Preliminary Study On The Molecular Mechanism Of CLEC3B Regulating Invasion And Metastasis Of Lung Cancer Cells And Its Clinical Application

Part 1 Molecular mechanism of C-Type Lectin Domain Family 3 Member B affecting invasion and migration of lung cancer cellsObjective:To investigate the gene changes of C-Type Lectin Domain Family 3 Member B(CLEC3B)in lung cancer,and to study the molecular mechanism that affects the invasion and migration of lung cancer cells.Exploring the potential of this gene as a diagnostic marker of lung cancer and to provides scientific basis for seeking molecular targeted drugs or clinicopathological diagnosis of lung cancer.Methods:(1)From January 2017 to August 2018,30 cases of lung cancer tissues were collected,tumor tissues and adjacent tissues were exfoliated on ice and transcriptome sequencing was performed.To detect the changes of gene expression in lung cancer tissues.GO(Gene ontology)was used to analyze the function and mechanism of abnormal genes,and Pathway analysis was used to further understand the involvement of genes in metabolic pathways and their specific biological functions.(2)The expression of CLEC3B in TCGA database was analyzed(LUAD:lung adenocarcinoma,total number of tumor tissue samples(T):483 cases,total number of paracancer tissue samples(N):347 cases;LUSD:lung squamous cell carcinoma,total number of tumor tissue samples(T):486cases,total number of paracancer tissue samples(N):338cases).The relationship between CLEC3B expression and overall survival of lung cancer patients was analyzed.(3)mRNA expression levels of CLEC3B in lung cancer tissues and adjacent tissues were detected by qRT-PCR.(4)The expression level of CLEC3B was detected by immunohistochemistry,and the patient’s clinical information was analyzed comprehensively.(5)The mRNA and protein levels of CLEC3B in common lung cancer cell lines(211H,95D,H441,A549)and human normal lung epithelial cells BEAS-2B were detected by qRT-PCR and Western blot,respectively.CLEC3B overexpressed lung cancer cell lines were established,and qRT-PCR and Western blot were used to verify the successful construction.(6)The effect of overexpressed CLEC3B on the proliferation function of lung cancer cells was detected by CCK-8 and trypan blue staining.(7)CLEC3B overexpression of lung cancer cell proliferation ability was determined by CLEC3B cloning test.(8)The migration and invasion ability of lung cancer cells overexpressed CLBC3B was determined in scratch test and Transwell invasion test.(9)The expression level of PI3K/Akt signaling pathway related proteins was detected by Western blot,and the effect of overexpressed CLEC3B on PI3K/Akt signaling pathway was investigated.(10)The expression levels of MAPK signaling pathway related proteins were detected by Western blot,and the effect of overexpressed CLEC3B on MAPK signaling pathway was investigated.(11)CLEC3B stable overexpressed A549 cell line constructed by lentivirus vector and its corresponding empty vector control cell line were inoculated subcutaneously in nude mice with Matrigel gel for tumor formation.The tumor size(volume)was measured,and the protein expression level of key signal molecules in tumor-bearing tissues was detected by Western blot,so as to further prove the inhibition of-CLEC3B on lung cancer progression and verify the in vitro molecular regulatory mechanism.Results:(1)The mRNA expression levels of a total of 3818 genes in tumor tissues were significantly changed,and the mRNA expression levels of 1530 genes were significantly increased(P<0.05),Ep:AFAP1,RP11,GRP110,MMP13,TCN1,GLB1L3,KCNQ3,TOX3,HABP2,SGPP2,TMPRSS4,STK32A,MROH6,FAM83A,IL37,ect.while mRNA transcription levels of 2288 genes were significantly reduced(P<0.05),Ep:RTKN2,CLEC3B,UPK3B,GPM6A,BTNL9,AATK,MYRF,GKN2,ITLN2,ARHGEF26,CRYAB,EGFL7,KCNK3,HIF3A,RASIP1,FOXF1,GRASP,GRK5,CLIC3,RAMP2,ect.This suggests that abnormal gene expression is closely related to the occurrence and development of lung cancer.GO analysis shows that overexpressed genes in cancer tissues are mainly involved in regulating cell biological processes and cell composition,while down-expressed genes in lung cancer tissues are mainly involved in regulating cell molecular functions.Pathway analysis showed that 94 out of 1530 up-regulated genes were involved in metabolic pathways,While 35 genes are involved in the regulation of the PI3K/Akt signaling pathway,the Cytokine-receptor interaction is the second step.Meanwhile,the analysis of 2288 down-regulated genes revealed that 54 genes regulated Neuroactive ligand-receptor interaction,MAPK signaling pathway,and also activated PI3K/Akt signaling pathway.It suggests that up-regulated genes and down-regulated genes may promote the occurrence and development of lung cancer by activating key signaling pathways in cell contents.