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Keywords:

  • HBXIP;
  • Lin28B;
  • TF II D;
  • miRNA-520b;
  • carcinogenesis

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

Hepatitis B X-interacting protein (HBXIP) is a novel oncoprotein and plays a key role in the development of breast cancer. However, its mechanisms of action are poorly understood. Lin28B functions as an oncogene in a variety of human cancers. In our study, we report that HBXIP acts with its partner Lin28B to contribute to carcinogenesis. Our data showed that the expression levels of HBXIP were significantly positively correlated with those of Lin28B in clinical breast cancer tissues. Then, we found that HBXIP was able to upregulate Lin28B in breast cancer MCF-7 cells. Chromatin immunoprecipitation assay (ChIP) and electrophoretic mobility shift assay (EMSA) revealed that HBXIP occupied the promoter region (−1199/-1073 nt) of Lin28B. Importantly, co-immunoprecipitation (Co-IP) and GST pull-down assay validated that HBXIP directly bound to the TATA-binding protein (TBP), a basal subunit of transcription factor TF II D complex. In addition, we discovered that Lin28B could block the downregulation of HBXIP via suppressing miR-520b which directly targeted HBXIP mRNA in the cells. In function, we demonstrated that HBXIP enhanced the proliferation of breast cancer cells through Lin28B in vitro and in vivo. Thus, we conclude that the oncoprotein HBXIP as a co-activator of TF II D transactivates Lin28B promoter via directly binding to TBP to upregulate the expression of Lin28B in promotion of proliferation of breast cancer cells, in which Lin28B maintains the high level of HBXIP through suppressing miR-520b in a feedback manner. Therapeutically, HBXIP may serve as a target of breast cancer.

Abbreviations
ChIP

chromatin immunoprecipitation

DMEM

Dulbecco's modified eagle medium

EMSA

electrophoretic mobility shift assay

HBXIP

hepatitis B X-interacting protein

miRNA

microRNA

miR-520

microRNA-520

nt

nucleotide

qRT-PCR

quantitative real-time PCR

RT-PCR

reverse transcription PCR

siRNA

small interfering RNA

TBP

TATA-binding protein

Hepatitis B X-interacting protein (HBXIP), a conserved ∼18KDa protein, is originally identified by its interaction with the hepatitis B virus X protein (HBx) and negatively regulates the activity of HBx and alters the replicative life cycle of the virus.[1] Expression of HBXIP mRNA occurs in nearly all tissues not limited to liver and is also in mice and other rodents.[2] The sequences of the HBXIP gene, containing a putative leucine zipper motif and two consensus phosphorylation sites for protein kinase C and casein kinase II, are well conserved among mammalian species.[1] HBXIP can regulate the duplication of centrosome and HBXIP-deficient Hela cells succumb eventually to apoptosis. In addition, a mouse model of liver regeneration experiment shows HBXIP is a critical regulator of hepatocyte cell growth in vivo.[3] Studies also report that HBXIP functions as a cofactor with survivin through forming a complex to suppress apoptosis.[2] Importantly, our group shows that HBXIP plays crucial roles in the development of breast cancer, serving as a key oncoprotein in cancer.[4-7] Recently, we show that HBXIP promotes the breast cancer cell growth by upregulating S100A4.[7] However, the mechanism by which HBXIP enhances the proliferation of breast cancer cells remains poorly understood.

Lin28B, a homolog of Lin28, originally identified in hepatocellular carcinoma conserves a cold shock domain and a pair of CCHC zinc finger domains.[8] Lin28B is a RNA-binding protein, which functions as an oncogene, selectively blocks miRNA let-7 maturation and promotes the proliferation of cancer cells.[9-12] Let-7 miRNA family members act as tumor suppressors by inhibiting the expression of oncogenes and key regulators of mitogenic pathways including RAS, MYC and HMGA2.[13] Growing evidence indicates that the elevated Lin28B protein is associated with the progression of several malignant tumors[8, 14-18] and with the poor clinical prognosis.[19] Lin28B, acting as one of the pluripotent factors involved in making induced pluripotent stem cells, may serve as a potential therapeutic target for cancer treatment.[15] Recently, many studies have reported that Lin28B may promote the proliferation through blocking the maturation of let-7 miRNAs in breast cancer cells.[20-22]

The transcription factor TF II D is composed of the TATA-binding protein (TBP) and at least 13 evolutionarily conserved TBP-associated factors (TAFs).[23, 24] The TBP component recognizes and binds to the TATA box to initiate assembly of the transcription complex.[25, 26] Interestingly, it has been reported that TF II D does not appear to be required for all transcription initiation events. Knockdown of TF II D subunits does not arrest transcription.[27] While, it has been reported that disassembly of TF II D in mice hepatocytes strikingly blocks embryonic liver development, but it has little affect on adult hypatocytes in which the transcription of ≤5% of genes are affected.[28] TF II D may also serve as a bridge or receptor of signals from enhancer-bound transcriptional activator proteins.[29]

In the present study, we investigated the mechanism by which HBXIP enhanced the proliferation of breast cancer cells. Our data show that the oncoprotein HBXIP, as a co-activator of TF II D via directly binding to TBP, transactivates the promoter activity of Lin28B. In turn, Lin28B maintains the high levels of HBXIP through suppressing miR-520b. The finding provides insight into the mechanism by which HBXIP enhances the proliferation of breast cancer cells.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

Clinical breast cancer tissues

Breast cancer tissue array (No. 08C14), comprising duplicates of 49 cases of primary breast carcinoma, was purchased from Xi'an Aomei Biotechnology (Xi'an, China). Total 31 paired breast carcinoma and adjacent normal breast tissues were collected from patients undergoing resection of breast carcinoma in Tianjin Tumor Hospital (Tianjin, China). The relevant characteristics of the studied subjects were shown in Supporting Information Table S1. The tissues were used for testing the expression levels of HBXIP mRNA or Lin28B mRNA. Informed consent was obtained from each patient, and the study was approved by the Institute Research Ethics Committee at Nankai University.

