD)and lung squamous cell adenocarcinoma(LSC)are the main histological types of NSCLC.The former accounts for about 40% of all lung cancers,while the latter accounts for about 25%.Today,the pathogenesis of LAD is still unclear,leading to the lack of markers for its early diagnosis as well as the high risk of recurrence and metastasis.Therefore,although the current treatment has improved significantly,the overall prognosis of LAD is still very poor,and the five-year average survival rate is only about 15%.It has very important clinical value in exploring some new and effective tumor markers or models to identify the risk of poor prognosis of LAD patients.Complement C1q/tumor necrosis factor-related protein 6(C1QTNF6)is a recently discovered member of the adipokine superfamily with highly conserved molecular structures.Many studies have found that C1QTNF6 is widely involved in a variety of inflammatory processes,and plays an important role in vascular inflammation,inflammatory kidney injury,rheumatoid arthritis and tumorigenesis and development.It has been reported that the change of C1QTNF6 expression levelcan affect the metastasis of hepatocellular carcinoma,colon cancer and ovarian malignant tumors,but the expression characteristics of C1QTNF6 in lung adenocarcinoma have not been explored.In vitro experiments have preliminarily shown that silencing the expression of C1QTNF6 in lung adenocarcinoma cells inhibits the growth and invasion of cancer cells,but this conclusion is not convincing due to the lack of in vivo experimental evidence.In order to fully identify the role of C1QTNF6 as a oncogene of lung adenocarcinoma,it is necessary to carry out animal experiments.Stable commercial human tumor transplantation models have been presently developed to study the effects of PRC gene knockout on the proliferation and metastasis of LAD.The successful homozygous mutants of nude mice can visually display the data of growth and metastasis of implanted tumors.This study aimed to find out the specific prognostic genes of LAD,and C1QTNF6 was selected as a candidate gene for prognosis prediction and personalized therapy in patients with LAD.The relationship between the expression of C1QTNF6 and pathological characteristics of lung adenocarcinoma was investigated,and the regulatory role of C1QTNF6 in the process of tumorigenesis was further studied in nude mice model to provide early diagnosis and prognostic treatment for lung adenocarcinoma.The results will provide significant theoretical basis for molecular targeted therapy strategy of lung adenocarcinoma.Materials and methods:1.RNA-seq expression profiles and related clinical information of 503 and 115 LAD patients were downloaded from TCGA and GEO databases,used as training and validation sets for constructing prognostic risk model,respectively;the differentially expressed genes(DEGs)were detected in 59 pairs of tumor tissues and adjacent tissues obtained from TCGA-LAD samples.2.Random forest algorithm was used to identify DEGs used for construction of prognostic risk model of LAD;multivariate COX regression method was used to score 503 samples,and to evaluate the correlation between risk score and clinical variables and survival time;survival difference between high-and low-risk groups was evaluated by Kaplan-Meier(KM)survival curve,and the difference in survival time between the two groups was identified by Chi-square evaluation.3.The significant DEGs between high-risk and low-risk group samples were screened using Voom in R-package limma.GO and KEGG pathways analysis were performed using online gene function annotation tool Web Gestalt,and a PPI network was constructed on basis of genes involved in construction of prognosis risk prediction model.4.The stable expression systems of small RNA interference(si-)and pcoverexpressionof C1QTNF6 were constructed;blank control,negative control(si-NC,pc-NC)were set up;lung adenocarcinoma cell lines SPCA 1 and A549 were selected as in vitro experimental vectors.5.Quantitative RT-PCR was used to validate the inhibitory or promoting effect of silencing or PC C1QTNF6 at the cellular level.MTT,clone formation,scratch test and Transwell migration test were used to detect the effects of C1QTNF6 silencing or PC treatment on the proliferation,migration