(2)Analysis of the expression of CLEC3B in TCGA database showed that compared with paracancerous tissues,the expression level of CLEC3B gene in lung cancer tissues was significantly reduced(P<0.05),and the overall survival of patients with CLEC3B high expression was significantly higher than that of patients with low expression of this gene(P<0.01).(3)The results of qRT-PCR showed that compared with paracancer tissues,CLEC3B significantly decreased in tumor tissues(P<0.01).(4)The expression level of CLEC3B was not significantly correlated with age and gender of patients and smoking history,but it was significantly correlated with tumor size of TNM stage of patients(P<0.05).(5)The mRNA expression level and protein expression level of CLEC3B gene in human normal lung epithelial cells BEAS-2B were significantly higher than those in other lung cancer tumor cell lines,while the expression level of CLEC3B gene in lung cancer cell line A549 was the lowest.In this study,CLEC3B cell line A549 with low expression was selected for functional research and mechanism exploration,and the overexpression of CLEC3B cell line A549 was successfully established.(6)Overexpression of CLEC3B at 24h and 48h did not significantly change the proliferation ability of A549 cells,but with the extension of culture time,overexpression of CLEC3B significantly inhibited the activity of A549 cells from 72h(P<0.05).(7)Compared with the blank control group(Vector),overexpression of CLEC3B significantly inhibited the number of clone formation of A549 cells(P<0.05).(8)Compared with the blank control group(Vector),overexpression of CLEC3B significantly inhibited migration of A549 cells(P<0.01),and overexpression of CLEC3B significantly inhibited the invasion ability of lung cancer cells A549,with an inhibition rate of 58%(P<0.01).Matrigel gel added to CLEC3B could significantly inhibit the invasion of A549 cells,with an inhibition rate of 48%(P<0.01).(9)Overexpressed CLEC3B can significantly inhibit the phosphorylation level of PI3K protein(p-PI3K)(P<0.05),and has no significant change in the total protein expression of PI3K(t-PI3K).Overexpression of CLEC3B significantly inhibited the phosphorylation of Akt(p-Akt)(P<0.05),and there was no significant change in the expression of total Akt(t-Akt).Grayscale analysis of protein conditions showed that overexpression of CLEC3B significantly inhibited the ratio of PI3K phosphate protein/PI3K total protein(p-PI3K/t-PI3K)(p<0.05),and significantly inhibited the ratio of phosphorylated Akt/Akt total protein(p-Akt/t-Akt)(p<0.05).(10)In A549 cells,overexpression of CLEC3B had no significant effect on phosphorylation of JNK(p-JNK),total JNK protein expression(t-JNK),phosphorylation of p38(p-p38)and total p38 protein expression(t-p38).Grayscale analysis of the protein bands also showed that there was no significant change in the ratio of phosphorylated JNK/total JNK protein(p-JNK/t-JNK)to phosphorylated p38/total p38 protein(p-p38/t-p38).On the other hand,overexpression of CLEC3B significantly reduced the level of phosphorylated ERK(p-ERK)(P<0.05).Grayscale analysis of the protein bands also showed that overexpression of CLEC3B significantly reduced the ratio of phosphorylated ERK/total ERK protein(p-ERK/t-ERK)(P<0.05).(11)In A549 cells,over-expressed CLEC3B(over-CLEC3B)significantly inhibited the proliferation ability of lung cancer cells in nude mice compared with Vector,both of which were statistically significant(P<0.01).Meanwhile,compared with the blank control group(Vector),the weight of overexpressed CLEC3B(over-CLEC3B)tumor tissues decreased(P<0.05).Western blot results showed that compared with the blank control group(Vector),the activity of ERK and Akt molecules in over-expressed CLEC3B subcutaneous tumor tissues of nude mice was significantly reduced(P<0.05),namely,the phosphorylation level was reducedConclusion:(1)CLEC3B gene is lowly expressed in lung cancer tissues and has a significant correlation with TNM grading in lung cancer patients.In addition,the overall survival of patients with high expression of CLEC3B was significantly higher than that of patients with low expression of this gene.This suggests that the expression level of CLEC3B may be an important predictor of survival in patients with lung cancer.