Immunohistochemistry

Immunohistochemistry assay was performed as described previously.[6] The slides were incubated with rabbit anti-HBXIP (or rabbit anti-Lin28B) antibody at 4°C for overnight. After incubation at room temperature for 30 min with biotinylated secondary antibody, the slides were incubated with streptavidin-peroxidase complex at room temperature for 30 min. Immunostaining was developed by using chromogen, 3,3-diaminobenzidine (DAB) and counterstained with Mayer's hematoxylin. The staining levels of HBXIP and Lin28B were classified into three groups using a modified scoring method based on the intensity of staining (0 = negative; 1 = low; 2 = high) and the percentage of stained cells (0 = 0% stained; 1 = 1–49% stained; 2 = 50–100% stained). A multiplied score (intensity score × percentage score) lower than 1 was considered to be a negative staining (−), 1 and 2 were considered to be moderate staining (+) and 4 was considered to be intense staining (++) as previously reported.[7]

Cell culture and treatment

Breast cancer cell line MCF-7 was cultured in RPMI medium 1640 (Gibco, Grand Island, NY), with 10% fetal calf serum (FCS). Breast cancer cell line MDA-MB-231 and human embryonic kidney cell line 293T cells were cultured in Dulbecco's modified Eagle's medium (Gibco) supplemented with heat-inactivated 10% FCS, 100U/ml penicillin, 100U/ml streptomycin and 1% glutamine at 37°C with 5% CO2. Stable cell lines were generated by transfecting plasmids pCMV-Tag-2B or pCMV-HBXIP into breast cancer MCF-7 cells with Lipofectamine 2000 and selected with G418 (Invitrogen). The engineered cell lines were as follows: MCF-7-pCMV (stably transfected pCMV-Tag-2B empty vector), MCF-7-HBXIP (stably transfected pCMV-HBXIP plasmid). Cells were collected and seeded in 6-well, 24-well or 96-well plates for 24 hr and then were transfected with plasmids or siRNAs. All transfections were performed with Lipofectamine 2000 according to (Invitrogen) manufacturer's instructions. The siRNAs used in our study were as follows: negative control (NC) siRNA; HBXIP siRNA-1: 5′-GCAGCUAAGGCAGCUAAG CUAACCUCUGTT-3′;[2] HBXIP siRNA-2: 5′-CGGAAGCGCAGUGAUGUUUdTdT-3′;[3] TBP siRNA: 5′-UUGAAUAGUGAGACGAGUUTT-3′;[30] Lin28B siRNA-1: 5′-GCAGAGAU CUCAGAACGGU-3′; Lin28B siRNA-2: 5′-GGAAGGAUUUAGAAGCCUA-3′.[31]

Plasmid construction

The 5'-flanking region (from −1417 to −28 nt) of Lin28B gene was cloned into the KpnI/HindIII site of promoterless luciferase construct pGL3-Basic vector (Promega). Then, the recombinant plasmids were used as the templates to clone the rest of the truncated promoter region (−1162/−503, −688/−32, −1356/−1073, −1325/−1179, −1199/−1073) of Lin28B with the same KpnI/HindIII site of the pGL3-Basic vector, respectively. The cDNA fragment of TBP was cloned into the BamHI/HindIII site of pCMV-Tag 2B vector and also cloned into the BamHI/HindIII site of pET-28a vector. The cDNA of HBXIP was subcloned into the pGEX-4T1 for GST-tagged recombinant protein expression. All primers are listed in Supporting Information Table S2.

RT-PCR, qRT-PCR and western blotting assays

Total RNA was extracted from the cells (or tumor specimens) using Trizol (Invitrogen) according to the manufacturer's protocol. For microRNA let-7 and miR-520b detection, total RNA was polyadenylated by poly (A) polymerase (Ambion, Austin, TX) as described previously.[32] Reverse transcription was performed using poly(A)-tailed total RNA and reverse transcription primer with ImPro-II Reverse Transcriptase (Promega) according to the manufacturer's protocol. For Lin28B mRNA level detection, the total RNA from those cells (or tissues) was directly reverse transcribed. The qRT-PCR was performed as described in the method of SYBR Premix ExTaqTMII Kit (Takara, Ohtsu, Japan). All primers are listed in Supporting Information Table S3. Western blot analysis was performed as previously described.[33] The primary antibodies used for western blot were rabbit polyclonal anti-HBXIP and anti-Lin28B (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Histone3 (Cell Signaling Technology, Beverly, MA) and mouse monoclonal anti-β-actin (Cell Signaling Technology).

Immunofluorescence staining

To determine the localization of Lin28B mediated by HBXIP, cells were placed on coverslips and fixed using 4% paraformaldehyde for 15 min. Coverslips were rinsed with PBS, and cells were permeabilized with 0.2% Triton X-100 for 30 min. Following PBS washes for three times, the cells were incubated with anti-Lin28B antibody (1:100, Santa Cruz Biotechnology) for 2 h. After being washed with PBS, cells were incubated with rhodamine-labeled goat anti-rabbit secondary antibody (1:100, Sigma) for 1 h and 4, 6-diamidino-2-phenylindole (30 nM) for 15 min in the dark. After three times washes with PBS, images were captured under a microscope using a digital camera and processed using Spot Advanced 4.5 software as previously described.[6]

Nuclear extraction

The nuclear extraction assay was performed using NE-PER@ nuclear and cytoplasmic extraction kit (Pierce). The MCF-7 cells (2× 106) transiently co-transfected with pCMV/NC siRNA, pCMV-HBXIP/NC siRNA or pCMV-HBXIP/Lin28B siRNA-2 were harvested, respectively. According to the manufacturer's instructions, the cytoplasmic and nuclear proteins were extracted, respectively, followed by western blot analysis to examine the expression levels of Lin28B in the nuclear extracts as well as in the cytoplasm.

