Our study has indicated a critical role ofmiR-145 in EMT and revealed its molecular mechanism. Our results suggest that miR145 targets genes essential for EMT, a prerequisite step for migration and invasion during metastasis. MiR145 targets Oct4 in cancercells, which in turn regulates the expression and function of b-catenin. Oct-4/b-catenin regulates the expression of Snail, ZEB1&2, three transcriptional factors in EMT. Consequently, tumor cells acquire properties ofmesenchymalcells, and become invasive and migratory. The results from immunohistochemical analysis of 41 clinical samples showed that miR-145 expression correlated with aggravation ofbreastcancer, supporting a role ofmiR-145 in EMT. Apparently, miR-145 is just one of the regulators. Slug and Twist, another two important transcriptional Figure 4. Snail, ZEB1, and ZEB2 are the downstream molecules ofmiR-145/Oct4 in MDA-MB-231, BT-549, ZR-75-30 and T47D cells. (A) MiR-145/Oct4 did not alter the expression of Slug and Twist. (B), (C) and (D) Real-time PCR and Western blot assay showed that miR-145/Oct4 regulated the expression of Snail, ZEB1 and ZEB2 both in mRNA level and protein level. Statistical analysis was performed by t test. Columns, mean of three separate experiments; Error bars show the SD of three independent experiments measured in triplicate. *, P,0.01, significant difference between two compared groups. (E) Western blot assay showed that down-regulation of b-catenin inhibited Oct-4 induced expression of ZEB1, ZEB2 and Snail in MDA-MB-231, BT-549, ZR-75-30 and T47D cells.
promotes epithelial-like properties in breastcancercells Next, we investigated the role ofmiR-203 in relationship with SNAI1 expression in breast carcinoma cells. In MCF7-SNAI1 cells, ectopically expressed SNAI1 lacks its natural 39UTR  and therefore, these cells are not suitable to study whether miR- 203 regulates SNAI1 messenger RNA (mRNA). The mesenchymalbreastcancer cell line HTB129 presents high levels of endogenous SNAI1 and low levels ofmiR-203 as compared toepithelial MCF7 cells [21,29]. HTB129 cells stably transfected with miR-203 (HTB129-miR203) exhibited a significant decrease in SNAI1 mRNA (Fig. 2A, Fig. S2). HTB129-miR203 cells lost their typical fibroblastic, dispersed phenotype and acquired a more compact and cohesive appearance (Fig. 2B). HTB129-miR203 cells further lost about 25% of their migratory and 15% of their invasive capacity (Fig. 2C, D). By performing MTT proliferation and AnnexinV apoptosis assays we excluded that observed inhibitions were due to decreased cell proliferation and/or programmed cell death (Fig. S3). Interestingly, previous studies in prostate cancer progression and metastasis showed that miR-203 expression not only controlled cell migration and invasion of prostate cancer cell lines, but also suppressed prostate cancer metastasis in vivo via repression of prometastatic targets such as ZEB2 [30,31]. HTB129 cells expressed high levels of ZEB1/2 factors, but in the present cellular context, miR-203 expression did not lead to significant decrease of ZEB2 mRNA. This may be due, in part, to incomplete miR-203-mediated repression of SNAI1 which has been shown previously to promote upregulation of ZEB factors . In addition, HTB129 cells co-expressed high levels of SNAI2 and TWIST1 (our unpublished data), two EMT inducers. Collectively, these factors may attenuate the effects ofmiR-203-mediated repression of SNAI1 in HTB129-miR203 cells. Accordingly, we did not detect a significant increase in expression of classical epithelial markers such as E-cadherin and keratin 18 in these cells (our unpublished observations). Altogether these results show that miR-203 significantly reduces SNAI1 expression and promotes epithelial-like features such as a more cohesive phenotype and reduced motility, motivating us to investigate whether miR-203 could directly target SNAI1.
