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/lp/wolters-kluwer-health/antitumor-and-off-target-effects-of-cholesterol-conjugated-let-7a-jK147D8ggp
- Publisher
- Wolters Kluwer Health
- Copyright
- Copyright © 2022 The Chinese Medical Association, Published by Wolters Kluwer Health, Inc.
- ISSN
- 2577-3585
- eISSN
- 2096-5672
- DOI
- 10.1097/jbr.0000000000000103
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- See Article on Publisher Site
Abstract
Objective: To explore the antitumor and potential off-target effects of systemically delivered cholesterol-conjugated let-7a mimics (Chol-let-7a) and control mimics (Chol-miRCtrl) on hepatocellular carcinoma in vivo. Methods: The antitumor effects of two intravenous dosing regimens of Chol-let-7a on heptocellular carcinoma growth were compared using an orthotopic xenograft mouse model. Off-targets were analyzed with histopathological and ultrapathological features of heparenal tissue and cells in the Chol-let-7a-, Chol-miRCtrl-, and saline-treated (blank) xenograft mice and normal control mice. Then, let-7a abundance in orthotopic tumors, corresponding paracancerous hepatic tissue, and normal liver tissue from healthy nude mice was examined by reverse transcription-polymerase chain reaction. The distribution of Chol-let-7a and Chol-miRCtrl in vivo was examined by whole-animal imaging and frozen-sections observation. The experiments were approved by the Institutional Research Board of Peking Union Medical College Hospital. Results: Continuous treatment with Chol-let-7a resulted in tumors that were 35.86% and 40.02% the size of those in the Chol- miRCtrl and blank xenograft group (P < 0.01 and P < 0.01, respectively), while intermittent dosing with Chol-let-7a resulted in tumors that were 65.42% and 56.66% the size of those in the Chol-miRCtrl and the blank control group, respectively (P < 0.05 and P < 0.05). In addition, some histopathological and ultrapathological features were only observed after treatment with the two cholesterol-conjugated molecules, however mild with intermittent dosing Chol-let-7a treatment, such as diffuse sinusoidal dilation and edema, primarily around the centrolobular vein in heptic tissues; mild hypercellularity with dilated capillary lumens in the renal tissue; and some organelle abnormalities found in heptic and renal cells. Furthermore, whole-animal imaging showed that Chol-let-7a and Chol-miRCtrl were predominantly distributed in the liver, kidney, and bladder regions after injection, and that the concentration of Chol-let-7a and Chol-miRCtrl in the kidney and the bladder decreased much slowly in the xenograft animals, especially in the Chol-miRCtrl group. Finally, RT-PCR analysis showed that let-7a levels were significantly increased in Chol-let- 7a-treated xenografts compared with Chol-miRCtrl group (P=0.003) and blank xenograft group (P=0.001); however, the level was only equivalent to 50.6% and 40.7% of that in paracancerous hepatic tissue and hepatic tissue in normal mice, respectively. Conclusions: Chol-let-7a, administered either continuously or intermittently, showed effective antitumor efficacy. Chol-let-7a had some off-target effects, such as mild acute hepatitis-like inflammation and non-specific drug-induced kidney injury. The intermittent dosing regimen resulted in less damage than the continuous regimen, while maintaining relatively satisfactory antitumor efficacy, which could be useful for the investigation and possible clinical use of miRNA treatment regimens in the future. Keywords: drug-induced renal injury, hepatic toxicity, in vivo off target effects, let-7 mimics, nonviral delivery vector Supplemental Digital Content is available for this article. JG and ML contributed equally to the writing of this article. a b c The National Population and Health Scientific Data Centre (Clinical Medicine), Department of Pathology, Department of Nuclear Medicine, Peking Union Medical Department of Nuclear Medicine, The Cancer College Hosptial, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), Beijing, Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital), Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences, e f National Laboratory of Medical Biology, Institute of Basic Medical Sciences, The Electron Microscope Laboratories Center, Institute of Hangzhou, Zhejiang Province, Department of Ultrasound, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical Basic Medical Sciences, College (PUMC), Beijing, China * Corresponding author: Jian Guan, the National Population and Health Scientific Data Centre (Clinical Medicine), Peking Union Medical College Hospital, CAMS/PUMC, No. 1, Shuaifuyuan Street, Dongcheng District, Beijing 100730, China. E-mail: gjpumch@126.com. Copyright © 2022 The Chinese Medical Association, Published by Wolters Kluwer Health, Inc. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Journal of Bio-X Research (2022) 5:181–196 Received: 5 March 2021; Accepted: 12 July 2021 Published online 24 August 2021 http://dx.doi.org/10.1097/JBR.0000000000000103 181 RESEARCH ARTICLE Journal of Bio-X Research Introduction information for the future study and potential clinical use of miRNA mimics. Hepatocellular carcinoma (HCC) is the fifth most common can- cer worldwide and the third most common cause of cancer mor- [1] tality. It is the second leading cause of cancer-related death to Materials and methods [2] lung cancer in China. There is a high recurrence rate of HCC Experimental animals and miRNAs after surgery, and chemotherapy and radiotherapy have limited [1,3] efficacy for HCC and are associated with serious toxicity. All in vivo experiments were performed in accordance with Thus, new therapeutic strategies for HCC are urgently needed, the Guide for the Care and Use of Laboratory Animals (NIH especially for advanced-stage patients. Publication No. 85-23, revised 1996) and the experimental ani- MicroRNAs (miRNAs) are endogenous, non-coding, small mal welfare ethics regulations of China, with the approval of RNAs that repress gene expression at the post-transcriptional the Institutional Research Board, Peking Union Medical College level by base pairing to the 3'-untranslated region of target mes- Hospital. All animal experiments were performed at the Centre senger RNAs, and are important mediators of carcinogenesis for Experimental Animal Research (SPF cleanliness, license [4–6] and predictors of clinical prognosis. Functional studies have No. SYXK (Jing) 2010-0021, Beijing, China), Institute of Basic confirmed that miRNAs act as tumor suppressors and are dys- Medical Sciences (IBMS), CAMS & PUMC. regulated in many types of cancer. Thus, modulating miRNAs BALB/C