Cholesterol-conjugated let-7amimics: antitumor efficacy on hepatocellular carcinoma in vitro and in a preclinical orthotopic xenograft model of systemic therapy
© Liu et al.; licensee BioMed Central Ltd. 2014
Received: 20 May 2014
Accepted: 23 October 2014
Published: 28 November 2014
A major challenge to the clinical utility of let-7 for hepatocellular carcinoma (HCC) therapy is the lack of an effective carrier to target tumours. We confirmed the high transfection efficiency of cholesterol-conjugated let-7a miRNA mimics (Chol-let-7a) in human HCC cells, as well as their high affinity for liver tissue in nude mice. However, their antitumor efficacy via systemic delivery remains unknown.
We explored the effects of Chol-let-7a on HCC in vitro and in vivo. Cell viability and mobility, let-7a abundance and the target ras genes was measured. Live-cell image and cell ultrastructure was observed. Antitumor efficacy in vivo was analyzed by ultrasonography, hispatholgogy and transmission electronic microscopy in a preclinical model of HCC orthotopic xenografts with systemic therapy.
Chol-let-7a inhibited the viability and mobility of HCC cells. Chol-let-7a was primarily observed in the cytoplasm and induced organelle changes, including autophagy. Mild changes were observed in the cells treated with negative control miRNA. Chol-let-7a reached HCC orthotopic tumours, significantly inhibited tumour growth, and prevented local invasion and metastasis. Compared to control tumours, Chol-let-7a-treated tumours showed more necrosis. Tumour cells showed no significant atypia, and mitoses were very rare after systemic Chol-let-7a therapy. Furthermore, let-7a abundance in orthotopic xenografts was coincident with a reduction in the expression of 3 human ras mRNAs and RAS proteins.
Chol-let-7a exerted significant antitumor effects by down-regulating all human ras genes at the transcriptional and translational levels. Chol-let-7a inhibited cell proliferation, growth, and metastasis, and mainly functioned in the cytoplasm. Chol-let-7a represents a potential useful modified molecule for systemic HCC therapy.
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third most common cause of cancer mortality and has high recurrence rates after surgery. Chemotherapy and radiotherapy for HCC show limited efficacy and serious toxicity [1, 2]. New therapeutic strategies are urgently needed, particularly for the treatment of advanced tumours.
MicroRNAs (miRNAs) are endogenous non-coding small RNAs that repress gene expression at the post-transcriptional level by base pairing to the 3′-untranslated region of target messenger RNAs, and they have been identified as important mediators of carcinogenesis and clinical prognosis [3–6]. The most recent findings regarding the role of miRNAs in HCC confirmed that they hold promise as new tools for diagnosis and therapy [7–11]. A recent study in C. elegans reports that the let-7 family negatively regulates let-60/RAS, and also that the let-60/RAS 3′-UTRs, including the 3′-UTRs of the human ras genes, contain multiple let-7 complementary sites (LCSs), which allow let-7 to regulate RAS protein expression . Furthermore, let-7 has been reported to inhibit tumour growth by down-regulating KRAS in some cancers, such as pancreatic carcinoma and lung cancer [13, 14]. Analysis with a computational screen showed that the human n-ras, k-ras, and h-ras mRNA 3′-UTRs have 9, 8, and 3 potential LCSs, respectively . Although ras proto-oncogenes produced by mutations in codons 12, 13, and 61 do not play major roles in hepatocellular carcinogenesis , abnormal activation of the RAS pathway occurs in human HCC, and activated (GTP-bound) Pan-RAS, HRAS, KRAS, and NRAS are significantly up-regulated in human hepatocarcinogenesis [16, 17]. Thus, we hypothesize that modulation of let-7 expression and its target RAS is a promising strategy for HCC treatment, because let-7 might suppress HCC tumour growth by down-regulating all human ras genes.
Recently, antitumor effects of synthetic miRNA mimics were confirmed in vitro and in vivo [18–20]. Hou et al. showed that intratumoural administration of cholesterol-conjugated PAK4 siRNA suppressed subcutaneous tumour growth in the SMMC-LTNM model . Trang and colleagues  found that synthetic miR-34a and let-7 mimics caused lung tumour reduction in mice. However, these mimics did not produced high miRNA levels in the liver tissues. We confirmed the significantly higher transfection efficiency of cholesterol-conjugated let-7a miRNA mimics (Chol-let-7a) in human HCC cells in vitro. Given the observed high affinity of Chol-let-7a for liver tissue in nude mice, we hypothesize that Chol-let-7a may be an ideal modified molecule for systemic HCC therapy.
