KDM5A silencing transcriptionally suppresses the FXYD3‐PI3K/AKT axis to inhibit angiogenesis in hepatocellular cancer via miR‐433 up‐regulation

Abstract Hepatocellular cancer (HCC) has been reported to belong to one of the highly vascularized solid tumours accompanied with angiogenesis of human umbilical vein endothelial cells (HUVECs). KDM5A, an attractive drug target, plays a critical role in diverse physiological processes. Thus, this study aims to investigate its role in angiogenesis and underlying mechanisms in HCC. ChIP‐qPCR was utilized to validate enrichment of H3K4me3 and KDM5A on the promotor region of miR‐433, while dual luciferase assay was carried out to confirm the targeting relationship between miR‐433 and FXYD3. Scratch assay, transwell assay, Edu assay, pseudo‐tube formation assay and mice with xenografted tumours were conducted to investigate the physiological function of KDM5A‐miR‐433‐FXYD3‐PI3K‐AKT axis in the progression of HCC after loss‐ and gain‐function assays. KDM5A p‐p85 and p‐AKT were highly expressed but miR‐433 was down‐regulated in HCC tissues and cell lines. Depletion of KDM5A led to reduced migrative, invasive and proliferative capacities in HCC cells, including growth and a lowered HUVEC angiogenic capacity in vitro. Furthermore, KDM5A suppressed the expression of miR‐433 by demethylating H3K4me3 on its promoterregion. miR‐433 negatively targeted FXYD3. Depleting miR‐433 or re‐expressing FXYD3 restores the reduced migrative, invasive and proliferative capacities, and lowers the HUVEC angiogenic capacity caused by silencing KDM5A. Therefore, KDM5A silencing significantly suppresses HCC tumorigenesis in vivo, accompanied with down‐regulated miR‐433 and up‐regulated FXYD3‐PI3K‐AKT axis in tumour tissues. Lastly, KDM5A activates the FXYD3‐PI3K‐AKT axis to enhance angiogenesis in HCC by suppressing miR‐433.


| INTRODUC TI ON
Hepatocellular cancer (HCC) is one of the most prevalent types of cancers with a high mortality rate worldwide. 1 Hepatocellular cancer is likely to arise with longstanding chronic liver disease, such as chronic viral hepatitis types B and C, alcohol abuse, aflatoxin exposure and non-alcoholic fatty liver disease 2 ; viral hepatitis B and C still remain the main causative agents to the development of HCC. 3 More importantly, HCC has been reported to belong to one of the highly vascularized solid tumours accompanied with hypervascularity and vascular abnormalities. 4 It was also reported that transarterial chemoembolization (TACE) is one of the therapeutic approaches that improves the survival rates of patients with HCC; however, only a limited number of randomized controlled trials have been performed on assessing the survival benefit of patients undergoing TACE. 5 Chemoresistance is still an ongoing issue that could induce the recurrence of disease. 6 Anti-angiogenic therapies have been utilized for HCC therapy, though the curative effect is very limited. 7 Therefore, the knowledge of molecular mechanisms in angiogenesis is required for HCC therapy.
A majority of the human genome cannot encode proteins, which usually turn to be non-coding RNAs such as microRNA, long non-coding RNA or circular RNA. 8 Increasing evidence demonstrates that miRNAs are implicated in the progression of HCC. For instance, miR-338-5p exerts inhibitory effects on the growth of HCC cells. 9 Although the role of miR-433 in HCC still remains exclusive, the physiological functions of miR-433 in cancer progression have already been explored 10 ; for instance, miR-433 represses non-small cell lung cancer progression by directly targeting smad2. 11 In gastric cancer tissues, miR-433 was found to be down-regulated which corresponds to a poor outcome and a lower overall survival rate. 12 To determine the upstream regulatory gene, ChIPBase database was used to predict the binding site between KDM5A and miR-134. Epigenetic regulation is essential for the dynamic regulation of gene expression. 13 The aberrant expressions of epigenetic modifiers are tightly associated with human diseases. 14 H3K4me3, a marker of active gene expression, is the substrate of lysine demethylase 5A (KDM5A). 15,16 KDM5A, an oncogene and a promising drug target, has been reported to contribute to tumorigenesis, metastasis and drug resistance by repressing gene expression through demethylating H3K4me3. [17][18][19] Moreover, a previous study has implied KDM5A being significantly up-regulated in HCC. 20 However, its role in HCC has not been well understood. A previous report demonstrated that FXYD3 was highly expressed in HCC tissues and was negatively correlated with overall survival rates. 12 The potential binding sites between miR-433 and FXYD domain-containing ion transport regulator 3 (FXYD3) were predicted by Starbase. FXYD3 belongs to a family of Na, K-ATPase regulators, which have a domain of FXYD. 21 The family includes seven members, and different members are expressed in certain tissues where the seven members exert diverse functions. 22 FXYD3 has been reported to be highly expressed in diverse cancer tissues and its aberrant up-regulation contributes to the development of breast cancer by activating PI3K-AKT signalling. 23,24 Aberrantly activated PI3K-AKT is frequently found in diverse cancers and is correlated with a worse prognosis and outcome. 25,26 Constituted activation of PI3K-AKT signalling may be attributed to the mutation occurring in PI3K or AKT, mutation or amplification of RTKs, or overexpression of other upstream genes. 23,27,28 PI3K/Akt pathway could further induce multidrug resistance in HCC. 29 Therefore, we hypothesize that KDM5A might be involved in HCC development by regulating the FXYD3-PI3K-AKT axis in an epigenetic manner through miR-433.

