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. 2008 Jul 2;3(7):e2557.
doi: 10.1371/journal.pone.0002557.

"VSports" Aberrant expression of oncogenic and tumor-suppressive microRNAs in cervical cancer is required for cancer cell growth

Xiaohong Wang  1 , Shuang Tang, Shu-Yun Le, Robert Lu, Janet S Rader, Craig Meyers, Zhi-Ming Zheng
Affiliations

"V体育官网" Aberrant expression of oncogenic and tumor-suppressive microRNAs in cervical cancer is required for cancer cell growth

Xiaohong Wang et al. PLoS One. .

Abstract

MicroRNAs (miRNAs) play important roles in cancer development. By cloning and sequencing of a HPV16(+) CaSki cell small RNA library, we isolated 174 miRNAs (including the novel miR-193c) which could be grouped into 46 different miRNA species, with miR-21, miR-24, miR-27a, and miR-205 being most abundant VSports手机版. We chose for further study 10 miRNAs according to their cloning frequency and associated their levels in 10 cervical cancer- or cervical intraepithelial neoplasia-derived cell lines. No correlation was observed between their expression with the presence or absence of an integrated or episomal HPV genome. All cell lines examined contained no detectable miR-143 and miR-145. HPV-infected cell lines expressed a different set of miRNAs when grown in organotypic raft cultured as compared to monolayer cell culture, including expression of miR-143 and miR-145. This suggests a correlation between miRNA expression and tissue differentiation. Using miRNA array analyses for age-matched normal cervix and cervical cancer tissues, in combination with northern blot verification, we identified significantly deregulated miRNAs in cervical cancer tissues, with miR-126, miR-143, and miR-145 downregulation and miR-15b, miR-16, miR-146a, and miR-155 upregulation. Functional studies showed that both miR-143 and miR-145 are suppressive to cell growth. When introduced into cell lines, miR-146a was found to promote cell proliferation. Collectively, our data indicate that downregulation of miR-143 and miR-145 and upregulation of miR-146a play a role in cervical carcinogenesis. .

