HBV Infection Promotes The Occurrence and Development of Hepatocellular Carcinoma Through Impairing The Inhibitory Effect of PPP2R5A on MAPK/AKT/WNT Signaling Pathway

Background: Reversible phosphorylation and dephosphorylation play important roles in cell function and cell signal transduction. PPP2R5A is responsible for specically regulating the catalytic function, substrate specicity and intracellular localization of the tumor suppressor phosphatase PP2A. Therefore, the abnormal expression and function of PPP2R5A may be related to the occurrence and development of tumors. Results: This study showed that PPP2R5A inhibited the proliferation and metastasis of hepatocellular carcinoma (HCC) cells, and acted as a tumor suppressor in HCC cells, but it had no signicant effect on cell cycle. Further research found that PPP2R5A exerted tumor suppressor ecacy by inhibiting the MAPK/AKT/WNT signaling pathway. Combined with analysis of clinical tissue samples and TCGA database, it was found that the expression of PPP2R5A in tumor tissues of Chinese HCC patients was down-regulated and signicantly correlated with the progression-free survival (PFS) of HCC patients. On the contrary, PPP2R5A showed an up-regulation trend in HCC cases in TCGA database although its effect on PFS was the same with that in Chinese HCC patients. HBV infection is the main pathogenic factor of HCC in China. It was found that hepatitis B virus (HBV) infection reduced the content of PPP2R5A in cells. Conclusions: It was concluded that HBV inhibited the initiation of the protective mechanism mediated by PPP2R5A, making the occurrence and progress of HCC more "unimpeded". This conclusion will further reveal the role of PPP2R5A in HBV-induced and HBV-unrelated HCC, therefore, providing clues for the prevention and treatment of the two types of HCC, respectively.


Background
China has a high incidence of hepatocellular carcinoma (HCC), while the vast majority of HCC are associated with HBV infection [1][2][3]. In recent years, the anti-HCC effects of molecular targeted drugs have attracted more and more attention and become one of the hot spots in anti-tumor research.
Reversible phosphorylation and dephosphorylation play important roles in the maintenance of cell homeostasis and the regulation of cell functions. About 50% of the serine/threonine dephosphorylation activity within cells is regulated by the phosphatase protein phosphatase 2A (PP2A). PP2A is a most abundant phosphatase in cells, and its expression accounts for 0.2-1% of the total cell protein [4][5][6][7]. Therefore, the expression level, substrate speci city, intracellular localization and enzyme activity of PP2A are closely related to cellular physiological state and pathological state. It is generally believed that PP2A is a tumor suppressor, which can negatively regulate cell division, inhibit protein synthesis and promote cell apoptosis, and is a potential target for tumor targeted therapy [8,9].
Protein phosphatase 2 regulatory subunit B' alpha (PPP2R5A) is one of the regulatory subunits of PP2A, which speci cally regulates the phosphatase activity, substrate speci city and intracellular localization of PP2A. It has been reported that PPP2R5A is involved in the negative regulation of tumor-related proteins such as Bcl2 [10], MAPK [11], AKT [12], c-Myc [13] and β-Catenin [14], and abnormalities of PPP2R5A are often accompanied by occurrence of various diseases such as adenocarcinoma and epithelial cancer [15][16][17][18]. It has been reported that lentiviral infection will increase the degradation of PPP2R5A to promote virus replication [19]. The analysis of HCC samples in the TCGA database found that PPP2R5A is upregulated in HCC patients, while the expression of PPP2R5A is signi cantly related to the prognosis of HCC. The HCC patients with high expression of PPP2R5A have a longer progression-free survival (PFS). The above clues suggested that PPP2R5A may exist as a tumor suppressor in the development of HCC.
This study will explore the role and molecular mechanism of PPP2R5A in HCC.

Methods
Cell lines and cell culture represents the average intensity. The median of the cancer tissue IHC score was selected to distinguish between the high and low expression of PPP2R5A. Combined with the pathological and follow-up data, the correlations between PPP2R5A expression level and clinical indicators and survival period were analyzed.

Western blot
Western blot was performed according to the conventional experimental methods. The antibodies used and the dilution ratio are shown in supplementary table 1 (Table S1).

CCK8 assay
The logarithmic growth phase cells were seeded in 96-well plates, the nal concentration was 5,000 cells /100 µL (3 replicate wells per group). After being cultured in an incubator for 48 h, cells of each well were added with 10 µL of CCK8 reagent and incubated at 37 °C for 2 h in dark, then placed in an enzyme-linked immunoassay instrument to detect OD 450 . The cell proliferation rate was calculated.

Colony formation assay
The logarithmic growth phase cells were seeded in 6-well plates, the nal cell density was 1,000 cells /well, and placed in an incubator for cultivation. After 14 days, cells were xed with 4% formaldehyde, then stained with crystal violet for 15-20 min, washed with PBS for 3-4 times, and then photographed and statistically analyzed.