| Ethical approval
The use of all specimens was agreed and approved by the Ethical Gaitherburg, MD, USA) was mixed with 4μg indicated plasmids or Lipofectamin2000 for 5 minutes at room temperature and were then mixed in a two-regent well for 20minutes before being added to a 6-well plate. After transfection, cells were cultured at 37°C in a saturated humidity atmosphere containing 95% air and 5% CO 2 ; the medium was replaced after 6 hours, and lysates were collected after 48 hours.

| Immunohistochemistry (IHC)
Formalin-fixed paraffin-embedded 5μm tissue sections were incubated at 60°C for 30 minutes and then deparaffinized in xylenes, rehydrated through a graded series of alcohol concentrations for 5 minutes and rinsed by water for 2 minutes. After antigen retrieval by 1mM Tris-EDTA (pH = 8.0) was performed, all sections were rinsed with phosphate buffer saline (PBS) for three times and left to block at room temperature in 3% H 2 O 2 -methanol for 10 minutes, followed by the coating of antibodies at room temperature for overnight at 4℃.

| Scratch assay
Each group of cells were seeded in a 6-well plate as 2.5 × 10 4 /cm 2 .
After cells were cultured for 24 hours, a scratch was made through the centre of each well using the 100 µL sterile pipette tip. The scratch was observed and imaged at the start and 24 hours following the scratch using a microscope. Image J software (1.48; National Institutes of Health) was used to measure the migrated distance. Cell

| Matrigel TM pseudo-tube formation
Matrigel (Shanghai Shanran Biological Technology Co., Ltd., Shanghai, China) was placed in a refrigerator at 4℃ overnight to be melted into yellow gel-like fluid. A total of 70 μL yellow gel-like fluid was added to pre-cooled 96-well plate by pre-cooled micropipette, followed by incubation at 37°C for solidification. After cells were transfected for 48 hours, they were starved for 1 hour and resuspended in DMEM to prepare for cell suspension. Cell suspension (1 × 10 5 cells/mL) was seeded into pre-coated 96-well plate and added with medium of different groups. The cell plate was then incubated at 37°C for 18 hours. Each group was set up with independent triplicates. Three contiguous fields of pictures were taken by an upright microscope (×100) (Leica, Weztlar, Germany), and the number of intact capillary lumens surrounded by cells was analysed by Image J software. This experiment was repeated for at least 3 times.

| Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA content was extracted from tissue samples and cells using a Trizol kit (Invitrogen). RNA was then reversely transcribed into

| KDM5A was highly expressed in HCC and is correlated with a poor prognosis in patients with HCC
A previous study has revealed an up-regulated expression of KDM5A in HCC. 20 With the attempt to further validate its expression in HCC, we performed prognosis analysis using GEPIA website, revealing that KDM5A was negatively correlated with the overall survival rate of patients with HCC ( Figure 1A). Therefore, KDM5A is correlated to a poor prognosis of patients with HCC. Furthermore, the expression of KDM5A was observed to be significantly increased in HCC tissues compared to that of benign tissues by analysing biopsy specimens through IHC and RT-qPCR ( Figure 1B,C) (P < .05). KDM5A expression level was much higher in HCC cell lines compared to that of normal liver cell lines ( Figure 1D) (P < .05). KDM5A was highly expressed in both HCC tissues and cell lines and is negatively correlated with HCC patient overall survival rates.

| KDM5A silencing suppresses HUVEC angiogenesis, HCC proliferation, migration and invasion capabilities
To investigate the role of KDM5A in HCC angiogenesis, we silenced ( Figure 2F) (P < .05). CD31 has been reported to play a vital role in angiogenesis (P < .05). We detected CD31 expression levels in hepatoma biopsy specimens and found CD31 was highly expressed in HCC tissues. Pearson's correlation analysis revealed that the level of CD31 expression was positively correlated with KDM5A ( Figure 2G). Angiogenesis assay suggested that the angiogenesis capacity of HUVECs was significantly suppressed when cultured in condition media from KDM5A-silenced Hep3B and MHCC97H cells ( Figure 2H) (P < .05). Taken together, our data revealed that silencing KDM5A could inhibit the progression of HCC by suppressing angiogenesis.

