Supplementary Material for: MiR-148b, MiR-152/ALCAM Axis Regulates the Proliferation and Invasion of Pituitary Adenomas Cells

<b><i>Background/Aims:</i></b> Aberrant expression of miRNA has been found in many tumor tissues to regulate the tumorigenesis by binding to the 3`- untranslated region (3`-UTR) of the target genes. The aim of this study is to investigate the role of miR-148b, miR-152/ALCAM axis in human pituitary adenomas (PAs). <b><i>Methods:</i></b> First, we detected the expression level of miR-148b-3p and miR-152 in human PAs samples by using qRT-PCR. Then we studied the role of miR-148b-3p, miR-152 on human PAs cell proliferation, invasion and apoptosis by using MTS assay, Transwell invasion assay and Annexin V/PI Staining Test. To study the relationship between miR-148b-3p, miR-152 and activated leukocyte antigen molecule (ALCAM), we overexpressed miR-148-3p or miR-152 by transfecting specific mimics. Lucifearase reporter assay was then performed to confirm the target. Next, we studied the biological functions of ALCAM in human PAs cells. Finally, the role of miR-148b-3p, miR-152/ALCAM axis in PAs cells was studied. <b><i>Results:</i></b> The expression level of miR-148-3p and miR-152 in invasive PAs samples was lower than those in noninvasive samples. Overexpression of miR-148b-3p, miR-152 could repress proliferation and invasion, and promote apoptosis. Moreover, miR-148b-3p and miR-152 could repress activated leukocyte antigen molecule (ALCAM) expression. Knockdown of ALCAM could repress proliferation and invasion and promote apoptosis. By contrary, overexpression of ALCAM promoted proliferation and invasion. Further, the rescue experiments indicated that overexpression of ALCAM significantly restored the proliferation, apoptosis, and invasion influenced by miR-148b-3p and miR-152. <b><i>Conclusions:</i></b> Our study suggests that miR-148b-3p, miR-152 may serve as suppressors in PAs through downregulating ALCAM expression. miR-148b, miR-152/ ALCAM axis may be a new therapeutic target in the future.

**背景与目的**：研究已证实，微小RNA（miRNA）的异常表达可通过结合靶基因的3'非翻译区（3'-UTR）调控肿瘤发生发展。本研究旨在探讨miR-148b、miR-152/活化白细胞黏附分子（ALCAM，activated leukocyte antigen molecule）轴在人垂体腺瘤（PAs）中的作用。
**研究方法**：首先，采用实时定量逆转录聚合酶链反应（qRT-PCR）检测人垂体腺瘤样本中miR-148b-3p与miR-152的表达水平。随后通过MTS实验、Transwell侵袭实验以及Annexin V/PI染色实验，探究miR-148b-3p、miR-152对人垂体腺瘤细胞增殖、侵袭与凋亡的调控作用。为明确miR-148b-3p、miR-152与活化白细胞黏附分子（ALCAM）之间的靶向调控关系，本研究通过转染特异性miRNA模拟物（mimics）过表达miR-148b-3p或miR-152，并采用萤光素酶报告基因实验验证二者的靶向结合关系。继而探究活化白细胞黏附分子（ALCAM）在人垂体腺瘤细胞中的生物学功能。最终明确miR-148b-3p、miR-152/ALCAM轴在垂体腺瘤细胞中的具体作用。
**研究结果**：侵袭性垂体腺瘤样本中miR-148b-3p与miR-152的表达水平显著低于非侵袭性垂体腺瘤样本。过表达miR-148b-3p或miR-152可抑制垂体腺瘤细胞的增殖与侵袭，并促进其凋亡。此外，miR-148b-3p与miR-152可下调活化白细胞黏附分子（ALCAM）的表达。敲低ALCAM的表达同样可抑制垂体腺瘤细胞的增殖与侵袭，并促进其凋亡；反之，过表达ALCAM则可促进垂体腺瘤细胞的增殖与侵袭。进一步的挽救实验结果显示，过表达ALCAM可显著逆转miR-148b-3p与miR-152对垂体腺瘤细胞增殖、凋亡及侵袭所产生的调控作用。
**研究结论**：本研究表明，miR-148b-3p与miR-152可通过下调ALCAM的表达，发挥垂体腺瘤抑癌因子的作用。miR-148b、miR-152/ALCAM轴有望成为未来垂体腺瘤治疗的新型靶点。