In this study, we explored the effects of Chol-let-7a on HCC tumour cells in vitro, as well as its antitumor efficacy in an in vivo preclinical model of HCC orthotopic xenografts, to evaluate its potential as a systemically administered drug in the treatment of HCC. In addition, we explored the effects of Chol-let-7a on ras gene expression at the transcriptional and translational levels.
Materials and methods
Cell culture and mice
HepG2 and SMMC7721 cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum (Invitrogen) and pen/strep (100 μg/mL). BALB/c nude (nu/nu) mice (6–7 weeks old, 20 ± 3 g) were purchased from the National Institutes for Food and Drug Control (lot number: 11400500001092; Beijing, China).
MTT cell proliferation assays
Cholesterol-conjugated let-7a mimics (Chol-let-7a) and the negative control miRNA (Chol-miRCtrl) were purchased from Ribobio (Guangzhou, China). Cells (5 × 103) were cultured in 96-well flat-bottomed plates. After 24 h of cell culture, cells were transfected with 50 nM Chol-let-7a or Chol-miRCtrl according to manufacturer instructions. Cells were cultured in 100 μL DMEM containing 10% FBS and 20 μL MTS reagent powder (Promega, Madison, WI, USA). Cells were harvested and seeded on 96-well flat-bottomed plates, which were incubated at 37°C for 4 h. After incubation for 1, 2, 3, 4, or 5 days, the absorbance at 550 nM was determined for each well.
Invasion and migration assay
Assays of invasion and migration were performed as described in previous report . For invasion assays, 5 × 104 cells in serum-free media were seeded into the upper chambers of a 24-well BioCoat Matrigel invasion chamber (Becton Dickinson Labware, Franklin Lakes, NJ, USA) with an 8-μm pore polycarbonate membrane coated with Matrigel. For migration assays, 5 × 104 cells were seeded into the upper chambers of a 24-well BioCoat control insert (Becton Dickinson Labware, Franklin Lakes, NJ, USA) with uncoated 8-μm pores in serum-free media. Medium with 10% FBS was added to the lower chambers as a chemoattractant. After 24 h of incubation, cells remaining on the upper surface of the membrane were removed with a cotton swab and cells that invaded through the membrane filter were fixed with 100% methanol, stained by hematoxylin and eosin, and photographed by soft BioLife DP under a microscope (Olympus BX40 with a DP70 digital camera, Tokyo, Japan). The number of invading or migrating cells was manually counted per high-power field for each condition (eight fields on each membrane were randomly selected).
Cells were grown to confluence in 25 cm2 cell culture flasks. Artificial wound tracks were created by scraping confluent cell monolayers with a pipette tip. After removal of the detached cells by gentle washing with PBS, the cells were fed with fresh complete medium and incubated to allow cells to migrate into the open area. The ability of the cells to migrate into the wound area was assessed at 24, 48, and 72 h after scratching by comparing the wound tracks in micrographs of 3 randomly selected wound areas.
Quantitative real-time PCR and reverse transcription PCR
Total miRNA from HCC cells or snap-frozen HepG2 xenografts was isolated using the mirVANA™ PARIS™ RNA isolation kit (Applied Biosystems, Carlsbad, CA, USA). RNA (10 ng) was reverse-transcribed with the miRNA Reverse Transcription Kit (Applied Biosystems) and let-7a specific primers (TaqMan miRNA assay, Applied Biosystems).
Total RNA was extracted from HCC cells or snap-frozen HepG2 xenografts using the IllustraRNA spin Mini RNA Isolation Kit (GE Healthcare UK Limited, Amersham Place, Little Chalfont, UK). cDNA was synthesized using SuperScript TM III First-Strand Synthesis SuperMix for quantitative real-time reverse transcription PCR (qRT-PCR; Invitrogen Corporation, Carlsbad, CA, USA) and primers specific for the 3 human ras genes (TaqMan miRNA assay, Applied Biosystems).
