MiR-486-5p represses chondrocyte multiplication but facilitates their deaths in congenital microtia through aiming at H3F3B


 Background

MicroRNAs have been reported to play important roles in the development of external ear. The present study was designed to explore the expression patterns and biological function of miR-486-5p in congenital microtia.
Methods

Cartilage tissue specimens were collected from congenital microtia cases who received reconstructive ear surgeries, while the normal cartilage tissues from other otology surgeries were adopted as normal controls. The relative expression of miR-486-5p was investigated by quantitative-real time polymerase chain reaction. The potential target of miR-486-5p was explored by TargetScanHuman 7.2 and dual-Luciferase reporter assay. MTT and flow cytometry assays were adopted to investigate cell proliferation and apoptosis, respectively.
Results

The expression of miR-486-5p was significantly up-regulated in microtia cartilage tissues. Knockdown of miR-486-5p in microtia chondrocytes enhanced proliferation and inhibited apoptosis. H3F3B might be a target of miR-486-5p, and miR-486-5p could regulate proliferation and apoptosis of microtia chondrocytes by targeting H3F3B. Moreover, the enforced expression of H3F3B could re-turned the function of miR-486-5p in microtia chondrocytes.
Conclusion

Up-regulation of miR-486-5p may inhibit microtia chondrocytes proliferation and promote apoptosis by targeting H3F3B.


Results
The expression of miR-486-5p was signi cantly up-regulated in microtia cartilage tissues. Knockdown of miR-486-5p in microtia chondrocytes enhanced proliferation and inhibited apoptosis. H3F3B might be a target of miR-486-5p, and miR-486-5p could regulate proliferation and apoptosis of microtia chondrocytes by targeting H3F3B. Moreover, the enforced expression of H3F3B could re-turned the function of miR-486-5p in microtia chondrocytes.

Conclusion
Up-regulation of miR-486-5p may inhibit microtia chondrocytes proliferation and promote apoptosis by targeting H3F3B.

Background
Microtia is a kind of congenital malformation in external and middle ear [1]. According to the statistics, the prevalence of microtia is about 0.83-17.4/10000 newborns [2,3] and its vary depends on race, gender, and region [4][5][6]. The severity of microtia is ranged from slight ear defect to complete absence of the ear [6]. The occurrence of microtia is a complex multifactorial disease in uenced by genetic and environmental factors [7,8]. In previous studies, the education level and age of mother, history of disease in early pregnancy, childbearing and abortion histories, smoking and drinking during pregnancy are the risk factors of microtia [9][10][11][12]. However, only a fraction of individuals exposure to the above risk factors suffer from microtia, so genetic factors play the vital role in microtia occurrence.
MicroRNAs (miRNAs) are a class of small non-coding RNA in the length of about 18-25 nucleotides [13].
They can regulate the expression of targeted mRNA through binding to 3'UTR of target gene in posttranscriptional level [14]. MiRNAs have been reported to be involved in multiple biological behaviors, such as cell proliferation, differentiation, cycle and apoptosis [15,16]. The abnormal expression of miRNAs are found in congenital microtia patients [17]. miR-486-5p is a common member of miRNAs family and widely studied in various diseases [18,19]. It is reported to participate in the proliferation, apoptosis and metastasis of cells and may be as the promising diagnostic and prognostic biomarkers for some diseases [20]. miR-486-5p can inhibit the proliferation and migration of chondrocytes through regulating some important genes and/or pathways [21]. It is also found to be possibly involved in the development and advancement of microtia [22]. However, the exact role of miR-486-5p in the occurrence of microtia and the relative molecular mechanism are rarely explored in the previous studies.
Therefore, in the present study, we aimed to investigate the expression level of miR-486-5p in the cartilage tissues of congenital microtia patients and reveal its promising mechanism in microtia progression.

Patients and samples
In this research, 112 congenital microtia cases were enrolled in the Otorhinolaryngologic Department of Plastic Surgery Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College and they conformed to the diagnostic criteria of congenital microtia. The cartilage tissues were obtained from congenital microtia patients receiving ear reconstruction and immediately put into liquid nitrogen, then transferred to -80℃ refrigerator. At the same time, 104 individuals with normal ear receiving other ear operations were as the controls and their cartilage tissues of ear were obtained in surgery. The handling and storage of samples in the controls were as the cases. The controls were frequency-matched with the cases in age and gender.
All subjects were Chinese Han population without any blood relationship each other. This study obtained the support of the Ethics Committee of Plastic Surgery Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College and the whole study research process was informed all subjects. Written consents were signed by all study subjects.