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

"VSports注册入口" Figures

Figure 1
Figure 1. Wobble digestion of miRNAs by Dicer in CaSki cells.
Arrows above the pre-miRNA indicate wobble-digestion sites on the pre–miR-21 in A and pre-miR-205 in B, producing products with additional nucleotides on the 3′ end. The sequences shown in red correspond to mature miRNA while that shown in black is the portion of pre-miRNA which is removed during processing.
Figure 2
Figure 2. miRNA expression profile in cervical cancer cells determined by northern blot analysis.
A. miR-193c expression in CaSki, HeLa, and 293 cells. CaSki and its subclones C-V and C-2 cells were compared with HeLa (HPV18+) and 293 cells (HPV−) for expression of miR-193c by northern blotting. Total RNA (30 µg) from CaSki, HeLa, and 293 cells was separated in a 15% denaturing polyacrylamide gel, blotted onto a Gene Screen-Plus membrane, and then probed separately with a sense (s) or an antisense (as) miR-193c probe. Subsequently, the membrane was reprobed with antisense probes for miR-21, miR-27a, miR-205, or U6, respectively. B. miRNA expression in cervical cancer–derived cell lines. Total cell RNA (30 µg) was extracted from various cell lines with or without HPV infection. Northern blotting was used to examine the expression of 10 miRNAs from eight cancer cell lines, two W12 cell subclones, HaCaT cells, normal cervix, and cervical cancer tissues (Ambion). C. Downregulation of miR-143 in cervical cancer tissues. Total RNAs from two pairs of normal (NT) cervix vs cervical cancer (Ca) tissues from clinical samples and one pair of normal cervix and cervical cancer purchased from Ambion were compared for the expression of miR-143 by northern blotting. U6 was also blotted as an internal control for sample loading in panel A, B, and C.
Figure 3
Figure 3. miRNA signatures in cervical cancer and normal cervical tissues by miRNA array analysis.
Total RNA (5 µg) isolated from age-matched normal cervical tissues (NT) and cervical cancer tissues (Ca) were analyzed by miRNA array analysis. The cancer tissue 1, 3, and 5 were infected with HPV16 and the cancer tissue 4 was infected with HPV45. A. A clustering graph was created using a hierarchical method from those miRNAs with signal density of more than 1000 and shows increased (red) or decreased (green) expression (P<0.05) of individual miRNA from 4 age-matched normal and cancerous cervical tissues (Table S1). The column heading indicates individual sample code in the assay. Each row shows the expression pattern of individual miRNA as a log2-transformed expression ratio, with the most closely related expression joined by a branch and clustered by an average-linked algorithm. Branch lengths reflect degrees of similarity between miRNA expression patterns. Color scale on the top of the panel represents the degree of expression based on a calibrated ratio. Green indicates lower expression compared to the mean (zero), black indicates expression equal to the mean, and red indicates higher expression compared to the mean. B. Verification of miRNA expression profile by northern blotting. Total cell RNA (30 µg) isolated from each tissue was probed with individual antisense oligo to each indicated miRNA. An additional band in each blot indicates a wobble product from Dicer digestion. C. Bar graphs show the relative levels of the examined miRNAs normalized to U6 RNA in each tissue in panel B.
Figure 4
Figure 4. miRNA expression from undifferentiated keratinocytes in monolayers and stratified and differentiated keratinocytes in raft tissues.
Raft tissues (cultures) with HPV18 infection were derived from human vaginal keratinocytes (HVKs) in monolayer cultures with HPV18 DNA transfection. Total cell RNA (5 µg each) extracted from each type of cells was compared in parallel for miRNA expression using miRNA array analyses. A. A clustering graph was created by a hierarchical method from those miRNAs with a signal density of more than 1000 obtained from three independent transfections in each group (Table S2). The graph shows decreased (green) or increased (red) expression (p<0.05) of individual miRNAs from undifferentiated HVKs in monolayers (Mono) versus stratified and differentiated HVKs in rafts. See other details in Fig. 3A. B. Verification of miRNA expression from undifferentiated HVKs in monolayers and stratified and differentiated HVKs in rafts by northern blotting. Total cell RNA (30 µg each) isolated from differentiated or undifferentiated HVKs was probed with selected miRNA probes. C. Bar graphs show the relative levels of the examined miRNAs normalized to U6 RNA in each sample in panel B. M = monolayer culture, R = raft.
Figure 5
Figure 5. Upregulation of miR-146a is cervical cancer-specific.
A. Total RNAs from HVKs in two rafts and two monolayer (Mono) cultures were compared with two cervical cancer (Ca) tissues for the expression of miR-146a by northern blotting. The expression levels of miR-21 and miR-26b were examined as miRNA controls. Small nuclear RNA U6 was also blotted as an internal control for sample loading. B. Bar graphs show the relative levels of the examined miRNAs normalized to U6 RNA in each sample in panel A. M = monolayer culture, R = raft.
Figure 6
Figure 6. Overexpression of miR-143 and miR-145 suppresses HeLa cell growth.
A. miR-143 and miR-145 are suppressive for HeLa cell growth. Arrows indicate the time points when the cells received a specific miRNA (30 nM) transfection. Viable cells in each group were counted at the indicated time points. Data represent the mean±SD of two experiments, each performed in triplicate. B. Relative levels of miR-143 and miR-145 in HeLa cells at 96 hrs after first transient transfection. Total cell RNA extracted was examined by miRNA ligation assay. A tRNA level in each sample was used as a loading control.
Figure 7
Figure 7. Overexpression of miR-146a promotes cell proliferation.
A. No expression of miR-146a in cervical cancer-derived cell lines and in a human skin-derived keratinocyte line, HaCaT cells. Total cell RNA was extracted from individual cell line and examined for miR-146a and miR-21 expression by northern blotting. U6 in each sample served as sample loading control. B. Relative level of miR-146a in HeLa and HCT116 cells at 96 hrs after the first transfection. Total cell RNA extracted from each group was examined by northern blotting. C and D. miR-146a promotes cell proliferation. Arrows indicate the time points when the cells received transfection with miR-146a or miR-control (30 nM). Viable cells in each group were counted at the indicated time points. Data represent the mean±SD of two experiments, each performed in triplicate.
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