Quantitative PCR was performed using RNU6 or GAPDH as a housekeeping control with an ABI Prism 7500 Sequence Detection System (Perkin-Elmer Applied Biosystems, Foster City, USA) and the Perkin-Elmer Biosystems analysis software in a manner consistent with the manufacturer’s instructions. Relative expression was calculated using the 2-ΔΔCTmethod .
HCC cells and tissues from snap-frozen HepG2 xenografts were lysed using RIPA lysis buffer (Applygen Technologies, Beijing, China). Proteins were quantified using a BCA protein kit (Applygen). Proteins (50 μg) were separated by SDS-PAGE and transferred to an Immobilon-P membrane (Millipore, Billerica, MA, USA). The membranes were blocked in 5% non-fat milk and incubated with primary antibodies. The membranes were washed in PBS-T (PBS and 0.1% Tween-20) and incubated with a peroxidase-conjugated secondary antibody (KPL, Gaithersburg, MD, USA), followed by development with a chemiluminescent substrate (Applygen). The Gel-Doc imaging system was used to scan images on Kodak film. Antibodies for KRAS, HRAS, and NRAS were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). GAPDH and beta-actin (β-action) antibodies were obtained from Proteintech (Chicago, IL, USA).
Transfection, live-cell imaging, and transmission electron microscopy
HepG2 and SMMC771 cells were labelled with GFP. Chol-let-7a and negative control mimics labelled with Cy5 were purchased from Ribobio (Guangzhou, China).
The GFP-labelled cells (2–3 × 104) were seeded in 8-well BD Falcon™ and BD BioCoat™ Culture Slides (Becton Dickinson Labware, Franklin Lakes, NJ, USA). After 48 h, cells were transfected with Cy5-labelled Chol-let-7a or the negative control mimics (Chol-miRCtrl).
For live-cell imaging, cells were continuously observed using a PerkinElmer UltraVIEW VoX-3D Live Cell Imaging System (Shanghai, China) from 24 to 72 h post-transfection. Digital images were produced using Volocity Demo software (version 5.4, 32-bit). Co-localization events were calculated using the Volocity Demo software as described in the manufacturer’s recommendations. The experiment was repeated 3 times and all samples for each individual experiment were scanned at 5 different locations.
For electron microscopy, cells were collected at 48 h and 60 h after transfection and were fixed with 2.5% glutaraldehyde for 30 min at room temperature, followed by 1.5 h in 2% OsO4. Samples were stained and examined with a transmission electron microscope (JEOL JEM 1010, Tokyo, Japan), and digital images were obtained with an Erlangshen ES1000W camera (Model 785, Gatan, Warrendale, PA, USA).
In vivo experiments
All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication nos. 80–23, revised 1996) and with the experimental animal welfare ethics regulations of China, with the approval of the Institution Animal Care and Use Committee of Peking Union Medical College Hospital. All animal experiments were performed at the Centre for Experimental Animal Research (CEAR), Institute of Basic Medical Sciences (IBMS), CAMS & PUMC.
Orthotopic xenograft model with nude mice and systemic therapy with Chol-let-7a
HepG2 cells (2 × 106) were injected directly into the livers of 20 nude mice. One week later, 18 mice with successfully engrafted HepG2 orthotopic xenografts were randomized into 3 groups of 6 animals each and examined by ultrasonography in a double-blinded manner (VisualSonics, Inc., Toronto, Ontario, Canada).
At the culmination of therapy, tumour tissues were harvested and preserved in 10% neutral buffered formalin for pathology observation. Fresh tumour tissues were snap-frozen for qRT-PCR, western blotting, or transmission electron microscopy.
Data are expressed as the mean ± SEM. All data analyses were performed with SPSS 16.0 software (IBM, Inc., Armonk, NY, USA). Analysis of variance (ANOVA) and Student’s t-test were used for statistical comparisons between groups. p <0.05 was considered to be statistically significant.
Chol-let-7areduced HCC cell growth and viability in vitro
Chol-let-7ainhibited the migration and invasion of HCC cells in vitro
We determined the effects of Chol-let-7a on HCC cell migration and invasion, which are 2 key steps in tumour metastasis. HepG2 and SMMC7721 cells were transfected with Chol-let-7a or with Chol-miRCtrl as a negative miRNA control. The transfected Chol-let-7a and Chol-miRCtrl cells and the parental cells were used in the migration and invasion assay 48 h post-transfection.