nude (nu/nu) mice (6-7 weeks old, 20 ± 3g) were [7,8] represents a promising new tool for cancer therapy. The let-7 purchased from the National Institutes for Food and Drug family of miRNAs function as typical miRNA tumor suppres- Control (lot No. 11400500001092; Beijing, China). The cho- sors in their ability to target multiple mRNAs, which makes lesterol-conjugated let-7a (Chol-let-7a) and negative control them interesting candidates for development as miRNA mim- miRNA ( Chol-miRCtrl) mimics were purchased from Ribobio [9] ic-based therapeutics. (Guangzhou, China). In recent years, both viral and nonviral delivery vehicles have been developed for the delivery of RNA, including the miRNA Orthotopic xenograft nude mouse model [10,11] gene or mimics in vivo. However, both types remain chal- HepG2 cells (2 × 10 ) were injected directly into the livers of 20 lenges for potential clinic use in the future. As an example nude mice. One week later, the formation of HepG2 orthotopic of viral delivery, the adeno-associated virus (AAV) vector for xenografts was confirmed by ultrasonography using a Vevo miRNA delivery has low efficiency in terms of miRNA expres- 2100 high-frequency ultrasound system (VisualSonics, Inc. sion, and provokes an immune reaction in response to the viral Beijing, China). Fifteen successfully engrafted mice were ran- proteins, and shows dose-dependent toxicity in patients, all of domized into three cohorts of five animals. which pose significant hurdles to its potential clinical use. In addition, AAV-mediated recombinant miRNA gene expression in target cancer cells decreases rapidly as the non-replicating Assessment of antitumor and off-target effects of [12] episomal viral genome is lost from the cells. Nonviral vehi- cholesterol-conjugated molecules administered cles that have been tested in vivo involve the use of miRNA continuously or intermittently mimic modifications or carriers that facilitate release from endo- somes, thereby improving delivery to target cells and potentially Two dosing regimens were designed to evaluate the antitumor increasing the therapeutic effects. Using nonviral delivery vehi- efficacy and off-target effects of Chol-let-7a and Chol-miRCtrl. cles, miRNA mimics, such as those mimicking the miR-122 and We first evaluated the effects of continuous delivery of Chol- '10' let-7 miRNA families, have shown effective anti-HCC efficacy let-7a on HepG2 orthotopic xenografts in nude mice. The [13,14] in vivo in preclinical studies. mice were injected with Chol-let-7a or Chol-miRCtrl (5nmol in Among the delivery system, some RNA-based drugs are 250 μL saline) via the tail vein every 3 days for 5 weeks, after modified with 3ʹ cholesterol that can aid the association of the which the tumor size (volume, mm) and changes in liver and siRNA to lipoprotein particles, lipoprotein receptors and trans- kidney tissues and cells were observed. Xenograft mice injected membrane proteins, and thus target the siRNA to specific tis- with the same volume of 0.9% physiological saline were used as [10] sues, such as the liver. In addition, as an essential component the negative xenograft control (blank) group. of animal cell membranes, and, cholesterol is an efficient trans- To observe the effects of the cholesterol-conjugated molecules membrane transporter and considered non-toxic. We previously on normal mice in the absence of tumor, 10 nude mice were ran- showed that cholesterol-conjugated let-7a (Chollet-7a) exerted domized to two groups of five animals each and injected with significant antitumor effects by down-regulating all three human Chol-let-7a or Chol-miRCtrl according to the dosing regimen ras genes in vivo, including n-ras, k-ras, and h-ras. However, our described above. in vitro experiments also suggested that cholesterol-conjugated To assess the effects of intermittent dosing, mice with orthot- miRNA mimics have potential off-target effects, as indicated by opic xenografts and normal nude mice were injected with Chol- morphological differences between cells treated with a negative let-7a, Chol-miRCtrl, or saline as above every 3 days for 2 control miRNA mimic (Chol-miRCtrl) and untreated tumor weeks, left uninjected for 1 week, and then injected every 3 days cells. Compared to untreated (blank) tumor cells, the Chol- for another 2 weeks. miRCtrl-treated cells exhibited decreased survival and viabil- At the end of the 5-week experimental period for all groups, [15] ity. The potential off-target effects that may occur in vivo tumor size was measured on three orthogonal axes (a, b, and c), remain unknown; and it is crucial for off-target effects to be and tumor volumes (mm ) were calculated according to the fol- [16] minimized when using this approach. In this study we investi- lowing formula: V = (abc)/2. Samples of tumor, liver, kidney, gated the antitumor efficacy and potential off-target effects of and other tissues from all animals were harvested and snap-fro- cholesterol-conjugated let-7a miRNA delivered by either a con- zen or preserved in 10% neutral buffered formalin rapidly for tinuous or an intermittent dosing regimen, to provide important further examination. 182 Journal of Bio-X Research RESEARCH ARTICLE Histopathological examination under light microscope the HepG2 cells were implanted. Serum samples were obtained from the tail vein at the beginning and end of the 4-week exper- All samples fixed in 10% neutral buffered formalin were rou- imental period, and the levels of four cytokines interleukin-6 tinely processed and embedded in paraffin; and were submitted (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF- for standard processing with hematoxylin and eosin staining α), and vascular endothelial growth factor (VEGF) were quan- (HE), Histopathological features was evaluated with HE sec- tified by multiplex assays. Cytokine levels (IL-6, IL-8, TNF-α, tions observed under histopathological examination under the and VEGF) in blood serum samples (200 μL) were quantified light microscope (LM) (OLYMPUS Corporation, Japan). with ELISA using a commercial kit (96-Well Plate MILLIPLEX MAP, Merck Millipore, Darmstadt, Germany) and a Lunimex Transmission electron microscopy ® 200 instrument (MILLIPLEX , Germany). The data were ana- lyzed using Milliplex Analyst software. Fresh and snap-frozen tissues were fixed with 2.5% glutaral- Serum samples were obtained prior to and after Chol-let-7a dehyde for 30 minutes at room temperature, followed by 1.5 or Chol-miRCtrl administration, and the levels of four cytokines hours in 2% OsO . Samples were stained and examined with were examined. The cytokine levels are shown in Additional a transmission electron