Chromatin immunoprecipitation assay

The chromatin immunoprecipitation assay (ChIP assay) was performed using the EpiQuikTM chromatin immunoprecipitation kit from Epigentek Group Inc and according to the published methods.[34, 35] Protein-DNA complexes were immunoprecipitated with HBXIP antibodies, with anti-RNA polymerase II as a positive control antibody and with mouse IgG as a negative control antibody. DNA collect by these antibodies was subjected to PCR analysis, followed by sequencing. Amplification of soluble chromatin prior to immunoprecipitation was used as an input control.

Luciferase reporter gene assay

Adherent cells (MCF-7, MDA-MB-231 and 293T cells) were seeded into 24-well plates and, respectively, transfected with the constructs containing different length fragments of Lin28B promoter or pGL3-Basic as a negative control, with the pRL-TK plasmid (Promega, Madison, WI) which was used as internal normalization. Cell extracts were harvested after 36 h and lysed using lysis buffer (Promega). Luciferase reporter gene assay was implemented using the Dual-Luciferase Reporter Assay System (Promega) as previously described.[33] All experiments were performed at least three times.

Electrophoresis mobility shift assay

The electrophoresis mobility shift assay (EMSA) protocol was described in detail.[33, 36] Nuclear extracts were prepared with MCF-7 cells. Probes were generated by annealing single strand oligonucleotides containing the different parts of Lin28B promoter. One µg anti-HBXIP antibody (Santa) or anti-TBP antibody (Santa) was incubated with nuclear extracts on ice for 30 min before probes were added into the binding buffer. Samples were incubated on ice for 1 h and then separated by electrophoresis on 6% nondenaturing poly-acrylamide gel, and then the gel was dried and subjected to autoradiography.

Co-immunoprecipitation assay

The co-immunoprecipitation (Co-IP) protocol was described in detail on previously published articles.[2] MCF-7 cells (2× 106) were harvested and lysed in a lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.3% Triton X-100, 1 mM protease inhibitor PMSF). The lysates were incubated with antibodies and protein G-conjugated agarose beads at 4°C for 2 h. The precipitates were washed eight times with ice-cold lysis buffer, resuspended in the PBS and resolved by SDS-PAGE followed by western blotting.

GST pull-down assay

To further confirm the interaction between HBXIP and TBP protein, a GST pull-down assay was assessed. The cDNA of HBXIP was cloned into pGEX-4T1 expression vector (pGEX-HBXIP), while the cDNA of TBP was cloned into pET-28a expression vector (pET-TBP), and the construct vectors (pGEX-HBXIP, pGEX-4T1 and pET-TBP) were transformed into E.coli strains BL21 (DE3), respectively. The detailed procedure was performed according to published protocols.[37, 38] Glutathione beads were recovered by a brief centrifugation and washed eight times with lysis buffer and the last wash used PBS before resolved by SDS-PAGE and detected by western blotting using the rabbit anti-TBP antibody (Cell Signaling Technology, Beverly, MA).

Colony formation assays

For colony formation assays, 500–1000 transfected cells were placed in six-well plates after 36–48 h transfection and maintained in complete medium for nearly 2 weeks. The colonies were fixed by methanol and stained with methylene blue as previously described.[33]

Analysis of cell proliferation

MCF-7-HBXIP cells were seeded onto 96 well plates (500 cells/well) for 24 h before transfection and 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay was carried out to assess cell proliferation using the Cell-Light TM EdU imaging detecting kit according to the manufacturer's instructions (RiboBio, China). In addition, the 3-(4,5-dimethylthiazol-2-yl) −2, 5-diphenyltetrazolium bromide (MTT) (Sigma) assays were used to assess cell proliferation every day from the 24 h until the 96 h after transfection as well.

Animal transplantation

All experimental procedures involving animals were in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publications nos. 80-23, revised 1996) and were performed according to the institutional ethical guidelines for animal experiment. The MCF-7-HBXIP cells transiently transfected with NC siRNA or Lin28B siRNA-2 and MCF-7-pCMV cells transiently transfected with NC siRNA were subcutaneously injected into the flanks of 4-week-old male BALB/c athymic nude mice, respectively. Tumor growth was monitored every four days. After 26 days, the mice were sacrificed, necropsies were performed and tumors were weighed. Tumor volume (V) was monitored by measuring the length (L) and width (W) with calipers and calculated with the formula (L× W2) × 0.5.[32]

Statistical analysis

Each experiment was repeated at least three times. Statistical significance was assessed by comparing mean values (±SD) using a Student's t test for independent groups and was assumed for p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***). Correlation between expression levels of HBXIP and Lin28B in tumorous tissues were explored using Pearson′s correlation coefficient. Lin28B expression in primary breast carcinoma and adjacent normal breast tissues were compared using a Wilcoxon signed-rank test.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

Lin28B has a significant positive correlation with HBXIP in clinical breast cancer tissues