significant unmet medical need. Recently, small RNA molecules known as miRNAs have emerged as post-transcriptional regulators of mRNA stability, and have been identified to enhance several aspects ofbreastcancer pathogenesis including metastasis , invasion , and self-renewal . Each of these phenotypes can be further modulated by the EMT program . miR-221/222 has previously been reported to promote the EMT by negatively regulating TRPS1 that leads to increased ZEB2 expression . Here, we found that miR-221/222 additionally targets ADIPOR1 and abrogates ADIPOR1 inhibition of the EMT. Our data show that miR-221/222 simultaneously targets two genes, ADIPOR1 and TRPS1, which likely allows for tight control of the EMT. Both the NF-kB/IL6/JAK2/STAT3 cascade, which we show here is modulated bymiR-221/222-mediated downregulation of ADI- POR1, and ZEB2 expression, which we have previously shown is Figure 1. ADIPOR1 is a direct target ofmiR-221/222. (A) qRT-PCR analysis of MCF10A cells, 48 hours post transfection with either miR-Control or miR-221/222 mimic. Data represent means of triplicates 6 SD. (B) ADIPOR1 mRNA expression data taken from microarray analysis of 32 luminal and 23 basal-like breastcancer cell lines. Data represented as boxplot, with whiskers indicating maximum and minimum values of the data set. (C) qRT- PCR analysis of ADIPOR1 mRNA expression versus miR-221 levels in 49 breastcancer cell lines. (D) qRT-PCR analysis of ADIPOR1 mRNA expression versus miR-221 levels in 27 primary breast tumors. (E) qRT-PCR analysis of ADIPOR1 mRNA expression versus miR-222 levels in 49 breastcancer cell lines. (F) qRT-PCR analysis of ADIPOR1 mRNA expression versus miR-222 levels in 27 primary breast tumors. (G) The predicted ADIPOR1 wild-type (WT) and mutant 3’UTR sequence for miR-221/222 is shown above. Below, MCF10A cells were co-transfected with the reporter constructs and an increasing titration ofmiR-221/222 mimic. Data represent means of triplicates 6 SD.
PRRX1 is a newly identified EMT (the epithelial-mesenchymaltransition) inducer and stemness regulator [24,25], both of which are closely related to radioresistence . In addition, abundant PRRX1 expression was correlated with poor prognosis and metastasis in CRC cases. Moreover, abundant expression of PRRX1 was also associated with poor clinical outcomes . Recent studies have also shown that PRRX1 played a pivotal role in pancreatic regeneration and carcinogenesis . In this study, we illustrated that miR-124 enhanced the sensitivity to radiation, both in CRC cells and human xenograft tumors. Moreover, PRRX1 is a novel, direct target ofmiR-124 that induces irradiation resistance. PRRX1 knockdown could sensitize cellsto ionizing radiation. In addition, the overexpression ofmiR-124 would cause the modulation of EMT and stemness-related genes expression, both of which are closely related with radioresistence. The restoration of PRRX1 could rescue the effects caused bymiR- 124. In this research study, we have illustrated that miR-124 sensitized colorectal cancercellsto radiation treatment to some extent by downregulating PRRX1. These findings have been uncovered for the first time, thereby illustrating how miR-124 plays a key role in inducing cells resistance to ionizing radiation.
To further investigate the roles of AKT and ERK1/2 pathways in miR-21 regulating EMT and CSC phenotype, the MDA-MB- 231/anti-miR-21 cells were transfected with miR-21 mimics at a concentration of 40 nmol for 72 h, and then the cells were treated with LY294002 (the inhibitor of PI3K-AKT) at 20 mmol/l or U0126 (the inhibitor of ERK1/2) at 10 mmol/l for 24 h. We confirmed that LY294002 treatment blocked re-expression ofmiR-21 inducing AKT activation (p,0.0001; Fig. 6A, E), while U0126 treatment abolished re-expression ofmiR-21 inducing ERK1/2 activation (p,0.0001; Fig. 6A, E). More interestingly, re- expression ofmiR-21 inducing EMT and CSC phenotype were inhibited by LY294002 (N-cadherin, p = 0.0005; Vimentin, p = 0.0005; alpha-SMA, p = 0.0008; E-cadherin, p = 0.0002; ALDH1, p,0.0001; CD44, p,0.0001; Fig. 6B, C, E) or U0126 (N-cadherin, p,0.0001; Vimentin, p = 0.0005; alpha-SMA, p = 0.0004; E-cadherin, p = 0.0003; ALDH1, p,0.0001; CD44, p = 0.0005; Fig. 6B, C, E) in the cells, indicated that both AKT and ERK1/2 pathways are requiring for miR-21 in inducing EMT and CSC phenotype, which further suggested that AKT and ERK1/2 pathways are two parallel downstream pathways ofmiR- 21 for regulating EMT and CSC phenotype. Furthermore, both LY294002 and U0126 have no proven effect on PTEN Figure 3. Antagonism ofmiR-21 induced the expression of PTEN, as well as inactivated AKT and ERK1/2. (A) Ectopic expression of PTEN mRNA in MDA-MB-231/anti-miR-21 cells and MDA-MB-231/control cells were verified by real time RT-PCR assay (p = 0.003). (B, C, D) Protein levels of PTEN, p-AKT, AKT, p-ERK1/2, and ERK1/2 in indicated cells were detected by Western blot analysis, and bands were semi-quantified using ImageJ software. GAPDH was used as loading control. (*indicates p,0.05).