| KDM5A suppresses miR-433 expression
Previous reports have demonstrated KDM5A suppressing downstream genes expression by binding to the promoter of these genes and demethylating H3K4me3. 30 According to the ChIPBase database, KDM5A could bind to the promotor region of miR-433 and its binding region was in chr14: 100 882 337-100 882 570, of which its binding site was analysed, e indicating that KDM5A might transcriptionally regulate miR-433 ( Figure 3A). RT-qPCR results revealed that miR-433 level was significantly up-regulated after inhibiting KDM5A ( Figure 3B). To further confirm our hypothesis, we analysed the expression of miR-433 in HCC tissues and found that the expression of miR-433 was down-regulated in HCC tissues compared with that of adjacent tissues ( Figure 3C) (P <.05). Moreover, ChIP assay F I G U R E 1 KDM5A was significantly up-regulated in HCC tissues and was negatively correlated with overall survival rates. A, the correlation between KDM5A expression and overall survival rates analysed by GEPIA. B, KDM5A expression levels in HCC tissues determined by IHC, ×400. C, KDM5A expression in HCC tissues and normal tissues determined by RT-qPCR, N = 110. D, KDM5A expression in Hep3B, MHCC97H and HHL5 determined by RT-qPCR, N = 3. *P < .05; **P < .01, compared to that of normal tissues. Data were shown as the mean ± standard deviation. Statistical comparisons were performed by Tukey's test-corrected one-way ANOVA when more than two groups were compared. The experiment was repeated 3 times

| KDM5A promotes the proliferative, migrative, invasive and HUVEC angiogenic properties of HCC cell angiogenesis by suppressing miR-433
To

| miR-433 negatively targets FXYD3
A previous report demonstrated FXYD3 was highly expressed in HCC tissues and is negatively correlated with overall survival rates. 12 On the contrary, miR-433 had been reported to suppress the progression of HCC. 12 Therefore, we assumed miR-433 might regulate the progression of HCC by targeting FXYD3. We found miR-433 might target FXYD3 and the potential binding sites on FXYD3 were identified by Starbase ( Figure 5A). Based on this, we performed dual luciferase assay and found that overexpressing miR-433 had significantly repressed the luciferase activity of

PI3K-AKT axis to inhibit HCC tumorigenesis via miR-433 up-regulation in vivo
To investigate the effect of regulating miR-433-FXYD3-PI3K-AKT signalling by KDM5A on HCC tumorigenesis, Hep3B and MHCC97H cells were subjected to subcutaneous injection in nude mice after being transfected with KDM5A shRNA. Based on this, we found that silencing KDM5A had significantly suppressed HCC tumorigenesis, tumour volume and tumour weight ( Figure 7A-C) (P < .05). We detected the presence of miR-433, FXYD3, p-AKT and p-p85 expressions in tumour tissues, which demonstrated that KDM5A silencing led to significant increase in miR-433 and reduction in expression F I G U R E 3 KDM5A suppressed miR-433 transcription. A, binding site of KDM5A predicted by ChIPBase. B, miR-433 expression levels after depleting KDM5A determined by RT-qPCR. C, miR-433 expression in HCC tissues and normal tissues determined by RT-qPCR. D, the enrichments of KDM5A and H3K4me2 on miR-433 promoter region in Hep3B cells determined by ChIP assay. E, the enrichment of KDM5A and H3K4me2 on miR-433 promoter region in MHCC97H cells determined by ChIP assay. *P < .05; **P < .01, compared to si-NC or adjacent tissues. Data were shown as the mean ± standard deviation. Statistical comparisons were performed by Tukey's test-corrected one-way ANOVA when more than two groups were compared. The experiment was repeated 3 times Results revealed that miR-433 was significantly down-regulated in clinical HCC tissues ( Figure 7F,G). Meanwhile, we also found that miR-433 was negatively correlated with angiogenesis ( Figure 7H).
Moreover, the overall survival rates of patients with HCC were positively correlated with the expression of miR-433 ( Figure 7I) (P < .05).
On the other hand, the protein levels of KDM5A, FXYD3, p-AKT and p-p85 were much higher in clinical HCC tissues compared to that of normal tissues ( Figure 7J). In conclusion, KDM5A promoted HCC tumorigenesis in vivo by regulating miR-433-FXYD3-PI3K-AKT signalling.

