N6-methyladenosine modified AFAP1-AS1 accelerates hepatocellular carcinoma proliferation and migration via miR-195-5p/KIAA1429 feedback loop

doi:10.21203/rs.3.rs-1975184/v1

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N⁶-methyladenosine modified AFAP1-AS1 accelerates hepatocellular carcinoma proliferation and migration via miR-195-5p/KIAA1429 feedback loop

https://doi.org/10.21203/rs.3.rs-1975184/v1

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Hepatocellular carcinoma (HCC) acts as a dominating malignant tumor with high-mortality rate and poor prognosis. Emerging evidence suggests that N⁶-methyladenosine (m⁶A) plays critical roles in HCC progression. However, function of m⁶A and lncRNA in HCC progression requires further clarification. In our study, we found that AFAP1-AS1, a m⁶A-modified lncRNA, substantially elevated in the HCC cells and correlated with poor prognosis. Interestingly, AFAP1-AS1 had m⁶A modification site and m⁶A writer KIAA1429 bound with the m⁶A site to enhance AFAP1-AS1 stability. Functionally, AFAP1-AS1 promoted HCC migration/ proliferation and knockdown of AFAP1-AS1 repressed the tumor growth. Mechanistically, AFAP1-AS1 up-regulated KIAA1429 expression through sponging miR-195-5p, forming positive feedback of KIAA1429/m⁶A/AFAP1-AS1/miR-195-5p. In summary, our study reveals a new mechanism for AFAP1-AS1-promoted HCC progression, contributing to overcoming HCC tumorigenesis.

N6-methyladenosine

hepatocellular carcinoma

AFAP1-AS1

KIAA1429

Although the death rate of malignant tumor is decreasing gradually in recent years, the incidence of liver cancer is increasing year by year, especially hepatocellular carcinoma (HCC)[1, 2]. As a common malignant tumor, HCC ranks fourth in the incidence of malignant tumors and second in the cause of death in China[3]. Cirrhosis caused by hepatitis B/C or alcohol is one of the leading causes of liver cancer[4]. Primary liver cancers are divided to three subtypes: hepatocellular carcinoma (HCC), bile duct cell carcinoma and mixed liver cancer[5]. HCC accounts for about 85% of primary liver cancer. New therapeutic options are available only if the explicit mechanisms underlying HCC.

N⁶-methyladenosine (m⁶A) regulation in various tumor processes has been an emerging central point of investigation[6, 7]. m⁶A is a class of internal modification of RNA in transcript. Generally, m⁶A acts as an essential regulation with the features of being dynamic and reversible modification. In HCC, the roles of m⁶A have been investigated[8, 9]. For instance, m⁶A methyltransferase METTL3 highly expressed in HCC and the m⁶A level remarkably increased. Clinically, METTL3 expression was related to worse overall survival and down-regulation of METTL3 remarkably attenuates the migration and proliferation[10]. Hypoxia triggers the METTL14 suppression via HIF-1α-dependent manner for HCC cells. METTL14 induces the m6 A modification of SLC7A11 mRNA at 5'-UTR via HIF-1α/METTL14/YTHDF2/SLC7A11 axis[11]. Thus, these evidences indicate that m⁶A participates the HCC tumorigenesis.

Herein, we found that the m⁶A modification was increased in HCC. A remarkable m⁶A-modified lncRNA, AFAP1-AS1, up-regulated in HCC cells and correlated to patients’ poor prognosis. Interestingly, AFAP1-AS1 had m⁶A modification site and m⁶A writer KIAA1429 bound with the m⁶A site to enhance AFAP1-AS1 stability in an m⁶A-dependent manner. AFAP1-AS1 promoted HCC proliferation and migration through sponging miR-195-5p, forming positive feedback of KIAA1429/m⁶A/AFAP1-AS1/miR-195-5p. In summary, our study reveals a new mechanism for AFAP1-AS1-promoted HCC progression, contributing to overcoming HCC tumorigenesis.

2.1. HCC cells

HCC cell lines (HCCLM3, Hep3B, Huh7) and normal liver epithelial cell line (THLE-3) were identified from the Chinese Academy Medical Sciences (Beijing, China) and cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented by 10% fetal bovine serum (FBS, Gibco, USA) in humidified atmosphere with 5% CO₂ at 37°C.

