Boosting effects of MiR-4262 on acute myeloid leukemia advancement via governing KLF6

Background: Purpose of this study was to explore the inuence of miR-4262 on the progression of acute myeloid leukemia (AML) and its molecular mechanism. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to assess the expression of miR-4262 in AML serum and cell lines. MTT, Transwell assays and ow cytometry were adopted to investigate the effect of miR-4262 on cell proliferation, invasion, migration and apoptosis abilities of HL-60 cells respectively. Luciferase reporter assay was conducted to reveal the target relationship of miR-4262 and KLF6. Western blot analysis was utilized to evaluate the expression level of proteins. Results: Relative expression of miR-4262 was up-regulated in AML serum and cell lines (P<0.05). miR-4262 expression was closely related to FAB classication (P=0.002) of AML patients. miR-4262 mimics could promotes the proliferation, invasion and migration of HL-60 cells, while miR-4262 inhibitor is obviously weakened these biological behaviors. Luciferase assay illustrated that miR-4262 can directly interact with KLF6 3’UTR. Up-regulation of miR-4262 could decrease KLF6 level, and increase EGFR level, while the down-regulation of miR-4262 showed the opposite results. Moreover, KLF6 gene knockdown reversed the results caused by miR-4262 inhibitor (P<0.05). Inhibition of KLF6 could signicantly promoted the proliferation, invasion and migration of HL-60 cells which is caused by miR-4262 inhibitor. Conclusions: miR-4262 was obviously increased in AML serum and cells, it promotes the progression of AML by regulating KLF6.

showed that miR-4262 is a cancer gene, which promotes the proliferation of skin malignant melanoma cells through KLF6 mediated EGFR inactivation and p21 up regulation [14]. miR-4262 is up-regulated in the bone marrow and serum of AML patients, which is a useful tool for predicting the development and prognosis of AML [15]. This suggested that miR-4262 plays an important role in AML. Unfortunately, miR-4262 in AML is still poorly elucidated. Therefore, it is necessary to verify the mechanism of miR-4262 in the development of AML.
In our research, we explored the expression pattern of miR-4262 in AML serum and cells, as well as investigated the target gene of miR-4262. In addition, the possible mechanism of miR-4262 in AML progression was also revealed.

Patients and specimens
This study collected 128 AML patients who visited PanYu Central Hospital. Meanwhile, 109 healthy volunteers were enrolled into the study as controls. The controls were frequently matched with cases both in age and gender No patients had received any treatment including radiotherapy or chemotherapy before diagnosis. Individuals younger than 18 years old, had other tumors, systemic diseases were excluded from this study. This study was approved by the Ethic committee of PanYu Central Hospital, and written informed consents were also signed by all patients or their family before sampling.
Blood specimens were taken from 128 AML patients and 109 healthy volunteers. Then the blood samples were put into blood collection tube of EDTA and centrifuged to collect leukocytes. Leukocytes were stored at -80℃ until used. Clinicopathological features of the patients were listed in Table 1, including age, gender, WBC, FAB classi cation, immunophenotype and lymphadenopathy.
Cell culture Human leukemia cell lines HL-60 and human embryonic kidney cell lines 293T were purchased from the American Type Culture Collection (ATCC; Rockefeller, MD, USA). Cells were cultured in RPMI 1640 media with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/mL penicillin and 10 mg/mL streptomycin). Then they were incubated at 37℃ humididied atmosphere containing 5% CO 2 for cell passage. The media was replaced every two days.

