| [Background]To date, lung cancer has the highest incidence rate of malignant tumors in the world[1]. Lung cancer is classified into small cell lung cancer and non-small cell lung cancer according to pathological classification, among which non small cell lung cancer (NSCLC) accounts for about 85% of all lung cancer. Over the past decade, lung cancer diagnosis and treatment has the epoch-making development, in addition to the three classical treatments such as surgery, radiotherapy and chemotherapy, gene target treatment is becoming the 4th methods, such as EGFR and ALK gene rearrangements and so on, lung cancer entered the new era, targeted medicine such as Gef itinib and Erlotinib、AZD9291 has been greatly improved the lung cancer survival rate. However, for Advanced Lung Cancer,5 year survival duration is still less optimistic[2]. Chemotherapy and radiotherapy are the main treatments for the patients with advanced lung cancer. After ten years of development, the new molecular targeted therapy has also become the first-line treatment.However response to chemotherapy is still unsatisfactory, progression free survival time (PFS) of the patients with advanced lung cancer is only 3-5 months, OS is about 8-10 months [3]. Precision Medicine Initiative is the era of medical treatment for lung cancer, and it’s remission rate is as high as 72% when patients with advanced lung cancer accepted molecular target therapy,5 years survival rate of patients with lung cancer has increased, but at present there are research show that EGFR-TKI treatment for lung cancer are resistant after take medicine about 11 months [3], In order to improve the survival time and quality of life of patients with lung cancer, science, epigenetics carried out like a raging fire in the protein group, From the genetic point of view, it is very important to find the new potential mechanism of lung cancer and the new therapeutic targets for the diagnosis and treatment of lung cancer.There are a large number of studies about miRNAs, they show that miRNAs will be become a new type of tumor markers. MiRNAs are divided into oncogenes and tumor suppressor genes, now research indicate there is a close relationship between miRNAs and mechanism of tumor genesis. Therefore, miRNAs and pathogenesis of lung cancer are a domain worthy to be penetratingly explored. It may provide a new target treatment for diagnosis and treatment of lung cancer. MiR-130a was a molecule miRNA that found in recent years, the study have shown that miR-130a can affect the activity of AR, MAPK signaling pathway, inhibit the proliferation of prostate cancer、 invasion and metastasis, and it played an important role in the function of tumor suppressor gene [4]. More studies have shown that miR-130a can also inhibit cell proliferation、 invasion and metastasis in breast cancer and.liver cancer [5]. At present, it is unclear that the expression and role of miR-130a in lung cancer. In this study, the role of miR-130a in the development of lung cancer was studied by immunoblotting, quantitative PCR, Transwell and luciferase reporter assay.[Objective]:to investigate expressed of miRNA molecules through microarray screened in lung cancer cells and the control group, and select miR-130a to do more study, detect its expression in human lung cancer tissue and analyze the relationship between clinical and pathological features of lung cancer, and more research is needed to study biological function and pathogenesis of miR-130a in lung cancer.[Methods]:1. To investigate the expression of miRNAs in lung cancer cell lines (A459 and Calu-3) and normal lung epithelial cell line (HBE) with Affymetrix chip. And qRT-PCR method was used to verify the expression of miRNAs in lung cancer tissues and cell, and confirmed result was accurate and reliable, and to select miR-130a to do next research. 2, quantitative real-time PCR (QRT PCR)was used for detection of mir-130a expression in human lung cancer tissues. miR-130a mimics were transfected into the lung cancer cell (A549 and Calu-3) through liposome method, to observe the effects of miR-130a on lung cancer cell growth, proliferation, invasion by EDU cell proliferation assay, flow cytometry, CCK-8 assay and Transwell experiments methods.3, Prediction and validation of the corresponding miR-130a target genes, and further study relationship between pathogenesis of lung cancer and miR-130a:putative target target gene prediction software was used to screen out the GOLPH3 gene may be the downstream target gene miR-130a. Then qRT-PCR and Western blotting were used to detect the influence of miRNA-130a on GOLPH3 and the corresponding protein levels in lung cancer cell lines. Finally, to verify the feedback regulation of miR-130a on GOLPH3 using luciferase reporter experiments, and further explore the regulatory mechanism of miR-130a in the pathogenesis of lung cancer.[Result]:1, Affymetrix microarray detection showed that there are 24 expression of upregulated miRNAs (fold change> 2) in the lung cancer cell(A459 and Calu-3) compared to normal lung epithelial cell(HBE), including hsa-miR-25, hsa-miR-222, hsa-miR-30e, hsa-miR-320e, hsa-mir-22, hsa-miR-16 etc; 19 expression down regulated miRNAs (fold change<0.5), such as hsa-mir-9, hsa-miR-130a, hsa-mir-21, has-mir-205 etc. Referring to the related literature, miR-130a had abnormal expression in a variety of tumors such as gastric cancer, liver cancer, we believe that miR-130a may be associated with tumor cell proliferation, apoptosis. But it was seldom reported that miR-130a expression and function research in non-small cell lung cancer (NSCLC). So, we choose miR-130a as the next research object. To make the results reliable, we used real-time quantitative RT-PCR to verify the expression of miR-130a in lung cancer tissues and cells, the results suggested that miR-130a expression were significantly lower in lung cancer tissues and cells than in the control group, the result were aligned with the microarray results, indicated that miR-130a expression was consistent with the trend. It suggested that miR-130a may play an important role in the development of lung cancer. 