2.2. Vector construction and transfection

Lentiviral shRNA vectors targeting AFAP1-AS1 (named as sh-AFAP1-AS1) were provided from GeneChem (Shanghai, China), and lentivirus particle was generated by co-transfecting the shRNA vectors/packaging plasmids into HEK293T packaging cells. The full length of AFAP1-AS1 was cloned into the expression vectors pcDNA3.1 and named AFAP1-AS1.

2.3. Quantitative real-time PCR (qRT-PCR)

Total RNAs were isolated from HCC cells using Trizol reagent (Invitrogen, USA) according to the manufacturer’s protocol. The kit of HiScript II (Vazyme, China) was used for cDNA was synthesis from RNA (5 µl RNA samples). For the qRT-PCR of mRNA or miRNA, StepOne Plus Real-Time PCR system (Applied Biosystems, USA) was used as manufacturer’s protocol. Standard controls were standardized by U6 and β-actin. All PCR primer was listed in Supplementary Table S1.

2.4. Western blotting

RIPA buffer was used to obtain the total cellular proteins, which was quantified by BCA analysis (Beyotime, Shanghai, China). Extracted protein (30 µg) was separated by SDS-PAGE (10%, sodium dodecyl sulphatepolyacrylamide gel electrophoresis) and then transferred to PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% skimmed milk in Tris-buffered saline plus Tween (TBST, 0.05 M Tris, 0.15 M NaCl, 0.2% Tween-20) for 1 h, and then incubated with primary antibodies (anti-KIAA1429, 1:1000, Catalog no.88358, Cell Signaling Technology) overnight, and then incubated with corresponding secondary antibody (Cell Signaling Technology, 1:1000). Lastly, proteins were detected using chemiluminescence system (BioRad, USA).

2.5. Cell counting kit‑8 assay

The proliferation of HCC cells was detected using cell counting kit‑8 (CCK-8) assay. With transfection of Hep3B and HCCLM3 cells, HCC cells were seeded on 96-well plates with concentration of 2×10⁴ cells and cultured at 0, 24, 48, 72 hours. Subsequently, the kit of cell counting kit-8 (10 µL) was added to each plate. After incubation at 37°C of 1 h, these absorbance values were measured at 450 nm.

2.6. Apoptosis assay

Apoptosis of HCC cells were analyzed by flow cytometry and subsequently detected as described previously[12]. Briefly, the transfected cell harvested by trypsinization. Subsequently, double staining by propidium iodide (PI)/fluorescein isothiocyanate (FITC)-annexin V was performed with FITC Annexin V-Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s recommendations. Cells death was determined by early apoptotic cells /apoptotic cells. The relative ratio apoptotic cells compared with control experiment.

2.7. Transwell assay

For the migration analysis, 6-well 8-µm chambers plates (Corning) were used. In brief, HCC transfected Hep3B and HCCLM3 cells were seeded in 6-well plates in concentration of 5×10⁴ cells. serum-free medium (200 µL) was added to upper chamber, and medium (600 µL) with 20% FBS was added to the lower chamber. After 48 h, the migrated cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet. Lastly, 5-fields were randomly selected to measure the migrating cells.

2.8. RNA m⁶A quantification assay

The m⁶A levels in HCC cells were detected by RNA m⁶A quantification kit (Abcam, Cat. ab185912) according to the protocols as previous studies. Briefly, RNA (200 ng) were incubated for 60 min with capture antibodies, and then detection antibody/enhancer solution were added. Lastly, HCC cells’ sample was incubated with 10 min developer solution. 450 nm wavelength absorbance was detected.

2.9. Subcellular fractionation location

For the RNA subcellular fractionation location analysis, PARIS Kit (Life Technologies, CA, USA) was performed to separate the nuclear/cytosolic fraction of HCC cells. qPCR was detected AFAFP1- AS1, U6 RNA and GAPDH in cytoplasm/nuclear fraction.

2.10. Luciferase Assay

To determine the effects of miR-195-5p on AFAP1-AS1 and KIAA1429 mRNA, psicheck2-based plasmid containing AFAP1-AS1 and KIAA1429 mRNA was constructed. These constructed plasmids were named psicheck2-AFAP1-AS1, psicheck2-KIAA1429. Besides, the mutants were constructed a psicheck2-AFAP1-AS1-mut or psicheck2-KIAA1429 vectors on 3′-untranslated region (3′-UTR), using Generate site directed mutagenesis system (USA, Invitrogen). Co-transfected cells with the psicheck2-based plasmids plus miR-195-5p mimics or miR-NC. After 24 h of transfection, Renilla Luciferase Assay System (Promega, Madison, WI) was performed to quantify the luciferase activity. The results were expressed as relative luciferase activity (firefly luciferase/Renilla luciferase).