(2)In lung cancer cell line A549,overexpression of CLEC3B significantly inhibited cell proliferation,clonogenesis,invasion and metastasis.(3)Molecular mechanism studies show that overexpression of CLEC3B can significantly inhibit the phosphorylation of PI3K Akt and ERK signaling molecules,and further inhibit the activity of PI3K/Akt and MAPK/ERK signaling pathways.(4)In vivo experiments showed that overexpression of CLEC3B inhibited phosphorylation of Akt and ERK signaling molecules in subcutaneous lung cancer tumors of nude mice.Part 2 Serum CLEC3B is used as a tumor marker to evaluate the curative effect of locally advanced non-small cell lung cancer after neoadjuvant chemotherapyObjective:To investigate the clinical value of serum CLEC3B expression level in the curative effect evaluation of neoadjuvant chemotherapy(NACT)in patients with locally advanced non-small cell lung cancer(NSCLC),and the specificity and sensitivity of CLEC3B was compared with other traditional tumor markers.Methods:Sixty patients with NSCLC initially diagnosed in Nantong tumor hospital from January 2018 to June 2018 were included as subjects,and 50 healthy patients were included as control group at the same time.(1)Patients with NSCLC were treated with neoadjuvant chemotherapy,and short-term efficacy was assessed after completion of chemotherapy.(2)Plasma samples of healthy individuals and NSCLC patients before and after NACT treatment were collected,and serum CLEC3B level was determined by ELISA assay.(3)The correlation between clinical characteristics and serum CLEC3B level was analyzed.(4)Serum levels of CYFRA21-1,CEA,NSE in NSCLC patients were determined by ELISA.cfDNA concentration was determined by qRT-PCR.(5)ROC curves of CYFRA21-1,CYFRA21-1,CEA,NSE and cfDNA were established to compare the sensitivity and specificity of each indicator.Result:(1)The short-term efficacy of NACT treatment in 60 Locally Advanced NSCLC patients was 0 patients with CR,32 patients with PR,26 patients with SD,and 2 patients with PD.(2)The results showed that the serum levels of CLEC3B of Locally Advanced NSCLC patients before NACT treatment was 42.04±10.39 ng/ml,which was significantly lower than that of healthy people(P<0.05).After two cycles of NACT treatment,the CLEC3B concentration of NSCLC patients was 68.26±15.53 ng/ml,which were significantly higher than those before chemotherapy(P<0.05).The CLEC3B level of patients with PR was 75.38±12.14 ng/ml,the CLEC3B level of patients with SD was 67.21±13.59 ng/ml,the CLEC3B level of patients with PD was 59.32±10.18 ng/ml Serum CLEC3B level in PD patients was significantly lower than that in PR patients(P<0.05).(3)To analyze the correlation between clinicopathological parameters and serum levels of CLEC3B in Locally Advanced NSCLC patients,we found that either before or after NACT treatment,the serum levels of CLEC3B with Locally Advanced NSCLC patients’ gender,age,tumor pathologic type,with or without smoking history,tumor size and location had no significant correlation(P>0.05).However,it was significantly correlated with lymph node metastasis,TNM clinical stage and differentiation degree of NSCLC patients(P<0.05).(4)The serum levels of CYFRA21-1,CEA,NSE and cfDNA in NSCLC patients before NACT treatment were significantly higher than those in healthy patients(P<0.05).The serum levels of CYFRA21-1,CEA,NSE and cfDNA in NSCLC patients after NACT treatment were significantly lower than those before treatment(P<0.05).(5)ROC curve was established to compare the sensitivity and specificity of various indicators.It was found that the area under the curve of CLEC3B was small,and its sensitivity and specificity were lowConclusion:Serum CLEC3B levels were significantly reduced in patients with Locally Advanced NSCLC and significantly increased after NACT treatment.It is of great clinical value to monitor the efficacy of NACT in patients with Locally Advanced NSCLC and expected to be a biological indicator for the prognosis assessment of Locally Advanced NSCLC patients,providing an auxiliary role for the formulation of personalized treatment plans in the later stage and improving the survival rate.