Ubiquitin ligase MARCH8 promotes the malignant progression of hepatocellular carcinoma through PTEN ubiquitination and degradation

Membrane‐associated ring‐CH‐type finger 8 (MARCH8) belongs to the MARCH family of membrane‐bound E3 ubiquitin ligases. The N‐terminus of MARCH family members contains the C4HC3 RING‐finger domain, which can bind to E2 ubiquitin‐conjugating enzymes, ubiquitinate substrate proteins, and thereby promote protein degradation through the proteasome pathway. The aim of this study was to determine the role of MARCH8 in hepatocellular carcinoma (HCC). We first analyzed the clinical relevance of MARCH8 based on The Cancer Genome Atlas database. Immunohistochemical staining was used to detect MARCH8 expression in human HCC samples. Migration and invasion assays were conducted in vitro. Cell apoptosis and cell cycle distribution were analyzed by flow cytometry. The expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN)‐related markers was evaluated in HCC cells through Western blot analysis. MARCH8 was highly expressed in human HCC tissues, and its high expression was inversely correlated with patients’ survival. Disrupting MARCH8 expression significantly inhibited the proliferation, migration, and cell cycle progression of HCC cells, while also promoting their apoptosis. In contrast, the overexpression of MARCH8 significantly enhanced cell proliferation. Mechanistically, our results showed that MARCH8 interacted with PTEN and suppressed the protein stability of PTEN by enhancing its ubiquitination level via the proteasome. MARCH8 also activated AKT in HCC cells and tumors. In vivo, overexpression of MARCH8 could promote the growth of hepatic tumors through the AKT pathway. MARCH8 may promote the malignant progression of HCC by promoting the ubiquitination of PTEN, thereby relieving the inhibitory effect of PTEN on the malignant phenotype of HCC cells.


| INTRODUCTION
Globally, the incidence of hepatocellular carcinoma (HCC) ranks sixth among all cancers, while the mortality rate ranks fourth. In 2018, an estimated 840,000 new cases of HCC were diagnosed worldwide, resulting in approximately 780,000 deaths. 1 In China, new HCC cases account for approximately 55% of the global total each year, and the country has the second highest HCCassociated mortality rate. 2 Over recent years, the treatment of HCC has made great progress. Curative treatments such as surgery, radiotherapy and chemotherapy, liver transplantation, immunotherapy, and targeted therapy have all contributed to improving patient prognosis. [3][4][5] Additionally, an improved understanding of the molecular mechanisms involved in HCC development and progression has led to the emergence of targeted drugs such as sorafenib, lenvatinib, and regorafenib that have greatly improved the choice of drugs for patients with advanced HCC. [6][7][8] However, the prognosis of patients with HCC remains poor, as does the 5-year survival rate. 9 These observations highlight the need to clarify the molecular mechanism underlying the malignant progression of HCC so as to identify therapeutic targets and develop more effective treatment strategies. In this study, we analyzed the expression of MARCH8 in HCC samples and its correlation with the classification and staging of HCC both in vitro and in vivo. The results indicated that MARCH8 was highly expressed in HCC and was closely related to the classification and staging of this disease. The results further showed that MARCH8 promoted the malignant progression of HCC through increased ubiquitination and subsequent degradation of PTEN. These findings suggest that MARCH8 may represent a novel therapeutic target for the treatment of HCC.

| Apoptosis and cell cycle analysis
Apoptosis was detected by PI/Annexin V-FITC staining assay kit.

| Statistical analysis
The experiments were performed for at least three independent repeats. The data were presented as mean ± SEM, excluding the figure part. Student's t-test and one-way analysis of variance, followed by Tukey's post hoc test, were used to compare the difference between two groups and more than or equal to three groups. p < 0.05 was considered statistically significant.