2.11. RNA stability

The RNA stability was analyzed using the RNA stability assay. HCC cells were treated by Act D, actinomycin D (2 µg/ml) and then collected in different time points. RNA was extracted by Trizol reagent (Invitrogen, Grand Island, NY, USA). mRNA levels were measured by qRT-PCR using oligo (dT) primers.

2.12. Mice tumor models

The animal study was approved by the Animal Care and Use Committee of Xinjiang medical university. BALB/c nude mice (aged 4–6 weeks) were purchased from the Model Animal Research of Slac Laboratory Animal Center (Shanghai, China). Mice were maintained in accordance with the institutional policies. Vector or AFAP1-AS1 transfected Hep3B cells (5×10⁶ cells per mouse, 5 mice per group) were collected and then subcutaneously injected to the mice to subcutaneous xenograft tumor model. The tumor size was evaluated by caliper every three days with following equation: 0.5×major axes×minor axes².

2.13. Statistical analysis

Statistical data were analyzed using SPSS software 19.0 (SPSS, Chicago, IL) and GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA). One way ANOVA testing or Student’s t-test was used for analysis according to actual conditions. The value p < 0.05 was regarded as statistical significance.

A m⁶A-modified lncRNA AFAP1-AS1 elevated in the HCC cells and correlated with poor prognosis. Interestingly, AFAP1-AS1 had m⁶A modification site and m⁶A writer KIAA1429 bound with the m⁶A site to enhance AFAP1-AS1 stability. Functionally, AFAP1-AS1 promoted HCC migration/ proliferation and knockdown of AFAP1-AS1 repressed the tumor growth. Mechanistically, AFAP1-AS1 up-regulated KIAA1429 expression through sponging miR-195-5p, forming positive feedback of KIAA1429/m⁶A/AFAP1-AS1/miR-195-5p.

3.1. AFAP1-AS1 up-regulated in hepatocellular carcinoma

In the HCC cohorts, we analyzed the AFAP1-AS1 expression as compared to normal tissue samples. Results found that AFAP1-AS1 expression level elevated in the liver hepatocellular carcinoma (LIHC) samples compared to normal samples (Fig. 1A). In HCC cell lines, AFAP1-AS1 expression was found to be up-regulated as compared to normal cells (Fig. 1B). Survival analysis by the Kaplan-Meier method and log-rank test was perforemd for survival curves. Survival analysis demonstrated that the high AFAP1-AS1 expression indicated the poor prognosis of HCC patients (Fig. 1C). Moreover, the survival analysis revealed that the high AFAP1-AS1 expression correlated to HCC patients’ poor prognosis (Fig. 1D). Taken together, these data suggested that AFAP1-AS1 level up-regulated in hepatocellular carcinoma.

3.2. KIAA1429 positively regulated the stability of AFAP1-AS1 in m⁶A-dependent manner

To investigate the potential genesis of AFAP1-AS1, we found that AFAP1-AS1 was a m⁶A-modified lncRNA (Fig. 2A). Then, we found that the m⁶A modification level up-regulated in the HCC cells as compared to normal cells (Fig. 2B). To investigate the potential regulator for AFAP1-AS1, we found that three m⁶A writers both up-regulated in HCC, especially KIAA1429 (Fig. 2C). Subsequently, correlation analysis revealed that AFAP1-AS1 positively correlated with KIAA1429 in HCC cohort (Fig. 2D). To investigate the roles of KIAA1429 on AFAP1-AS1, the overexpression of KIAA1429 was constructed and the transfection efficiency was tested using western blot (Fig. 2E). Then, Act D administration was performed and results indicated that KIAA1429 overexpression promoted the AFAP1-AS1 expression (Fig. 2F). Taken together, these data suggested that KIAA1429 positively regulated the stability of AFAP1-AS1 in m⁶A-dependent manner.