A chamber-based cell migration assay revealed that the number of Chol-let-7a-treated HCC cells that migrated through the membrane was significantly lower than the number of Chol-miRCtrl-treated cells (p <0.05) or parental cells (blank) (p <0.05) that migrated through the membrane (HepG2, 148.3 ± 7.02 (Chol-let-7a) vs. 203.0 ± 5.29 (Chol-miRCtrl) vs. 214.67 ± 11.67 (blank); SMMC7721, 155.67 ± 6.66 (Chol-let-7a) vs. 218.33 ± 9.45 (Chol-miRCtrl) vs. 230.67 ± 7.02 (blank)) (Figure 1C). There were no significant differences between the 2 control groups (p >0.05), indicating that Chol-let-7a suppressed HCC cell migration.
We also used an in vitro wound healing assay to measure cell migration (data not shown). Healing speed was slower and gaps were wider in the Chol-let-7a-treated HepG2 and SMMC7721 cells at each time point (24, 48 h, and 72 h) in comparison with their respective control groups. At 48 h, most gaps in the 2 control cell groups were completely closed; whereas the gaps in the Chol-let-7a-treated cells remained open. Consistent with the results from the chamber-based cell migration assay, these data indicated that Chol-let-7a inhibited HCC cell migration.
Next, we evaluated the ability of HCC cells to pass through the extracellular matrix (ECM) in a Boyden chamber invasion assay. We found significantly fewer invading cells in the Chol-let-7a-treated group in comparison with the 2 control groups (Figure 1D) (66.33 ± 4.73 (Chol-let-7a) vs. 91.33 ± 3.21 (Chol-miRCtrl) vs. 97.00 ± 5.29 (blank); Chol-let-7a vs. the 2 control groups, both p <0.05), and similar results were observed with SMMC7721 cells (73.00 ± 5.29 (Chol-let-7a) vs. 91.33 ± 3.21 (Chol-miRCtrl) vs. 103.33 ± 4.73 (blank); Chol-let-7a vs. the 2 control groups, both p <0.05). Thus, it appears that Chol-let-7a affects cell migration and invasion.
Up-regulated let-7a down-regulated ras/RAS expression in HCC cells
We measured let-7a levels by quantitative real-time PCR 48 h after transfection with Chol-let-7a and Chol-miRCtrl. Using miRNA-specific primers, let-7a up-regulation in comparison with parental HCC cells and Chol-miRCtrl-treated control cells was confirmed in Chol-let-7a-treated cells (see Additional file 1A). Next, we analysed the expression of let-7 target ras genes at the transcriptional and translational levels 48 h after transfection. Western blotting revealed a marked decrease in KRAS, HRAS, and NRAS protein abundance in the Chol-let-7a-treated HepG2 and SMMC7721 cells (see Additional file 1B). Quantitative real-time PCR (qRT-PCR) was used to measure k-ras, h-ras, and n-ras transcript abundance in the Chol-let-7a-treated HCC cells, and these 3 ras genes were found to be reduced by Chol-let-7a treatment (see Additional file 1C). These results verified our hypothesis that Chol-let-7a would inhibit the transcription and translation of all 3 human ras genes in vitro.
Continuous observation of the images showed that cell proliferation and mobility decreased in the Chol-let-7a- and Chol-miRCtrl-treated cells. In addition, images revealed that cell viability differed between the 2 control groups (Figure 2). There were no significant differences in proliferation and cell mobility between the 2 control groups immediately after Chol-miRCtrl transfection. However, the Chol-miRCtrl-treated cells exhibited poorer survival than the parental tumour cells (blank) from 39 h after transfection, and few living cells with GFP fluorescence were observed 72 h after treatment, whereas the parental cells were still active with respect to proliferation, growth, and mobility at this time point.
Long-term treatment produced significant ultrastructure modifications. In the cytoplasm of Chol-let-7a-treated cells, mitochondria, heterolysosomes, and RER were vacuolated and showed irregular and unclear contours and structures (see Additional file 3B), and apoptotic and necrotic cells were clearly observed 60 h after treatment. In the Chol-miRCtrl group, a few cells underwent death. However, interestingly, cellular morphology did not show characteristics associated with apoptotic cells. Apoptotic nuclear changes, such as nuclear shrinkage and nuclear fragmentation, were barely observed in Chol-let-7a-treated cells. In comparison with the rapid changes in cytoplasmic organelles, nuclear damage was strikingly delayed after Chol-let-7a-treatment (see Additional file 3C).