Cell transfection
When the cell density reached approximately 70% con uency, the cells were transfected. KLF6 siRNA, KLF6 siRNA-NC, miR-4262 mimics, miR-4262 mimics NC (negative control), miR-4262 inhibitor, miR-4262 inhibitor NC were purchased from GenePharma (Shanghai, China). They were transfected into HL-60 cells by Lipofectamine TM 2000 (Invitrogen, CA, USA) according to the manufacturer's instruction, then incubated at 37℃ humidi ed atmosphere with 5% CO2 for 6h. Medium was replaced and continuing cultivated for 48h. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify whether the transfection was successful.
qRT-PCR miRNA from leukocytes was performed with the miRNeasy TM RNA isolation kit (Qiagen, Valencia, CA, USA). RNAs of cells were extracted using Trizol method (TaKaRa, Japan). RNA was reverse-transcribed into cDNA by PrimerScript RT reagent kit (Takara, Dalian, China) according to the instruction. PCR ampli cation was conducted by SYBR Premix Ex TaqTM II kit in ViiATM 7 real-time uorescent quantitative PCR system. Expression of KLF6 were normalized to GAPDH, and miR-4262 was normalized to U6. Relative expression of miR-4262 was analyzed by 2 -∆∆CT method. Every sample was measured at least three times.
MMT assay MTT method was used to detect the proliferation of HL-60 cells. Cells transfected with miR-4262 mimics, miR-4262 mimics NC, miR-4262 inhibitor, miR-4262 inhibitor NC were adjusted to 2×10 4 /mL and inoculated on 96-well plates. They were cultured at incubator containing 5% CO 2 at 37℃ for 0h, 24h, 48h and 72h respectively. 50μl MTT solution (5mg/mL) were added to cells, then continuously incubated for 4h. Next step, 150μl 20% SDS was added to every well, incubation for overnight at room temperature. Cell proliferation was determined by MTT cell proliferation kit (Cayman Chemical) following the manufacturer's instruction. MTT enzyme-linked immunometric meter was used to measure OD value (490nm).

Transwell assays
Transwell assays were used for the detection of the migration and invasion abilities of HL-60 cells.
Transwell chambers were pre-coated by matrigel (BD Biosciences) and 50μl serum-free medium with BSA were added to the upper compartment, 37˚C dehydration for 2h. Then upper chamber was added 200μl cell suspension and the lower chamber was 500μl DMEM media with 10% FBS. Then cells were incubated at 37˚C incubator with 5% CO 2 for 48h. The invasive cells were stained with 0.1% crystal violet for 30 min. Under the microscope the cells were counted in 7 random sights.
Meanwhile, without matrigel transwell assay was conducted to measure the migrative ability of HL-60 cells.

Apoptosis Analysis
Forty-eight hours after transfection, cells were harvested, stained with propidium iodide and anti-annexin-V antibody (Annexin V-FITC Apoptosis Detection kit, BD Biosciences, San Jose, CA, USA) following the manufacturer's protocol, and stained cells were detected by ow cytometry. The experiments for the apoptosis assay were performed at least three times.