Bmi-1 has been shown to be an upstream regulator of the PI3K/Akt pathway [13,18,19], which plays a critical role in regulation of the EMT process and cell migration. Our data shows that Bmi-1 knockdown is correlated to reduced phosphorylation of Akt after the IR treatment (Fig. 3A, shBmi-1 vs. NC or WT). To determine whether Akt mediates IR-induced Bmi-1-reg- ulated migration ofbreastcancercells, we analyzed the effect of PI3K inhibitor LY294002 or AKT 1/2 kinase inhibitor AKT I on the migration of the same set ofbreastcancer cell lines after IR. Our data demonstrated that while LY294002 or AKT I inhibited the induced migra- tion of the wild-type (WT) and the non-targeting lentivirus infected (NC) breastcancercells at day 7 post-IR, neither LY294002 nor AKT I affected the migration of Bmi-1 knockdown (shBmi-1) breastcancercells (Fig. 4, shBmi-1 vs. NC or WT). These results indicate that Bmi-1 regulates IR-induced EMT and migration ofbreastcancercells through activation of PI3K/ AKT signaling.
A proposed critical step in the conversion of primary tumors to metastases is attributed to the process known as epithelial-to- mesenchymaltransition (EMT). EMT is a remarkable example of cellular plasticity that involves the dissolution ofepithelial tight junctions, the intonation of adherens junctions, the remodeling of the cytoskeleton, and the loss of apical-basal polarity [46,47]. Our data reveals that miR-7 inhibits EMT which promotes the conversion of highly invasive breastcancercells with mesenchymal characteristic to the cells with epithelial properties. Transforming growth factor-b (TGF-b) has emerged as a key regulator of EMT in late-stage carcinomas, where it promotes invasion and metastasis . Cicchini et al. (2007) have demostrated that FAK is required for the TGF-b induced EMT in hepatocytes . Figure 7. miR-7 inhibits breastcancer cell metastasis to mice lungs. (A) H&E stained sections of lungs isolated from mice that received tail vein injection ofmiR-7 or control infected MDA-MB-435s cells (scale bar: 100 mm). (B) Numbers of lung micrometastases per section in individual mice (each data point represents a different mouse; n = 7 mice per group). (C) Expression of human HPRT mRNA relative to mouse 18S rRNA in the lungs of the tumor-bearing mice.
The mammalian E2A gene is located at chromosome 19 and is non-tissue-specifically and ubiquitously expressed in a wide range of cell types. Through alternative splicing, the E2A gene encodes two isoforms, E12 and E47 (collectively referred as E2A proteins), which both have the basic-Helix-Loop-Helix (bHLH) domain and could regulate gene transcription by binding to the E-box DNA sequences, CAGGTG. Though widely expressed, E2A is not essential in some organogeneses like skeletal or cardiac myogen- esis, erythropoiesis, chondrogenesis, and neurogenesis  but plays an important role in the development and differentiation of B [2,3] and T lymphocytes . E2A deficient mice showed an arrest at the pro-B cell stage during B cell development  and transgenic expression of either E12 or E47 could partially rescue the B lymphopoiesis initiation; similarly, E2A deficiency led to a block at the earliest stage of T cell development  and disturbed thymocyte positive selection . Moreover, E2A has been found to be involved in some cellular activities including cell differentiation , proliferation , apoptosis , cell cycle  and epithelial- mesenchymaltransition (EMT) .