microscope (JEOL JEM 1010, Tokyo, Table 1 (http://links.lww.com/JR9/A31). Japan), and digital images were obtained with an Erlangshen ES1000W camera (Model 785, Gatan, Warrendale, PA, USA). Statistical analysis In vivo whole-animal imaging and distribution analysis Data are expressed as the mean ± SEM. All data analyses were Fluorophore-labeled synthetic Chol-let-7a or Chol-miRCtrl performed using SPSS 16.0 software (SPSS, Chicago, IL, USA). (RiboBio, Guangzhou, China) was administered via the tail Student's t-test was used for statistical comparisons between vein. Two mice with HepG2 orthotopic xenografts were injected groups. P < 0.05 was considered to be statistically significant. with Cy5-labeled Chol-let-7a, two mice with HepG2 orthotopic xenografts were injected with Cy5-labeled Chol-miRCtrl, two Results normal nude mice were injected with Cy5-labeled Chol-let-7a, and two normal nude mice were injected with Cy5-labeled Antitumor efficacy of Chol-let-7a in orthotopic xenograft Chol-miRCtrl. Whole-animal images were recorded at 15, models 45, 60, and 100 minutes, and 19, 24, 48, and 72hours after Orthotopic tumor sizes were evaluated at the end of 5 weeks of treatment using the NightOWL LB 983 in vivo imaging system treatment with the continuous or intermittent dosage regimen (Berthold, Beijing, China). Digital images were produced and (Fig. 1). The tumor size (volume, mm ) in the continuous-dosage analyzed with Indigo software (Berthold). Chol-let-7a-treated xenograft group was 152.15 ± 38.43, which was 35.86% (P < 0.01) and 40.02% (P < 0.01) the size of the In vitro con foca and phase fluorescence microscopy tumors in the two control groups (Chol-miRCtrl, 424.34 ± 60.10; blank xenograft group, 380.23 ± 74.83). At the end of the inter- Two pairs of xenograft mice and two pairs of normal mice were mittent treatment regimen, the tumor sizes in the Chol-let-7a- injected with Cy5-labeled Chol-let-7a or Chol-miRCtrl (5nmol treated, Chol-miRCtrl-treated, and blank xenograft group were in 250 μL saline buffer). Fresh orthotopic tumor and liver tissues 163.66 ± 25.81, 250.16 ± 44.91, and 288.85 ± 38.59, respectively; were snap-frozen 2 hours after injection of Chol-let-7a or Chol- the tumors in the Chol-let-7a treated mice were significantly miRCtrl. Frozen sections with a thickness of 10-20 μm were smaller than those in the Chol-miRCtrl-treated and blank xeno- observed and imaged using a confocal laser scanning micro- graft groups, at 65.42% (P = 0.012) and 56.66% (P = 0.031), scope (OLYMPUS FV1000, OLYMPUS Inc., Shanghai, China) respectively. There was no significant difference in tumor size in and a phase fluorescence microscope (Leitz, Laborlux 12). the Chol-miRCtrl and blank xenograft groups (P = 0.23). Quantification of let-7a miRNA expression Effects of Continuous administration of Chol-let-7a or Total miRNA was isolated from snap-frozen HepG2 xeno- TM Chol-miRCtrl on the liver and kidney in xenograft and graft tumor and paracancerous liver tissue using a mirVANA TM normal mice PARIS RNA isolation kit (Applied Biosystems, Carlsbad, CA, USA). RNA (10 ng) was reverse-transcribed using a miRNA LM and transmission electron microscopy analysis of Reverse Transcription Kit (Applied Biosystems) and let-7a-spe- hepatic tissues and cells cific primers (TaqMan miRNA Assay, Applied Biosystems). Hepatic tissues from xenograft mice and control mice were Quantitative real-time PCR was performed with an ABI observed after continuous administration of Chol-let-7a, Chol- Prism 7500 Sequence Detection System (Perkin-Elmer Applied miRCtrl and saline only (blank) for 5 weeks. LM analysis (Fig. 2) Biosystems, Foster City, USA) and the Perkin-Elmer Biosystems showed diffuse, chronic, nonspecific inflammation in the liver analysis software according to the manufacturer's instructions. parenchyma of all three xenograft mice. Spotty and focal necrosis RNU6B was used as a housekeeping control gene. Relative −∆∆CT [17] and mass lesions/confluent necrosis were observed. The necrosis expression was calculated using the 2 method. was milder in the Chol-let-7a-treated group than in the two xeno- graft control groups. Significant diffuse sinusoidal dilation and Determination of serum cytokines edema, primarily around the centrolobular vein, were observed Twelve normal nude mice and twelve mice with HCC orthotopic in the Chol-let-7a-and Chol-miRCtrl-treated groups. Higher tumors were injected with either Chol-let-7a or Chol-miRCtrl magnification showed sinusoidal endothelial cell hyperplasia in (n = 6 per group) every 3 days for 4 weeks, beginning 1 week after the Chol-let-7a-, and Chol-miRCtrl-treated xenograft mice; and 183 RESEARCH ARTICLE Journal of Bio-X Research Figure 1. Orthotopic tumor growth and metastasis inhibited by systemic treatment with Chol-let-7a. To assess antitumor efficacy in vivo, we evaluated the effects of Chol-let-7a, administered via a continuous or intermittent dosage regimen, on HepG2 orthotopic xenografts in nude mice. Orthotopic tumor size was evaluated at the end of 5 weeks. Gross specimen analysis showed that both dosing regimens inhibited tumor growth (area in the yellow band) and metastasis. (A) Continuous dosing regimen. (B) Intermittent dosing regimen. significant centrolobular vein congestion and necrosis was pre- from these mice, diffuse sinusoidal dilation was observed after dominantly observed in the Chol-miRCtrl and blank xenograft Chol-let-7a- and Chol-miRCtrl treatment. However, the inflam- groups (Fig. 3A). In addition, focal irregular sinusoidal dilation mation and necrosis were significantly milder than in the xeno- with congestion was found primarily in the Chol-miRCtrl and graft mice (Fig. 2). LM analysis of the hepatic tissue showed blank xenograft groups, along with some leukocyte infiltration. chronic inflammation and fibrosis, along with nonspecific focal Sinusoidal and perisinusoidal lumen dilation in the hepatic tis- degeneration and necrosis or focal or spotty apoptotic hepa- sue from Chol-let-7a- and Chol-miRCtrl-treated was confirmed to-cytes (Fig. 2). Discontinuous central lobular veins and bile by transmission electron microscopy (TEM) (Fig. 3B). The tis- ducts accompanied by non-specific reactive cell proliferation sue samples from some of these mice exhibited disrupted hepatic and inflammatory cell infiltration were occasionally observed. sinusoid structure (3/5 Chol-let-7a-treated mice; 4/5 Chol- miRCtrl-treated mice) and hepatic sinusoid fibrosis (2/5 Chol- LM and TEM analysis of renal tissues and cells let-7a-treated mice; 3/5 Chol-miRCtrl-treated mice). In addition, some organelle abnormalities, such as enlarged, irregular, or Renal tissues