| D ISCUSS I ON
Angiogenesis describes the formation of new blood vessels from preexisting ones, which is a complicated physiological process forms the basis for solid tumour growth. 31,32 VEGF has been regarded as the major pro-angiogenic regulator and a drug target for anti-angiogenic therapy. 33 More importantly, a previous report mentioned that HCC belongs to one of the highly vascularized solid tumours and is accompanied with hypervascularity and vascular abnormalities. 4 The efficiency of anti-angiogenesis therapy on the treatment of HCC is very limited. 7 Increasing evidence demonstrates that miRNAs are critical for the regulation of angiogenesis. 34 Accumulated evidence F I G U R E 4 Depletion of KDM5A up-regulated miR-433 to suppress HCC angiogenesis and progression. A, the expression levels of miR-433 and KDM5A after restoration of miR-433 in KDM5A-silenced Hep3B and MHCC97H cells determined by RT-qPCR. B, the effect of miR-433 restoration on the migrative capacity of KDM5A silenced Hep3B and MHCC97H cells determined by scratch assay. C, the effect of miR-433 restoration on invasive capacity of KDM5A silenced Hep3B and MHCC97H cells determined by transwell assay. D, the effect of miR-433 restoration on proliferative capacity of KDM5A silenced Hep3B and MHCC97H cells determined by EDU assay. E, the effect of miR-433 restoration on angiogenesis of KDM5A silenced Hep3B and MHCC97H cells determined by pseudo-tube formation assay. *P < .05; **P < .01, compared to si-NC+ inhibitor-NC. #P < .05; ##P < .01, compared to si-KDM5A+ inhibitor-NC. Data were shown as the mean ± standard deviation. Statistical comparisons were performed by Tukey's test-corrected one-way ANOVA when more than two groups were compared. The experiment was repeated 3 times Numerous studies revealed that epigenetic restructuring is tightly intertwined with cancer initiation and development, 35 as the alternation of the epigenetic modifications in tumour cells might lead to resistance to typical therapeutic strategies. 36 As to the role of epigenetic factors in favouring angiogenesis, a recent report demonstrated EZH1, a methyltransferase, to promote angiogenesis by catalysing H3K27me3. 37 In-depth investigation of the molecular mechanism of down-regulating miR-433 in HCC, we noticed that KDM5A, a demethylase for H3K4me2/3, is predicted to a directly bind to the miR-433 promoter region. In the present study, KDM5A regulated the expression of miR-433 by removing H3K4me3 on its promoter region. However, the role of KDM5A in HCC angiogenesis remains largely unknown. We analysed HCC biopsy specimens  and found KDM5A expression was significantly increased in HCC tissues, but was negatively correlated with miR-433, the results of which were consistent with a previous study. 20 The expression of KDM5A is negatively correlated with the overall survival rates of patients with HCC. In vitro assays demonstrated that silencing KDM5A up-regulates miR-433 to reduce migrative and invasive capacities, growth and suppress angiogenesis. Moreover, the up-regulation of miR-433 is correlated with the proliferative, migrative, invasive and HUVEC angiogenic capacities in HCC cells. miR-433 was found to be down-regulated, which indicates a poor outcome and limited overall survival rates. 12 Interestingly, overexpressing miR-433 inhibited focus formation in vivo and inhibits the oncogenic phenotype development of HCC. 12 In the subsequent study, we found that miR-433 negatively targeted FXYD3. FXYD3, majorly found in the breast, colon, stomach and prostate, is able to interact with Na, K-ATPase b subunit and regulates the transport properties of Na, K-ATPase. 38 FXYD3 has been reported to be highly expressed in several types of cancers, including breast cancer, and is related to the survival rate and metastasis. 24 FXYD3 has been reported to be a clinical diagnosis marker of HCC. Based on this, restoration of FXYD3 expression rescues the inhibited oncogenic phenotype of HCC caused by KDM5A silencing.
The up-regulated expression level of FXYD3 in HCC is associated with HCC clinicopathological characteristics, serving as a potential prognostic marker for HCC. 39 Furthermore, our data revealed that

| CON CLUS IONS
Our data suggested that KDM5A directly binds to the promotor region of miR-433 and suppresses its expression by removing H3K4me3. The down-regulation of miR-433 up-regulates FXYD3 levels in HCC tissues, which leads to the aberrant activation of PI3K-AKT signalling, contributing to the angiogenic process of HCC ( Figure 8). Importantly, selective inhibitor of KDM5A has been developed. Based on our studies, we hypothesized that KDM5A inhibitor treatment may be beneficial to traditional anti-angiogenic therapy.
Therefore, future experiments on this speculation are warranted to increase the efficiency of anti-angiogenic therapy in HCC.

ACK N OWLED G EM ENTS
We acknowledge and appreciate our colleagues for their valuable suggestions and technical assistance for this study.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest. Writing-review and editing.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets generated and/or analysed during the current study are available from the corresponding author on reasonable request.