Up-regulated let-7a down-regulated ras/RAS expression after systemic delivery
We analysed the expression of RAS proteins by western blotting and observed marked decreases in KRAS, HRAS, and NRAS abundance in Chol-let-7a-treated xenografts (Figure 4B). Similarly, deregulated mRNA expression of k-ras, h-ras, and n-ras was also investigated by qRT-PCR. The expression of n-ras was inhibited most significantly by Chol-let-7a (p <0.01), and expression levels of h-ras and k-ras (p <0.05) were also reduced (Figure 4C). These results suggest that Chol-let-7a successfully carried let-7a mimics into target HCC tumour cells and suppressed all 3 human ras genes at the transcriptional and translational levels, which were in accordance with our in vitro results.
Chol-let-7ainhibited growth and metastasis of HCC orthotopic xenografts after systemic delivery
Beginning 2 weeks after Chol-let-7a treatment, inhibition of tumour metastasis was observed by ultrasonography. Metastases within the liver are shown in Figure 5B. Local invasion and metastasis to the spleen were inhibited in the Chol-let-7a-treated group (data not shown).
Under light microscopy, small and large necrosis foci were observed in tumour tissues from all 3 groups, but more necrosis was observed in the Chol-let-7a-treated xenografts, and necrosis was also observed in capillary-rich areas. In contrast, significant necrosis was typically observed in the central tumour area in the control groups. These necrotic features were confirmed under TEM (Figure 5D). In addition, Chol-let-7a-treated tumour cells showed no significant atypia, and mitoses were very rare per unit of measurement in most areas in comparison with the control groups (Figure 5E). Immunohistochemical staining for Ki-67 and ultrastructure changes in HCC cells in the orthotopic xenografts showed similar features (data not shown). These results suggest that Chol-let-7a inhibits tumour growth by promoting cell death and inhibiting cell proliferation.
We confirmed the significant antitumor efficacy of Chol-let-7a on HCC, and in particular its significant effect on HepG2 orthotopic xenografts after systemic delivery in a preclinical animal model. Chol-let-7a effectively carried let-7a mimics to target tumours in vivo and inhibited tumour growth, metastasis within the liver, and local invasion and metastasis to the spleen. Significant increases in let-7a miRNA abundance were observed in Chol-let-7a-treated xenografts. Moreover, let-7a abundance in HepG2 orthotopic xenografts was coincident with a reduction in the expression of 3 human ras mRNAs and RAS proteins. These results suggest that Chol-let-7a inhibits HCC cell growth by regulating all 3 human ras genes at the transcriptional and translational levels.
We found some different features in the orthotopic xenograft tissues after Chol-let-7a systemic therapy in comparison with the 2 control groups. All tumour tissues contained small and large necrosis foci, but more necrosis was observed in the Chol-let-7a-treated xenografts, and necrosis was also observed in capillary-rich areas. In contrast, significant necrosis was typically observed in the central areas of tumours in the control groups. The histopathological features of Chol-let-7a-treated xenografts may have been induced by the type of Chol-let-7a transportation used in this study. Tumour cells in capillary-rich areas could be more susceptible than other cells to systemically administered Chol-let-7a molecules. In addition, well-differentiated tumour cells with no significant atypia and only very rare mitoses were observed after Chol-let-7a therapy. These results suggest that Chol-let-7a inhibited tumour growth by inhibiting cell proliferation and promoting cell death.
We confirmed that Chol-let-7a entered cells and functioned primarily in the cytoplasm based on morphology and ultrastructure analysis. This result was consistent with the potential functional basis of let-7a, which involves the inhibition of target ras genes at the transcriptional and translational levels. In vitro, we observed the effects of Chol-let-7a on HCC cells by living cell image analysis and transmission electron microscopy. Both results suggested that Chol-let-7a entered cells and functioned primarily in the cytoplasm. The red fluorescence that indicated Chol-let-7a and Chol-miRCtrl was primarily focused in the cytoplasm. TEM revealed that Chol-let-7a damaged some cytoplasmic organelles, but only slight changes in nuclear morphology were observed. Cellular nuclear morphology did not show characteristics associated with apoptotic cells even at 60 h after Chol-let-7a therapy, when long-term treatment had produced significant ultrastructure modifications in the cytoplasm. Apoptotic nuclear changes such as shrinkage and fragmentation were barely observed in Chol-let-7a-treated cells, including those in which mitochondria, heterolysosomes, and RER were vacuolated and showed irregular and unclear contours and structures. In comparison with the rapid changes in cytoplasmic organelles, nuclear damage was strikingly delayed after Chol-let-7a-treatment. Autophagocytic activity was observed in Chol-let-7a- treated cells. Therefore, we suggest that autophagy may be an important mechanism through which Chol-let-7a produces antitumor effects .
