Upregulation of miR-181c inhibits chemoresistance by targeting ST8SIA4 in chronic myelocytic leukemia

Chemotherapy resistance frequently drives tumor progression. Increased expression of ST8SIA4 has been reported in diverse carcinomas and highly correlates with leukemia multidrug resistance (MDR). MicroRNAs (miRNA) are widely recognized as key players in cancer progression and drug resistance. Here, to explore whether miRNA modulates the sensitivity of chronic myelocytic leukemia (CML) to chemotherapeutic agents and regulates ST8SIA4 expression, we analyzed the complete miRNA expression profile and found a subset of miRNAs specifically dysregulated in adriamycin-resistant CML cell line K562/ADR and its parent cell line K562. Compared with three pairs of CML cell lines and 38 clinical samples of peripheral blood mononuclear cells (PBMC) of CML patients, miR-181c expression was down-regulated in drug-resistant cell lines and CML/MDR samples. Altered expression levels of miR-181c influenced the MDR phenotypes of K562 and K562/ADR. Reporter-gene assay showed that miR-181c directly targeted and inhibited the ST8SIA4 expression, as well as miR-181c was inversely correlated with the levels of ST8SIA4 expression in CML cell lines and samples. Moreover, ST8SIA4 could reverse the effect of miR-181c on drug resistance in K562 and K562/ADR cells in vitro. Upregulation of miR-181c sensitized K562/ADR cells to adriamycin in vivo through directly suppressing ST8SIA4 expression. Further investigation showed that miR-181c mediated the activity of phosphoinositide-3 kinase (PI3K)/AKT signal pathway, and inhibition of PI3K/Akt in K562 cells counteracted miR-181c-mediated MDR phenotype. These data revealed an important role for miR-181c in the regulation of chemoresistance in CML, and suggested the potential application of miR-181c in drug resistance treatment.


INTRODUCTION
Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by BCR-ABL fusion gene [1].Over the past two decades, major advances have been achieved in the treatment of cancers thanks to the highly potent antineoplastic drugs.However, chemoresistance is still a significant obstacle for successful chemotherapy of CML patients.
MicroRNAs (miRNAs) are a class of regulatory non-coding RNAs of 19-25 nucleotides which act by targeting specific messenger RNAs (mRNAs) for degradation or inhibition of translation through base pairing to partially or fully complementary sites [2].It has been demonstrated that miRNAmediated gene regulation was involved in multiple biological processes, such as cell proliferation, migration and invasion, differentiation, survival, and tumorigenesis [3].It has been reported that miRNAs play important roles in chemotherapeutic resistance [4], highlighting miRNAs as potent therapeutic targets or chemoresistant modulators in cancer treatment.Ectopic expression of miR-370 sensitized K562 cells to homoharringtonine and partially targeted FoxM1 by inducing apoptosis [5].Additionally, inhibition of miR-21 by antagomiR-21 markedly increased

Research Paper
apoptosis induced by imatinib in CML [6], and forced expression of miR-217 sensitizes dasatinib-resistant K562 cells to dasatinib [7].More recently, miR-17 and miR-20a have been demonstrated to be involved in resistance of the leukemia cells to chemotherapeutic drug VP-16 mediated by BIM-S [8].
Sialic acids are negatively charged nine-carbon carboxylated monosaccharides on glycosylated proteins and lipids formed due to post translational modification [9].Most cell surface glycans are highly sialylated and often involved in cell-cell and/or cellextracellular matrix interaction [10].High level of ST3Gal III sensitizes ovarian cancer cells to Taxo [11].Endogenous expression of ST6GalNAc I in CML cell line K562 was associated with the expression of the STn O-glycan related to a lack of response to chemotherapy [12].In many tumors, continued activation of phosphatidylinositol-3-OH kinase (PI3K)-Akt pathway has been implicated as a mechanism of resistance to cytotoxic chemotherapy agents [13,14].ST8SIA4 has been reported to enhance the chemoresistance in leukemia by phosphorylating and activating of Akt at Ser473 and Thr308 specifically and increasing the activity of PI3K/AKT signaling pathway [15,16].
By combining microarray analyses and real-time PCR, we found that miR-181c levels remained low in K562/ADR cells but became markedly higher in K562 cells.Other study has reported that upregulation of miR-181c contributes to chemoresistance in pancreatic cancer.Based on the above, we selected miR-181c for analysis.By miRNA target prediction algorithms, and experimental validation approaches, the objective of this study was to reveal the molecular mechanisms of miR-181c in adriamycin-resistant CML cancer.

