| The incidence and motality of lung cancer is very high in malignant tumors. Without typical early symptoms, screening sensitive markers and effective early diagnosis, most patients are in middle or advanced stage and miss effective surgical treatment when diagnosed by a doctor. Radiotherapy and chemotherapy do not specifically kill tumor cells and may cause several side effects. Poor prognosis of lung cancer is universal even after complete resection, many patients die of recurrence and metastasis. Patients with a similar lung cancer may experience very different clinical outcomes, it is often very difficult to predict prognosis. Although tumor-node-metastasis(TNM) classification allows diagnosis of the tumor, it provides little therapeutic biological information, such as the metastatic potential or the sensitivity or resistance of the tumor to radio therapy and chemotherapy.Recently, effects of MicroRNA (miRNA) on the occurrence, development, diagnosis and treatment of lung cancer, especially NSCLC draw much attention. miRNAs, a class of small non-protein-coding RNAs, are evolutionarily conserved, endogenous, small, noncoding RNA molecules of about 22 nucleotides in length, which is almost exist in all eucaryon cells. Recent evidence has shown that miRNA may take part in several processes and play an important role in the initiation and progression of a number of cancers. Further study of miRNAs in tumor can bring new diagnostic plans and therapeutic targets.Objective:Real Time PCR method was used to detect the expression of miR-204-5p in carcinoma tissues and para-carcinoma tissues harvested from patients with surgical resection for non-small cell lung cancer. Detailed biological functions of miR-204-5p in A549 and HCC827 cell line were identified according to a series of experimental methods in vitro. Targets of miR-204-5p were predicted by bioinformatics, and validated with experimental biology for its functions.Methods:1. The relative expression levels of miR-204-5p were tested in 30 paired specimens tissues of non-small cell lung cancer. The statistical analysis was performed for the expression differences of miR-204-5p in carcinoma tissues and para-carcinoma tissues, as well as in different pathological stagings of non-small cell lung cancer.2. The relative expression levels of miR-204-5p were tested in BEAS-2B, A549 and HCC827 cells. A549 and HCC827 cells were transfected with miR-204-5p mimic to overexpress miR-204-5p, and then a series of functional experiments were performed. The influence of miR-204-5p overexpression on cell proliferation, migration and invasion capabilities of A549 and HCC827 cells were analyzed by means of CCK-8 test, colony formation, flow cytometry cell cycle detection and cycle apoptosis detection, wound healing assays, cell migration and invasion assays with transwell chamber method;3. Online software was used for target gene prediction of miR-204-5p, and verification of mRNA and protein expression variation for predicted target gene was carried out by using Real Time PCR, Western Blot and other tests. The test of dual luciferase report was used for identification of miR-204-5p target sequences, the role of miR-204-5p target genes was validated by rescue experiments ulteriorly.Results:1) miR-204-5p was quantified in human lung cancer tissue by qPCR. Out of 30 paired lung cancer tissues and para-carcinoma tissues,27 (90%) cancer tissues presented with miR-204-5p in down-regulated pattern while comparing with para-carcinoma tissues. The mean miR-204-5p expression level in cancer tissues was about 1/2 of that in normal adjacent tissue. However, no difference was detected when the lung cancer cases were stratified by histological stage.2) BEAS-2B cells presented with miR-204-5p in low level while comparing with A549 and HCC827 cells. To better characterize the role of miR-204-5p in lung cancer, we conducted gain of function analysis by transfecting non-small lung cancer cell line A549 and HCC827 with chemically synthesized miR-204-5p mimic. We found that over expression of miR-204-5p (50nM) could weaken the capacity of proliferation, colony formation and cell cycle in A549 and HCC827 cells. However, no effect on cycle apoptosis, wound healing assays, cell migration and invasion assays was found.3) MiR-204-5p target gene is CCND1. Overexpression of miR-204-5p can inhibit cell proliferation in non-small cell lung cancer A549 and HCC827 cell lines.Conclusion:We have shown miR-204-5p to be a novel suppressor of NSCLC for proliferation through its negative regulation of cyclin D1 in A549 and HCC827. |
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