3.3. AFAP1-AS1 promoted the proliferation and migration of HCC

To test the function of AFAP1-AS1 on HCC cells, AFAP1-AS1 overexpression and knockdown were constructed in HCCLM3 or Hep3B cells (Fig. 3A). CCK-8 proliferative assay demonstrated that AFAP1-AS1 overexpression promoted the proliferation of HCCLM3 cells, and AFAP1-AS1 knockdown repressed the proliferation of Hep3B cells (Fig. 3B, 3C). sh-AFAP1-AS1-1# was used in following assays. Migration assay indicated that AFAP1-AS1 overexpression promoted the migration of HCCLM3 cells, and AFAP1-AS1 knockdown inhibited migration of Hep3B cells (Fig. 3D). Apoptosis assay showed that AFAP1-AS1 overexpression reduced the apoptosis, while AFAP1-AS1 knockdown increased the apoptosis (Fig. 3E). In vivo mice assay indicated that AFAP1-AS1 knockdown repressed the tumor growth (Fig. 3F). Taken together, these data suggested that AFAP1-AS1 promoted the proliferation and migration of HCC.

3.4. AFAP1-AS1 targeted miR-195-5p/KIAA1429 feedback loop

The subcellular localization analysis revealed that AFAP1-AS1 mainly located in the cytoplasm, rather than nucleus, including Hep3B and HCCLM3 (Fig. 4A). To screen the potential target miRNAs for AFAP1-AS1, we detected several miRNAs using RT-PCR, including miR-424-5p, miR-655-3p, miR-512-3p, miR-195-5p and miR-15a-5p. Results indicated that miR-195-5p showed remarkable up-regulation upon AFAP1-AS1 knockdown (Fig. 4B). The wild type and mutant sequences were constructed, including AFAP1-AS1 3’-UTR WT and Mut (Fig. 4C). Luciferase assay indicated that miR-195-5p significantly bound with AFAP1-AS1 wild type 3’-UTR (Fig. 4D). In the liver hepatocellular carcinoma (LIHC) samples, KIAA1429 expression was significantly up-regulated compared to normal samples (Fig. 4E). Then, the wild type and mutant sequences were constructed, including KIAA1429 3’-UTR WT and Mut (Fig. 4F). Luciferase assay indicated that miR-195-5p significantly bound with KIAA1429 wild type 3’-UTR (Fig. 4G). Moreover, AFAP1-AS1 overexpression up-regulated the expression of KIAA1429 protein level (Fig. 4H). Taken together, these data suggested that AFAP1-AS1 targeted miR-195-5p/KIAA1429 feedback loop (Fig. 5).

Presently, researchers have investigated various mechanisms of tumorigenesis for HCC upon epigenetics. Here, our study explored the important functions of RNA epigenetics modification. A critical lncRNA, AFAP1-AS1, was found to be up-regulated in the HCC tissue samples and cells.

Firstly, the high-expression level of AFAP1-AS1 closely correlated to the poor prognosis of HCC patients. This result combined with the existing high level of expression, we found that AFAP1-AS1 had the possibility of promoting tumorigenesis. Previously, many papers have indicated that AFAP1-AS1 might act as oncogene in human cancers[13]. For example, in colorectal cancer, AFAP1-AS1 is notably increased, and AFAP1-AS1 knockdown inhibits the malignant behavior of cancer cells through targeting miR-195-5p/WISP1 axis[14]. Moreover, FAP1-AS1 is upregulated in oral squamous cell carcinoma and positively correlated to tumor stage and lymph node metastasis and pathological grade. Moreover, AFAP1-AS1 down-regulation decreased the proliferation of cancer cells in vitro[15]. Thus, these evidences suggest the critical roles of lncRNA AFAP1-AS1 in human cancer.

KIAA1429, acts as an m⁶A methyltransferase (‘writer’), was found to be up-regulated in the HCC tissue samples and cells[16, 17]. And this is a very interesting point that KIAA1429 bound with the m⁶A methylated site in AFAP1-AS1 genomic sequences, and KIAA1429 promotes the RNA stability of AFAP1-AS1 upon Act D administration. For the characteristic that KIAA1429 promote RNA stability, literature has illustrated this tendency in several cancers. For example, in gastric cancer, KIAA1429 catalyzes the m⁶A modification site on LINC00958 loci and then LINC00958 interacts with GLUT1 mRNA via the m⁶A-dependent manner to enhance GLUT1 mRNA transcript stability[18]. In colorectal carcinogenesis, overexpression of KIAA1429 increases the cellular proliferation, colony formation and migration, and KIAA1429 increases the SIRT1 expression via regulating mRNA stability by m⁶A-dependent manner[19]. From these results, we concluded that KIAA1429 could promote the RNA stability via m⁶A-dependent manner.