MiRNA-181c is downregulated in chemoresistant CML cell lines and CML/MDR patients
A screen to identify miRNAs involved in adriamycin resistance was performed in K562 and K562/ADR cell lines, which were made adriamycin-resistant by continuous exposure to adriamycin in vitro.We identified 41 miRNAs to be dysregulated at least 5-fold in resistant compared to adriamycin sensitive parental cells (Table 1).Supervised hierarchical clustering using the 41 differentially expressed miRNAs clustered cell lines according to miRNA expression rather than doxorubicin response (Figure 1A).We found that miR-181c levels remained low in K562/ ADR cells but became markedly higher in K562 cells.We further confirmed miR-181c expression using real-time PCR.As shown in Figure 1B, the expressions of miR-181c were decreased in K562/ADR, KU812/ADR, KCL22/ADR cells.To identify the expression level of miR-181c in CML patients, the PBMC isolated from CML patients was also analyzed.The PBMC were first divided into two groups, CML without MDR and CML/MDR.The frequency of P-gp positivity was 57.9% (22 of 38) in the CML patients.As shown in Figure 1C, miR-181c expression was increased in CML patients compared with that in CML/MDR patients.We hypothesized that miR-181c might functionally regulate therapy response.We therefore focused on its role in chemoresistance regulation.

MiR-181c signature impacts chemoresistance of K562 and K562/ADR cells in vitro
The role of miR-181c in chemoresistance was characterized by endogenously silencing miR-181c in K562 cells.As shown in Figure 2B, transient transfection of antagomiR-181c in K562 cell resulted in an increase in cell viability to ADR, VCR and Taxol.Moreover, increased IC50 values for chemotherapeutic agents were observed, after downregulation of miR-181c (Figure 2C).In addition, flow cytometry analysis showed that inhibition of miR-181c led to a decrease in the apoptosis rate (Figure 2D).The role of miR-181c in chemoresistance was further examined by upregulation of miR-181c.K562/ ADR cells were transiently transfected with miR-181c or mimic control.Quantitative RT-PCR confirmed that the transient transfection of miR-181c effectively enhanced the expression of miR-181c (Figure 3A).Conversely, transient transfection of miR-181c resulted in a significant decrease in cell viability of K562/ADR cells to ADR, VCR and Taxol (Figure 2B).Furthermore, overexpression of miR-181c sensitized cells to chemotherapy, as indicated by a decrease in the IC50 (Figure 3C).Moreover, flow cytometry analysis confirmed that miR-181c significantly increased the apoptosis of these cancer cells upon drug treatments (Figure 3D).Collectively, these results indicated that downregulation of miR-181c promotes chronic myelocytic leukemia cell resistance to chemotherapeutic drugs in vitro.Recently, the phosphatidylinositol-3-kinase (PI3K) also has been implicated in the signalling events initiated by NCAM [15].ST8SIA4 seems to be the major polyST of the adult brain [16].Moreover, constitutive activation of PI3K/Akt is associated with the expression of ST8SIA4 [17,18].In addition, our previous study has demonstrated it was involved in the development of multidrug resistance in human leukemia [19,20].So, among the search results, ST8SIA4 captured our attention.Then, we investigated whether ST8SIA4 was a target of miR-181c in CML cells.Luciferase reporter constructs were made, containing the putative binding sites of ST8SIA4-wt-3'UTR(222-243) regions, ST8SIA4-wt1-3'UTR(2117-2140) regions or the mutant 3'-UTR regions (mut:ST8SIA4-mut-3'UTR(222-243), mut1:ST8SIA4-wt1-3'UTR(2117-2140)) of these transcripts within miR-181cbinding seed regions.