Our laboratory has focused on the investigation of mechanisms of HBXIP in promotion of the development of breast cancer. HBXIP is an important oncoprotein, which promotes the proliferation of breast cancer cells.[4, 7] Lin28B, as an oncogene, plays an important role in promoting the growth of variety of human cancers.[15-17] Thus, we tried to explore whether the expression of HBXIP was correlated with that of Lin28B. We tested the expression levels of HBXIP and Lin28B by immunohistochemical assays (Fig. 1a) using tissue microarray containing 49 cases of breast carcinoma samples and 13 cases of breast normal tissues. The data indicated that the positive rate of HBXIP was 77.6% (38/49), in which the positive rate of Lin28B was 94.7% (36/38). However, there was no Lin28B positive section found in the remaining 11 sections of no HBXIP staining. Besides, the combined staining had the grading preferences. The combined staining was 5/13 sections at grade I, 15/18 sections at grade II and 10/10 sections at grade III, respectively. Then, we examined the mRNA levels of HBXIP and Lin28B in the clinical breast carcinoma and their adjacent normal tissues (n = 31) by qRT-PCR assays, respectively. The results demonstrated that the expression levels of HBXIP and Lin28B in tumors were obviously higher than those in their adjacent normal tissues (Figs. 1b and 1c). Moreover, we analyzed the correlation between HBXIP and Lin28B in above tumor tissues by qRT-PCR assay. The data showed that the expression levels of Lin28B had a significant positive correlation with those of HBXIP in breast cancer tissues (p < 0.01 Wilcoxon signed-rank, Fig. 1d). Taken together, the expression levels of Lin28B are significantly positively associated with those of HBXIP in breast cancer tissues.

image

Figure 1. Lin28B has a significant positive correlation with HBXIP in clinical breast cancer tissues. (a) The expression levels of HBXIP and Lin28B were determined in the breast carcinoma and adjacent normal breast tissues by immunohistochemical staining using tissue microarray. (b and c) The relative mRNA expression levels of HBXIP and Lin28B were detected, respectively, by qRT-PCR in clinical breast carcinoma specimens and adjacent normal tissues (n = 31) (*p < 0.05, **p < 0.01, Wilcoxon signed-rank test). (d) The correlation of mRNA levels of HBXIP and Lin28B was examined by qRT-PCR assay in 31 pairs breast carcinoma tissues (p < 0.01, r = 0.7675, Pearson's correlation).

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HBXIP upregulates Lin28B and increases its nuclear import

We have recently reported that HBXIP as a coactivator of STAT4 upregulates the expression of S100A4 oncogene.[7] Therefore, we hypothesized that HBXIP may regulate Lin28B through activating transcription factors in promotion of proliferation of breast cancer cells. Interestingly, we observed that the mRNA and protein levels of Lin28B were markedly elevated by transiently transfecting HBXIP in a dose-dependent manner in breast cancer MCF-7 cells exhibited low levels of HBXIP (Figs. 2a and 2b). Conversely, the expression of Lin28B was reduced when HBXIP was silenced by siRNA-1 and siRNA-2, respectively, in breast cancer MDA-MB-231 cells exhibited high levels of HBXIP (Fig. 2b). It has been reported that Lin28B predominantly functions in the nucleus.[9] Intriguingly, immunofluorescence assays showed that the Lin28B localized in the nucleus was significantly reduced and the expression levels of Lin28B was also decreased when the endogenous HBXIP was knockdown by siRNA in MDA-MB-231 and MCF-7-HBXIP cells (Fig. 2c). The nuclear localization of Lin28B increased strikingly when the ectopic HBXIP was over-expressed in MCF-7 cells (Fig. 2d), while the western blot analysis indicated that the expression level of Lin28B in the nucleus was significantly increased when the ectopic HBXIP was over-expressed and decreased when the endogenous HBXIP was knockdown by siRNA in MCF-7 cells (Fig. 2e), suggesting that HBXIP is able to upregulate the expression of Lin28B and promote the nuclear entry of Lin28B in breast cancer cells. Lin28B is a RNA-binding protein, which selectively blocks the maturation of miRNA let-7 family members in breast cancer.[8, 9, 12, 31, 39] To further validate whether HBXIP had effect on let-7 miRNAs via Lin28B, we first tested the effect of HBXIP on let-7 miRNAs. The qRT-PCR showed that the over-expression of HBXIP greatly reduced the expression of let-7 miRNAs, such as let-7a, let-7f and let-7g, in MCF-7 cells (Fig. 2f). Importantly, the reduction of let-7 miRNAs mediated by HBXIP could be rescued by the knockdown of Lin28B using Lin28B siRNA-2 (Fig. 2f). The similar results were also observed in MDA-MB-231 cells (data not shown). Meanwhile, the efficiency of HBXIP over-expression or knowndown and Lin28B knowndown were tested in the cells by RT-PCR, qRT-PCR and western blotting assays (Supporting Information Fig. S1; Fig. 2b), respectively. The data support that HBXIP upregulates Lin28B. Thus, we conclude that HBXIP upregulates Lin28B and increases its nuclear entry in breast cancer cells.