Mature miRNAs are generated via a two-step processing by Drosha and Dicer. The initial processing that occurs in the nucleus is catalyzed by Drosha. The Drosha complex cleaves pri-miRNA into pre-miRNA. To further verify whether miR-361 prevents the processing of Drosha. We applied Drosha to assay the levels of pri- miR-484 and pre-miR-484. Our data showed that enforced expression ofmiR-361 prevented the reduction of pri-miR-484 and inhibited the increase of pre-miR-484 induced by Drosha, and knockdown ofmiR-361 had an opposite effects (Figures 2F and 2G). These data suggest that miR-361 prevents the processing of pri-miR-484 by Drosha into pre-miR-484 in nucleus. To further test whether the miR-361 recognition element on pri-miR-484 was responsible for miR-361 binding and inhibition of processing, we applied a biotin-avidin pull-down system to assess the direct binding ofmiR-361 to pri-miR-484. The cardiomyocytes were transfected with biotinylated miR-361, biotinylated mutant miR- 361 and biotinylated negative control (NC) (Figure S1E). Then the cells were harvested for biotin-based pull-down assay. Pri-miR-484 was co-precipitated, and the levels of pri-miR-484 in the pull- down complexes was analyzed by the qRT-PCR (Figure 2H). As shown in Figure 2H, pri-miR-484 was significantly enriched in the miR-361 pull-down products as compared to the biotinylated mutant miR-361 group and negative control group, indicating that miR-361 can directly bind to pri-miR-484 in vivo. We also employed inverse pull-down assay to test whether pri-miR-484 could pull down miR-361, a biotin-labeled-specific pri-miR-484 probe was used. The results showed that pri-miR-484 (Figure S1F) and miR-361 could be co-precipitated (Figure 2I). Taken together, it appears that miR-361 is able to directly bind to pri-miR-484 and prevent the processing of pri-miR-484 by Drosha into pre-miR- 484.
Cells were harvested two days after being passaged at about 70– 80% confluence. Total cell lysate was collected and RNA was extracted using an RNeasy kit (Qiagen). cDNA was synthesized using Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Real-time PCR reaction mixes were prepared using Power SYBR Green (Applied Biosystems, Foster City, CA), and run on the 7300 Real-time PCR System (Applied Biosystems) using the following program: 95uC for 10 min, 95 uC for 30 s, and 60uC for 1 min, for 40 cycles. Results were analyzed using the Comparative ddCt method and melting curves were performed to ensure product specificity. Experiments were done in technical triplicates and were repeated at least twice independently. GAPDH gene expression was measured as endogenous control. Primers were custom ordered (Eurofins MWG Operon, Huntsville, AL) using the following sequences:
In order to further investigate whether MSCs in inflammation microenvironment were exerting their effects through induction of EMT, a number of specific genetic markers of EMT were examined in HCC tissues. E-cadherin present in most epithelialcells is a calcium-dependent transmembrane glycoprotein. Vi- mentin has been consistently associated with mesenchymaltransition in epithelialcells [29,30]. E-cadherin down-regulation and Vimentin up-regulation are commonly observed in more invasive basal cancer subtypes and have been positively correlated with poor prognosis in breastcancer patients . Interestingly, our results showed that HCC undergoing EMT, which labeled by down-regulation of E-cadherin and up-regulation of Vimentin, were associated with a shorter CFI and a worse OS. Further investigations demonstrated that induction of EMT was correlated with SSEA-4 expression in HCC tissues, which revealed that the major channel of MSCs in inflammation microenvironment increased HCC metastatic potential may be EMT induction, the multivariate Cox regression analysis indicated that both MSCs and EMT induction could predict HCC metastasis and re- currence.
