miR-106a represses the Rb tumor suppressor p130 to regulate cellular proliferation and differentiation in high-grade serous ovarian carcinoma

Abstract

The degree of differentiation in human cancers generally reflects the degree of malignancy, with the most undifferentiated cancer being also the highest grade and the most aggressive. High-grade serous ovarian carcinomas (HGSOC) are poorly differentiated and fast-growing malignancies. The molecular mechanisms underlying the poor differentiation of HGSOC has not been completely characterized. Evidence suggests that miRNA, miR are dysregulated in HGSOC. Therefore, we focused on those miRNAs that are relevant to tumor differentiation. Expression profiling of miRNAs in HGSOC, indicated miR-106a and its family members were significantly upregulated. Upregulation of miR-106a was further validated by real-time reverse transcriptase PCR (qRT-PCR) and miRNA in situ hybridization in a large cohort of HGSOC specimens. Overexpression of miR-106a in benign and malignant ovarian cells significantly increased the cellular proliferation rate and expanded the side-population fraction. In particular, SKOV3 cells with miR-106a overexpression had significantly higher tumor initial/stem cell population (CD24- and CD133-positive cells) than control SKOV3 cells. Among many miR-106a predicated target genes, p130 (RBL2), an retinoblastoma (Rb) tumor suppressor family member, was not only confirmed as a specific target of miR-106a but also related to tumor growth and differentiation. The importance of mir-106a and RBL2 was further demonstrated in vivo, in which, SKOV3 cells overexpressing miR-106a formed poorly differentiated carcinomas and had reduced RBL2 levels. To our knowledge, this is the first study of miR-106a mediating proliferation and tumor differentiation in HGSOC.

Implications: The current study suggests that the RB tumor suppressor pathway is a critical regulator of growth and differentiation in HGSOC.

Figure 1

Expression analysis of miR-106a in ovarian carcinoma. A, photomicrographs illustrate consecutive sections of normal fallopian tube (FT) epithelium (arrow), STIC (arrow head), and invasive high-grade serous carcinoma (double arrow heads) by hematoxylin and eosin stain (H&E, left), Immunostain for P53 (middle) and miRNA in situ hybridization for miR-106a (right). Strong immunoreactivity for P53 represents the accumulation of the mutant P53 in STIC and invasive high-grade serous carcinoma. B, the histobars represent the mean values of relative miR-106a expression in 117 HGSOC and 30 fallopian tube (normalized by RNA loading control of U6) by miRNA in situ hybridization. C, the relative miR-106a and miR-106b expression detected by miRNA profiling analysis (N = 5) in normal fallopian tube (light gray), STIC (dark gray), and HGSOC (black). **, P < 0.01.

Figure 2

miR-106a promotes normal and malignant ovarian/ fallopian tube epithelial cell proliferation. A, growth curves illustrate the significant differences in cell proliferation from day 2 to 5 in normal fallopian tube secretory epithelial cell line (FTE187; left) and ovarian cancer cell line (SKOV3; right) with and without miR-106a overexpression (see Materials and Methods). B and C, cell-cycle analysis by the cell flow cytometer reveals that a significant increase of S-phase cell population in FTE187 and in SKOV3 with miR-106a overexpression (C) in comparison with those without miR-106a overexpression (B; P < 0.05).

Figure 3

miR-106a overexpression increases stem cell-like (side-population, SP) population. The side-population, major population of normal (FTE187; A) and malignant (SKOV3; C) cell lines were analyzed by flow cytometer (see Materials and Methods). Circled areas represent the side-populations. Overall, a 2- to 4-fold increase of side-population cells were observed in four cell lines with miR-106a overexpression. CD133 and CD24 expression were counted by flow cytometry in FTE187 (B) and in SKOV3 (D and E). Cells with miR-106a overexpression significantly increase CD24- and CD133-positive cell population in comparison with those without miR-106a overexpression (P < 0.05).

Figure 4

Tumor suppressor gene RBL2 is specifically targeted by miR-106a. A, RT-PCR analysis reveals an inverse correlation of endogenous RBL2 with miR-106a expression in normal (FTE187 and T29) and malignant (SKOV3, HEY, OV-90, and OVCAR3) cell lines. U6 is used as an RNA loading control. B, two predicted binding sites of the miR-106a family in RBL2 3′-UTR and their sequence. C, histobars illustrate the relative luciferase expression in T29 cells cotransfected with wild-type and mutant miR-106a–binding sites with (gray bars) and without (black bars) miR-106a overexpression. Vector stands for luciferase transfection without RBL2 3′-UTR. D, Western blot analysis reveals that introducing stable miR-106a overexpression in FTE187 cells results in significant reduction of RBL2 and P21 (well known target gene of miR-106a, used as positive control). miR-106a and U6 expression are shown below. Histobars represent the average levels of RBL2 and P21 expression with and without miR-106a expression. *, P < 0.05; **, P < 0.01. E, blocking RBL2 expression promotes cell proliferation as did with miR-106a overexpression. F, miR-106a–targeted gene expression was further validated by blocking miR-106a expression with anti-miR-106a in OV-90 cells; blocking miR-106a expression inhibits cell proliferation compared with control cells.

Figure 5

miR-106a overexpression leads to faster growth and poor differentiation of tumor massin xenografts of nude mice. A, photographs illustrate an example of larger tumor masses in xenografts of SKOV3 cell lines with miR-106a overexpression, in comparison with the tumors without miR-106a overexpression. B, growth curves for tumor volumes in xenografts of nude mice with SKOV3 cell line with and without miR-106a overexpression (each consists of 8 mice), measured from day 14 to 35. C, photomicrographs illustrate the histology (top), Ki-67, RBL2, CD133, and CD24 expression (by immunohistochemistry stain, middle and lower) in xenografts of SKOV3 cell lines with [miR-106a (+)] and without [miR-106a (−)] miR-106a overexpression. SKOV3 tumors without miR-106a overexpression show well-differentiated tumor growth, characterized by mostly glandular growth pattern, low Ki-67 index (low cell proliferation rate), and high level of RBL2 expression. Although SKOV3 tumors with miR-106a overexpression show poorly differentiated and solid growth patterns, high Ki-67 index and lower RBL2 expression. D, the statistical analysis of mean (wide histobars) and standard errors (small T-bars) between tumors with (T) and without miR-106 (C) overexpression in the order of tumor volume,% of glandular component, Ki-67 index, RBL2, CD133, and CD24 expression. *, P < 0.05; **, P < 0.01.

Figure 6

miR-106a, miR-106b, and RBL2 expression in HGSOCs and fallopian tube. Real-time RT-PCR analysis of miR-106a (A), miR-106b (B), and RBL2 (C) expression in individual panels and combined comparison (D). The relative expression of each gene was normalized by either U6 (for miRNA) or actin (for RBL2).