| Expression of MARCH8 is related to the occurrence and malignant progression of HCC
In this study, we first used TCGA to analyze the RNA-seq expression data for MARCH8 in HCC and adjacent samples. We found that the promoter methylation level of MARCH8 gene was reduced in HCC tissues compared with normal tissues ( Figure 1A). The expression of MARCH8 was significantly higher in HCC tissues than in adjacent samples ( Figure 1B). We then collected clinical samples of 15 pairs of HCC and control adjacent tissues and assessed the protein expression level of MARCH8 using immunohistochemistry. Consistent with TCGA data, we found that the level of MARCH8 in cancer tissues was significantly higher than that in adjacent tissues ( Figure 1C,D). These data indicated that the low methylation level of MARCH8 gene and high expression of MARCH8 may be closely related to the occurrence of HCC.  Figure 2C,D). Moreover, in a colony formation assay, the knockdown of MARCH8 led to the formation of significantly fewer clones when compared with that of the control group ( Figure 2E). We further found that the migratory ability of MARCH8-depleted HCC cells was significantly weaker than that of the control group as evidenced by a Transwell assay ( Figure 2F).

| MARCH8 promotes the cell proliferation and migration of HCC cells
To verify the tumor-promoting role of MARCH8, we over-

| Knockdown of MARCH8 significantly promotes cell apoptosis and cell cycle arrest
The knockdown of MARCH8 could inhibit the proliferative and migratory abilities of HCC cells (Figure 2). To test whether MARCH8 depletion regulates cell apoptosis, we used flow cytometry and PI/ Annexin V-FITC staining to detect the apoptosis level of HCC cells. We found that, compared with the siCtrl treatment group, the apoptosis level was significantly increased in HCC cells transfected with siMARCH8 ( Figure 3A,B). Then, the cell cycle was checked by PI staining and flow cytometric analysis. The results showed that siMARCH8-transfected cells were arrested at the G0/G1 phase ( Figure 3C,D). These results suggest that MARCH8 depletion promotes cell apoptosis and induces cell cycle arrest at the G0/G1 phase.
Next, we employed RT-qPCR to measure the expression of genes related to these processes. Compared with the controls, we found that the expression of the cell proliferation-and cell cycle-related genes cyclin D1 and cyclin E1 was significantly downregulated in MARCH8-depleted SKHEP1 and MHCC97H cells ( Figure 3E). The expression of E-cadherin, an epithelial-mesenchymal transitionrelated gene that is associated with cell migration, was significantly upregulated, and that of N-cadherin was significantly downregulated, in the siMARCH8-transfected group when compared with the controls ( Figure 3E). These changes in the expression levels of these marker genes were consistent with the cell phenotype changes observed under the same treatment.

| MARCH8 interacts with PTEN and inhibits PTEN protein stability
The above results indicated that MARCH8 has an important role in HCC. However, its mechanism of action is still unclear. As an E3 XU ET AL. showed that PTEN is a potential MARCH8-interacting protein ( Figure 4A,B). We then performed CoIP and immunoblotting assay and found that there was an interaction between MARCH8 and PTEN in 293T cells ( Figure 4C). Furthermore, the expression of PTEN protein level was increased in MARCH8-depleted cells ( Figure 4D).
By contrast, MARCH8 overexpression suppressed the protein expression of PTEN in SKHEP1 and MHCC97H cells ( Figure 4D).
We also found that MARCH8 knockdown reduced, while its overexpression enhanced the phosphorylation level, but not the protein expression of AKT in SKHEP1 and MHCC97H cells ( Figure 4D). In addition, MARCH8 overexpression or knockdown had no effect on the mRNA expression of PTEN and AKT (Supporting Information: Figure S1). These results suggested that MARCH8

| CONCLUSIONS
In conclusion, our study provided the first evidence that MARCH8 played a tumor-promoting role in the development of HCC.
Importantly, MARCH8 interacted with PTEN and promoted the degradation of PTEN, which was frequently downregulated in HCC patients. Thus, this study identified MARCH8 as a novel modulator of PTEN and a promising drug target for HCC patients.

AUTHOR CONTRIBUTIONS
Yingchen Xu designed and performed experiments, analyzed data, and wrote the paper. Dongxin Zhang performed experiments. Jiajun Ji initiated the study and analyzed data. Lijun Zhang initiated the study, organized, designed, and wrote the paper.