Based on gene expression profiling, breastcancer have been classified into four main subtypes - luminal A, luminal B, Her-2+ enriched and basal-like. This classification is used to direct the use of targeted therapies such as endocrine-based therapy for hormone receptor positive (HR+) breastcancer and Her2-based therapy such as trastuzumab, lapatinib and pertuzumab for Her2-enriched cancers [34–35]. The use of this molecular-based approach in the treatment ofbreastcancer has revolutionized how we treat breast Figure 4. Panepoxydone inhibited NF-kB: (A) Immunoblotting of IkB and pIkBa showed accumulation of IkBa (inactive) and down-regulation of pIkBa (active) in PP treated breastcancercells. This indicates the potential of PP in keeping NF-kB in the active state. (B) Bar diagram indicate the increased IkB/pIkB ratio in all breastcancercells after PP treatment. * indicates statistically significant difference between PP treated and untreated cells at p,0.05 (*), p,0.01(**), and, p,.001(***) levels by student’s t-test. (C) Localization of NF-kB was done on MCF-7, MDAMB-231, MDAMB-468 and MDAMB-453 breastcancercells that were fixed, permeabilized and labeled with anti-p65 subunit of NF-kB then nuclei were stained with DAPI. Controls cells were compared tocells treated with the top dose of PP (D3). Controls cells showed increased expression of NF- kB in the nucleus, whereas following treatment with PP, NF-kB accumulated in the cytoplasm, indicating decreased activity.
To confirm this hypothesis, we first examined the expression ofmiR-30c and found that the level of expression was significantly decreased in doxorubicin- resistant cell lines MCF-7/ADR and MDA-MB-231/ADR, compared with their corresponding parental cell lines MCF-7 and MDA-MB-231, respectively, which indicated that reduced miR-30c levels may be associated with doxorubicin resistance in breastcancer. To further investigate the function ofmiR-30c, we predicted binding sites for miR-30c in the YWHAZ 39-UTR by bioinformatic analysis. Western blot assay indicated that increased expression ofmiR-30c might have an impact on YWHAZ expression. Furthermore, we demonstrated that YWHAZ was a target gene ofmiR-30c by luciferase reporter assay. Then, we transfected the chemically synthesized miR-30c mimic oligonucleotides or YWHAZ siRNA into MCF-7/ADR cells. Results showed that restoration ofmiR-30c or inhibition of YWHAZ in MCF-7/ADR cells sensitized MCF-7/ADR cellsto doxorubicin. Importantly, experiments in vivo showed that doxorubicin significantly inhibited the growth of tumor cells in the miR-30c overexpression group, but not in the negative control group, which strongly confirmed our argument. Consistent with these findings, we also found that overexpression ofmiR-30c led to downregulation of YWHAZ and a more active signaling through the p38MAPK pathway, which contributed to reversing doxorubicin resistance in MCF-7/ ADR cells.
Until now, a handful of studies have identified specific miRNAs involved in human tumorigenesis and tumor progression , , . Therefore, we believe more effort should be made, not only towards the identification of relevant miRNAs but also to identify the specific mechanisms through which they accomplish their specific functions, particularly with regard to the oncogenesis of different types of tumors , , . )n this study, we used qRT‐PCR and )S( to show that miR‐ was frequently down‐regulated in gastric cancer, and % out ofof the gastric cancer had . ‐fold reduced expression ofmiR‐ as compared to their corresponding non‐tumorous tissues. )ntriguingly, we found that lower expression ofmiR‐ tended to have more advanced TNM stage stage )/)) vs. stage )))/)V, P= . , suggesting that low expression ofmiR‐ is associated with gastric cancer progression. Kaplan‐Meier survival analyses revealed that patients whose primary tumours displayed low expression ofmiR‐ had a shorter OS and RFS in gastric cancer. Further studies showed that overexpression ofmiR‐ suppressed gastric cancer cell proliferation and invasion, in vitro. The data from the current study suggests that miR‐ is important for gastric cancer initiation and progression and that down‐regulated miR‐ contributes to lymph node‐metastasis and tumor progression in gastric cancer patients.