Using a bioinformatics approach, present study was performed to conduct the genome-wide comparison of HCC[20, 21]. Moreover, the prediction tools indicated that there were remarkable m⁶A modified sites on AFAP1-AS1 RNA sequences. KIAA1429 targeted the m⁶A modified sites on AFAP1-AS1 RNA sequences, thereby up-regulating the RNA stability of AFAP1-AS1 towards Act D treatment. In turn, we found that AFAP1-AS1 positively regulates KIAA1429 expression through sponging miR-195-5p, suggesting the lncRNA/miRNA/mRNA axis. For the interaction within miR-195-5p and AFAP1-AS1/KIAA1429, we run the online target-prediction database for searching possible candidates, including StarBase (https://starbase.sysu.edu.cn/), miRBase (https://www.mirbase.org/), TargetScan (https://www.targetscan.org/vert_80/). For the inquiry that why did we choose KIAA1429 instead of METTL3 or WTAP as m⁶A modifiers, the most important determinant was that KIAA1429 showed a remarkable regulation on AFAP-AS1 expression, rather than METTL3 or WTAP. Different lncRNA are regulated by different m⁶A writers, thus, m⁶A-installed AFAP-AS1 was regulated by KIAA1429 in this particular tumor.

In conclusion, the present research aimed to investigate the roles of m⁶A and lncRNA in HCC progression. A critical m⁶A writer KIAA1429 modified the lncRNA AFAP1-AS1 via m⁶A modification manner. In turn, AFAP1-AS1 regulates KIAA1429 via a positive feedback loop of miR-195-5p/KIAA1429 (Figure 5). These data suggests a novel therapeutic strategy and novel insight for HCC.

Conflict of interest

All authors declare no conflicts of interest

Acknowledgement

No.

Data Availability Statement

No research data shared.

Funding declaration

No funding was received

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No competing interests reported.

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N⁶-methyladenosine modified AFAP1-AS1 accelerates hepatocellular carcinoma proliferation and migration via miR-195-5p/KIAA1429 feedback loop

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. HCC cells

2.2. Vector construction and transfection

2.3. Quantitative real-time PCR (qRT-PCR)

2.4. Western blotting

2.5. Cell counting kit‑8 assay

2.6. Apoptosis assay

2.7. Transwell assay

2.8. RNA m⁶A quantification assay

2.9. Subcellular fractionation location

2.10. Luciferase Assay

2.11. RNA stability

2.12. Mice tumor models

2.13. Statistical analysis

3. Results

3.1. AFAP1-AS1 up-regulated in hepatocellular carcinoma

3.2. KIAA1429 positively regulated the stability of AFAP1-AS1 in m⁶A-dependent manner

3.3. AFAP1-AS1 promoted the proliferation and migration of HCC

3.4. AFAP1-AS1 targeted miR-195-5p/KIAA1429 feedback loop

4. Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

N6-methyladenosine modified AFAP1-AS1 accelerates hepatocellular carcinoma proliferation and migration via miR-195-5p/KIAA1429 feedback loop

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. HCC cells

2.2. Vector construction and transfection

2.3. Quantitative real-time PCR (qRT-PCR)

2.4. Western blotting

2.5. Cell counting kit‑8 assay

2.6. Apoptosis assay

2.7. Transwell assay

2.8. RNA m6A quantification assay

2.9. Subcellular fractionation location

2.10. Luciferase Assay

2.11. RNA stability

2.12. Mice tumor models

2.13. Statistical analysis

3. Results

3.1. AFAP1-AS1 up-regulated in hepatocellular carcinoma

3.2. KIAA1429 positively regulated the stability of AFAP1-AS1 in m6A-dependent manner

3.3. AFAP1-AS1 promoted the proliferation and migration of HCC

3.4. AFAP1-AS1 targeted miR-195-5p/KIAA1429 feedback loop

4. Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

N⁶-methyladenosine modified AFAP1-AS1 accelerates hepatocellular carcinoma proliferation and migration via miR-195-5p/KIAA1429 feedback loop

2.8. RNA m⁶A quantification assay

3.2. KIAA1429 positively regulated the stability of AFAP1-AS1 in m⁶A-dependent manner