and cells from xenograft and normal mice treated swollen mitochondria, autophagic bodies, and multilamellar continuously with Chol-let-7a or Chol-miRCtrl were then bodies, were observed in the hepatocyte cytoplasm (4/5 Chol-let- assessed by LM and TEM. 7a-treated mice; 4/5 Chol-miRCtrl-treated mice) (Fig. 3C). LM analysis of renal tissue showed mild inflammatory cell infil- The same experiment was performed in normal nude mice to tration and interstitial alterations, such as intertubular blood cap- exclude the effects of xenograft tumor growth. In hepatic tissue illary dilation and intertubular hemorrhage, in all three xenograft 184 Journal of Bio-X Research RESEARCH ARTICLE Figure 2. Light microscope images of liver sections from xenograft and normal mice after continuous treatment with Chol-let-7a or Chol-miRCtrl. The images show focal necrosis and mass lesions in the xenograft mice, and diffuse sinusoidal dilation in the Chol-let-7a- and Chol-miRCtrl-treated xenograft mice and normal mice under hematoxylin-eosin staining. Scale bars = 100 mm. groups. These effects were relatively mild in the Chol-let-7a-treated observed in the Chol-miRCtrl-treated and blank xenograft con- group. Dilated glomerular lumens, renal cell dissociation, and trol mice. No significant congestion was observed in the Chol- renal tubule necrosis were seen in all three xenograft groups under let-7a-treated group. However, mild hypercellularity with dilated high magnification. Focal segmental sclerotic glomeruli were occa- patent capillary lumens was observed in the Chol-let-7a- and sionally observed in the blank xenograft mice. Congested glomer- Chol-miRCtrl-treated mice. Congestion and hypercellularity were uli and intertubular blood capillary dilation were predominantly observed in the Chol-miRCtrl-treated and blank xenograft mice. 185 RESEARCH ARTICLE Journal of Bio-X Research Figure 3. Liver histopathology and ultrastructural features after continuous therapy. (A) Light microscopy findings under high magnification. The images show dilated sinusoids. Also shown are sinusoidal endothelial cell hyperplasia (white arrows); mild focal and spotty apoptotic cells (yellow arrows); and inflammatory cells (orange arrows) under hematoxylin-eosin staining. Scale bars = 20 μm. (B) Transmission electron microscopy (TEM) images of hepatic sinusoidal struc- tures. The images show edematous and dilated sinusoidal and perisinusoidal lumens with structural damage and mild fibrosis of the hepatic sinusoids (red arrows) in the Chol-let-7a- and Chol-miRCtrl-treated groups. Also shown are leukocytes (yellow arrows) in the Chol-miRCtrl-treated and blank control groups. Scale bars = 2 μm. (C) Ultrastructure of liver parenchymal cells after continuous treatment with Chol-let-7a (TEM). Here shows some organelle abnormalities, enlarged, irregular, swollen mitochondria (Mit); autophagic bodies (Aut), and multilamellar bodies (MLB) observed in the hepatocyte cytoplasm. Scale bars = 200 nm. The features described above were confirmed by TEM (Fig. 4). and blank xenograft mice; inflammatory cells were also more Congestion was primarily observed in the glomerular capillar- prevalent in the glomeruli in the two control groups. As shown ies and proximal tubule interstitia in the Chol-miRCtrl-treated in Figure 4A and B, some features were similar in the xenograft 186 Journal of Bio-X Research RESEARCH ARTICLE mice after Chol-let-7a and Chol-miRCtrl treatment. The fea- Let-7a distribution and abundance in xenograft tumors tures and their incidence rate in Chol-let-7a- and Chol-miRCtrl- and hepatic tissues after Chol-let-7a treatment treated mice were: glomerular features such as dilated lumens Whole-animal imaging and frozen section analysis and endocapillary membrane hyperplasia (5/5, 5/5) and focal [5] Whole-animal imaging of nude mice and xenograft mice was foot process effacement and focal basement membrane thick- performed to observe the distribution of Chol-let-7a and Chol- ening (3/5, 2/5). In addition, inclusion bodies (4/5, 5/5) were miRCtrl in vivo, and the overall fluorescence levels of Cy5- readily apparent in the glomerular capillary endothelial cells labeled Chol-let-7a and Chol-miRCtrl in the liver and kidney at (Fig. 4A and B). The tubule cells exhibited abnormal mitochon- [7] different time points were analyzed using Indigo software. The dria (Mit) (4/5, 4/5), multilamellar bodies (MLBs) (3/5, 3/5), whole-animal images showed high concentrations of fluores- and autophagic bodies (Aut) (3/5, 4/5) (Fig. 4A and C). However, cence predominantly distributed in the liver, kidney, and blad- dilated and destroyed interstitial structures with inflammatory der from 15 to 100 minutes post-injection (Fig. 6A and B), after cell infiltration, as well as mild tubulitis, were observed in all which they began to decrease. At 19 hours after administration, three xenograft groups (Fig. 4A). the fluorescence was nearly undetectable in the liver, and fluores- In normal mice, the congestion and inflammation were mild. cence levels in the kidney and bladder region began to decrease. However, hyperplasia of the glomerular endocapillary cell mem- Beginning at 19 hours after administration, differences in fluo- brane (3/4, 3/4) and the presence of inclusion bodies in glomerular rescence levels and distribution among the groups became appar- capillary endothelial cells were observed in Chol-let-7a- and Chol- ent. The patterns of fluorescence levels in the liver, kidney, and miRCtrl-treated mice (3/4, 3/4) (Fig. 4A). Inflammatory cells were bladder showed similar trends in normal and xenograft mice occasionally observed in the dilated glomerular lumen in Chol- injected with Chol-let-7a and Chol-miRCtrl. However, the con- let-7a and Chol-miRCtrl-treated normal mice (2/4, 1/4) (Fig. 4A). centrations of the fluorescently labeled Chol-let-7a and Chol- The LM and TEM findings regarding off-target effects of miRCtrl in the kidney and bladder decreased much more slowly in this cholesterol-conjugated delivery system are summarized in the xenograft mice. Moreover, the fluorescence patterns in the liv- Tables 1 and 2. ers and kidneys of the xenograft mice differed over time between the Chol-let-7a and Chol-miRCtrl groups. The fluorescence sig- Effects of intermittent administration of Chol-let-7a or nal was chiefly concentrated in the hepatorenal region from 15 to Chol-miRCtrl on the liver and kidney in xenograft and 100 minutes. However, the Chol-let-7a fluorescence concentration normal mice was much stronger than the Chol-miRCtrl signal, especially in the Next, we examined the effects of injection with Chol-let-7a, liver (Fig. 6C and D). As measured in