and invasion of lung adenocarcinoma cells.Flow cytometry Annexin-V(APC staining)and flow cytometry PI staining were used to detect the changes of apoptosis and cell cycle of LAD cells after silencing or PC treatment of C1QTNF6,respectively.Western blot was used to examine the relationship between C1QTNF6 silencing or PC treatment and the expression of apoptotic regulatory proteins and cell cycle regulatory factors in LAD cells SPCA1 and A549.6.The subcutaneous tumor model of BALB/c nude mice was established,and thenC1QTNF6 silenced or PC treated human LAD cell lines SPCA1 and A549 were subcutaneously injected to observe the growth rate and volume of subcutaneous tumors in nude mice,respectively.7.qRT-PCR,Western blot and immunohistochemistry were used to detect C1QTNF6 transcription and expression in subcutaneous tumor.HE staining and TUNEL staining were used to demonstrate the effect of interfering with the expression of C1QTNF6 in subcutaneous tumors on the growth and metastasis of human lung adenocarcinoma cells.Quantitative RT-PCR and Western blot were used to reveal the effects of silencing the expression of C1QTNF6 on the positive regulatory protein of metastasis MMP-2,MMP-9 and the positive regulatory factors of apoptosis Caspase-3 and Caspase-9 in LAD cells.The positive and negative effects of C1QTNF6 on cell metastasis and apoptosis of LAD solid tumors were explored.Results:1.Compared to the adjacent normal tissue,a total of 18,567 DEGs were detected in LAD tissues,of which 15,483 were highly expressed in cancer tissues,while the rest were significantly decreased;totally 279 genes significantly related to the survival time of LAD patients were obtained through univariate COX regression analysis;according to the ranking of contribution degree,the top four genes including FAM83 A,MYO1E,C1QTNF6 and ERO1 L were finally chosen to establish a prognostic risk model.Moreover,t;according to the FPKM value of four genes and the survival status of the patients,the following formula was constructed to calculate the prognostic risk score of LAD patients: The risk score = 0.00635 * FAM83 A +0.02114 * MYO1 E + 0.04234 * C1QTNF6 + 0.00634 * ERO1 L.2.Using the four-gene prognostic risk model,LAD patients in training set and validation set were both divided into high-risk group and low-risk group according to risk score.Univariate and multivariate COX regression analyses between risk scoreand clinical variables showed that only risk score and clinical stage(Stage)were found to be significantly correlated with survival of LAD patients in training set,and only risk score was significantly correlated with patient survival time in the validation set;KM curve analysis found that there were significant differences in survival time between high and low risk groups in the two datasets.3.Compared with the low-risk group of TCGA-LAD patients,118 significantly up-regulated and 175 down-regulated were identified in the high-risk group.GO enrichment showed that DEGs were mainly involved in extracellular matrix organization(including extracellular matrix organization,extracellular structure organization,collagen fibril organization),circulatory system development(including circulatory system development,blood vessel development,vasculature development,angiogenesis),cell adhesion and cell migration,indicating that these prognostic differences may be related to the proliferation and metastasis of cancer cells.KEGG pathway enrichment analysis also showed that these DEGs were significantly enriched in focal adhesion,ECM receptor interaction,protein digestion and absorption pathways,all of which were closely related to tumors.4.To further reveal the effectiveness of the four-genes prognostic risk model,a protein interaction network of the four genes was built.The number of proteins interacting with MYO1 E,ERO1L,C1QTNF6 and FAM83 A was 266,154,10 and 8,respectively.GO functional enrichment analysis showed that the interacting proteins of these four genes were mainly involved in cell cycle process(including M/G1 transition of mitotic cell cycle,ribosome biogenesis,regulation of centrosome duplication and mitotic cell cycle).Specifically,MYO1 E interacting proteins mainly regulated cell cycle,ERO1 L interacting proteins play important roles