Chunhua Wei, Qian Cai, Ruiguang Zhang, Xiaorong Dong, Gang Wu

Oncology department, union   hospital of huazhong university of science and technology

Objective:Over the past decades, Lung cancer has located the first place of world-wide tumorigenic incidence and been the leading reason of death caused by cancers, and the subtype of non-small cell lung cancer (NSCLC) takes 80%-85%.what's more, brain metastasis is the highest reason of poor prognosis, recurrence, and death in NSCLC patients. It is therefore essential to unravel the biomakers and molecular mechanisms that govern disease progression to metastasis, while previous studies haven't shown functional evidence in tumor development. Recently MicroRNAs (miRs), endogenous non-coding RNAs, are reported to be involved in tumourigenesis. Aberrant DNA hypermethylation contributes to cancer progression. Our study is to explore predictive biomakers and molecular mechanisms in NSCLC brain metastasis patients. Method: RNA extracted from 122 patients’ blood, including brain metastasis from 62 newly diagnosed NSCLC patients and non- brain metastasis from 60 NSCLC patients, was analysed by real-time PCR to detect the differential expression in several MicroRNAs. Cox regression analysis and microarray profiling were performed to select miR-330-3p as a target. Lentivirus over expressing and down regulating miR-330-3p respectively was transfected into A549 and Hcc827 NSCLC cells. RT-PCR was applied to examine transfective rate and analyse DNMT1, DNMT3A, DNMT3B levels in groups. Western blot was used to explore molecular mechanisms at protein levels. In vitro, wound healing assay, trnswell, Proliferation, apoptosis and cell cycle assays were performed to demonstrate the effect of miR-330-3p on NSCLC cells. Measurement of global DNA methylation levels was used to illustrate whether miR-330-3p affect tumorgenisis. In vivo, tumor xenograft model was established seperatelly infecting cells ubcutaneously into nude mice forearm and the heart. In vivo animal imaging technology was applied. Bcl-2, caspase-3, PCNA and CyclinD1 was immunohistochemically verified. Result: Real-time PCR demonstrated miR-330-3p played a marked role in NSCLC brain metastasis compared with NSCLC patients without brain metastasis. In vitro assays exhibit miR-330-3p can promote proliferative, migrative and invasive in A549 and Hcc827 cell lines. Flow cytometry, western blot and Immunohistochemical manifest that miR-330-3p shows an anti-apoptosic role in lung cancer cells. Global DNA methylation levels in A549 and Hcc827 cell lines Over expressing miR-330-3p were up-regulated and those cells after down-regulating miR-330-3p showed low global c.RT-PCR indicates that DNMT1 and DNMT3A are positive correlated with miR-330-3p, that is DNMT1 and DNMT3A remarkbly affect DNA methylation levels. In vivo assays suggest that miR-330-3p can induce the tumorigenic and metastasic ability. Conclusion: Our work led to the identification of a novel functional pathway, controlled by miR-330-3p and including its direct targets BMI1, GRIA3, SOSTDC and AGTR2 as well as multiplesurface proteins, that coordinates metastasis formation in a lung cancer metastasis model, represented by the A549 and Hcc 827 cell lines. Considering these results, we can conclude that the pathway including miR- and its modulated genes is able to coordinate NSCLC progression and could be considered for therapeutic intervention.

Key Words: miR-330-3p  DNA methylation  GRIA3