RNA extraction and QRT-PCR assay
Totol RNA of cartilage tissue in congenital microtia patients and normal individuals were isolated using Trizol reagent (BBI, Kitchener, Ont., Canada) following the producer's speci cation. Then total RNA was as the template to synthesize cDNA using the RevertAdiTM First Strand cDNA Synthesis Kit (Ferments, Glenn Burnie, Md., USA). qRT-PCR reaction was conducted by the SYBR Green PCR master mix (Applied Biosystems, USA) on the 7300 Real-Time PCR System (Applied Biosystems, USA). U6 was as the internal reference to normalize the expression of miR-486-5p. Its relative expression was counted by 2 -∆∆Ct method. Every samples were repeated at least 3 times.
Cell culture Firstly, we isolated cartilage cells from cartilage tissue of congenital microtia cases through 0.25% trypsin and 0.02% collagenase . When the single free cell was observed under an inverted microscope, DMEM media was added to dilute collagenase solution for termination of the digestion, ltration, centrifugation at 1500r/min for 5min. Abandon supernatant, resuspended and putting into 37℃, 5% CO 2 incubator.
Nutrient solution was replaced three days at a time.

Cell transfection
When the cells were in logarithmic phase, they were conducted transfection. miR-486-5p mimics, inhibitors and corresponding negative control (NC) were respectively transfected into cartilage cells applying Lipofectamine 2000 (Invitrogen, CA, USA) following the producers' protocol. Then they were cultured at 37℃ containing 5% CO 2 for 48h. The transfection e ciency was detected using qRT-PCR method.

Cell proliferation assay
Cell proliferation ability was checked by MTT assay following the standardized protocol (Roche). After transfection, cartilage cells were inoculate in 96-well plates and the density of cells was adjusted to 2×10 4 /well. All groups were incubated for oh, 24h, 48h and 72h. 50μl MTT reagent (5mg/mL) was added into every well, then 37℃ incubation for 4h. The proliferation ability were determined using MTT cell proliferation kit (Cayman Chemical) based on the instruction. MTT enzyme-linked immunometric meter was used to measure OD value (570nm) so as to indicate the proliferation ability of cells .

Apoptosis assay
Cells were seeded into 24 well plates in the density of 1×10 6 /ml and incubated at 37℃, 5% CO 2 incubator overnight. Added 10μL PI and 400μL Annexin V-FITC (BD Biosciences, Detroit, MI, USA) in one after another to cells, incubated for 20min at room temperature, dark. Then ow cytometer was used to analyze the situation of cell apoptosis.

Double luciferase report assay
To explore the mechanisms of miR-486-5p in congenital microtia, TargetScanHuman 7.2 (updated March 2018) database analysis was used to investigate its targeted mRNAs. We found that miR-486-5p could bind to 3'UTR of H3F3B gene. To verify the target relationship between miR-486-5p and H3F3B, luciferase reporter assay was adopted. The 3'UTR region of H3F3B gene (H3F3B-wt), as well as the mutant 3'UTR region which did not contain the complementary sequence of miR-486-5p (H3F3B-mut), was synthesized and ligated into the luciferase reporter vector pGL3 (Promega, USA) following the production instruction. Then H3F3B-wt and H3F3B-mut were respectively cotransfected with miR-486-5p mimics or mimic-NC into cartilage cells by Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scienti c, Inc.). After incubation for 48h, the transfected cells were harvested, and their luciferase activity was evaluated using dual-luciferase reporter assay system (Promega Corporation) following the instruction.

Western blot
Cartilage tissues were lysed using radio immunoprecipitation assay (RIPA) buffer (Thermo Scienti c, Belmont, MA, USA) at 4℃ for 30min. Proteins of cartilage tissues were extracted and isolated by 10% sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE). They were transferred onto a PVDF membrane (Roche) and then were blocked by nonfat milk at 4˚C for overnight. Then it was incubated by primary antibodies anti-H3F3B (1:1000) (Abcam, Cambridge, UK) at 4℃ for overnight, GAPDH was as the internal reference, then were incubated at room temperature for 1.5h using second antibody (1:2000, Abcam, Cambridge, UK). The target band of protein was showed using ECL Western blotting kit (Millipore, Boston, MA, USA) according to the producers' instruction.
Statistical analysis SPSS 18.0 software (SPSS Inc., Chicago, IL, USA) was used for data statistics analysis and gures were plotted by GraphPad Prism (GraphPad, San Diego, CA, USA) software. All experiments were repeated at least in triplicate and the data were expressed using mean±SD (standard deviation). The difference between groups was compared by two-tailed Student's t-test or χ² test. P<0.05 was considered as the statistically signi cant difference.