Cell Cycle
The cells in the logarithmic growth phase were collected and xed with pre-cooled 75% alcohol, and placed at 4 °C overnight. The cells were centrifuged to remove alcohol, washed twice with PBS, added with 200 µL PI (BD Company, USA) staining solution to resuspend, then placed in dark to react for 30 minutes, and tested on the machine.

Transwell assay
The transwell chambers were coated with matrigel, and 5 × 10 4 cells were seeded into 200 µL serum-free DMEM in the upper chamber. 500 µL of media containing 20% FBS were added to the lower chamber of transwell. Then the plate was placed in incubator for static culture. After 48 h, the upper chamber cells were wiped off, the migrated and invasive cells were stained with crystal violet for 15 minutes, and photographed and recorded under a microscope.

Statistical analysis
The data were analyzed by SPSS 17.0 software package and expressed as mean ± standard deviation (Mean ± SD). Analysis of variance (ANOVA) was used to analyze the data. p < 0.05 indicates statistical signi cance. The statistical graphs were made with Graphpad Prism 5 software.

Results
Low expression of PPP2R5A in HCC tissue is signi cantly related to poor prognosis of patients In order to explore the expression level of PPP2R5A in Chinese HCC patients, immunohistochemistry (IHC) detection was performed on tissue microarrays containing cancer and adjacent tissues from 110 HCC patients. The results showed that the expression of PPP2R5A in 77 cases (70%) of cancer tissues was lower than that in the adjacent tissues, and the expression of PPP2R5A in 25 cases (22.7%) did not change signi cantly. The expression of PPP2R5A in 8 cases (7.3%) of cancer tissue was higher than that in adjacent tissues (Fig. 1a, b). The expression of PPP2R5A in each sample of tissue microarray was scored by ImageScope (Aperio) software. The results of statistical analysis showed that the expression score of PPP2R5A in HCC tissues was signi cantly lower than that in adjacent tissues, and the difference was statistically signi cant (p < 0.001) (Fig. 1c). Western Blot was used to detect the content of PPP2R5A in the total proteins of 12 pairs of HCC and adjacent tissues. It was found that the content of PPP2R5A protein in HCC tissues was lower than that in adjacent tissues (Fig. 1d). The above results indicated that the expression of PPP2R5A in HCC tissues of Chinese patients is down-regulated. Among the 366 HCC samples in the TCGA database, about 24% (87/366) of the tumor tissues showed high expression of PPP2R5A (Fig. S1) (http://www.cbioportal.org/), and others showed the same expression levels of PPP2R5A between HCC tissues and adjacent tissues.
After scoring HCC tissue microarray, the median score of cancer tissue (0.24) was selected as the criterion for distinguishing the high and low expression levels of PPP2R5A. Combined with pathological and follow-up data, the correlation between the expression level of PPP2R5A and the survival time of HCC patients was analyzed. The higher expression level of PPP2R5A corresponds to longer progression-free survival (PFS) and overall survival (OS) (p < 0.05) (Fig. 1e). Analysis of samples from the TCGA database also showed that the high expression of PPP2R5A corresponds to a long PFS (Fig. S1).
In summary, PPP2R5A is generally down regulated in cancer tissues of Chinese HCC patients, and it is signi cantly positively correlated with the PFS of HCC patients.
PPP2R5A inhibits the proliferation of HCC cells PPP2R5A was knocked down or overexpressed in HCC QGY-7701 and Hep3B cells. Western blot results showed that knockdown or overexpression of PPP2R5A was effective in both cell lines (Fig. 2a). The results of colony formation assay showed that the number of colonies of HCC cells overexpressing PPP2R5A was signi cantly less than that of the control group, while the number of colonies of HCC cells that were knocked down in PPP2R5A expression was signi cantly more than that of the control group ( Fig. 2b). CCK8 assay con rmed that the overexpression of PPP2R5A caused a decrease in the proliferation of QGY-7701 and Hep3B cells, while knockdown of PPP2R5A caused an increase in the proliferation of the two HCC cell sublines (Fig. 2c).
Western Blot results showed that the overexpression of PPP2R5A resulted in the down-regulation of cell proliferation related indicators such as Bcl-2 and c-Myc. The knockdown of PPP2R5A yielded the opposite results (Fig. 2d, e). The changes of protein molecules were consistent with the inhibition of cell proliferation by PPP2R5A.
PPP2R5A inhibits the metastasis of HCC cells but has no effect on cell cycle The effects of PPP2R5A on cell cycle were detected by ow cytometry. The results showed that PPP2R5A had no signi cant effect on cell cycle (Fig. 3a). The results of wound healing and transwell assays showed that the high expression of PPP2R5A inhibited the migration and invasion of HCC QGY-7701 and Hep3B cells, while the knockdown of PPP2R5A signi cantly promoted HCC cell metastasis (Fig. 3b, c).