ST8SIA4 is a target of miR-181c in CML cells
Forced miR-181c expression decreased luciferase activity, and this suppression was reversed by the mutation of the target sequences in the 3'-UTR of ST8SIA4 (Figure 4A).Western blotting analysis showed that the inhibition of miR-181c increased ST8SIA4 expression levels in K562 cells.Whereas, ectopic expression of miR-181c by transient transfection decreased the protein expression of ST8SIA4 in K562/ADR cells (Figure 4B).Furthermore, analysis of miR-181c and ST8SIA4 expression in CML patients by Spearman's correlation analysis showed a significant inverse correlation (r= -0.7355, P =0.000; Figure 4C) Taken together, the results indicated that miR-181c targets ST8SIA4 to repress its expression by binding to 3'-UTR regions.

ST8SIA4 reverses the effect of miR-181cmediated chemoresistance in K562 and K562/ ADR cells
To assess the possible role of ST8SIA4 in the chemoresistance-mediating capability of miR-181c, we performed functional loss assays by co-transfecting ST8SIA4-shRNA plasmid and antagomiR-181c in K562 cells.Western blotting analyses showed a decrease of ST8SIA4 protein in antagomiR-181 transfected clone (K562-antagomiR-181c) (Figure 5A), indicating the effective inhibition of the wild-type protein.
While suppression of miR-181c expression promoted chemoresistance, knockdown of ST8SIA4 reversed antagomiR-181c-mediated sensitivity of the K562 cells to ADR, VCR and Taxol (Figure 5B).Re-introducing the wild-type form of ST8SIA4 in K562/ADR cells led to a marked increase in the protein expression upon transfection with the miR-181c compared to control (Figure 5C).As shown in Figure 5D, while miR-181c expression induced inhibitory effects on chemoresistance, recombinantly expressed ST8SIA4 reversed miR-181c-mediated sensitivity of K562/ADR cells to drug treatment (Figure 5D).Equally important, knockdown of ST8SIA4 copied the phenotype of high miR-181c levels by sensitizing cells to chemotherapeutic agents [19].These results suggested that ST8SIA4 is responsible for mediating the effects of miR-181c on chemoresistance in CML cell lines.

MiR-181c regulates chemoresistance via ST8SIA4 dependent PI3K/AKT signaling pathway
It has been reported that inhibition of PI3K/AKT signaling has proven to be an efficient way to attenuate the resistance of chemotherapy [36].Our previous results showed that PI3K/AKT pathway is activated in K562/ ADR cells compared with its parental cells K562, and ST8SIA4 was positive regulators of PI3K/AKT pathway.In present study, given that the expression of ST8SIA4 was down-regulated by miR-181c in CML cells (Figure 4), we further investigated whether dysregulation miR-181c altered the activation of PI3K/AKT pathway.As shown in Figure 6A, a significant increase in PI3K p110α, phospho-Akt 308, phospho-Akt 473 and NF-κB protein was observed when miR-181c was inhibited in K562 cells.By contrast, there was no change in the total amount of Akt protein, demonstrating a truedecrease in phosphorylation status.In addition, after overexpression of miR-181c in   K562/ADR cells, we observed a noticeable decrease in protein expression of PI3K110α, p-Akt 308, p-Akt 473 and NF-κB (Figure 6B).Together, the data confirmed that augmented PI3K/AKT pathway expression accompanying miR-181c deficiency was also associated with activation of PI3K/AKT.
To further support the role of PI3K/AKT signaling in regulation chemoresistance by miR-181c, Akt shRNA was utilized in K562 cells.The protein levels of the main signal molecules of PI3K/AKT pathway were analyzed by western blotting.Our results indicated that in K562-antagomiR-181c cells, the protein levels of PI3K/AKT pathway were decreased in Akt shRNA treatment group compared to control group (Figure 6C).In addition, inhibition of PI3K/AKT pathway made the K562-antagomiR-181c cells susceptible to chemotherapy (Figure 6D).These data indicated the involvement PI3K/ AKT pathway in suppression of drug resistance by miR-181c.