Transcription factors are key molecules that finely tune gene expression in response to injury. We focused on the role of a transcription factor, Foxn1, whose expression is limited to the skin and thymus epithelium. Our previous studies showed that Foxn1 inactivity in nude mice creates a pro-regenerative environment during skin wound healing. To explore the mechanistic role of Foxn1 in the skin wound healing process, we analyzed post-injured skin tissues from Foxn1::Egfp transgenic and C57BL/6 mice with Western Blotting, qRT- PCR, immunofluorescence and flow cytometric assays. Foxn1 expression in non-injured skin localized to the epidermis and hair follicles. Post-injured skin tissues showed an intense Foxn1-eGFP signal at the wound margin and in leading epithelial tongue, where it co-localized with keratin 16, a marker of activated keratinocytes. This data support the con- cept that suprabasal keratinocytes, expressing Foxn1, are key cells in the process of re-epi- thelialization. The occurrence of an epithelial-mesenchymaltransition (EMT) was confirmed by high levels of Snail1 and Mmp-9 expression as well as through co-localization of vimen- tin/E-cadherin-positive cells in dermis tissue at four days post-wounding. Involvement of Foxn1 in the EMT process was verified by co-localization of Foxn1-eGFP cells with Snail1 in histological sections. Flow cytometric analysis showed the increase of double positive E- cadherin/N-cadherin cells within Foxn1-eGFP population of post-wounded skin cells iso- lates, which corroborated histological and gene expression analyses. Together, our findings indicate that Foxn1 acts as regulator of the skin wound healing process through engage- ment in re-epithelization and possible involvement in scar formation due to Foxn1 activity during the EMT process.
cells were suspended in serum-free stem cell medium containing DMEM/F12 (1:1) supplemented with B27 (Life Technologies, Gaithersburg, MD), 20 ng/ml EGF (Biomol International, Plymouth, PA), 20 ng/ml fibroblast growth factor (Biomol International, Plymouth, PA), and 100 m g/ml gentamy- cin. Approximately 150–200 cells per well were seeded in an ultra low-attachment 96-well plate (Corning Inc, Lowell, MA). 3- Cl-AHPC (1.0 m M) was added the day after cells were plated or after 7 days of sphere formation. Spheres were photographed and measured utilizing an Olympus microscope (OLYMPUS CKX41) and Olympus microscope digital camera with DP2- BSW software (Olympus soft imaging solutions GmbH, Germany). All statistics were performed using VassarStats web statistical software (Richard Lowry, Poughkeepsie, NY, USA). One-way analysis of variance (ANOVA) was performed to detect any differences between groups of sphere control, 3-Cl AHPC treated spheres. If the result of the ANOVA was significant (**P,0.01 vs control), pair wise comparisons between the groups were made by a post-hoc test (Tukey’s HSD procedure). The significance level was set at **P,0.01 vs control and *P,0.05 vs control. Square brackets were used in the figures to indicate treatments that are significantly different from the control.
opment. However, hurdles exist to the eventual translation of 20(S)-Rg3 to clinical use. One of them entails the lack of knowledge still about the molecular target(s) 20(S)-Rg3 directly interacts with to induce the various biological effects seen in this study. This paucity of 20(S)-Rg3 targets hinders the elucidation of precise upstream signaling events leading to the inhibition of hypoxia-induced EMT, and poses technical challenges to struc- ture-based function improvement of 20(S)-Rg3 for even better therapeutic efficacy in vivo. Moreover, since 20(S)-Rg3 is active against a variety of human cancers [29,30,45], it remains to be seen whether or not this ginsenoside also blocks EMT in other well-characterized tumor models. Our work reported here lays Figure 7. 20(S)-Rg3 suppressed ovarian cancer intraperitoneal dissemination in vivo. Mice injected i.p. with SKOV3 cells underwent 20(S)- Rg3 treatment for 30 days. (A) The body weights of nude mice with metastatic tumors were monitored every other day. Mice in 20(S)-Rg3 group lost less body weight than those given PBS. The overall tumor weight (B), the number of metastases (C) and the volume of ascites (D) were measured. Data are presented as the means (g) 6 SD (n = 7 per groups). *P,0.05, **P,0.01, for t-test(picture A, B and C) and Wilcoxon test (picture D), respectively. (E) The representative images of xenografts of spleen fascia and diaphragm. Those xenografts in 20(S)-Rg3-treated group were obviously smaller in size than those in control group.