HBV infection leads to the down-regulation of PPP2R5A expression
From the above results, it can be known that the expression of PPP2R5A in the cancer tissues from 70% of Chinese HCC patients is down-regulated and shows a certain tumor suppressor effect. Among the 366 HCC samples from TCGA database, about 24% of cancer tissues showed high expression of PPP2R5A (Fig. S1). Considering that there are great differences in the regional pathogenic factors of HCC, the majority of HCC patients from China are HBV positive, it is speculated that the down-regulation of PPP2R5A expression may be related to HBV infection.
In HCC and normal liver tissues, the expression of PPP2R5A in HBV-positive tissues is lower than that in HBV-negative tissues (Fig. 4a). The results of HCC tissue microarray analysis showed that the expression of PPP2R5A was not signi cantly correlated with gender, age, AFP value and TNM staging of HCC patients, but was signi cantly correlated with the expression of HBsAg and pathological grade (p < 0.05) ( Table 1). Western Blot found that the content of PPP2R5A in HepG2.2.15 (HBV+) cells was lower than its parental HepG2 (HBV-) cells (Fig. 4b), suggesting that the expression of PPP2R5A might be inhibited by HBV infection. Western Blot results showed that PPP2R5A was generally low expressed in HCC QGY-7701, PLC/PRF/5, and Hep3B cell lines that were integrated by HBV in cell genome (Fig. 4c). It is concluded from the above results that HBV infection down-regulates the content of PPP2R5A in hepatocytes. However, the speci c mechanism through which HBV regulates the content of PPP2R5A remains unclear and needs to be further studied.

PPP2R5A exerts tumor suppressor e cacy by inhibiting the MAPK/AKT/WNT signaling pathway
The MAPK and AKT signaling pathway play an important role in the process of tumor invasion and metastasis [20,21]. The activation of WNT signaling pathway is also more common in cancer development [22,23]. PPP2R5A is a regulatory subunit of intracellular phosphatase PP2A, responsible for the speci c regulation of the enzymatic function, substrate speci city and intracellular localization of the tumor suppressor phosphatase PP2A, and likely to participate in the regulation of the above-mentioned signaling pathways.
Western Blot was used to detect the phosphorylation levels of Raf, ERK and AKT in HCC cells after knocking down or overexpressing PPP2R5A. The results showed that the overexpression of PPP2R5A signi cantly reduced the phosphorylation levels of Raf, ERK and AKT, and down-regulated the expression of β-Catenin in cells. While the knockdown of PPP2R5A obtained the opposite results. The p-RAF, p-ERK, and p-AKT are all upstream factors that regulate β-Catenin. It is speculated that PPP2R5A inhibits the activation of the WNT pathway by inhibiting the MAPK and AKT signaling pathways, and promotes the degradation of β-Catenin, thereby exerting a tumor suppressor e cacy (Fig. 5a, b).