image

Figure 2. HBXIP upregulates Lin28B and increases its nuclear import. (a) The mRNA levels of HBXIP and Lin28B were tested by RT-PCR in MCF-7 cells transiently transfected with pCMV (0.8 μg) or pCMV-HBXIP (0.4, 0.8 μg). (b) The expression levels of HBXIP and Lin28B were examined by western blot analysis in MCF-7 cells transiently transfected with pCMV-HBXIP or in MDA-MB-231 cells treated with NC siRNA, HBXIP siRNA-1 and siRNA-2 (70 nM), respectively. (c) The localization of Lin28B in the cells was observed in MDA-MB-231 and MCF-7-HBXIP cells transiently transfected with NC siRNA, HBXIP siRNA-1 and siRNA-2 (70 nM) by immunofluorescence staining, respectively. (d) The localization of Lin28B in the cells was observed in MCF-7 cells transiently transfected with pCMV (0.5 μg), pCMV-HBXIP (0.5 μg), NC siRNA (70 nM) and HBXIP siRNA-2 (70 nM), respectively. (e) The expression levels of Lin28B were measured by western blot analysis in the nucleus or cytoplasm of MCF-7 cells transiently transfected with pCMV (0.5 μg)/NC siRNA (70 nM), pCMV-HBXIP (0.5 μg)/NC siRNA(70 nM) or pCMV (0.5 μg)/HBXIP siRNA-2 (70 nM), respectively. (f) The relative expression levels of let-7 miRNAs were detected by qRT-PCR in MCF-7 cells treated with pCMV-HBXIP (0.5 μg)/NC siRNA (70 nM) or pCMV-HBXIP (0.5 μg)/Lin28B siRNA-2 (70 nM), respectively.

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HBXIP activates the core promoter (−1199/−1073 nt) of Lin28B

Our published report demonstrated that HBXIP was able to activate the S100A4 promoter through transcription factor STAT4.[7] Accordingly, we supposed that HBXIP might be involved in the transcriptional regulation of Lin28B. As expected, chromatin immunoprecipitation (ChIP) assays showed HBXIP could occupy the promoter of Lin28B (Fig. 3a), and luciferase reporter gene assays indicated that the activity of Lin28B promoter (−1417/−28 nucleotides region) could be markedly increased by HBXIP in MCF-7 cells (Fig. 3b), suggesting that HBXIP upregulates Lin28B expression through activating Lin28B promoter. Next, we tried to map the core promoter region of Lin28B. We identified that the core region of Lin28B promoter was localized in the −1199/−1073 nt region of Lin28B gene (Fig. 3c), and the luciferase activities of the core promoter region could be elevated by HBXIP in a dose-dependent manner or decreased by knockdown of HBXIP in MCF-7 cells and 293T cells, respectively (Figs. 3d and 3e). Thus, we conclude that HBXIP upregulates Lin28B via activating the core promoter (−1199/-1073 nt) of Lin28B.

image

Figure 3. HBXIP activates the core promoter (−1199/−1073 nt) of Lin28B. (a) The interaction between HBXIP and Lin28B promoter was detected by ChIP assay. The data presented are from three independent experiments, and the ChIP products were validated by DNA sequencing. (b) The relative luciferase activities of Lin28B promoter were tested by Dual Luciferase System in MCF-7 cells transiently transfected with pCMV-HBXIP (80 ng). (c) The relative activities of different regions of Lin28B promoter were examined by Dual Luciferase System in MCF-7 cells transiently transfected with different fragments (100 ng), respectively. (d and e) The relative activities of the core promoter (−1199/−1073nt) of Lin28B were detected by Dual Luciferase System in MCF-7 cells or 293T cells transiently transfected with pCMV-HBXIP or HBXIP siRNA-2 (70 nM), respectively. *p < 0.05, **p < 0.01, Student's t-test. All experiments were performed at least three times.

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HBXIP stimulates Lin28B promoter via directly binding to TBP subunit of TF II D

Next, we unraveled the mechanism by which HBXIP activates Lin28B promoter. We used DNA fragment of −1199/−1073 nt region of Lin28B promoter as the probe. EMSA indicated that there was an interaction between nuclear proteins and the probe (Fig. 4a, lane 3). While, a supershift band was observed when HBXIP antibody was added (Fig. 4a, lane 4), suggesting that HBXIP is able to bind to the −1199/−1073 nt region of Lin28B promoter. However, the purified HBXIP protein alone failed to bind to the −1199/−1073 nt fragment in vitro (Supporting Information Fig. S2a), implying that HBXIP indirectly interacts with Lin28B promoter in the cells. To explore the interaction site between HBXIP and Lin28B promoter, we decomposed the −1199/−1073 nt region into five overlapping DNA fragments, and the overlapping region contains eight nucleotides between the adjacent two fragments (Supporting Information Fig. S2b). Finally, EMSA assay identified that the DNA segment covering −1178/−1148 nt region of Lin28B promoter was responsible for interacting with HBXIP (Supporting Information Fig. S2b). Next, using promoter analysis program (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/prom omenu.cgi?dirDB=TF_8.3&Option=3&idCon=128523136200), we predicted the putative recognition motifs of three transcription factors with high scores, such as GATA-1,[40] TF II D,[41] and GR-β,[42] in the −1178/-1148 nt region of Lin28B promoter. EMSA assay showed that the interaction between nuclear extracts of MCF-7 cells and the probe of −1178/-1148 promoter region of Lin28B was disrupted when the binding site of TF II D was mutated in the promoter. However, the mutant binding site of the GATA-1 or GR-β failed to affect the interaction (Fig. 4b), suggesting that TF II D is responsible for the interaction between the nuclear extracts and Lin28B promoter. Additionally, the luciferase activities of Lin28B promoter were markedly decreased when the TF II D binding site was mutated in the cells (Fig. 4c). Thus, we speculated that HBXIP might activate the Lin28B promoter through interacting with TF II D. To test this speculation, we investigated whether HBXIP could bind to TF II D. Because the transcription factor TF II D is a complex composed of the TBP and TAFs, and the TBP component recognizes and binds to the TATA-box to initiate assembly of the transcription complex.[25, 26] Thus, to elucidate the relationship between HBXIP and TF II D, we focused on the relationship between HBXIP and TBP as a substitute of TF II D. Next, the Co-IP assays were performed and the results revealed that HBXIP and TBP could bind to each other in MCF-7 cells (Fig. 4d, left panel). To further verify whether the combination between HBXIP and TBP is direct or not, GST pull-down assays were performed. The protein of TBP and HBXIP was expressed, respectively, in bacteria. We found that the TBP protein with His-tag could be pulled down by GST-HBXIP, but not by the control GST alone (Fig. 4d, right panel), suggesting that HBXIP directly binds to TBP protein in the cells. These data suggested that HBXIP directly bound to TF II D. Next, we asked whether the promoter activity of Lin28B is dependent on the interaction between HBXIP and TBP. Luciferase reporter gene assays showed that over-expression of HBXIP or TBP resulted in the enhanced activity of the core promoter, while the core promoter activity was decreased when HBXIP or TBP was knocked down by HBXIP siRNA-2 or TBP siRNA, respectively (Fig. 4e). Importantly, we also found that the elevated activities of Lin28B promoter mediated by ectopic HBXIP (or TBP) could be abolished by the TBP siRNA (or HBXIP siRNA-2) in MCF-7 cells (Fig. 4e). Moreover, EMSA assay showed that there was a weaker band when TBP antibody was added (Supporting Information Fig. S2c, lane 3) compared to the controls containing no antibodies (Supporting Information Fig. S2c, lane 2). These data strongly demonstrate that the promoter activity of Lin28B is dependent on the interaction between HBXIP and TBP, suggesting that HBXIP functions as a co-activator of transcription factor TF II D. Taken together, we conclude that HBXIP, as a co-activator of TF II D, stimulates Lin28B promoter through directly binding to TBP subunit of TF II D.