Total RNA was purified using the RNeasy mini kit (Qiagen, Valencia, CA), and cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad) with 0.5 m g of total RNA according to the manufacturer’s recommendations. qPCR was performed with pre- designed primers selected from PrimerBank  and ordered from Integrated DNA Technologies (Coralville, IA). RT qPCR was performed using the Applied Biosystems 7900 real-time PCR system. All qPCR reactions were carried out in a final volume of 15 m l containing 1X of SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA), 300 nM of each gene specific primers, 100 ng cDNA, in sterile deionized water. The standard cycling condition was 50uC for 2 min, 90uC for 10 min, followed by 40 cycles of 95uC for 15 s and 62uC for 1 min. The results were analyzed using SDS 2.3 relative quantification manager software. The comparative threshold cycles values were normalized for GAPDH reference genes. qPCR was performed in triplicate to ensure quantitative accuracy.
OSCC is one of the leading causes ofcancer-related deaths worldwide . Its high invasiveness and frequent recurrence are the major reasons for treatment failures and a poor prognosis . Accumulating data demonstrate that a CSCs hypothesis has been bolstered by the clinical observation that malignant tumors are relatively resistant to chemo-radiotherapies [35,36]. OSCC- derived CSCs have been known to have the capacity to promote tumor progression and metastasis, and also contribute to radio- resistance and chemo-resistance [37,38]. Thus, it is necessary to unravel the underlying mechanisms of the CSC pathway in OSCC and to further evaluate the therapeutic possibilities of CSC- targeted therapy clinically. It is generally accepted that several Figure 4. Oct4 downregulation abrogate the cancer stemness in OSCC-CSCs. (A) Representative images of tumor sphere formation ability of control-GFP or Oct4-overexpressing OSCCs. (B) Single cell suspensions of OSCC-CSCs were transduced with sh-Luc or sh-Oct4 lentivirus for 3 days. Total proteins prepared from infected cells were prepared and analyzed. The silencing effect ofOct4 shRNA in OSCC-CSCs was validated translationally by western blotting (OECM1 (left panel) and SAS (right panel)). Immunoblotting against anti-Oct-4 or anti-GAPDH antibodies was performed as indicated. The amount of GAPDH protein of different crude cell extracts was referred to as a loading control, and for further quantification. (C) OSCC-CSCs were first infected with sh-Oct-4-1, sh-Oct-4 -2 or sh-Luc lentivirus for 3 days, and then further cultivated under the serum-free defined selection medium. The cellular morphology of OSCC-CSCs treated with sh-Luc or Oct-4-shRNA lentivirus was examined. (D) To elucidate the capability of cell invasiveness (upper panel) and anchorage independent growth (lower panel) of OSCC-CSCs, OSCC-CSCs with Oct-4 down-regulation, single cell suspension of OSCC-CSCs infected with Oct-4-specific shRNA or control sh-Luc lentivirus for three days were plated onto transwells coated with matrigel and soft agar, respectively, and analyzed as described in the Materials and Methods section. Results are means 6 SD of triplicate samples from three experiments. (E) Representative tumors of control and Oct4-knockdown OSCC-CSCs were generated, and the tumors were then dissected from the subcutaneous space of recipient mice (n = 3). (F) Kaplan–Meier survival analysis further indicated the mean survival rates of animals transplanted with control and Oct4-knockdown OSCC-CSCs.
Cholangiocarcinoma remains difﬁcult to detect in its early stages, which leaves patients with limited treat- ment options due to the late diagnosis and high rates of metastasis. In addition, response rates to chemotherapy in cholangiocarcinoma patients remain low, primarily due to the development of drug resistance (11). Chemoresis- tance (e.g., to doxorubicin) remains a big problem in the clinic, and may result from the induction of EMT in cancercells. Indeed, the EMT process has previously been Figure 4. Salinomycin (SAL) reduced the doxoru- bicin-induced expression of AMP-activated protein kinase family member 5 (ARK5) in chol- angiocarcinoma RBE and Huh-28 cells after treatment with doxorubicin (DOX), doxorubicin plus SAL, or SAL alone.