| BackgroudLung cancer is a significant disease with major health implications, currently reported as the most commonly diagnosed cancer and the most common cause of cancer death. About 80% of lung carcinomas fall under the classification of non-small cell lung cancer (NSCLC) with adenocarcinoma as the most common subtype. Lung adenocarcinoma has the heterogeneity of clinical, radioavtivity, molecular and histological. Although we have significant progress in the awareness of lung adenocarcinoma from tumorigenesis to treatment for many years, it remains one of the most common fatal cancer and survival rates are not significantly improved. At present, the TNM stage is still the main basis for judging prognosis of lung adenocarcinoma, but the prognosis of tumor remain vary greatly in the same stage with clinical observation and follow-up. With the deepening of molecular mechanism of malignant tumors and the rapid development of molecular biology and bioinformatics, individual genetic differences in susceptibility to cancer prognosis, treatment development and tumor biological behavior is gradually revealed. Clinical pathological and molecular characteristics of academic study and clinical practice of lung cancer which can assess and early warn lung adenocarcinoma, particularly early stage postoperative recurrence and metastasis are the research focus.It is noteworthy that the pleural effusion is one of the most common clinical symptoms in patients with lung adenocarcinoma. Patients may be no symptoms with little effusions in the early stage, but patients often have difficulty in breathing and coughing and their quality of life also decrease with more effusions in the advanced stage. It means the process has been the advanced stage and the average survival period is only about four months when patients with lung adenocarcinoma appear malignant pleural effusions. The diagnosis of adenocarcinoma cells in pleural effusion is a very important factor to the treatment and prognosis of patients. However, as the primary sites of tumors being unclear, the atypia of tumor cells, the diagnostic level of pathologists or the process of slicing, the cytomorphologic distinction between metastatic adenocarcinoma and reactive mesothelial cells is not always straightforward and is sometimes extremely challenging.Insulin-like growth factor II mRNA-binding protein 3 (IMP3) is a member of the insulin-like growth factor II mRNA-binding protein (IMP) family that consists of IMP1, IMP2, and IMP3. IMP family members play an important role in RNA trafficking and stabilization, cell growth, and cell migration during the early stages of embryogenesis. IMP3 is expressed in a variety of tumors with variable positivity and considered as an oncofetal protein. It is closely realted with the proliferation, Invasion, metastasis and prognosis of tumors. Direct knockdown of the CD44 transcript mimicked the effect of IMPs on invadopodia, and we infer that CD44mRNA stabilization may be involved in IMP-mediated invadopodia formation. The current literatures on IMP3 expression in lung adenocarcinoma is limited and there is no literature examining IMP3 expression in the precursor lesion. The previous literatures reported that IMP3 expression was associated with histologic differentiation, histological subtypes and distant metastasis in lung adenocarcinoma tissues. However, clinical specimens of these studies were only detected by immunohistochemical staining and lacked overall survival analysis of patients with lung adenocarcinoma. The relationship between IMP3 re-expression and high invasion of lung adenocarcinoma cell lines remained to be further validated in the cellular level. In addition, there are few literatures reporting the unility of IMP3 in the differentiating metastasis adenocarcinoma (MAC) from reactive mesothelial cells (RMs) in pleural effusion. The unility of IMP3 for differential cytological diagnosis between metastatic adenocarcinoma of lung (LAC) and non-lung (NLAC) origin remain unclear.Objective1.To investigate IMP3 expression in human lung adenocarcinoma tissues, and examined its correlation with clinicopathological factors and prognosis of patients.2. To construct cell sublines with stable knockdown of IMP3 gene and detected their invasion and proliferation in cellular leve.3. To explore the diagnostic usefulness of the combination with IMP3, CK5/6 and TTF1 in differentiating among reactive mesothelial cells, malignant mesothelioma, metastatic adenocarcinoma of lung and non-lung origin in pleural effusion.Methods1. Relationship between IMP3 expression and clinicopathological characteristics and prognosis of patients with lung adenocarcinomaTissues microarray including 95 lung adenocarcinoma and 75 normal lung tissues were detected by IMP3 Immunohistochemical (IHC) staining and the results of IMP3 expression were divided into negative group and positive group. The relationship between IMP3 expression and clinicopathological characteristics of patients and survival analysis were investigated.2. Knockdown of IMP3 by shRNA inhibits invasion and proliferation of lung adenocarcinoma cell lines(1) IMP3 expression in lung adenocarcinoma cell linesIMP3 expression in mRNA and protein level in lung adenocarcinoma cell lines and immortalized bronchial epithelial cell were detected by Real-time PCR and Western blot; then the location of IMP3 expression in A549 and H322 were detected by immunocytochemistry (ICC) staining.