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Figure 4. HBXIP stimulates Lin28B promoter via directly binding to TBP subunit of TF II D. (a) The interaction between Lin28B promoter (−1199/−1073 nt) probe and HBXIP was tested by EMSA. (b) The interaction between nuclear proteins and Lin28B promoter (−1178/−1148 nt) probe with the mutant binding sites of GATA-1, TF II D or GR-β was detected, respectively, by EMSA. (c) The luciferase activities of the Lin28B core promoter with wild type (or mutant binding sites of TF II D) were, respectively, determined by luciferase reporter gene assays in MCF-7 cells transfected with pCMV-HBXIP (80 ng). *p < 0.05, **p hx003C 0.01, Student's t-test. All experiments were performed at least three times. (d) The interaction between HBXIP and TBP was detected by Co-IP assays in MCF-7 cells in vivo. The direct interaction between recombinant GST-HBXIP and His-TBP proteins was detected by GST pull-down assay and western blot analysis in vitro. (e) The luciferase activities of Lin28B core promoter (−1199/−1073nt) were detected by luciferase reporter gene assays in MCF-7 cells transiently transfected with HBXIP siRNA-2 (70 nM) (or TBP siRNA), pCMV-HBXIP (100 ng) (or pCMV-TBP), pCMV-HBXIP (100 ng)/TBP siRNA (70 nM) (or pCMV-TBP/HBXIP siRNA-2), respectively.

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Lin28B maintains high levels of HBXIP via suppressing miR-520b targeting HBXIP mRNA

Many studies show that the feedback regulation is an important regulation manner in cancer.[43-45] Thus, we supposed that Lin28B might in turn regulate the expression of HBXIP. Strikingly, we observed that the expression of HBXIP at the levels of mRNA and protein was significantly decreased by Lin28B siRNA-2 in MCF-7-HBXIP cells using qRT-PCR and western blot analysis (Figs. 5a and 5b), suggesting that HBXIP may be regulated by Lin28B. Next, we speculated the underlying mechanism. Recent study has unraveled that miR-520b inhibits the expression of HBXIP through targeting HBXIP mRNA.[6] To demonstrate the effect of Lin28B on the expression of miR-520b, we performed the transfection using Lin28B siRNA-1 or siRNA-2 in MCF-7-HBXIP or MDA-MB-231 cells, respectively. Interestingly, we found that the knockdown of Lin28B by Lin28B siRNA-1 or siRNA-2 resulted in the upregulation of miR-520b by qRT-PCR assay in the cells (Figs. 5c and 5d). Thus, we conclude that Lin28B maintains high level of HBXIP through suppressing miR-520b targeting HBXIP mRNA in breast cancer cells.

image

Figure 5. Lin28B maintains high levels of HBXIP via suppressing miR-520b targeting HBXIP mRNA. (a) The relative mRNA levels of Lin28B and HBXIP were examined by qRT-PCR assay in MCF-7-HBXIP cells transiently transfected with NC siRNA or Lin28B siRNA-2 (80 nM). (b) The protein levels of Lin28B and HBXIP were detected by western blot in MCF-7-HBXIP cells transiently transfected with NC siRNA, Lin28B siRNA-1 and Lin28B siRNA-2 (80 nM), respectively. (c and d) The relative expression levels of miR-520b were detected in MCF-7-HBXIP or MDA-MB-231 cells transiently transfected with NC siRNA, Lin28B siRNA-1 (40, 80 nM) or Lin28B siRNA-2 (40, 80 nM). All experiments were performed at least three times.