Western blot
Transfected cells were lysed using RIPA buffer (Thermo Scienti c, Belmont, MA, USA) at 4˚C for 30min.
Cells proteins were extracted and separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane (Roche) by electroblotting. Nonfat milk was used to block PVDF membrane at 4˚C for overnight. Then it was incubated by primary antibodies at 4℃ for overnight, including anti-KLF6 (1:300), anti-EGFR (1:400) (Abcam, Cambridge, MA, USA), and anti-GAPDH (1:800; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), which was used as the internal reference. Then they were incubated 1.5h at room temperature using second antibody (1:2000, Abcam, China). Target band of protein was showed using ECL Western blotting kit (Millipore, Boston, MA, USA).
Statistical analysis SPSS 18.0 software (SPSS Inc., Chicago, IL, USA) was used for data statistics analysis and GraphPad Prism (GraphPad, San Diego, CA, USA) software was applied for plotting. All experiments were repeated at least in triplicate and the data were expressed using mean±SD (standard deviation). Difference between groups was compared by Student's t-test (Mann-Whitney U test) or χ² test. Two-tailed P<0.05 indicated statistically signi cant difference.
Results miR-4262 expression was up-regulated in AML patients and HL-60 cells QRT-PCR results showed that relative miR-4262 level in AML patients serum was signi cantly increased, compared with healthy controls (P<0.001, Figure 1A). Similarly, miR-4262 expression level in HL-60 cells was also signi cantly higher than that in 293T cells (P<0.05, Figure 1B).
Relationship between miR-4262 expression and AML clinical features 128 AML patients were divided into two groups according to the median expression level of miR-4262: low expression (n=63) and high expression (n=65) groups. miR-4262 expression was closely associated with FAB (French-American-British) classi cation (Table 1, P=0.002) of AML patients, but it's not related to age, gender, white blood cell count (WBC), immunophenotype and lymphadenopathy (P>0.05). miR-4262 transfection e ciency in HL-60 cells Relative expression level of miR-4262 was measured by qRT-PCR in HL-60 cells transfected by miR-4262 mimics, miR-4262 inhibitor and corresponding negative controls to detect the transfection e ciency. The results showed that the relative expression level of miR-4262 in miR-4262 mimics group was signi cantly higher than that in miR-4262 mimics NC group. miR-4262 expression level in miR-4262 inhibitor group was signi cantly decreased, compared with the miR-4262 inhibitor NC group (P<0.05, Figure 2). miR-4262 regulated the proliferation, migration, invasion and apoptosis of HL-60 cells MTT assay showed that the proliferation ability of HL-60 cells was signi cantly increased in miR-4262 mimics group, while that in miR-4262 inhibitor group was signi cantly reduced, respectively compared with the corresponding controls (P<0.05, Figure 3A). Transwell assays indicated that the migration and invasion number of HL-60 cells in miR-4262 mimics group were signi cantly higher than that in miR-4262 mimics NC group. Moreover, miR-4262 inhibitor signi cantly reduced the migration (P<0.05, Figure 3B) and invasion (*, P<0.05, **, P<0.01, Figure 3C) number of HL-60 cells. In addition, ow cytometry assays suggested that miR-205 mimics group signi cantly reduced the apoptosis ability of HL-60 cells, while that of miR-4262 inhibitor group was signi cantly increased, respectively compared with the corresponding controls (*, P<0.05, **, P<0.01, Figure 3D). Therefore, miR-4262 could in uence the biological behaviors of AML cells.
KLF6 is identi ed as a target gene of miR-4262 Targetscan 7.2 software found that miR-4262 speci cally targeted 3'UTR region of KLF6. In this study, the cells transfected with KLF6-3'UTR-WT, miR-4262 mimics had signi cantly lower luciferase activity; when the binding site was mutated, the above inhibition was lost ( Figure 4). So, miR-4262 could play a role in AML by combining with KLF6 promoter, and KLF6 is identi ed as a target gene of miR-4262. miR-4262 regulated EGFR expression in HL-60 cells miR-4262 mimics could signi cantly inhibit the protein level of KLF6, while miR-4262 inhibitor could signi cantly enhance the protein level of KLF6 ( Figure 5). Meanwhile, EGFR protein expression levels were signi cantly up-regulated in HL-60 cells with miR-4262 mimics transfection, but signi cantly decreased in HL-60 cells transfected by miR-4262 inhibitor ( Figure 5), respectively compared with the controls.

miR-4262 promotes HL-60 cells proliferation, invasion and migration through KLF6 mediated EGFR
When miR-4262 inhibitor + KLF6 siRNA was transfected, the expression of KLF6 protein in HL-60 cells decreased signi cantly, while the expression of EGFR protein increased signi cantly ( Figure 6). Moreover, we found that the reduced proliferation, migration and invasion ability of HL-60 cells caused by miR-4262 inhibitor was reversed by KLF6 knockdown (Figure 7 A-C). The inhibition of KLF6 expression was also signi cantly reversed the enhanced the apoptosis ability of HL-60 cells caused by miR-4262 downexpression (Figure 7 D).