(2) Construction of cell sublines with stable knockdown of IMP3Stable knockdown lentiviral vectors for IMP3 were provided by the Shanghai GeneChem (China), and the lentiviral supernatant was synthesized by GeneChem. A549 and H322 were transfected by lentiviral supernatant (experimental group and control group), and the cell sublines were namely respectively A549-shIMP3 and H322-shIMP3, all of which were tagged by GFP including control, and then sorted for GFP expression on a Flow cytometric sorting (FACS) aria. Transfection efficancy was confirmed by real-time PCR and Western blot.(3)Knockdown of IMP3 inhibits invasion and proliferation of lung adenocarcinoma cellsThe migration and invasion ability were examined by transwell migration assay, and proliferation rate were examined by CCK8 and flow cytometry.3. The diagnostic usefulness of the combination with IMP3, CK5/6 and TTF1 in differentiating among RMs, LAC and NLAC.Pleural effusion specimens were made into cell blocks. IMP3, CK5/6 and TTF1 expression in RMs (32), LAC (51) and NLAC (25) were detected by immunocytoche-mistry (ICC) and conventional PCR.4. Statistical analysisThe SPSS software 13.0 was used for the statistical analysis. Measurement data are expressed by mean ± standard deviation (x±s). The Ⅹ2 test was used to assess the association between categorical variables. Single factor survival analysis was used by log-Rank and Kaplan-Meier curve, Multivariate analysis of prognosis by Cox model. assessed using the one-way ANOVA for assays with Real-time PCR. The results of Flow cytometry, Transwell and CCK8 by using two sample t tests. Differences are considered statistically significant at p< 0.05.Results1. Relationship between IMP3 expression and clinicopathological characteristics and prognosis of patients with lung adenocarcinomaImmunostaining demonstrated that IMP3 was absent from all 75 non-tumor lung tissues. In contrast, IMP3 expression was observed in 39 of 95 in lung adenocarcinoma tissues and appeared different expression level including weak, moderate and strong positive. The results of IMP3 immunostaining in lung adenocarcinoma tissues were divided into two groups:IMP3 negative and IMP3 positive. IMP3 was positive in 19.0%(4/21)of well grade,42.6%(20/47) of moderate grade and 55.6%(15/27) of poor grade (P=0.037). The positivity of IMP3 expression in Ⅲ～Ⅳ- stage was higher than that in I or II stage in patients with lung adenocarcinoma (P=0.034). IMP3 expression was significantly related to Lymph node metastasis (P=0.011); In contrast, little relationship was seen between IMP3 expression and age (P=0.659), gender (P=0.785) and tumor size (P=0.489). The median survival time of all the patients was 62.95 months and the survival rate of five years was 54.7%. The Kaplan-Meier method was used to demonstrate that IMP3 expression (P=0.000), TNM stage (P=0.000) and lymph node metastasis (P=0.001) were associated with clinical prognosis of patients with lung adenocarcinoma. Upon the univariate analysis with the cox proportional hazards model, TNM stage (P=0.000), lymph node metastasis (P= 0.002) and IMP3 expression (P=0.000) were associated with overall survival. Multivariate analyses revealed that TNM stage (HR, 2.338; 95% CI:1.393-3.925; P=0.001) and IMP3 expression (HR,2.310; 95% CI: 1.192-4.476; P=0.013) was an independent predictor of an poor prognosis.2. Knockdown of IMP3 by shRNA inhibits invasion and proliferation of lung adenocarcinoma cell lines(1) IMP3 expression in lung adenocarcinoma cell linesWe used Real-time PCR and Western blot to analyze IMP3 expression in 16HBE and lung adenocarcinoma cells:A549, GLC-82, H358, H322 and SPC-A1. The results showed that the IMP3 expression level in lung adenocarcinoma cell lines was relatively higher than that in 16HBE (P=0.001). Only H358 was less lower, but no statistically significant (P>0.05). In addition, we detected IMP3 expression in A549 and H322 by Immunocytochemistry (ICC) staining. The IMP3 Immunostaining by ICC was also concentrated cytoplasmic in cell lines, which consisted with tissues staining by IHC.(2) Construction of cell sublines with stable knockdown IMP3Lentiviral vectors constitutively knockdown of IMP3 labeled with GFP were stably transfected into A549 and H322 cells along with a control vector labeled with GFP. GFP+cells were selected by Flow cytometric sorting. The efficiency of transfection was measured by Real-time PCR and Western blot. The result showed that the IMP3 expression both in mRNA and protein levels were significantly down-regulated compared with controls.