The Roles And Molecular Mechanisms Of Let-7c And APRIL In The Occurrence And Development Of Non Small Cell Lung Cancer

Lung cancer is the predominant cause of cancer-related mortality in Chinese population. It is classified into two major pathological categories: small cell(SCLC)and non-small cell(NSCLC) lung cancer, and over 80% of mortality cases are NSCLC. Although early diagnosis and standardized treatment of NSCLC have been rapidly developed, the 5-year survival rate is only 15%.Micro RNAs(mi RNA) are small noncoding RNAs derived from long,endogenously expressed primary RNA(pri-mi RNA) molecules and functionally conserved from worms to humans. Mi RNAs could affect a multitude of biological processes, including cell proliferation, survival, differentiation, and motility, by negative regulation of the gene expression at the translational level. This is carried out by the stabilization of m RNA transcripts via post-transcriptional gene silencing thereby inhibiting the translation process or cleavage of their target m RNAs.Mi RNAs control various cellular processes such as cell death, differentiation, and development and are implicated in human diseases, including cancer. Mi RNA is extremely stable in the tissue, stool, plasma, and other fluids and can be quantified in minuscule sample sizes, qualifying it as an excellent potential biomarker for cancer diagnosis and treatment. Several reports have demonstrated that the aberrant expression of specific mi RNAs is associated with tumorigenesis and have beenimplicated in promoting proliferation and metastasis of tumor cells. The Let-7 family of micro RNAs, the second mi RNA after lin-4, is designated according to the phenotype of a Let-7 deficient C. elegans mutant. Till date, thirteen Let-7 family precursor mi RNAs have been described. Current evidence suggests that Let-7 seems to be downregulated in various cancers, including lung, liver, and prostate. Aberrant Let-7 expression was reported to associate with cancer initiation, progression, and aggressiveness. Moreover, the restored expression may be useful in cancer therapeutics. Let-7c is one of the members of the Let-7 family and maps to 21q11-21,a region frequently deleted in lung cancer. It is poorly expressed in various types of cancer. However, the underlying mechanism of Let-7c-mediated NSCLC remains unclear, mainly due to the samples size in the studies.APRIL(a proliferation-inducing ligand), is a member of the tumor necrosis factor(TNF) superfamily. It was discovered as a cytokine that could stimulate cellular differentiation, proliferation, apoptosis, and immune reaction. APRIL is predominantly expressed in the tumor tissues harbored in pancreatic cancer, gastric cancer, colon carcinoma, and by normal immune cells such as B cells, monocytes,macrophages, and dendritic cells. APRIL potentially binds two receptors, namely,TACI(transmembrane activator and CAML interactor) and BCMA(B-cell maturation antigen), and was presumed to be involved in B- or T-cell activation, differentiation,and regulated humoral immunity. The comprehensive reports confirmed that APRIL and its receptors(BCMA and TACI) were overexpressed in NSCLC. Consecutively,APRIL could also induce proliferation in human lung carcinoma cell line, A549, via signal-regulated MAP kinases pathway. This suggested that APRIL, BCMA, and TACI are potentially involved in tumor formation and development.In the present study, we evaluated the expression level of mi R-Let-7c, APRIL,BCMA, and TACI in NSCLC tissues, paired normal tissues, and peripheral blood. We also assessed the clinical relevance of these biomarkers. In addition, the effect of APRIL and its receptors, BCMA and TACI, on cell proliferation and migration in the human NSCLC cell lines in vitro was also explored. The primary objective of the study was to investigate the putative mechanism of mi R-Let-7c and APRIL in NSCLC and to identify a novel biomarker for its diagnosis and treatment.Part? Expression of mi R-Let-7c in NSCLC and its value in diagnosis of the patients with NSCLCMethods(1) Freshly-frozen tumor(30 squamous cell carcinomas and 46adenocarcinomas) and peritumoral tissue samples were collected from 76 patients with NSCLC for our case-control study. Also, the peripheral blood samples were collected from 76 NSCLC cases and 180 healthy controls.(2) Real-time quantitive PCR was performed to detect the expression level of mi R-Let-7c in the 76 cases of NSCLC tumor and peritumoral tissue samples.(3) Real-time quantitive PCR was used to detect the relative expression level of mi R-Let-7c in the plasma of NSCLC and healthy controls.(4) Expression level of let-7c was quantified by q RT-PCR in A549 and H1650 cells. H1650 and A549 cell lines were transiently transfected with mi R-let-7c plasmids and empty vector as control. The cell migration and cell cycle of NSCLC cells influenced by let-7c was detected by MTS assay, cell scratch test and CCK8 analysis.(5) The obtained results were analyzed using SPSS19.0. Each test was repeated three times, and the data were expressed as (?)±S. RT-PCR data to detect the differences among tissues or peripheral blood were compared by one-way ANOVA.P<0.05 was considered statistically significant.Results(1) The relative level of mi R-Let-7c in NSCLC carcinoma tissues was markedly lower than that in the peritumoral normal tissues(P<0.05).(2) The low expression of mi R-Let-7c in NSCLC tumor tissue was not related to the age, gender, tumor size, and pathological classification(P>0.05), but closely correlated with the clinical staging, lymph node metastasis, and histological grade(P<0.05).