MiR-181c decreases chemoresistance of K562/ ADR cells in vivo
The promotive effect of miR-181c on CML cell chemoresistance was further examined in vivo.Mice were inoculated subcutaneously (1 × 10 7 K562/ADR cells per mouse) in the right flank.One week later, the mice were randomly divided into four groups (n = 6/ group, mimic control group, mimic control+ADR group, miR-181c group, miR-181c+ADR group).Mice were intratumorally injected with mimic control or miR-181c mimic three times per week for three weeks,combining with intraperitoneal injection of adriamycin (7mg/kg) weekly or PBS.The tumor volumes were decreased in the miR-181c group,as compared to the mimic control group (Figure 7A).Significantly, the combined miR-181c and adriamycin treatment markedly restricted the tumor growth to low volumes.These results suggested that miR-181c decreased adriamycin resistance and tumor growth, while injection of miR-181c sensitized K562/ADR cells to adriamycin treatment and inhibited tumor growth.Meanwhile, tumors injected with miR-181c had decreased Ki67, ST8SIA4, PI3K p110α, p-Akt 308, p-Akt 473 and NF-κB expression, analyzed by immunohistochemistry (Figure 7B and 7C).These results further suggest that miR-181c and its downstream PI3K/AKT pathway play important roles in controlling K562/ADR cells adriamycin sensitivity in vivo.