(3)Knockdown of IMP3 inhibits invasion and proliferation of lung adenocarcinoma cellsThe invasion ability of cell sublines were detected by Transwell migration assay at 6h, 24h and 48h, respectively. The upper chamber was 100ul cell suspension and the lower chamber was loaded with 500 μ1 of RPMI-1640 containing 10% FBS. The results of migration assay showed that A549-shIMP3 had significant reduced migrated cells compared with A549-control, and all the time effects, group effects and timexgroup effects of A549 cell sublines were significantly different; In addition, the results that the migrated cells of H322-shIMP3 were also significantly less than H322-control, and all the time effects, group effects and timexgroup effects of H322 cell sublines were significantly different. At the same time, we detected cells proliferation after downregulated IMP3 by CCK8 in vitro. The results of CCK8 showed that the proliferation ability of A549-shIMP3 cells were weaker than A549-control cells. All the time effects, group effects and timexgroup effects of cell sublines were significantly different; In addition, the results that the proliferation ability of H322 cell sublines was consisted with A549 cell sublines. The cycle of cell sublines were detected by flow cytometer. The results showed that the cell numbers of G1 phase in experiment group were significantly higher than the control group. In contrast, the cell numbers of S phase in experiment group were significantly lower than the control group.3. The diagnostic usefulness of the combination with IMP3, CK5/6 and TTF1 in differentiating among RMs, LAC and NLAC in pleural effusion.(1) IMP3, CK5/6 and TTF1 expression in 32 RMs,76 MAC in pleural effusion were detected by ICCThe results showed that the positivity of CK5/6 staining in RMs was higher than in MAC(78.1% VS 14.5%, P=0.000); The positivity of IMP3 staining in MAC was higher than in RMs(88.2% VS 9.4%, P=0.000). Immunostaining for detection of CK5/6 was generally concentrated cytoplasmic staining in RMs and negative staining in MAC; In contrast, cytoplasmic staining of IMP3 was often observed in MAC but negative staining in RMs.We further evaluated IMP3 and TTF1 staining in MAC, and found that the positivity of IMP3 staining in MAC was higher than TTFl staining (88.2% VS 52.6%, P=0.000). In addition, we evaluated the expression of IMP3 and TTF1 in LAC and NLAC. IMP3 staining was no statistically significant in LAC and in NLAC (88.2% VS 88.0%, P> 0.05); Unlike IMP3, The positivity of TTF1 staining in LAC was higher than in NLAC (76.5% VS 4.0%, P=0.000) Immunostaining for detection of IMP3 was generally concentrated cytoplasmic staining both in LAC and NLAC; Nevertheless, nuclear staining of TTF1 was often observed in LAC, but negative staining in NLAC.(2) The sensitivity and specificity using a combination of CK5/6 and IMP3 for detecting RMs and MACThe staining pattern of CK5/6+ /IMP3 - using ICC had a higher specificity than CK5/6+ ICC alone (98.3% VS 85.5%), but a lower sensitivity for detecting RMs (71.5% VS 78.1%). The staining pattern of IMP3+/CK5/6-using ICC had a higher specificity than IMP3+ alone (98.2% VS 91.6%), but a lower sensitivity for detecting MAC (75.4% VS 88.2%).(3) The DNA fragments of CK5/6 and IMP3 in RMs and MAC in pleural effusion were detected by C-PCRThe 487bp DNA fragments of IMP3 were expected to be amplified 6/9 in MAC specimens showed negative in ICC; and the 394bp DNA fragments of CK5/6 was expected to be amplified 4/7 in RMs specimens showed negative in ICC; As with ICC staining, the 487bp DNA fragments of IMP3 in 6/6 of LAC and in 6/6 of NLAC specimens were detected by conventional PCR. In contrast, almost all 447bp DNA fragments of TTF1 (6/6) were showed positive expression in LAC specimens but scarcely expression (0/6) in NLAC specimens.Conclusion1. Immunostaining for detection of IMP3 was generally concentrated cytoplasmic staining in lung adenocarcinoma tissues and negative staining in normal lung tissues.2. IMP3 expression was significantly related to histologic grade, TNM stage and lymph node metastasis. In contrast, little relationship was seen between IMP3 expression and age, gender and tumor size.3. IMP3-positive specimens had a poorer survival rate than did those patients with IMP3-negative specimens, IMP3 expression could be an independent prognostic factor for clinical prognosis of patients with lung adenocarcinoma.4. Silencing IMP3 gene could inhibit the invasion and proliferation of lung adenocarcinoma cells.5. The combination with IMP3, CK5/6 and TTF1 had an important role in differentiating among reactive mesothelial cells, malignant mesothelioma, metastatic adenocarcinoma of lung and non-lung origin in pleural effusion.Innovation points1. The relationship between IMP3 expression and invasion and clinical prognosis of patients with lung adenocarcinoma was investigated in tissue and cell levels.2. We propose the use of a fine decision tree consisted of IMP3, CK5/6 and TTF1 for differentiating among reactive mesothelial cells, malignant mesothelioma, metastatic adenocarcinoma of lung and non-lung origin in pleural effusion and could provide some referenced value to clinical diagnosis。... |
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