fluorescence arbitrary units, Chol-miRCtrl, or a saline treatment as blank control on the liv- Chol-let-7a fluorescence in the xenograft liver was 4.76 times that ers and kidneys of xenograft and normal mice, and compared seen in Chol-miRCtrl xenograft mice, and 9.90 and 7.55 times the findings to those from mice that received the continuous that seen in Chol-let-7a-and Chol-miRCtrl-treated normal mice, dosing regimen. respectively, at 100 minutes. At 19hours after administration, the Inflammation and mass necrosis were observed in the two fluorescence decreased rapidly, and was nearly undetectable at control xenograft mouse groups (Chol-miRCtrl-treated and 48 hours in normal mice, but was still present at relatively high blank xenograft); mild inflammation and focal and spotty levels in the kidneys and bladders of both xenograft groups. The necrosis were observed in the Chol-let-7a-treated mice. Under Chol-let-7a and Chol-miRCtrl fluorescence levels in the xenograft TEM, organelle abnormalities were occasionally observed in the kidneys were 5.0938 and 4.0938 fluorescence arbitrary units, hepatic parenchymal cells in all xenograft mice. However, only respectively, which corresponds to 4.77 and 4.08 times of those of mild, non-specificinflammation was observed in normal mice the normal controls, respectively (Fig. 6D). after injection of Chol-let-7a or Chol-miRCtrl. To further compare the fluorescence distribution in orthot- Diffuse sinusoidal dilation and edema and mild sinusoidal opic tumors and adjacent hepatic tissue, frozen sections col- endothelial cell hyperplasia were observed in the Chol-let-7a- lected 2 hours post-injection of Chol-let-7a or Chol-miRCtrl and Chol-miRCtrl-treated xenograft and normal mice (Fig. 5A), were observed under confocal and phase fluorescence micros- but at milder levels than those observed in the mice that received copy. In the xenograft mice, fluorescence was observed in both the continuous dosing regimen. the orthotopic tumor tissues and hepatic tissues, and the inten- Analysis of the renal tissues and cells revealed a similar sity was stronger in the tumor tissue than in the corresponding trend. Mild glomerular inflammation and interstitial lesions normal hepatic tissue (Fig. 6E). were observed in the renal tissues of all of the xenograft mice Next, fluorescence signals were observed in orthotopic under LM, and the changes were mild in the Chol-let-7a-treated tumor and other organ tissue samples taken 2 hours after injec- group. However, significant congestion was observed in the tion of Chol-let-7a or Chol-miRCtrl into normal and xenograft glomeruli and tubular interstitia in the Chol-miRCtrl-treated mice, using confocal and phase fluorescence microscopy. Strong group and the blank xenograft group (Fig. 5B). TEM analysis fluorescence signals were primarily observed in the xenograft showed significant deposition of blood cells in the dilated glo- tumors, livers, and kidneys from normal and xenograft mice; merular capillaries in the Chol-miRCtrl-treated mice and blank there was almost no fluorescence seen in other organs, such as xenograft mice (Fig. 5B). Mild membrane hyperplasia of the the heart, lung, spleen, and pancreas (Fig. 6F). This was consis- glomerular capillary walls and the presence of inclusion bod- tent with the results from the whole-animal imaging. ies in the glomerular capillary endothelial cells were observed in the Chol-let-7a- and Chol-miRCtrl-treated xenograft mice RT-PCR results (4/4, 3/4). Mild dilation of the capillary lumens was seen in the Using miRNA-specific primers and RT-PCR, we examined Chol-let-7a- and Chol-miRCtrl-treated normal mice (3/4, 2/4) let-7a abundance in orthotopic tumor tissue, paracancerous (Fig. 5B). Occasional irregular organelles were observed in the hepatic tissue from xenograft mice after 5 weeks of Chol-let-7a, proximal tubular cells (data not shown). 187 RESEARCH ARTICLE Journal of Bio-X Research Figure 4. TEM Images of the kidney ultrastructure after continuous treatment. Renal tissues and cells from xenograft and normal mice treated continuously with Chol-let-7a or Chol-miRCtrl were assessed by TEM. Compared to those in normal mice, congestion was primarily observed in the glomerular capillaries and proximal tubule interstitia in the Chol-miRCtrl-treated and blank xenograft mice; inflammatory cells were also more prevalent in the glomeruli in the two control groups. Some features were similar in the xenograft mice after Chol-let-7a and Chol-miRCtrl treatment. (A) Glomeruli and proximal tubules. The images show inclusion bodies in the glomerular endothelial cells (short red arrows), leukocytes (yellow arrows), and macrophages (orange arrow) in the interstititia, and incom- plete interstitial structure (areas between two long thin red arrows). Scale bars = 2 μm. (B) Glomerular changes. Membrane hyperplasia in glomerular capillary cells with bridge structure (wide black arrow) and grid structure (thin black arrow); inclusion bodies (red arrow) in the endothelial cells (En); and focal podocyte (Po) foot process effacement (yellow arrows). Scale bars = 0.5 μm. (C) Changes in proximal tubule cells. Enlarged and irregular mitochondria with vacuoles (white arrows), irregular lysosomes (orange arrow), autophagy (blue arrow), and multilamellar bodies (yellow arrow). Scale bars = 1 μm. TEM = Transmission electron microscopy. Chol-miRCtrl- and saline (blank) treatment, and heptic tissue not significantly up-regulated in the paracancerous hepatic tis- from normal mice (Fig. 6G). The let-7a levels were significantly sue compared to hepatic tissues from normal mice (P = 0.691). higher in Chol-let-7a-treated xenografts (Chol-let-7a vs Chol- miRCtrl, P = 0.003; Chol-let-7a vs blank xenograft, P = 0.001); Serum cytokines they were 4.35 and 5.26 times those of the Chol-miRCtrl- In the xenograft mice, IL-6 (P< 0.05), TNF-α (P< 0.05), and treated and blank xenograft controls, respectively. There was no VEGF (P < 0.05) levels were significantly lower in the Chol-let- significant difference between the two xenograft control groups 7a-treated group than in the Chol-miRCtrl and blank xenograft (Chol-miRCtrl vs Blank xenograft, P = 0.184). However, the groups (Additional Fig. 1A, http://links.lww.com/JR9/A31). let-7a levels in the orthotopic xenografts were still much lower Serum IL-8 levels were also decreased in the Chol-let-7a-treated than those seen in the paracancerous hepatic tissue (50.6%) and group, but the differences were not significant (P > 0.05). hepatic tissue in normal mice (40.7%). Moreover, let-7a was 188 Journal of Bio-X Research RESEARCH ARTICLE Figure 4. Continued The levels of IL-6, IL-8, TNF-α, and