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HBXIP promotes the proliferation of breast cancer cells through Lin28B

Next, we evaluated whether Lin28B was involved in the HBXIP-enhanced proliferation of breast cancer cells. EdU incorporation assay and MTT assay indicated that the knockdown of HBXIP or Lin28B by siRNAs resulted in approximately five-fold reduction of the proliferation of MCF-7-HBXIP cells relative to the control group, respectively (Fig. 6a and Supporting Information Figs. S3a and S6b). Moreover, colony formation assays revealed that the overexpression of HBXIP by transient transfection led to a significant increase of the colony numbers in MCF-7 cells, but the increase was abolished by the addition of Lin28B siRNA-2 (Fig. 6c and Supporting Information Fig. S3b), while the knockdown of Lin28B resulted in nearly five-fold reduction in colony numbers in MCF-7-HBXIP cells, compared to control (Fig. 6d and Supporting Information Fig. S3c). The data strongly suggest that HBXIP promotes the proliferation of breast cancer cells via Lin28B in vitro.

image

Figure 6. HBXIP promotes the proliferation of breast cancer cells through Lin28B in vitro and in vivo. (a) The effect of HBXIP siRNA-2 (100 nM), Lin28B siRNA-1 (100 nM) or Lin28B siRNA-2 (100 nM) on the proliferation of MCF-7-HBXIP cells was detected by EdU incorporation assays. (b) The effect of Lin28B knockdown on the cell proliferation of MCF-7-HBXIP cells transiently transfected with NC siRNA (100 nM) or Lin28B siRNA-2 (100 nM) compared to the MCF-7-pCMV cells transfected with NC siRNA (100 nM) was measured by MTT assays. (c) The effect of Lin28B knockdown by Lin28B siRNA-2 (100 nM) on the growth of MCF-7 cells transfected transiently with pCMV-HBXIP (0.5 μg)/NC siRNA (100 nM) or pCMV-HBXIP (0.5 μg)/Lin28B siRNA-2 (100 nM) was tested respectively by colony formation assays. (d) The effect of Lin28B knockdown by Lin28B siRNA-2 (100 nM) on the growth of MCF-7-HBXIP cells was detected by colony formation assays. (e) The curves of tumor growth. (f) The average tumor weight. Values are means of tumor weight ± SD (*p < 0.05, **p < 0.01, Student's t-test).

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To further determine the effect of Lin28B on the proliferation of MCF-7-HBXIP cells in vivo, we performed the tumor formation assay in mice. The data showed that MCF-7-HBXIP cells displayed a significant increase in both volumes and the average weights of the tumors relative to MCF-7-pCMV cells (Figs. 6e and 6f). While, the MCF-7-HBXIP cells transfected with Lin28B siRNA-2 exhibited significant depressed tumor growth (Supporting Information Fig. S3d). Meanwhile, the expression levels of HBXIP and Lin28B in the tumor tissues from mice were tested by western blotting analysis (Supporting Information Fig. S3e). The relative gray values were calculated by Quantity One software (Bio-Rad) (Supporting Information Fig. S3f). The data showed that the expression levels of HBXIP were correspondence to the tumor size in mice. Interestingly, the silence of Lin28B resulted in the downregulation of HBXIP in the tissues, suggesting that Lin28B is responsible for maintaining the high level of HBXIP. Thus, we conclude that HBXIP significantly promotes the proliferation of breast cancer cells via Lin28B both in vitro and in vivo.

Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

Growing evidence shows that HBXIP is an important oncoprotein. Our data strongly support that HBXIP functions in the development of breast cancer.[4, 5, 7] Previous report unraveled that HBXIP functioned in the cytoplasm.[2] Intriguingly, some studies showed that HBXIP functioned in the nucleus as well.[3, 5, 7] However, the mechanism by which HBXIP promotes the proliferation of breast cancer cells remains unclear. Many studies indicated that Lin28B, acting as a repressor of let-7 miRNAs maturation,[9, 10, 12, 20] was able to promote cancer cell progression.[17, 22] Thus, we are interested in whether Lin28B is involved in the HBXIP-enhanced proliferation of breast cancer cells. In our study, we investigate whether HBXIP functions with another oncoprotein Lin28B in carcinogenesis.

We initially found that the expression levels of Lin28B were positively correlated with those of HBXIP in breast cancer tissues. Next, HBXIP was able to upregulate Lin28B in breast cancer MCF-7 and MDA-MB-231 cell lines. Importantly, we discovered that HBXIP promoted the nuclear entry of Lin28B in the cells. It has been reported that Lin28B mainly functions in the nucleus to block the maturation of let-7 miRNAs,[9, 12] and let-7 miRNAs suppresses the tumorigenicity in breast cancer cells.[46] Accordingly, we demonstrated that HBXIP downregulated the expression of let-7 miRNAs via Lin28B, which further validated that HBXIP upregulates Lin28B in the cells. We further investigated the mechanism by which HBXIP upregulated Lin28B. Our data revealed that HBXIP was able to occupy the Lin28B promoter and stimulate its promoter activation. Next, we identified that the promoter core region of Lin28B located at −1199/-1073 nt. Intriguingly, we found that the HBXIP-enhanced activities of Lin28B promoter were abolished when the binding site of TF II D was mutated. Due to the purified HBXIP protein could not bind to the DNA sequence of Lin28B promoter, we supposed that HBXIP might activate the promoter of Lin28B through binding to TF II D. It has been reported that the transcription factor TF II D is composed of the TBP and at least 13 evolutionarily conserved TAFs.[23, 34] and the TBP component initiates the assembly of the transcription complex.[25, 26] Besides, TF II D can serve as a bridge or receptor of signals from enhancer-bound transcriptional activator proteins.[29] In our study, we demonstrated that HBXIP activated the promoter of Lin28B through directly binding to TBP, a basal subunit of TF II D complex. Thus, HBXIP as a co-activator of TF II D activates Lin28B transcription. This finding is consistent with our recent report that HBXIP may function as a co-activator.[7]