DISCUSSION
MiRNAs can regulate various biological processes in tumorigenesis, metastasis and chemoresistance.In the current study, we reported that specific miRNA expression signatures characterized and contributed to the MDR phenotypic of CML cell lines.Among them, we validated miR-181c, which was downregulated in chemoresistant CML cell lines and CML/MDR patients.Furthermore, miR-181c could exert chemoresistant functions and impede CML drug resistance, partly through targeting ST8SIA4 and its downstream PI3K/AKT pathway both in vitro and in vivo.Our data provide a insight into the function of miR-181c in regulating ST8SIA4 and drug resistance in CML cells.
The miR-181 family contains four miRNAs (miR-181a/b/c/d).Recent studies showed that miR-181 also play a vital role in chemotherapeutic resistance.Restoration of miR-181a expression could sensitize K562/A02 and HL-60/Ara-C cell lines to chemotherapeutic agents [21,22].MiR-181b enhanced resistance to the anticancer drug doxorubicin in hepatocellular carcinoma (HCC) [23], and functioned as a tumor suppressor in AML chemoresistance [24].In the present study, we performed miRNA expression profiling in adriamycin-sensitive cell line K562 and adriamycinresistant cell line K562/ADR.The expression of miR-181c was decreased in ADR-resistant cell lines (K562/ ADR, KU812/ADR, KCL22/ADR) as well as in CML/ MDR patients.Moreover, we found that inhibition of miR-181c may enhance resistance to the anticancer drugs of K562 cells.Overexpression of miR-181c sensitized K562/ADR cells to chemotherapy both in vitro and in vivo.Our findings were supported by investigations in another study.Contrary to our results, miR-181c contributed pancreatic cancer cell resistance to chemotherapeutic drugs by inactivating the Hippo signaling pathway [25].Down-regulation of miR-181c was found in imatinib-resistant CML patients, compared with imatinib-responders patients [26], which was consistent with our study.These results additionally demonstrate that miR-181c may be a reasonable approach to improving or prolonging drug sensitivity in CML.
Altered mRNA expression levels of sialyltransferases in different cancers are reported as potential targets for therapeuticapproaches [11,[27][28][29].Sialyltransferases of the mammalian ST8SIA family catalyze oligo-and polysialylation of surface-localized glycoproteins and glycolipids through transfer of sialic acids from CMP-sialic acid to the nonreducing ends of sialic acid acceptors [30].Our previously showed that the sialyltransferase ST8SIA4, encoded N-acetylgalactosaminide a-2, 8-sialyltransferase IV (ST8SIA4), was critical for CML multidrug resistance [19,20].In addition, miRNAs that target glycosylation enzymes have been identified.Vaiana et al demonstrated that MGAT4A, an N-acetylglucosamintltransferase that installed the β-1, 4 branch of N-glycans, was directly regulated by miR-424 in multiple mammary epithelial cell lines [31].MiR-200a might target ST3GAL3 and ST3GAL4 sialyltransferases, which potentially involved in antithrombin sialylation, were 85% lower in neonates in comparison with adults [32].MiR-199a targeted ST6GAL1 and reduced both the sialylation and the protein level of Necl-2 [33].Herein we demonstrated that miR-181c targets the 3' UTR of the sialylation related gene ST8SIA4, suggesting the enzyme may play a role in cancer chemoresistance.Thus, further exploration of the interaction between miR-181c and ST8SIA4 in alternate MDR system of CML was warranted.We showed that downregulation of miR-181 contributes to high levels of ST8SIA4.While, forced expression of miR-181c resulted in a significant decrease of ST8SIA4.We also observed a significant inverse correlation between miR-181c and ST8SIA4 levels in CML patients.Furthermore, silencing of the ST8SIA4 gene reversed antagomiR-181c-mediated sensitivity of the K562 cells to ADR, VCR and Taxol.Overexpression of ST8SIA4 in K562/ADR cells increased chemoresistance after miR-181c introduction.Taken together, our findings suggest that miR-181c mediates CML chemoresistance at least in part by functionally targeting ST8SIA4.
Numerous studies have demonstrated that activation of the PI3K/Akt signaling pathway was essential to the development and progression of most cancer types and associated with nearly all aspects of the malignant phenotype of cancer, such as uncontrolled proliferation, resistance to cell death, invasiveness, angiogenesis and metastasis [34,35].PI3K/AKT inhibition correlated down-regulation of NF-kappaB activity and inhibition P-gp function in murine lymphoma cell lines [36].Inhibition of the PI3K/mTOR pathway was a promising therapeutic approach in patients with acute lymphoblastic leukemia [37].Recent evidences have indicated that miRNAs were involved in the regulation of PI3K/AKT signaling pathways.For example, overexpression of miR-22 in CLL B cells switched on PI3K/AKT, leading to downregulation of p27 (-Kip1) and overexpression of Survivin and Ki-67 proteins [38].Ectopic expression of miR-206 mimics inhibited cisplatin resistance, decreased the migration and invasion in cisplatin-resistant lung adenocarcinoma cells, partly due to inactivation of MET/ PI3K/AKT/mTOR signaling pathway [39].Our recent study showed that downregulation of ST8SIA4 expression suppressed the activity of PI3K/AKT pathway.In the present study, we found miR-181c-ST8SIA4 axis regualted PI3K/AKT in CML cell lines.MiR-181c inhibitor enhanced phosphorylation of PI3K/AKT in K562 cells.In contrast, miR-181c suppressed the phosphorylation of PI3K/AKT in K562/ADR cells.Further investigation detected Akt shRNA inhibited the phosphorylation of PI3K/AKT in K562-antagomiR-181c cells, subsequently leading to a decrease in chemoresistance.Other studies also reported that ectopic expression of miR-181a leads to AKT phosphorylation, enhancing cell proliferation and inducing cell resistance to chemotherapy in T-cell leukemia/lymphoma [40].These findings may indicate that miR-181 functions as both an oncomir and tumorsuppressive miRNA, depending on the tumor type and the subtypes of miR-181.These results provides a possible mechanism linking miR-181c, PI3K/AKT pathway, drug resistance, by which altered expression of ST8SIA4 leads to drug resistance in CML cell lines.In addition, we also assessed the anti-tumor effect of miR-181c in an adriamycin-resistant in vivo mice model.We found that overexpression of miR-181c enhanced the K562/ADR cell sensitivity to adriamycin and inhibited the PI3K/ AKT pathway and ST8SIA4 expression in vivo.Therefore, these results further demonstrated in vivo that miR-181c inhibiting ST8SIA4 and its downstream PI3K/AKT pathways is one potential mechanism to overcome drug resistance in CML.
In conclusion, our study demonstrated that miR-181c was downregulated in adriamycin-resistant chronic myelocytic leukemia cell lines and CML/MDR patients.In addition, we showed that miR-181c regulated chemoresistance both in vitro and in vivo by targeting ST8SIA4 and mediating its downstream PI3K/AKT.Therefore, activation of miR-181c or inactivation of its target gene pathway may be a potential strategy to reverse drug resistance in human CML.

Primary patient samples
Thirty-eight previously untreated CML patients were included in this study.All patients were diagnosed with CML from 2013 to 2015 at First Affiliated Hospital of Dalian Medical University (Dalian, China) according to the current World Health Organization criteria.The diagnosis of CML was based on cytomorphology, cytochemistry, multiparameter flow cytometry, immunology, molecular genetics and cytogenetics.All the participants were provided written informed consent and the study were approved by and the institutional ethics committees.Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Hypaque and were further cultured in plastic dishes to remove adherent cells at 37°C for 24 h.The PBMC were divided into two groups, CML without MDR and CML/MDR.The frequency of P-gp positivity was 57.9% (22 of 38) in the CML patients.The clinical data of enrolled 38 patients was given in Supplementary Table S1.