(3) The relative level of mi R-Let-7c in the plasma of NSCLC was lower than that in the healthy controls(P<0.05).(4) The expression of mi R-Let-7c in the tissue was not related to the age, gender,tumor size, clinical staging, histological grade, and pathological classification(P>0.05), but closely correlated with the lymph node metastasis and histological grade(P<0.05).(5) The plasma mi R-Let-7c exhibited a superior diagnostic accuracy for NSCLC(72%sensitivity and 78% specificity). Receiver operating characteristic(ROC) curve was constructed, and its result showed that the area under the curve(AUC) was0.714.(6) The preoperative plasma mi R-Let-7c level was significantly lower than that in the preoperative ones(P<0.05).(7) Low expression of let-7c was showed in A549 and H1650 cells, and the cell proliferation was inhibited when A549 and H1650 cells were transfected with let-7c mimics.Part ? Expression of APRIL and its receptors(TACI and BCMA)in NSCLC: mechanisms and clinical significanceMethods(1) The expression level of APRIL and its receptors(BCMA and TACI) were detected by immunohistochemistry in 76 cases of NSCLC tumor and peritumoral tissue samples.(2) H1299 and A549 cell lines were transiently transfected with BCMA- and TACI-sh RNA plasmids and empty vector as control, using Lipofectin. The cell migration influenced by soluble APRIL was detected by cell scratch assay.(3) The transiently transfected H1299 and A549 cell lines with BCMATACI-sh RNA plasmids using Lipofectin, and empty vector as control were used for the evaluation of the cell proliferation and apoptosis affected by soluble APRIL and were detected by CCK-8 assay.(4) Real-time quantitive PCR was used to investigate the expression level of APRIL and its receptors(BCMA and TACI) in the peripheral blood of NSCLC and healthy controls.(5) A549 cell line was transiently transfected with BCMA-TACI-sh RNA plasmids using Lipofectin, and empty vector as control. The phosphorylation level of ERK1/2 MAP kinase as a result of soluble APRIL was detected by Western blot.Results(1) The result of immunohistochemistry showed that APRIL was mainly displayed in the cytoplasm of NSCLC tumor cells. The positive rate of APRIL in NSCLC tumor tissues(71.7%, 54/76) was significantly higher than that in the peritumoral normal tissue(10.5%, 8/76)(P<0.05).(2) The positive expression rate of APRIL in immunohistochemistry was not related to the age, gender, clinical staging, and pathological classification(P>0.05),but closely related to the tumor size, lymph node metastasis, and histological grade(P<0.05).(3) The positive expression rate of BCMA in immunohistochemistry was not related to the age, gender, clinical staging, tumor size, histological grade, and pathological classification(P>0.05), but rather correlated with the lymph node metastasis(P<0.05).(4) The positive expression rate of TACI in immunohistochemistry was closely correlated with the pathological classification(P<0.05).(5) The results of cell scratch assay showed that the soluble APRIL could promote cell migration. However, the migration ability of H1299 and A549 cells transfected with BCMA-TACI-sh RNA was inhibited as compared to the control groups.(6) The results of CCK-8 assay showed that the soluble APRIL could promote cell proliferation. However, the proliferation ability of H1299 and A549 cells transfected with BCMA-TACI-sh RNA was inhibited as compared to the sh RNA control groups(P<0.05).(7) The relative level of APRIL m RNA in the peripheral blood of NSCLC was higher than that in the healthy controls(P<0.05). The expression of APRIL m RNA was not related to the age, gender, tumor size, histological grade, and pathological classification(P>0.05), but tightly correlated with the lymph node metastasis andclinical staging(P<0.05). Also, the expression of TACI m RNA was correlated with the lymph node metastasis(P<0.05) while that of BCMA m RNA was correlated with the pathological classification(P<0.05).(8) The soluble APRIL protein could increase the phosphorylation level of ERK1/2 MAP kinase in A549 cells(P<0.05). However, the phosphorylation in A549 cells transfected with BCMA-TACI-sh RNA was inhibited when compared to the control groups.(9) The specificity and sensitivity of combined real-time quantitive PCR detections of mi R-Let-7c in plasma and APRIL m RNA in peripheral blood were82.22%(148/180)and 59.21%(45/76), respectively.Conclusions(1) The high mi R-Let-7c expression in tissue and peripheral blood is associated with the development and invasion of NSCLC.(2) Mi R-Let-7c may be a potential molecular biomarker for metastasis, diagnosis,and prognosis of NSCLC.(3) The high expression of APRIL and its receptors(TACI and BCMA) in the tissue is associated with the development, metastasis, and invasion of NSCLC.(4) The elevated APRIL m RNA level in peripheral blood is associated with the development, metastasis, and invasion of NSCLC. This suggested APRIL m RNA as a novel biomarker for diagnosis and prognosis of NSCLC.(5) In vitro, soluble APRIL could promote the proliferation of lung cancer cells by increasing the phosphorylation of ERK1/2 MAP kinase and may contribute to the occurrence and development of NSCLC.(6) The sensitivity of real-time quantitive PCR detection of mi R-Let-7c in plasma together with the APRIL m RNA was higher than that of the RT-PCR for mi R-Let-7c and APRIL m RNA alone in the diagnosis of NSCLC.