in the synthesis and assembly of intracellular nucleic acid substances,C1QTNF6 interacting proteins are involved in histone modification and cell cycle process,and FAM83 A interactingproteins are closely related to telomere replication process.5.The apoptotic rates of SPCA1 and A549 cells in si-C1QTNF6 group were 16.7%,9.23%,significantly higher than those of pc-C1QTNF6 groups 1.84%,1.40%.Western blot results showed that the expression of caspase-3,caspase-9 and Bax increased significantly in si-C1QTNF6 group,while the expression of Bcl-2decreased significantly.6.The results of cell cycle showed that compared with blank control and NC group,the proportion of G1-phase cells in SPCA1 cells and A549 cells in si-C1QTNF6 group was significantly higher than that in other phases(P< 0.05),while significantly lower than that pc-C1QTNF6 group(P< 0.05).Subsequently,Western blot results showed that the expression of Cyclin D1 in pc-C1QTNF6 group of the two cell lines was significantly increased,while the expression of cell cycle inhibitor P27 was significantly inhibited.The expression trend of Cyclin D1 and P27 in the si-C1QTNF6 group was opposite to that in the pc-C1QTNF6 group.7.The results of subcutaneous tumor growth experiment showed that silencing the expression of C1QTNF6 could significantly reduce the growth rate of SPCA 1 and A549 cells in nude mice,and the volume of tumors decreased synchronously.The growth rate and volume of tumors increased significantly when treated with C1QTNF6 overexpression.The results showed that the growth of lung adenocarcinoma subcutaneous tumors was slowed down and the weight was reduced after the inhibition of C1QTNF6 expression.HE staining showed that silencing C1QTNF6 could significantly reduce the number of lung metastases in solid tumors,while pc-treated C1QTNF6 could significantly increase the number of lung metastases in tumors.8.The results of qRT-PCR and Western blot showed that the expression of C1QTNF6 was weakest in the subcutaneous tumors of si-C1QTNF6 group and strongest in thesubcutaneous tumors of pc-C1QTNF6 group,suggesting that the animal model with LAD cell transfection was stable and reliable.Further,immunohistochemical staining showed that the signal intensity of anti-C1QTNF6 antibody was the weakest in LAD tissue of si-C1QTNF6 group,while the highest in that of PC group with statistical significance.9.TUNEL staining of apoptotic cells showed that the number of brown positive cells in the si-C1QTNF6 group was significantly higher than that in the control group and the pc-C1QTNF6 group,suggesting that silencing the expression of C1QTNF6 could induce the apoptosis of subcutaneous tumors.Quantitative analysis of qRT-PCR and Western blot showed that compared with blank control and negative control,the expression of apoptotic promoters Caspase-3 and Caspase-9 increased in tumor tissues silenced by C1QTNF6,and the expression of MMP-2 and MMP-9 involved in tumor invasion and transfection decreased;PC treatment of C1QTNF6 inhibited the expression of Caspase-3 and Caspase-9 but promoted the expression of MMP-2 and MMP-9 with statistically significant difference.Conclusion:1.The risk model based on MYO1 E,ERO1L,C1QTNF6 and FAM83 A genes can better classify LAD patients into high-risk group and low-risk group,and can successfully predict the prognosis of patients independently of their clinical variables.2.Interference of C1QTNF6 gene expression can significantly promote apoptosis of LAD SPCA1 and A549 cells,delay cell cycle progression,block cell proliferation and migration,and thus inhibit the development of lung adenocarcinoma.In this study,the effects of C1QTNF6 silencing on the proliferation and migration of LAD cells was investigated,and it is preliminarily confirmed that C1QTNF6 is a candidate oncogene for lung adenocarcinoma,providing a theoretical basis for molecular targeted therapy of lung cancer.3.In vivo interference C1QTNF6 gene expression can significantly promote apoptosis of tumor cells and inhibit proliferation and metastasis of lung adenocarcinoma cell lines.Animal model experiments showed that C1QTNF6 could promote the proliferation and metastasis of lung adenocarcinoma,and could be used as a new gene marker for molecular targeted therapy of lung adenocarcinoma.