2, In the comparison of different tissue types, expression level of miR-130a were no significant difference between small cell lung cancer and non-small cell lung cancer(P> 0.05); miR-130a expression were lower(0.68±0.13) in the non small cell lung carcinoma than in the pulmonary adenocarcinoma (P<0.05); in addition, miR-130a expression were significantly lower in the lymph node transfer shift group (N1) patients (0.41±0.06) than that without lymph node metastasis (NO) (P<0.05);and miR-130a expression were increased with lung cancer clinical stage Ⅲ/Ⅳ tissue in patients(0.52 ± 0.14), there was significantly lower than that in Ⅰ/Ⅱ stage (P<0.05). In vitro experiment, after miR-130a mimic transfected, CCK-8 results showed that the transfection group can inhibit the proliferation of lung cancer cells compared the control group; EdU results suggested that lung cancer cell proliferation decreased significantly in the transfected group; Transwell cell indicated the ability of migration and invasion of lung cancer cells have obvious effects in the transfection group than in the control group; clone formation experiment results indicated cloning the formation of reduced capacity in the transfected cells decreased significantly compared with the control group; and cell apoptosis of experimental results indicated that flow cytometry, lung cancer cell clone formation rate were far lower in the transfected group than that In the control group; the lung cancer cells in the transfected group appeared block in the G1/S phase, which showed that the expression of miR-130a was increased and the growth of lung cancer cells was inhibited in the regulation of Gl/S phase transition. Subcutaneous tumor of nude mice experimental results showed that the tumor forming ability were significantly decreased in the nude mice injected with the transfected cells compared with the control group.3. from the bioinformatics software, we predict GOLPH3 may be one of the target genes of mir-130a, Western blotting was used to detect the expression of GOLPH3 protein in lung cancer/tumor adjacent tissue and lung cancer cell/normal bronchial epithelial cell.The expression of GOLPH3 protein in lung cancer tissue was significantly higher than that in the adjacent tissues. Then the Western blotting and qRT-PCR were used to detect the levels of GOLPH3 protein and mRNA, and the pGL3-GOLPH3-Mut plasmid was constructed successfully, in the wild type GOLPH3 recombinant plasmid;compared to miR-130a mimics after transfection(0.61± 0.11), fluorescence intensity ratio was significantly lower in the GOLPH3 mutant in the transfected with negative control(1.27±0.05), p<0.01; the recombinant plasmid, and fluorescence intensity ratio in the transfected with negative control (1.33±0.06)was no significant difference compared to miR-130a mimic after transfection(1.30±0.05), p>0.05;and also found that the expression level of miR-130a protein was significantly inhibited after combination of GOLPH3 3’-UTR to GOLPH3 regulation, but there had no significant influence on mRNA lever. It confirmed that miR-130a could inhibit the activity of the reporter gene containing the binding site, but it had no effect on the mutant group and the control group.It shows that the prediction results of bioinformatics software are accurate and reliable. CCK-8 experiments showed that lung cancer cells transfected with GOLPH3-Mut pGL3- 3’-UTR and mimics miR-130a can partly eliminate the inhibitory effect of miR-130a on the proliferation of lung cancer cells. The expression of GOLPH3 gene in lung cancer cells and tissues were detected by westernblotting, the expression of Cyclin E1, CyclinDl, GAPDH were decreased obviously, suggested that its expression were correlated with the expression of miR-130a. The expression of GOLPH3 and WIS、β catenin were significantly increased in lung cancer group cncer group after the 3’-UTR transfected miR-130a mimics and pGL3 GOLPH3-Mut, It suggested that miR-130a might affect the development of lung cancer through the WNT/WIS pathway. [Conclusion]:1. From miRNA microarray anlysizes and RT-PCR results, they confirmed the expression of miR-130a in lung cancer tissues/cells were decreased significantly compared with the control group, suggested that there had a certain correlation between miR-130a and Pathogenesis of lung cancer.2. With the expression of miR-130a increased, it inhibited the proliferation, migration and invasion of lung cancer cells in vitro and in vivo, and indicated miR-130a play an important role in the process of proliferation, invasion and apoptosis of lung cancer.3 miR-130a and target gene GOLPH3 had feedback ralationship, miR-130a may affect the pathogenesis of lung cancer through the WNT/WIS pathway, miR-130a need to further study, and it is expected to become a new target for lung cancer gene therapy. |
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