VEGF in the Chol-let-7a- used in combined regimens to decrease the dosage of each med- and Chol-miRCtrl-treated normal mice were not significantly icine used, thereby reducing their untoward effects and avoiding different from those seen in the normal control mice (P > 0.05) drug resistance. In addition, some drugs are administered inter- (Additional Fig. 1B, http://links.lww.com/JR9/A31). Moreover, mittently to facilitate tissue and cell repair between doses. Based we found that IL-6, IL-8, TNF-α, and VEGF levels were signifi- on this idea, we designed an intermittent dosing regimen for cantly increased in the xenograft-only mice in comparison with miRNA administration. Our results suggest that both continu- the normal control mice (Additional Fig. 1C, http://links.lww. ous and intermittent Chol-let-7a dosing regimens inhibit tumor -5 com/JR9/A31) (IL-6, P = 3.885 × 10 ; IL-8, P = 0.0076; TNF-a, growth, although the continuous dosing regimen exhibited -4 P = 3.72 × 10 ; VEGF, P = 0.0467). a greater capacity to shrink the tumors and resulted in fewer intrahepatic and distal (spleen and colon) metastases. Discussion Antitumor efficacy of Chol-let-7a delivered continuously Potential characteristic morphological features of Chol- or intermittently let-7a off-target effects on the liver and kidney Previous studies have primarily focused on identifying an effec- Safety is an important aspect of drug delivery systems tive delivery system for miRNA-based anticancer therapies, intended for clinic use. The liver and the kidney often [11] which are considered a major obstacle to their clinical use. show signs of toxicity and injury as a result of exposure [18] In clinical practice, a variety of anti-tumor drugs are usually to drugs, xenobiotics, or chemicals. Thus, understanding Table 1 Liver histopathology and ultrastructural changes in normal and xenograft mice after treatment Histopathological findings TEM features Sinusoidal structure Inflammation Sinusoidal Inflammation Irregular Disrupted Cell Group features Necrosis dilation Congestion features organelles Dilation structures hyperplasia† Chol-let-7a-treated normal mice Mild Focal /spotty Diffuse Mild Mild (leukocyte) Yes Yes Yes, mild/less Yes Chol-miRCtrl-treated normal mice Mild Focal /spotty Diffuse Mild/Severe Mild (leukocyte) Yes Yes Yes, mild/less Yes Xenograft with no treatment Severe Severe Irregular Severe Severe (leukocyte) No Mild Yes Yes Focal Cho/-lef-7a-treated xenograft Mild/severe Mild/severe Diffuse Mild/Severe Mild Leukocyte Yes Yes Yes Yes Chol-miRCtrl-treated xenograft Severe Severe Diffuse Severe Severe (leukocyte Yes Severe Yes Yes and macrophage) *Organelle abnormalities in parenchymal cells. Sinusoidal endothelial cell hyperplasia. TEM = transmission electron microscopy. 189 RESEARCH ARTICLE Journal of Bio-X Research Table 2 Kidney histopathology and ultrastructural changes in normal and xenograft mice after treatment Histopathological findings TEM features Glomeruli Tubule and tubular interstitia Dilated lumen Dilated Membrane Inclusion Congestion Dilated Group Inflammation Congestion of glomeruli lumen† hyperplasia† bodies† Congestion Tubulitis Dilation Incomplete Cho/7ef-7a-treated normal mice Mild Irregular, mild Yes Yes Yes, mild Yes Mild Mild Mild Mild Chol-miRCtrl-treated normal mice Mild Irregular, mild Yes Yes Yes, mild Yes Mild Mild Mild Mild Xenograft with no treatment Severe Severe Severe/focal Focal No No Severe Severe Severe Focal severe Cho/7ef-7a-treated xenograft Mild/severe Mild Yes Mild Yes, mild Yes Mild Mild Mild Mild Chol-miRCtrl-treated xenograft Severe Severe Yes Yes Yes, mild Yes Yes, severe Yes, severe Yes, severe Yes, severe † ‡ *Membrane hyperplasia of the glomerular capillary wall. Inclusion bodies in endothelial cells. Dilated and incomplete tubular interstitia. TEM=transmission electron microscopy. the features and potential mechanism of hepatotoxicity and All these features found in xenograft mice treated with nephrotoxicity of novel therapeutics is critical for pharma- Chol-let-7a or Chol-miRCtrl exhibited tissue and cell fea- ceutical drug development. Consistent with this, whole-an- tures that are consistent with some common necroinflam- imal imaging showed that Chol-let-7a and Chol-miRCtrl matory patterns of drug-induced liver injuries, such as acute primarily accumulated in the liver and kidney. Thus, we ana- (lobular) hepatitis, chronic (portal) hepatitis, and zonal lyzed these organs in detail to evaluate the potential off-tar- coagulative necrosis, were not characteristic features result- get effects of Chol-let-7a. ing from Chol-let-7a or Chol-miRCtrl. Some of the changes Histopathology is recognized as the single most appropriate were accompanied by spotty and focal parenchyma cell [14] screen for evidence of liver and kidney injury. Cell injury can degeneration, apoptosis, or necrosis. Furthermore, these fea- be noted by electron microscopy examination. Thus, the fea- tures were aggravated in the corresponding Chol-miRCtrl- tures of liver and kidney tissues and cells after treatment with treated xenograft groups. Congestion and severe acute the cholesterol-conjugated molecules were observed under LM hepatitis with mass necrosis were mainly accompanied by and TEM to identify the effects of xenograft tumor growth, the xenograft tumor growth. In addition, congestion and inflam- delivery system, and up-regulated let-7a expression. Features mation were less severe in the Chol-let7a-treated xenograft that were only observed in Chol-let-7a- and Chol-miRCtrl- group. To confirm this hypothesis, we examined serum cyto- treated mice, and not found in blank xenograft mice, were con- kines from xenograft and healthy nude mice to analyze the sidered to represent damage induced by Chol-let-7a therapy. potential effects of xenograft growth and Chol-let-7a on the Contrary, some histopathological and ultrapathological features immune state of nude mice, and to clarify the off-targets that consistent with the growth of xenograft, especially found from some of the immuno-related changes of Chol-let-7a in in blank xenograft mice, were not off-targets of the cholester- xenograft mice. Serum cytokine changes in xenograft mice ol-conjugated molecules, and normal mice after Chol-let-7a and Chol-miRCtrl sug- We summarized and compared the features among the gested that the immune status of the xenograft nude mice xenograft groups, as well as the differences between the affected mostly from the tumor growth. The levels of four xenograft and normal groups. These results in this study cytokines IL-6, IL-8, TNF-α, and VEGF in the Chol-let-7a- showed that the diffuse simusoidal dilation and edema, and