Discussion
PPP2R5A has been reported to be involved in the regulation of signaling pathways of tumorigenesis [24]. Studies have shown that PP2A negatively regulated the cell anti-apoptotic factor Bcl2, PPP2R5A assisted PP2A in its functions and promoted the occurrence of apoptosis [11,25]. Yamamoto et al. reported that PPP2R5A helped PP2A locate the important proto-oncoprotein β-Catenin in the WNT pathway by binding to the scaffold protein Axin, and promoted β-Catenin degradation [26]. Rodriguez et al. found that the lack of function of the PPP2R5A protein family led to the hyperfunction of the protein kinase B (AKT) family and promoted the activation of the WNT pathway and the occurrence of tumors [12,26]. In addition, studies have shown that after inhibited the expression of PPP2R5A by microRNA-218, the WNT pathway would be signi cantly activated [27]. PPP2R5A can also assist PP2A to localize to c-Myc protein and promote the dephosphorylation and degradation of c-Myc protein [24]. These oncoproteins play an important role in the unlimited proliferation, in ltration and metastasis of tumor cells, and cell death resistance.
It is worth noting that some studies have found that viral infection could affect the expression of PPP2R5A. After treated cells with ceramide or double-stranded RNA, eukaryotic translation initiation factor 2 alpha kinase 2 (PKR) itself activated and phosphorylated the 28th serine of PPP2R5A, which enhanced the inhibitory effect of PP2A on protein synthesis and induction of apoptosis, thereby inhibiting viral infection [28,29]. However, hepatitis C virus (HCV) mRNA can promote Staufen1 to competitively inhibit the binding of viral mRNA and PKR, and block the autophosphorylation activation of PKR [30], which can lead to the decline of PPP2R5A expression and function. The Vif protein produced by lentiviral infection can recruit Cullin-5 (CUL5) E3 ubiquitin ligase to induce the ubiquitination and degradation of PPP2R5A [19,31]. These examples demonstrated the antagonism of PPP2R5A and viral infection in the co-evolution of virus-host. However, the effect of HBV infection on PPP2R5A has not been reported yet.
Based on the above results, the molecular mechanism of PPP22R5A in the occurrence and development of HCC is speculated as follows (Fig. 6): PPP2R5A exerts a tumor suppressor e cacy in hepatocytes. Under HBV-uninfected circumstances, when hepatocytes become cancerous, as one of the protective mechanisms, cells will up-regulate the expression of PPP2R5A. The high expression of PPP2R5A can inhibit the proliferation and metastasis of HCC cells. Therefore, the results of survival analysis showed that the higher the expression of PPP2R5A, the higher the PFS of HCC patients. But HBV is precisely the "nemesis" of PPP2R5A. When HCC patients are infected with HBV, HBV can down-regulate the content of PPP2R5A in cells, leading to a weakening of cellular self-protection mechanism. At this time, the expression of p-RAF, p-ERK, and p-AKT is up-regulated, and the activation of MAPK and AKT signaling pathways leads to an increase of β-Catenin, the key molecule of WNT signaling pathway, and an activation of WNT signaling pathway. The continuous activation of MAPK/AKT/WNT signaling pathways can promote cell proliferation and metastasis, thereby promoting the occurrence and development of HCC.
Combined with the fact that 85%-90% of HCC in China is closely related to HBV infection, it may be that HBV infection weakens the self-protection function of hepatocytes and increases the possibility of cell canceration.

Conclusions
PPP2R5A acted as a tumor suppressor in HCC cells. HBV inhibited the initiation of the protective mechanism mediated by PPP2R5A, making the occurrence and progress of HCC more "unimpeded". This conclusion will further reveal the molecular mechanism of HBV-induced HCC and provide clues for the prevention and treatment of HCC induced by HBV infection.

Declarations
Ethics approval and consent to participate The study has been approved by the Committee on Ethics of Medicine, Navy Military Medical University (Shanghai, China), and the patients have signed informed consents.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information les. Further details are available from the corresponding author upon request.

Competing interests
The authors declare that they have no competing interests  The low expression of PPP2R5A is signi cantly related to the poor prognosis of HCC. a. IHC results showed that the higher, lower and comparable expression of PPP2R5A were found in HCC tissues compared with peritumor tissues in the tissue microarray. Case 1: tumor>peritumor; Case 2: tumor≈peritumor; Case 3: tumor<peritumor. Bar=50 μm; b. Statistical analysis of the expression of PPP2R5A in HCC tissue samples relative to the adjacent tissues; c. IHC score results showed that the expression of PPP2R5A in HCC tissues is lower than that of adjacent tissues (***, p<0.001); d. Western blot of PPP2R5A in the total proteins of 12 pairs of HCC and adjacent tissues and semi-quantitative statistical analysis; e. Survival analysis showed that low expression of PPP2R5A was related to the poor prognosis of HCC patients.

Figure 3
PPP2R5A inhibits the migration and invasion of HCC cells. a. The effect of PPP2R5A on cell cycle; b. Wound healing assay to detect the effect of PPP2R5A on cell migration, and the statistical analysis; c.
Transwell assay to detect the effect of PPP2R5A on cell invasion, and the statistical analysis. (*, p<0.05; **, p<0.01; ***, p<0.001) Figure 4 HBV infection leads to the down-regulation of PPP2R5A. a. The differences in the expression of PPP2R5A in liver tissues of HBV(+) and HBV(-), Bar=50 μm; b. Western Blot of PPP2R5A in HepG2.2.15 HBV(+) and HepG2 HBV(-) HCC cells, and the bottom is the semi-quantitative statistical bar graph; c. Western Blot of PPP2R5A in HBV(+) and HBV(-) HCC cells, and the semi-quantitative statistical bar graph is shown below.
a. PPP2R5A inhibits the activation of WNT signaling pathway by inhibiting MAPK and AKT signaling pathways. b. PPP2R5A acts as a tumor suppressor both in hepatocytes and HCC cells. Without HBV infection, when hepatocytes become cancerous, as one of the protective mechanisms, the cells will upregulate the expression of PPP2R5A. The high expression of PPP2R5A can inhibit the proliferation and metastasis of HCC cells. When HCC patients are infected by HBV, HBV can down-regulate the content of PPP2R5A in cells. It leads to the reduction of PPP2R5A that exerts a tumor suppressor e cacy and the weakening of this cellular self-protection mechanism, thereby promoting the occurrence and development of HCC.

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