MicroRNA-1238 Regulates Cell Proliferation By Targeting LHX2 In Non-small Cell Lung Cancer

Background and Objective: Lung cancer, includes nearly 85% non-small cell lung cancer(NSCLC), is the most common cancer worldwide. Previously studies have reported the transcription factor LIM homeobox gene 2(LHX2) plays a critical role in primary tumor growth. However, it is still elusive that the epigenetic mechanism underlied the role of LHX2. MicroRNAs regulate gene expression in the form of binding to the 3’-untranslated region(3’UTR) of specific target genes. Besides, studies have detected that microRNAs expression are changed in many human cancers. This study aimed to explore the epigenetic mechanisms of LHX2 abnormal expression in NSCLC regulated by microRNAs. The ultimate goal is to provide an exciting new theoretical basis for treatment of NSCLC.Methods:(1) To research the relative level of LHX2 mRNA and protein in NSCLC, quantitative real-time PCR and Western Blot were initially performed in four NSCLC cells and HBE, besides q-PCR performed 50 paired NSCLC tissue specimens of LHX2 mRNA level.(2) A549 and LTEP-α-2 cells were transfected with si-LHX2-1,si-LHX2-2 and their corresponding negative controls to detect NSCLC proliferation using CCK-8 assay and colony formation(3) Firstly, we predicted the potential microRNAs targets of LHX2 3’UTR by bioinformatics software, we screened miR-1238. Secondly, we performed dual-luciferase assay to verify whether LHX2 is a direct target gene of miR-1238 in NSCLC. Finally, A549 and LTEP-α-2 cells were transfected with miR-1238 mimics and their corresponding negative controls to detect the relative expression of LHX2 mRNA and protein in using quantitative Real-Time PCR and Western Blot.(4) Detected the relative expression of the miR-1238 in HBE and four NSCLC cell lines and 50 paired NSCLC tissue samples by using quantitative real-time PCR, meanwhile, we performed regression analysis of the miR-1238 andLHX2 expression to explore their correlation.(5) A549 and LTEP-α-2 cells were transfected with miR-1238 mimics and their corresponding negative controls to detect NSCLC proliferation using CCK-8 assay and colony formation.Results:(1) a. Both the levels of LHX2 m RNA and protein were obviously increased in A549, LTEP-a-2, H460 and H1299 cells when compared with HBE. b. The LHX2 mRNA levels were significantly higher in 50 paired NSCLC tissues when compared with the corresponding non-tumor tissues(p<0.05). In a word, the level of LHX2 mRNA is increased in NSCLC cells and tissues.(2) Knockdown LHX2 expression significantly reduced cell clone numbers in A549 and LTEP-α-2 cells, as a word, LHX2 promoted NSCLC growth.(3) miR-1238 decreased LHX2 mRNA and protein level by directly targeting the seed sequence of LHX2 3’UTR in NSCLC cells through Dual Luciferase Reporter Assay experiment and miR-1238 mimics transient transfection in A549 and LTEP-a-2 cells.(4) miR-1238 was decreased in A549,LTEP-a-2, H460 and H1299 cell lines when compared to that in HBE. Meanwhile, the expression level of miR-1238 was found to be decreased in 50 paired NSCLC tissue specimens compared with the paired noncancerous lung tissues. In conclusion, the level of miR-1238 is decreased in NSCLC cells and tissues. Furthermore, regression analysis uncovered that the ratio of miR-1238 and LHX2 mRNA level was negative correlated in 50 paired tissues.(5) miR-1238 expression significantly inhibited cell viability in A549 and LTEP-α-2 cell, as demonstrated by CCK-8 assay and colony formation.Conclusion: miR-1238 level is decreased in NSCLC, LHX2 mRNA and protein level were increased in NSCLC, miR-1238 can decrease the level of LHX2 by directly binding to the LHX2 3’UTR seed sequences in NSCLC cells. Besides, the ratio of miR-1238 and LHX2 mRNA level was negatively correlated in NSCLC tissues.miR-1238 can inhibit the growth of tumor while LHX2 can promote tumor progression according to biological function test. Our findings demonstrating that miR-1238 may be an suppressor and play a critical role in decreasing LHX2 expression in NSCLC.The study of down-regulation of LHX2 that mediated by miR-1238 may increase ourcomprehending of the mechanisms of NSCLC carcinogenesis and provide a theoretical basis for the diagnosis and treatment of NSCLC.