RNA extraction and real-time PCR
Total RNAs for array analysis were extracted using Trizol (Invitrogen) and RNAeasy mini kit (Qiagen) or RNA was precipitated with isopropanol and glycogen (Invitrogen) according to the manufacturer's instructions.cDNA were synthesised from RNA using a TaqMan miRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA), and quantified the expression levels of miR-181c using a miRNA-specific TaqMan MiRNA Assay Kit (Applied Biosystems).The expression of miRNA was defined based on the Ct, and relative expression levels were calculated as 2 -[(Ct of miR-181c) − (Ct of U6)] after normalization with reference to expression of U6 small nuclear RNA.

Cell viability assay
The cell viability was monitored using the Cell Counting Kit-8 (CCK8) (Dojindo Molecular Technologies,

Figure 1 :
Figure 1: Expression of miR-181c is associated with chemoresistance in chronic myelocytic leukemia.A. Comparison of miRNA expression in K562/ADR and its parental cells by using the Exiqon Human miRNA microarray.Expression levels of miR-181c in K562 cells were higher than those in K562/ADR cells.B. A validation experiment was carried out using qRT-PCR.The relative levels were normalized to U6 snRNA.Expressions of miR-181c in parental K562, KCL22 and KU812 cells were higher compared to their ADRresistant cells (*p<0.05).C.Moreover, chemoresistant cancer samples have significantly lower levels of miR-181c (*p<0.05).

Figure 2 :
Figure 2: Knockdown of miR-181c promotes drug resistance in vitro.A. qRT-PCR was performed to measure miR-181c levels in K562 cells transfected with the antagomir control or antagomiR-181c.Decreased miR-181c levels were observed in the antagomiR-181-transfected cells compared to the control (*p<0.05).B. K562 cells were treated with ADR, VCR and Taxol for 48 h.Cell viability was determined using a CCK8 assay.More cells survived in antagomiR-181c-transfected group (*p<0.05).C. Knockdown of miR-181c increased IC50 values of K562 cells to ADR, VCR, and Taxol (*p<0.05).D. K562 cells were treated with ADR, VCR and Taxol, followed by analysis of apoptosis.There were fewer cells undergoing apoptosis in the antagomiR-181-transfected group (*p<0.05).

Figure 3 :
Figure 3: Upregulation of miR-181c decreases cell chemoresistance in vitro.A. qRT-PCR was performed to measure miR-181c levels in K562/ADR cells transfected with the mimic control or miR-181c.Increased miR-181c levels were observed in the miR-181transfected cells compared to the control (*p<0.05).B. K562/ADR cells were treated with ADR, VCR and Taxol for 48 h.Cell viability was determined using a CCK8 assay.Lower cells survived in miR-181c-transfected group (*p<0.05).C. Ectopic expression of miR-181c sensitized K562/ADR cells to ADR, VCR and Taxol (*p<0.05).D. K562/ADR cells were treated with ADR, VCR and Taxol, followed by analysis of apoptosis.There were more cells undergoing apoptosis in the miR-181c-transfected group (*p<0.05).

Figure 5 :
Figure 5: The silence or overexpression of ST8SIA4 reversed the effect of miR-181c on drug resistance.A, C. Western blotting analysis of the protein levels of ST8SIA4 in cells transfected with specific ST8SIA4-shRNA or ST8SIA4, respectively.(B) ST8SIA4 silencing reversed the drug resistance caused by the downregulation of miR-181c in K562 cells (*p<0.05).D. Re-expression of ST8SIA4 could antagonize miR-181c mediated chemo-sensitivity in K562/ADR cells (*p<0.05).

Figure 7 :
Figure 7: MiR-181c sensitizes K562/ADR cells to chemotherapeutic drugs in vivo. A. Tumor volumes of tumors in the mimic control, mimic control + ADR, miR-181c and miR-181c+ADR groups were measured on indicated days.Data presented are the mean ± SD B, C. Expression levels of Ki67, ST8SIA4 and PI3K/Akt signaling molecules in K562/ADR-mimic control group and K562/ADR-miR-181c group were analyzed by immunohistochemistry (*p<0.05).