We previously examined the antitumor effect of Chol-let-7a on HCC by using intratumoural administration in a subcutaneous xenograft model. Results showed that intratumoural administration of Chol-let-7a reduced tumour growth; however, cell phenotype and morphology in most areas of the subcutaneous xenografts showed no such changes, and these areas showed actively growing cells with high rates of mitosis (data not shown). Therefore, Chol-let-7a produces better inhibitory effects when it is systemically administered. Because of the high affinity of Chol-let-7a for liver tissue in nude mice (data not shown) and the convenience of systemic administration, Chol-let-7a represents a potential useful modified molecule for systemic HCC therapy.
However, the delivery system may have off-target effects, as indicated by the differences observed between the Chol-miRCtrl cells and the parental cells. Cell viability differed between the 2 control groups. The Chol-miRCtrl-treated cells exhibited poorer survival than the parental tumour cells from 39 h after transfection, and few living cells with GFP fluorescence were observed 72 h after treatment, whereas the parental cells were still active with respect to proliferation, growth, and mobility at this time point. This result suggests that Chol-miRCtrl can reduce the viability of some cells. We compared the effects of Chol-let-7a and Chol-miRCtrl on HepG2 cells at 3 different doses. In comparison with the 25 nM and 50 nM doses, 100 nM Chol-miRCtrl slightly slowed cell growth 72 h after transfection (see Additional file 4A), but there were no differences in cell growth between the treatment groups at 48 h (see Additional file 4B). These results suggest that dosage and prolonged action time contribute to the off-target effects of Chol-miRCtrl.
Ultrastructure features also differed between the 2 control groups. Some organelle changes observed in the Chol-let-7a-treated cells were also found in the Chol-miRCtrl-treated HCC cells under TEM. In addition, autophagy was observed in some Chol-miRCtrl treated tumour cells that under death, indicating that autophagocytic activity could also be a potential factor induces off-target effects. Given the observed high affinity of Chol-let-7a for liver tissue in nude mice, we hypothesize that Chol-let-7a may has potential off-target effects primary in liver tissue when it is administered systemically as a therapeutic molecule. In future studies, we will investigate off-target effects of Chol-let-7a in preclinical animal models.
We confirmed the significant antitumor efficacy of Chol-let-7a on HCC, and in particular its significant effect on HepG2 orthotopic xenografts after systemic delivery in a preclinical animal model. Chol-let-7a effectively carried let-7a to target tumours in vivo and inhibit tumour growth by inhibiting cell proliferation and promoting cell death. In addition, Chol-let-7a can inhibit HCC cell growth by regulating all 3 human ras genes at the transcriptional and translational levels. Moreover, we confirmed Chol-let-7a entered cells and functioned primarily in the cytoplasm, and autophagy may be an important mechanism through which Chol-let-7a produces antitumor effects. Taken together, Chol-let-7a represents a potential useful modified molecule for systemic HCC therapy. However, further studies of Chol-let-7a-produced off-target effects when it is systemically administered are required.
We thank Mrs. Huimin Zhao, Wenyu Hao, and Huanxian Cui from the Centre for Experimental Animal Research (CEAR), Institute of Basic Medical Sciences, CAMS/PUMC, as well as Xiao Yang (VisualSonics, Inc., Beijing, China) and Yi Gao (Berthold, Beijing, China) for the technical support. We also thank Dr. Wei-Min Tong for his constructive suggestions in support of this study. We thank Dr. Xingyi Hang and Ms. Yuxing You for their assistance with the statistical analysis. This work was supported partly by the Scientific Data Sharing Program funded by the Chinese Ministry of Science (2004DKA20240-2013, JG, JC).
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