and Chol-miRCtrl-treated normal mice were not signifi- the sinudoidal endothelial cell hyperplasis were related to cantly different from those seen in the normal control mice Chol-let-7a and Chol-miRCtrl treatment. This was con- (P > 0.05). However, the serum levels of IL-6, IL-8, TNF-a, firmed in the Chol-let-7a- and Chol-miRCtrl-treated normal and VEGF in the blank xenograft group were significantly mice. The characteristic changes in normal hepatic tissues increased in the blank xenograft mice in comparison with and cells after administration of the cholesterol-conjugated the normal control mice. molecules; however not found in blank xenograft mice, were diffuse sinusoidal dilatation with or without structural Analysis of potential off-target effects of Chol-let-7a to damage and sinusoidal lining cell hyperplasia. Similarly, liver dilation of the glomerular lumen and endocapillary mem- brane hyperplasia or edematous glomerular capillary and MiRNAs target multiple molecules, which could cause side tubular interstitium, often accompanied by inclusion bodies effects at the molecular level in vivo. In this study, we ana- in the glomerular endothelial cells, were primarily induced lyzed the risks of up-regulating let-7a expression in mouse liv- by injection of the cholesterol-conjugated molecules, Chol- ers. First, we confirmed that let-7a was effectively delivered let-7a and Chol-miRCtrl. to the xenografted tumor and liver tissues, through in vivo However, all of these features, which were found after Chol- whole-animal imaging and in vitro frozen section analysis. let-7a treatment, are non-specific changes that are also produced Under confocal and phase fluorescence microscopy, Chol- [18] by other drugs and treatments, including the relative charac- let-7a fluorescence was observed in both the orthotopic tumor teristic feature, diffuse sinusoidal dilatation in hepatic tissues, tissues and adjacent liver tissues. The Chol-let-7a fluorescence has also been reported in an oral contraceptive-induced liver signal was stronger than the Chol-miRCtrl fluorescence signal [19] injury. Similarly, characteristic changes in the renal tissues in the orthotopic tumor tissues. We then used RT-PCR to semi- and cells have also been reported as drug side effect. In addition, quantitatively detect let-7a levels in the tumor and adjacent the liver and kidney effects were less severe in the intermittent hepatic tissues, and found that let-7a was significantly up-reg- dosing regimen group compared with the continuous regimen. ulated in the tumor tissues but only slightly up-regulated in the 190 Journal of Bio-X Research RESEARCH ARTICLE Figure 5. Changes in the liver and kidney after intermittent treatment with Chol-let-7a or Chol-miRCtrl. Mice with orthotopic xenografts and normal nude mice were injected with intermittent dosing of Chol-let-7a, Chol-miRCtrl, or saline (blank), and compared the effects of the molecules on hepatic and renal tissues and cells under light microscope (LM) and transmission electron microscopy (TEM). Here show the findings of histopathological and ultrastructures features in livers and kidneys under LM and TEM to those from mice that received the continuous dosing regimen. The results suggest that features are similar however at milder levels after intermittent dosing treatment than those observed in the mice that received the continuous dosing regimen. (A) Histopathology of the liver after continuous and intermittent therapy (LM). The images show areas of relatively normal liver tissue with no mass necrosis under hematoxylin-eosin staining. Scale bars=100 μm. (B) Glomeruli and tubular interstitia in xenograft and normal mice after intermittent therapy (TEM). Scale bars=2 μm. 191 RESEARCH ARTICLE Journal of Bio-X Research Figure 5. Continued paracancerous hepatic tissues. These results suggest that there kidney decreased slowly and were still partially present at 72 is no major risk of let-7a up-regulation in healthy liver tissue hours post-injection. We believe that the delayed excretion having off-target effects. was due primarily to kidney injury and inflammation, which resulted from orthotopic tumor growth. A previous study reported that kidney parenchyma is Potential explanation for the delayed urinary excretion of especially vulnerable to the pro-inflammatory response cholesterol-conjugated molecules seen in the xenograft to infection, and thus tissue damage, because it receives mice [14] approximately 25% of the cardiac output. Serum cytokine The specific pattern of kidney toxicity and injury of a drug, levels in this study confirmed this to some extent. In nor- xenobiotic, or chemical is dependent on many factors, such mal mice, cytokine levels were not altered by Chol-let-7a or as its physiochemical properties (eg, low intrinsic solubility), Chol-miRCtrl treatment. The abundance of four cytokines dose, toxicokinetic properties, renal clearance profile, met- was significantly increased in the xenograft mice compared [20] abolic attributes, and length of exposure. In this study, with the normal control mice. However, the cytokine lev- we were unable to determine the mechanism of the damage els were significantly reduced after Chol-let-7a treatment. induced by the conjugates. We assessed morphological and In addition, hepatic inflammation and necrosis were also ultrastructural tissue changes, as well as conjugate distribu- decreased in the Chol-let-7a-treated xenograft mice. Thus, tion and excretion, to determine why excretion of the cho- the cytokine levels seemed to be consistent with tumor size, lesterol-conjugated molecules was delayed. There were no degree of inflammation, and necrosis in the three xenograft obvious differences in the Chol-let-7a and Chol-miRCtrl flu- groups. These results further suggest that the up-regula- orescence in the liver and kidney of normal mice. However, tion of cytokines and inflammation were primarily associ- in the xenograft mice, especially the Chol-miRCtrl and blank ated with xenograft growth, rather than Chol-let-7a. Thus, xenograft control groups, the fluorescence signals in the the inhibition of kidney-mediated excretion seen in the 192 Journal of Bio-X Research RESEARCH ARTICLE Figure 6. let-7a distribution and abundance levels in xenograft tumors and hepatic tissues after Chol-let-7a treatment. The distribution of Chol-let-7a and Chol-miRCtrl in vivo was examined by whole-animal imaging and frozen-sections under confocal and phase fluorescence microscopy observation. let-7a abundance in orthotopic tumors, corresponding paracancerous hepatic tissue, and normal liver tissue from healthy nude mice was semi quantitative detected