Results
Up-regulated expression of miR-486-5p in cartilage tissues of congenital microtia We measured the expression level of miR-486-5p in cartilage tissues of congenital microtia patients and the controls by qRT-PCR. The results showed that miR-486-5p expression level was obviously upregulated in cartilage tissues of congenital microtia patients, compared with the controls (P < 0.001, Fig. 1).

The baseline data of congenital microtia patients and normal controls
We compared the baseline information of congenital microtia patients and normal controls by χ² test. The average age of the two groups were respectively 15.26 ± 10.23 and 16.61 ± 9.86 years old. There was no signi cant difference between the two groups in age (P = 0.239). The number of males and females in congenital microtia patients were 51 and 61, 43 and 59 in normal controls, the difference was not signi cant (P = 0.619). We didn't nd the difference between the two groups in body mass index (BMI), either (P = 0.567). The data were showed in Table 1.

BMI: body mass index
The in uence of miR-486-5p on the proliferation and apoptosis of cartilage cells in congenital microtia In this study, we transfected miR-486-5p mimics and mimic-NC into cartilage cells from cartilage tissues of congenital microtia patients. We found that the proliferation ability of cartilage cells was obviously lower in miR-486-5p mimics transfection group than that in the mimic-NC group (P < 0.05, Fig. 2).
H3F3B was as the target gene of miR-486-5p The targeted relationship of miR-486-5p with H3F3B was predicted by TargetScanHuman 7.2 website (Fig. 4A) and veri ed through double luciferase report assay. The results showed that miR-486-5p inhibitor signi cantly enhanced the luciferase activity of cartilage cells with H3F3B-wt in comparison with inhibitor-NC co-transfection group (P < 0.01, Fig. 4B). However, in cartilage cells with H3F3B-mut, there was no obvious different in luciferase activity between miR-486-5p inhibitors and inhibitor-NC groups (P > 0.05). Moreover, we found that the knockdown of miR-486-5p could remarkably promote the expression of H3F3B in cartilage cells (P < 0.001, Fig. 4C). Additionally, H3F3B mRNA and protein levels were found the signi cantly down-regulated expression in cartilage tissues of congenital microtia patients, compared with individuals with normal ear (P < 0.001, Fig. 4D).
miR-486-5p regulated the proliferation and apoptosis of cartilage cells in congenital microtia through H3F3B In our research, we also detected whether miR-486-5p mimics inhibited proliferation and promote apoptosis of cartilage cells trough directly targeting H3F3B. Firstly, we conducted the overexpression of H3F3B by pcDNA3.1 vector (OE-H3F3B) and empty-vector (OE-NC) was as the control. miR-486-5p mimics and OE-H3F3B was respectively and together transfected into cartilage cells. The results showed that H3F3B overexpression signi cantly reversed the inhibited proliferation ability resulted from miR-486-5p mimics transfection (P < 0.05, Fig. 5A). Furthermore, enhanced H3F3B expression also obviously reversed the promoted role of apoptosis caused by miR-486-5p overexpression in cartilage cells of congenital microtia (P < 0.01, Fig. 5B and C).