The feedback regulation is an important regulation manner in cancer.[43, 44] Intriguingly, we observed that the expression levels of HBXIP were significantly reduced by the knockdown of Lin28B. Thus, we supposed that Lin28B was capable of regulating HBXIP in a feedback manner. Due to Lin28B regulates let-7 miRNAs, thus Lin28B may regulate HBXIP through microRNAs. Our study showed that HBXIP could be downregulated by miR-520b through targeting HBXIP mRNA.[6] Importantly, our data indicated that the knockdown of Lin28B resulted in the elevated expression of miR-520b in MCF-7-HBXIP and MDA-MB-231 cells, suggesting that Lin28B maintains the high level of HBXIP through suppressing miR-520b targeting HBXIP mRNA. This finding is consistent with the reports that Lin28B functions as a miRNA suppressor.[11, 47, 48] It has been reported that Lin28B promotes the proliferation of cancer cells.[9-12] Our results showed that the downregulation of Lin28B was able to significantly block the HBXIP-enhanced proliferation of breast cancer cells in vitro and in vivo. Thus, we conclude that the two oncoproteins, such as HBXIP and Lin28B, display the coaction to promote growth of cancer cells.

In summary, we show a model that the oncoprotein HBXIP promotes the proliferation of breast cancer cells through upregulating Lin28B (Supporting Information Fig. S4), in which HBXIP, as a coactivator of TF II D, upregulates Lin28B expression through directly binding to TBP, a basal subunit of TF II D. In turn, Lin28B maintains the high levels of HBXIP via suppressing the miR-520b targeting HBXIP mRNA in a feedback manner. Therefore, our findings provide new insights into the mechanism of HBXIP in the development of cancer. Therapeutically, HBXIP may serve as a target in breast cancer.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

The authors thank Tianjin Tumor Hospital to provide the breast cancer specimens.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
ijc28154-sup-0001-suppfig1.tif427KFigure S1 The efficiency of HBXIP over-expression/knockdown and Lin28B knockdown assay. (a) The mRNA levels of HBXIP were examined by RT-PCR in MCF-7 cells co-transfected transiently with pCMV (0.5 μg)/NC siRNA (70 nM), pCMV-HBXIP (0.5 μg)/NC siRNA (70 nM) and pCMV (0.5 μg)/HBXIP siRNA-2 (70 nM), respectively. (b) The mRNA levels of Lin28B were detected by qRT-PCR in MCF-7 cells co-transfected transiently with pCMV-HBXIP (0.5 μg)/NC siRNA (80 nM) and pCMV-HBXIP (0.5 μg)/Lin28B siRNA-2 (80 nM), respectively.
ijc28154-sup-0002-suppfig2.tif2921KFigure S2 Identification of interaction between HBXIP and core region of Lin28B promoter. (a) The interaction between the purified HBXIP proteins and Lin28B promoter (−1199/-1073 nt region) was examined by EMSA assays. (b) The fragment of −1199/-1073 nt was divided into 5 segments, each contains 30 nucleotides and the adjacent two segments have 8 nucleotides overlapping. Then, EMSA was performed with HBXIP antibody. (c) EMSA assays were used to test the interaction between HBXIP (or TBP) and segment 2.
ijc28154-sup-0003-suppfig3.tif4356KFigure S3 HBXIP promotes the proliferation of breast cancer cells through Lin28B in vitro and in vivo. (a) The image of effect of HBXIP or Lin28B on the proliferation of breast cancer cells. The proliferation of MCF-7-HBXIP cells was examined by EdU incorporation assay after treatment with NC siRNA (100 nM), HBXIP siRNA-2 (100 nM), Lin28B siRNA-1 (100 nM) and Lin28B siRNA-2 (100 nM), respectively. (b) The effect of Lin28B knockdown by Lin28B siRNA-2 (100 nM) on the growth of MCF-7 cells transfected transiently with pCMV-HBXIP (0.5 μg)/NC siRNA (100 nM) or pCMV-HBXIP (0.5 μg)/Lin28B siRNA-2 (100 nM) was tested respectively by Colony Formation Assays. (c) The effect of Lin28B knockdown by Lin28B siRNA-2 (100 nM) on the growth of MCF-7-HBXIP cells was detected by Colony Formation Assays. (d) The tumor images from the mice injected MCF-7-pCMV cells transfected with NC siRNA, or MCF-7-HBXIP cells transfected with NC siRNA (100 nM) or with Lin28B siRNA-2 (100 nM), respectively. (e) The expression levels of HBXIP and Lin28B in the tumor tissues from nude mice were tested by western blot analysis. (f) The gray value of western blot of the expression levels of HBXIP and Lin28B in the tumor tissues from nude mice, the columns were calculated according to results by Quantity One software (Bio-Rad, USA).
ijc28154-sup-0004-suppfig4.tif181KFigure S4. A model displays the oncoprotein HBXIP up-regulates Lin28B via activating TFⅡD in promoting the proliferation of cancer cell. The oncoprotein HBXIP directly binds to the TBP, a basal subunit of TFⅡD complex, to activate the Lin28B promoter. Then, the enforced expression of Lin28B in turn maintains the high levels of HBXIP via suppressing miR-520b, which directly targets HBXIP mRNA.
ijc28154-sup-0005-supptbl1.doc89KSupplementary Table S1. Clinical characteristics of breast cancer from microarray samples.
ijc28154-sup-0006-supptbl2.doc33KSupplementary Table S2. Primer sequences for cloning Primers.
ijc28154-sup-0007-supptbl3.doc36KSupplementary Table S3. Primer sequence for ChIP and quantitative real-time PCR.
ijc28154-sup-0008-suppinfo.doc30KSupporting Information

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