by reverse transcription-polymerase chain reaction (RT-PCR). Whole-animal imaging and frozen-sections observation suggest that the cholesterol-conjugated molecules target to xenograft tumor and liver tissues. RT-PCR confirmed the situation. Whole-animal imaging suggests that kidney function of the xenograft mice was affected; excretion of the cholesterol-conjugated molecules was delayed. (A) Cy5-labeled Chol-let-7a and Chol-miRCtrl distribution observed from belly (Ventral whole-animal images). (B) Cy5-labeled Chol-let-7a and Chol-miRCtrl distribution observed from back (Dorsal whole-animal images). The signal strength increases as the color shifts from purple to blue, green, yellow, and red. (C, D) Overall fluorescence levels in the liver (C) and kidney (D) of normal and xenograft animals. The representative average overall signal levels from all groups are shown (n=2 mice per cohort). Chol-let-7a-N: Chol-let-7a-treated normal mice; Chol-miRCtrl-N: Chol-miRCtrl-treated normal mice; Chol-let-7a-X: Chol-let-7a-treated xenograft mice; Chol-miRCtrl-X: Chol-miRCtrl-treated xenograft mice. (E) Chol-let-7a and Chol-miRCtrl distribution in orthotopic tumor tissue and liver tissue. Sections taken 2 hours after injection with Chol-let-7a or Chol- miRCtrl were observed by confocal and phase fluorescence microscopy. The fluorescent confocal micrographs show Chol-let-7a or Chol-miRCtrl in the orthot- opic tumor tissue and tumor-adjacent liver tissue. Original magnification: 40×. (F) Chol-let-7a and Chol-miRCtrl distribution in orthotopic tumor and other organ tissue in normal and xenograft mice. Fluorescence was observed 2 hours after injection with Chol-let-7a or Chol-miRCtrl. 0: tumor; 1: liver; 2: kidney; 3: heart; 4: lung; 5: spleen; 6: pancreas. The signal strength increases as the color shifts from purple to blue, green, yellow, and red. (G) Let-7a abundance in xenograft tumor tissue and adjacent liver tissue as determined by real-time PCR. Using miRNA-specific primers, relative let-7a levels were calculated using the compara- −ΔΔCT tive cycle threshold (CT) method (2 method), with let-7a expression normalized to U6. The results shown represent the mean and standard error from three ** independent experiments. P<0.01 (Student's t-test). 193 RESEARCH ARTICLE Journal of Bio-X Research Figure 6. Continued xenograft mice most resulted from tumor growth, not tox- period allowed us to roughly estimate the in vivo distribu- icity of the cholesterol-conjugated molecules. Moreover, the tion and metabolism of these molecules, as well as observe results showed that Chol-let-7a treatment could alleviate the delay in urinary excretion, it did not enable us to track renal injury by down-regulated cytokines indirectly through the complete roadmap of these molecules in the tumor and inhibiting tumor growth. hepatic tissues. Owing to the limitations of the experimental conditions, serum cytokines were examined using methods and devices Limitations designed for clinical use. In addition, the samples from each group were pooled for testing; thus, the results may not reflect In this study, the modification with cholesterol-conjugated mol- the actual situation. ecules may function as miRNA, as well as ds-siRNA that could not be divided completely. We only analyzed and discussed based on miRNA functions. Conclusions Fluorescence patterns were only analyzed beginning at 100 minutes post-injection and continuing until 19 hours postin- Both continuous and intermittent dosing with Chol-let-7a jection, owing to experimental limitations. Although this time resulted in successful delivery of the conjugate to xenograft 194 Journal of Bio-X Research RESEARCH ARTICLE Figure 6. Continued tumor tissues, as well as up-regulation of let-7a levels, and sig- YX performed ultrasonic analysis. XL, WD, and YH per- nificantly inhibited tumor growth and metastasis. formed electronic microscopic analysis. LZ, XD, TY, SG, Chol-let-7a treatment induced some non-specific liver and XL, and YL performed molecular imaging, western blot, kidney damage, including diffuse sinusoidal dilatation with or immunohistochemi-cal staining, hematoxylin-eosin stain- without structural damage and sinusoidal lining cell hyperplasia ing, and analysis. All authors read and approved the final in livers, and mild membrane hyperplasia in dilated or edema- manuscript. tous glomerular capillaries and the tubular interstitium, often accompanied by the presence of inclusion bodies in the glomer- Financial support ular endothelial cells. Chol-let-7a did not seem to have any severe off-target effects. This work was supported partly by the National Human and In addition, it alleviates the inflammation and congestion caused Health Scientific Data Sharing Platform, Clinical Center of by the tumor itself by inhibiting tumor growth. China (No. 2004DKA20240-2014), and National Program Using an intermittent dosing regimen to reduce the damage funded by the Ministry of Science and Technology of China associated with Chol-let-7a while maintaining satisfactory anti- (No. 2015KJRK1L01). The content is solely the responsibility tumor efficacy could be a useful strategy in future studies, and of the authors and does not represent the official views of the potentially clinical use. funding agencies. Acknowledgments Institutional review board statement The authors thank Mrs. Huimin Zhao, Wenyu Hao, and The experiments were approved by the Institutional Research Huanxian Cui from the Centre for Experimental Animal Board of Peking Union Medical College Hospital. 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. Conflicts of interest We also thank Dr. Wei-Min Tong for his constructive suggestions in support of this study. The authors thank Dr. Xingyi Hang and The authors declare that they have no conflicts of interest. Ms. Jiahui Liu for their assistance with statistical analysis. Editor note: JG is an Editorial Board member of Journal of Bio-X Research. She was blinded from reviewing or making decisions on the manuscript. The article was subject to the Author contributions journal's standard procedures, with peer review handled inde- JG initiated the project. JG and ML analyzed data, wrote pendently of this Editorial Board member and their research the manuscript, and performed most of the experiments. groups. 195 RESEARCH ARTICLE Journal of Bio-X Research Figure 6. Continued [10] Bajan S, Hutvagner G. RNA-Based therapeutics: from antisense oligo- References nucleotides to miRNAs. Cells 2020;9:137. [1] Bray, F, Ferlay, J, Soerjomataram, I, et al. 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Journal of Bio-X Research – Wolters Kluwer Health
Published: Dec 24, 2022