Screening And Functional Study Of MicroRNAs Related To Human NSCLC Metastasis

Background and Objective Lung cancer is one of the most common malignant tumors in the world, which has high morbidity and mortality. According to WHO, in 2005 the total mortality of lung cancer was 30.83 in one hundred thousand and the mortality of male patients was 41.343 in one hundred thousand, while the female was 19.84 in one hundred thousand. As a result, lung cancer total mortality ranked first in all the malignant tumors. Due to the high malignancy of lung cancer, most patients were already in advanced stage and lost the opportunities of surgical treatment when they were diagnosed. Radiotherapy and chemotherapy do not specifically kill tumor cells and may cause severe side effects, so seeking new approaches of diagnosis and treatment are still the hotspots. Lung cancer metastasis is a multi-step and multi-period process and it depends primarily on the invasive ability of lung cancer cell which involves tumor cell proliferation, adhesion, migration, invasion, cell matrix degradation and so on. Multiple tumor suppressor gene inactivation and overexpression of oncogenes are involved. To date, a large amount of research has covered the development and progression of tumor, while the molecular mechanism in each step is still not elucidated, therefore, searching novel genes and molecules associated with lung cancer invasion and metastasis and investigating their mechanism are considered to be of great importance for the improvement of survival rate and the rational design of therapeutic agents.MicroRNAs (miRNAs) are a group of small non-coding RNA (approximately 21-25nt) which widely exists in eukaryotic cells. MiRNAs can downregulate gene expression by imprecisely binding to complementary sequences in the 3’untranslated region (3’UTR) of their target mRNAs. Although miRNAs account for only a very minor fraction of genome, they are pivotal regulators of development and cellular homeostasis. In recent years, increasing studies have shown that miRNAs were involved in the process of tumor invasion and metastasis. Zhang JG et al confirmed that miR-21 was overexpressed in tumor tissues compared to adjacent non-tumor tissues in lung cancer. Notably, patients with advanced clinical TNM stage or distal metastasis demonstrated higher miR-21 expression than those without them. Tumor tissues showed an inverse correlation between miR-21 and PTEN protein. MiR-21 inhibitor transfection increased the activity of a luciferase-reporter which contains a PTEN-3’-UTR construct and increased PTEN protein but not PTEN-mRNA levels in NSCLC cell lines. Finally, miR-21 inhibitor-transfected cells exhibited markedly reduced cell growth and invasion. He X and his colleagues revealed a novel regulatory mechanism for let-7a in the control of cellular proliferation and lung carcinogenesis in A549 cells in vitro and in vivo. The regulatory mechanism may be explained in pari by le-7a-induced suppression of NIRF through NIRF 3’UTR and elevation of p21 (WAF1). So there is a practical significance on the study of miRNAs differential expression and biological function. It will contribute to a better understanding of tumor development, invasion and metastasis.Method and Results This study covered the following three parts:1. MicroRNAs differential expression profiles were screened out from high and low metastasis potential human large cell lung cancer cell lines L9981/NL9980. Meanwhile microRNAs arrays were carried out in lung cancer tissues with N2 lymph nodes metastasis and the early lung cancer tissues without lymph nodes metastasis as a control group, and then we compared the differential miRNAs between N2 lymph node tissues and matched primary lung cancer tissues. Differential expression of microRNAs was verified by using real-time PCR.2. We constructed a recombinant eukaryotic expression vector pcDNA3.1(+)-miR-132 containing human miR-132 and hygro resistance gene and transfected it into L9981 cell line which expressed lower endogenous miR-132. After screening a few weeks, a stable cell line L9981 which overexpressed the mature miR-132 was successfully selected. The distribution and localization of miR-132 was determined by Locked nucleic acid (LNA) in situ hybridization. The ability of proliferation, soft agar colony formation, migration and invasion were studied among the stable cell line group, L9981 transfected with pcDNA3.1(+)-Hygro group and blank control group.3. By bioinformatics analysis, some potential candidate target genes of miR-132 are obtained. Luciferase reporter vectors fused with target gene 3’UTR were constructed and dual-luciferase reporter assays were carried out to test the effects of miR-132 on target genes. In order to study the regulatory effect of miR-132 on target genes and investigate the mechanism of miR-132 in inhibiting the growth of L9981 cell, the expression levels of target gene protein in stable cells which overexpress miR-132 were detected by Western blot.Conclusions 1. A cluster of differential expression miRNAs were screened out between high and low metastasis potential human large cell lung cancer cell lines L9981/NL9980. There were obvious differential expression miRNAs between lung cancer tissues with N2 lymph nodes metastasis and early lung cancer tissues without lymph nodes metastasis. A class of differential expression miRNA was obtained from N2 metastasis lymph nodes tissues and matched primary lung cancer tissues. It is postulated that the screened cluster of miRNAs might be involved in lung cancer invasion and metastasis.2. In situ hybridization showed that MiR-132 was distributed in cytoplasm as red fine particles. There were much more red particles in L9981 overexpressing miR-132 than in L9981 transfected with pcDNA3.1(+)-Hygro. There was no effect of miR-132 on cell proliferation, but it did affect L9981 morphology and malignant phenotype. And migration and invasion ability decreased after L9981 was transfected with miR-132.3. Dual-luciferase reporter assay showed that MiR-132 downregulated MMP16 expression through MMP163’UTR. This finding suggests that the effect of miR-132 on malignant phenotype of L9981 cells in vitro may be in part due to miR-132 induced suppression of MMP16. Our work reveals a novel regulatory mechanism for miR-132 in affecting lung cancer cell migration and invasion ability...