Discussion
AML is a highly invasive malignant disease of hematopoietic system, which is characterized by proliferation enhancement, differentiation retardation and apoptosis disorder [16,17]. Accumulated data show that miR-4262 plays an important role in various human diseases [18,19]. AML has the highest mortality, and abnormal expression of miRNAs is involved in the pathogenesis of AML [20]. Therefore, we analyzed the effect of miR-4262 on the biological behavior and progression of AML in this study.
Previous studies have shown that miR-4262 is up-regulated in non-small cell lung cancer and glioma tissues, and participates in biological processes such as cell proliferation, migration and apoptosis [21,22]. In our study, we proved that the expression of miR-4262 was signi cantly up-regulated in AML patients and cell lines. This is consistent with the results of a previous study [15]. We also found that the miR-4262 mRNA expression was closely related to FAB classi cation. That is also consisten with previous study. Han et al. Indicated that increased miR-4262 expression was obviously associated with FAB classi cation and cytogenetics [15]. So, miR-4262 may be a novel potential therapeutic strategy for AML.
Expression of miR-4262 in breast cancer tissues and cell lines is increased, which plays the role of oncogene and promotes the proliferation and invasion of breast cancer cells through directly targeting KLF6 and KLF15 [23]. However, Weng et al showed that the expression of miR-4262 in colon cancer was signi cantly decreased, which may be due to the different expression trends of miR-4262 in different diseases [18]. In our study, miR-4262 mimics could promotes the proliferation, migration and invasion, as well as inhibits the apoptosis of AML cells, while miR-4262 inhibitor has the opposite results. In addition, previous studies have shown that a large number of miRNAs are up-regulated in AML, including miR-216b, miR-23a-3p and miR-210 [24][25][26], and they play an important role in the process of AML. Therefore, miR-4262 might play an important role in the development and progression of AML.
Wang et al. demonstrated that miR-4262 targets the 3'-UTR region of KLF6 and KLF15 mRNA [23]. In our research, we con rmed that KLF6 is the target gene of miR-4262 by luciferase reporter gene analysis. In addition, the overexpression of miR-4262 can inhibit the expression of KLF6, while the transfection of miR-4262 has the opposite effect. A previous study found that KLF6 was down regulated in AML [27]. This suggests that miR-4262 may play a role in AML through KLF6.
Zhang et al. Proved that miR-4262 promotes the proliferation of human cutaneous malignant melanoma cells through KLF6-mediated EGFR inactivation and p21 upregulation [14]. Zhuang et al. found that low expression of GLUT3 could promote apoptosis and chemosensitivity of AML cells through EGFR signal [28]. A previous study found that in lung adenocarcinoma, whether in vivo or in vitro, KLF6 is negatively regulated by the activated EGFR signal, and inhibition of EGFR signal can increase KLF6 expression [29]. In our study, we found that the expression of miR-4262 was positively correlated with the expression of EGFR, and the inhibition of KLF6 could signi cantly promoted the progression of HL-60 cells with miR-4262 down-expression. This indicated that miR-4262 promotes the AML process by targeting KLF6 to regulate the expression of EGFR.
Several shortcomings in this study should be not avoided, although we obtained some achievements. Firstly, the sample size was relatively small, which may reduce the accuracy of our results. Secondly, only one cell line was used in this study, so this result should be veri ed in other cell lines. Moreover, in vivo experiment was not conducted to verify the relative results. Therefore, further studies are needed to verify our results with well-design and large sample size.

Conclusions
In conclusion, the expression of miR-4262 is signi cantly increased in AML patients and cell lines. The subjects had been informed the objective. Certainly, written consents were signed by every subject in this study.

Consent for publication
We obtaining permission from participants to publish their data.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.     In uence of miR-4262 expression on biological behaviors of HL-60 cells. A, MMT assay showed that miR-4262 mimics signi cantly increased the proliferation of HL-60 cells and miR-4262 inhibitor reduced cell proliferation; B, Transwell analysis indicated that miR-4262 mimics obviously enhanced the migration of HL-60 cells and miR-4262 inhibitor inhibited cell migration; C, miR-4262 mimics obviously increased the invasion of HL-60 cells and miR-4262 inhibitor decreased cell invasion. D, Flow cytometry assay suggested that miR-4262 mimics obviously decreased the apoptosis of HL-60 cells and miR-4262 inhibitor enhanced cell apoptosis. *P<0.05, **P<0.01.

Figure 4
Luciferase activity assay. KLF6 was as the target binding site of miR-4262; *, P<0.05. Western blot analysis for expression of KLF6 and EGFR in HL-60 cells. miR-4262 mimics could inhibit relative KLF6 expression and enhance EGFR expression levels than that in corresponding controls. miR-4262 inhibitor could promote relative KLF6 expression and suppress EGFR expression levels than that in corresponding controls. *, P<0.05, **P<0.01.

Figure 6
Expression of KLF6 and EGFR were examined by western blot analysis in co-transfected HL-60 cells. In miR-4262 inhibitor + KLF6 siRNA cells, KLF6 expression was decreased signi cantly, while EGFR expression was increased signi cantly*, P<0.05, **P<0.01. Figure 7 miR-4262 promotes the biological behaviors of HL-60 cells through KLF6 mediated EGFR. A, MMT assay demonstrated that cell proliferation was signi cantly enhanced in miR-4262 inhibitor+si-RNA group than in miR-4262 inhibitor+siRNA-NC group; B, Migration of HL-60 cells was obviously decreased in miR-4262 inhibitor+siRNA group; C, Invasion of HL-60 cells was obviously decreased in miR-4262 inhibitor+siRNA group; D, Apoptosis ability of HL-60 cells was obviously increased in miR-4262 inhibitor+siRNA group. **P<0.05 and **P<0.01.