Discussion
In the current study, we explored the expression level of miR-486-5p in cartilage tissues of congenital microtia. The results showed that miR-486-5p expression was obviously up-regulated in cartilage tissues of congenital microtia in comparison to normal ear. In previous study, Li reported that 6 miRNAs were signi cantly up-regulated expression in the microtic group, compared with the normal controls, including miR-486-5p [22]. In the study of Wei, miR-486-5p expression was also found to be signi cantly higher in congenital microtia than that in the normal controls [17]. Our results were consistent with the previous studies. Hence, miR-486-5p plays an important role in the development of congenital microtia.
Microtia is a consequence of genetic factors combined with environmental factors [23]. So far, a number of genetic factors have been discovered to participate in the onset of microtia, including multiple miRNAs.
For instance, miR-193b-3p can promote the synthesis of chondrocyte extracellular matrix (ECM) in microtia patients [24]. In the research of Li, 6 up-regulated miRNAs and 5 down-regulated miRNAs were identi ed in microtic patients [22]. Moreover, some environmental factors are also reported to be involved in microtia occurrence, such as season and sex [25]. However, in our study, we found that there was no signi cant association between age, gender or BMI and microtia etiology.
Meanwhile, in our study, we also explored the possible mechanism of miR-486-5p in uencing microtia development. The results showed that miR-486-5p overexpression signi cantly inhibited the proliferation ability of chondrocytes in microtia, moreover, it also promoted the apoptosis of chondrocytes. miR-486-5p may regulate the progression of microtia through in uencing its chondrocyte proliferation and apoptosis.
In previous study, miR-486-5p was considered to inhibit the proliferation and migration of chondrocytes in osteoarthritis [21]. It also inhibited in ammatory response, ECM degradation and apoptosis of nucleus pulposus cells [26]. So, our outcomes were similar to the published articles.
Usually, miR-486-5p is found to take part in the development of disease through targeting some genes. Liu reported that miR-486-5p inhibited cell proliferation and induced apoptosis of leukemia cells by targeting FOXO1 [27]. It also promoted the cell proliferation, migration, invasion of endometrial carcinoma by through targeting MARK1 [28]. H3F3B plays the key role in the maturation and differentiation of chondrocyte [29] and serves as a target gene of miR-486-5p according to the report [30] and database analysis. However, whether miR-486-5p in uences the progression of microtia through targeting H3F3B is rarely explored in previous study. Our study found that H3F3B expression was remarkably down-regulated in microtia patients in comparison to normal controls. miR-486-5p knockdown inhibited the luciferase activity in chondrocytes with H3F3B-wt, but not H3F3B-mut cells. Moreover, the knockdown of miR-486-5p signi cantly enhanced H3F3B expression, which indicated H3F3B was a target gene of miR-486-5p. in addition, the rescue assays showed that H3F3B overexpression obviously reversed the inhibited role caused by miR-486-5p mimics transfection for chondrocyte proliferation and promoted role for the apoptosis. Hence, miR-486-5p inhibited the proliferation and promoted the apoptosis of cartilage cells in congenital microtia through targeting H3F3B.
Although our study obtained some achievements about the expression pattern of miR-486-5p in congenital microtia and the potential mechanism, several shortcomings should be paid attention. Firstly, the sample size was relatively small, which might cause the result deviation. Only one population was included in this study, the practicability was limited. Multiple target genes of miR-486-5p are reported, but only H3F3B was explored in our study. Besides, in vivo experiment was not conducted to verify our results. Therefore, further studies are needed to overcome the above limitations and verify the present outcomes.

Conclusions
In 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. The expression level of miR-486-5p in cartilage tissue of congenital microtia and normal ear. *** P<0.001.

Figure 3
The apoptosis of cartilage cells of congenital microtia was checked by ow cytometry. The results showed that the apoptosis ability of cartilage cells was obviously higher in miR-486-5p mimics group than that in mimic-NC group. *** P<0.001.

Figure 4
The targeted relationship between miR-486-5p and H3F3B. The 3'UTR of H3F3B has the binding site of miR-486-5p (A). miR-486-5p inhibitors can obviously increase luciferase activity in cartilage cells with H3F3B-wt, compared with inhibitor group, however, the difference was not signi cant in cartilage cells with H3F3B-mut (B). The knockdown expression of miR-486-5p resulted in increased expression of H3F3B in cartilage cells of congenital microtia (C). The expression of H3F3B mRNA and protein in cartilage tissues of congenital microtia and normal ear. Lane 1 and 2: normal ear; Lane 3 and 4: congenital microtia (D). **: P<0.01, ***: P<0.001. Figure 5 miR-486-5p regulated the proliferation and apoptosis of cartilage cells in congenital microtia patients. The results showed that H3F3B overexpression reversed the inhibited role of miR-486-5p mimics transfection for the proliferation of cartilage cells (A). H3F3B overexpression expression also signi cantly reversed the promoted role of miR-486-5p mimics for the apoptosis of cartilage cells (B, C). *: P<0.05, **: