Zyxin promotes hepatocellular carcinoma progression via activation the AKT/mTOR signaling pathway

doi:10.21203/rs.3.rs-2239977/v1

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Zyxin promotes hepatocellular carcinoma progression via activation the AKT/mTOR signaling pathway

https://doi.org/10.21203/rs.3.rs-2239977/v1

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To investigate the regulatory effect and specific mechanism of the actin-interacting protein zyxin (ZYX) in hepatocellular carcinoma (HCC). HCC is one of the most common malignant tumors in the world which occurrence and development areregulated by multiple genes. We found that the expression of ZYX in HCC tissues was significantly higher than that in normal liver tissues. The results of cell proliferation assay, scratch test and transwell assay showed that high expression of ZYX promoted the proliferation, migration and invasion of hepatoma cell lines (PLC/PRF/5, HCCLM3), inhibiting the expression of ZYX reduced the proliferation, migration, and invasion of hepatoma cells(SK HEP-1, Huh-7). Further analysis found that the expression of cell cycle-related proteins, cell migration and invasion-related proteins were changed when the expression of ZYX changed. Xenograft models showed similar results. The AKT/mTOR signaling pathway is a classic pathway ofcancer development. We found that the phosphorylation level of AKT/mTOR protein was up-regulated with increasing ZYX expression and down-regulated with decreasing ZYX expression. While the addition of the AKT inhibitor MK2206 counteracted the proliferation, migration and invasion of HCC cells with increasing ZYX expression, the AKT activator SC79 also restored the proliferation, migration and invasion of HCC cells with decreasing ZYX expression. Therefore, we speculate that the expression of ZYX may promoting the progression of HCC by activating AKT/mTOR signaling pathway, thereby. This is also the first time to find the mechanism of ZYX in HCC, indicating that ZYX is a possible new target for HCC treatment.

zyxin

hepatocellular carcinoma

AKT/mTOR

proliferation

migration

invasion

Liver cancer is one of the most malignant tumors in the world, with the sixth highest incidence and the third highest mortality rate in the world[1]. Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Although there are many mature treatments for HCC such as radical surgery, targeted drugs, and interventional therapy, its early clinical symptoms are insidious, the course of the disease progresses rapidly, and it is easy to be treated after treatment. Recurrence and metastasis, poor prognosis, and mortality remains high[2, 3] .

The occurrence and development of HCC is a complex biological process involving the dysfunction of various cancer-related molecules and genes. In recent decades, the research on the molecular mechanism of HCC diagnosis and treatment has never stopped, but the key factors of its pathogenesis are still unclear[4]. In recent years, increasing evidence shows that ZYX plays a crucial role in various biological processes[5]. The actin-interacting protein ZYX is a LIM domain protein that has been detected in the cytoplasm and nucleus, and can translocate between these two compartments[6]. ZYX is a component of focal adhesions, responsible for the interaction between cells and the extracellular matrix, and is also involved in the organization and regeneration of the cytoskeleton[7]. Studies have shown that ZYX has pro-proliferative and invasive effects in various cancers including glioblastoma[8] and colorectal cancer[9]. There is no relevant report on its role in hepatocellular carcinoma, so we explored the expression and biological functions of ZYX in HCC.

It has been reported that the expression of ZYX in HCC tissues is significantly higher than that in normal liver tissues[10]. We verified this conclusion through molecular informatics analysis and molecular biology experiments, and found that ZYX can promote the phosphorylation of AKT/mTOR signaling pathway proteins. The focal adhesions where ZYX is located are the centers of various signaling pathways, including the classic cancer pathway involving AKT and the mTOR[11–13], but the role of ZYX has not been clearly demonstrated in this context. In this study, we verified the effect of abnormal expression of ZYX on the AKT/mTOR pathway in HCC, and confirmed that ZYX promotes the progression of the cancer through this pathway.

2.1 Bioinformatic analysis

Microarray data GSE14520, comprising 22 HCC tissues and 21 normal liver tissues, was downloaded from the Gene Expression Omnibus(GEO) database (http://www.ncbi.nlm.nih.gov/geo/) on the National Center for Biotechnology Information(NCBI) website. Expression matrix was formed with the raw counts of each RNA of each sample. We compared data from.

HCC tissue and normal liver tissue to identify differentially expressed RNAs. The threshold was set at |log₂FC|>1. The expression level of differentially expressed RNAs was log2(*+1)-transformed for further analysis. Heat maps and volcano plots were drawn to analyze RNAs showing differential expression. StarBase V3.0 and GEPIA were used to analyze the expression of ZYX in HCC and normal liver tissues. The Human Protein Atlas was used to analyze the overall survival in HCC patients with or without ZYX.

2.2 Patients and specimens

HCC tissues and matched normal adjacent tissues were obtained from 17 patients undergoing surgery for HCC in the Department of Hepatobiliary Surgery at the Affiliated Hospital of Southwest Medical University. None of the patients had received adjuvant therapy. The fresh tissues were cut into 1-mm³ pieces and rinsed immediately with cold saline. One part of the fresh tissues were snap-frozen in liquid nitrogen and stored at -80°C for tissue protein extraction, while the other part were formalin-fixed and paraffin-embedded for HE staining and immunohistochemical (IHC). Final diagnosis was confirmed by two experts based on the biopsy specimens. The patients provided their written informed consent to participate in this study. The studies involving human participants were reviewed and approved by the Clinical Trial Ethics Committee of Affiliated Hospital of Southwest Medical University (approval number: KY2019053).

2.3 Cell culture

The human HCC cells, including BEL-7402, SK HEP-1, Huh7, MHCC97H, HCCLM3, PLC/PRF/5, and normal human liver cell line QSG-7701, were purchased from the cell bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cell lines were cultured in high-glucose DMEM medium (Gibco, Shanghai, China) supplemented with 10% fetal bovine serum (Catalog No. ZQ500-A, Zhong Qiao Xin Zhou Biotechnology, Shanghai, China) and 1% penicillin and streptomycin (Catalog No. C0222, Beyotime Bio, Shanghai, China). All the cells were grown in a humidified environment at 37 ̊C with a condition of 5% CO2 and 95% air.

2.4 Preparation of lentivirus and screening of stable strains

Short hairpin RNA (shRNA) targeting human ZYX (shZYX) was purchased from Sangon Biotech (Shanghai, China). The shZYX oligonucleotide sequences were as follows: shZYX-1: 5'-CTGGGTCACAACCAAATCAAA-3'; shZYX-2: 5'-CTTCCACATGAAGTGTTACAA-3'. For stable transfection of plasmids, lentivirus were generated by co-transfecting 293T cells with shRNA plasmids and packaging plasmids (pLP1, pLP2 and pLP/VSVG). Cell supernatants were harvested 48h after plasmid transfection and incubated with hepatoma cells for 48h. Infected cultures were then switched to selection medium supplemented with puromycin (4µg/ml) for 48 h to screen for stable cell lines. shZYX-1 and shZYX-2 were used as disruptors of ZYX expression, and shNC was used as a negative control.

The forward amplification primer sequence of ZYX is 5'- acctccatagaagattctagaATG GCG GCC CCC CGC CCG − 3' and the reverse amplification primer sequence is 5'- ttcgaattcgctagctctagaTCA GGT CTG GGC TCT AGC AGT G -3'.The full sequence of ZYX were constructed by PCR and cloned into the pCDH vector.The lentivirus was obtained by co-transfecting the pCDH plasmid with ZYX sequence and the packaging plasmid into 293T cells. HCC cells were infected with lentivirus and screened as described above. Finally HCC cell lines stably overexpressing ZYX were obtained. PCDH-ZYX is expressed as the overexpression of ZYX, and Vector is the control. PCDH-ZYX is expressed as the overexpression of ZYX, and Vector is the control.

2.5 Cell proliferation

Cells were seeded in 96-well plates at a density of 2000 cells per well along with 100ul of cell culture medium per well. To measure cell proliferation, 10 µl of Cell Counting Kit-8(CCK-8; Catalog No. C0038, Beyotime Bio, Shanghai, China) was added to each well after 24h, 48h, 72h, 96h, and 120h, respectively, the mixture was incubated for 2h at 37 ̊C. The absorbance of all wells was assessed at 450 nm by a microplate reader. 0.5ul of MK2206(Catalog No. SF2712-10mM, Beyotime Bio, Shanghai, China) and 1.64ul of SC79(Catalog No. SF2730-10mM, Beyotime Bio, Shanghai, China) were added per milliliter of DMEM to achieve the final concentrations of MK2206 and SC79 in DMEM to 2.5 and 5ug/ml, respectively. DMEM with MK2206 or SC79 was used for the above experimental operations.

2.6 EdU staining

A Click™ EdU-555 Cell Proliferation Kit (Catalog No. C0075S, Beyotime Bio, Shanghai, China) was used for the cell proliferation assay. Cells were seeded in 6-well plates and when they reached 70% confluence, the medium was removed and 2 ml of complete medium containing 10 µmol/L EdU was added and incubated at 37°C for 2 hours. Media was removed, cells were washed 3 times with phosphate buffered saline (PBS), and after fixation with 4% formaldehyde for 15 min at room temperature, the cells were washed 3 times with PBS again and permeabilized with 0.3% Triton X-100 for 15 min. After three more PBS washes, cells were incubated with Click Additive Solution for 30 min. Nuclei were stained with Hoechst-33342.

2.7 Cell cycle and apoptosis

1 × 10⁵ cells of PLC/PRF/5 and SK HEP-1 respectively were harvested and washed with PBS, then fixed them in 70% ethanol at 4°C overnight. The next day, samples were incubated in a solution of 10 mg/ml of RNase and 1 mg/ml of propidium iodide (Catalog No. C1052, Beyotime Bio, Shanghai, China) at 37°C for 30 min in the dark. The DNA content was determined using flow cytometry (BD Biosciences). For cell apoptosis, PLC/PRF/5 and SK HEP-1 cells were digested with EDTA-free trypsin and collected in 1.5ml EP tubes. The cells were washed with pre-cooled PBS, and were collected by centrifugation at 300g, 4°C for 5min. Apoptosis analysis of SK HEP-1 cells using Annexin V-FITC/PI Apoptosis Detection Kit (Catalog No. 40302ES20, YEASEN, Shanghai, China), 100 µL Binding Buffer was used to resuspend cells, added 5 µL Annexin V-FITC and 10 µL PI Staining Solution, mixed gently. Incubate the reaction at room temperature for 10 min in the dark. Then additional 400 µL of Binding Buffer was added. Annexin V-PE/7-AAD Apoptosis Detection Kit (Catalog No. 40310ES20, YEASEN, Shanghai, China) was used to detect apoptosis of PLC/PRF/5-Vector and PRF/PLC/5-PCDHZYX. Resuspended cells in 100 µL Binding Buffer, added 5 µL Annexin V/PE and 10 µL 7-AAD staining solution, mixed well, incubated at room temperature for 10 minutes in the dark, and then added 400 µL Binding Buffer. Analysis was performed using flow cytometry (BD Biosciences).

2.8 Colony formation

Cells were seeded in 6-well plates at a density of 1000 cells per well, cultured at 37°C for 10 days. The medium was discarded, PBS was used to wash the colonies. Then the colonies were fixed with 4% paraformaldehyde and stained with crystal violet. Colonies were photographed, counted and statistically analyzed.

2.9 Scratch test

Cells were seeded in 6-well plates. Upon reaching 90% confluence, scratches were evenly made with a 200 µl pipette tip and photographed under a microscope. The scratched cells were cultured in serum-free medium, and the scratch repair was monitored by taking pictures under a microscope at 24 h and 48 h after scratching, respectively.

2.10 Transwell assay

For the migration assay,a 24-well transwell was used (Corning, Shanghai, China). The cells were resuspended in serum-free medium and seeded in transwell chambers at 5 × 10⁴ cells per well. In the lower chamber, complete medium containing 10% fetal bovine serum was used as an inducer. After incubation for24h, the unmigrated cells in the upper chamber were wiped off with a cotton swab, and migrated cells on the underside of the filter were fixed in 4% paraformaldehyde and stained with crystal violet, and then photographed and counted. For the invasion assay, membranes were coated with Matrigel (BD Biosciences, Shanghai, China); the other steps were the same as in the migration assay.

2.11 Tumor xenografts

Ten 4-week-old male BALB/c nude mice were purchased from Chongqing Tengxin Biotechnology Company (Chongqing, China) and housed in a temperature and humidity-controlled laboratory animal room. Mice were randomly divided into two groups: shNC and shZYX. For the xenograft model, 5 × 10⁵ SK HEP-1-shNC or SK HEP-1-shZYX-2 cells suspended in 0.1 ml serum-free medium were injected subcutaneously into the right axilla of nude mice. The experiment lasted 24 days, the mice were sacrificed at the end of the experiment, the tumors were removed and weighed, the length and width of the tumors were measured, and the values were plugged into the formula: size = length × (width)²/2 to calculate tumor size. Part of the tumor tissue from each group was fixed in 4% paraformaldehyde, and then subjected to IHC staining analysis. The rest of the tissue protein was extracted for WB detection. Animal studies were reviewed and approved by the Southwest Medical University Laboratory Animal Ethics Committee (approval number: 20211119-062).

2.12 RNA extraction, reverse transcription and quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from hepatoma cells using RNA-easy Isolation Reagent (Cat. #R701-01, Vazyme, China) according to the manufacturer’s instruction. Master Mix (Cat. Q712-02, Vazyme, China) was used for reverse transcription, and the resulting cDNA was used for qRT-PCR. The total qRT-PCR reaction was 20 µl, including 2 µl of sample cDNA, 0.4 µl each of forward and reverse primers, 7.2 µl of enzyme-free water, and 10 µl of Taq Pro Universal SYBR qPCR Master Mix. The expression of each gene was normalized to β-actin expression. The primer sequences were ZYX: forward 5'-TCTCCCGCGATCTCCGTTT-3', reverse 5'-CCGGAAGGGATTCACTTTGGG-3';β-actin: forward 5′-ATCGTGCGTGACATTAAGGAGAAG-3′, reverse 5′-AGGAAGGAAGGCTGGAAGAGTG-3′.

2.13 Western blot

An appropriate amount of cells and tissues were collected into 1.5ml EP tubes, and the tissues and cells were lysed using RIPA lysis buffer (Catalog No. P0013B, Beyotime,China) supplemented with protease inhibitors(Catalog No. P1005, Beyotime, China) and phosphoprotease inhibitors(Catalog No. P1081, Beyotime, China), and the samples were placed on ice for 1 hour with discontinuous vortexing. The lysates were centrifuged at 13000 rpm for 10 min at 4 ̊C. The supernatant of lysates was harvested and protein concentration was quantified using a BCA protein assay kit (Catalog No. P0012, Beyotime, China). Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5% nonfat milk in Tris-buffered saline containing Tween 20 (TBST) for 1h, washed using TBST thrice, and incubated with primary antibody overnight at 4°C. On the second day, the membranes were washed with TBST thrice for 10min each time, incubated with the secondary antibody for 1h at room temperature, washed with TBST thrice for 10min each time, and finally, detected by the enhanced chemiluminescence detection system (Catalog No. P0018FS, Beyotime, China).

2.14 Hematoxylin–eosin(HE) and immunohistochemistry(IHC) staining

The tissue was embedded in paraffin and cut into thin slices with a thickness of 5 µm, after baking the tissue sections were invaded into xylene for dewaxing followed by rehydration with graded alcohol (100%, 100%, 95%, 80% and 70%) solutions. Then HE staining was done using a commercial kit (Catalog No. C0105, Beyotime, China).

For the IHC, the procedures of dewaxing and rehydration were the same as HE staining. Then, antigen retrieval was performed by heating slides in the microwave for 20 min in 0.01M citrate buffer (pH 6.0), and 3% hydrogen peroxide was added for 10 min to quench peroxidase activity. Primary antibodies ZYX (1:100), MMP-2 (1:200), and Ki-67 (1:2000) were added, and the sections were kept overnight at 4°C. The following day, the secondary antibody was incubated at room temperature for 1h, and after washing with PBS, sections were stained with diaminobenzidine and counterstained with hematoxylin. Sections were dehydrated and sealed, then examined using a microscope (Olympus, Chengdu, China).

2.15 Statistical analysis

Experiments carried out in triplicates were analyzed with statistics and shown in the figures. Inter-group differences were assessed for significance using the two-tailed Student’s t test. Data were expressed as mean ± standard deviation. p < 0.05 was considered statistically significant.

3.1 ZYX is overexpressed in HCC tissue

Analysis of the GSE14520 dataset revealed that 710 genes were significantly up-regulated and 896 genes were significantly down-regulated in HCC tissues compared with normal liver tissues (Fig. 1A). ZYX was one of the most significantly up-regulated genes (Fig. 1B). StarBase V3.0 database and GEPIA database also confirmed the high expression of ZYX in HCC tissues (Fig. 1C, D). Overall survival from the Human Protein Atlas showed that the HCC population with low ZYX expression had a better prognosis than the HCC population with high ZYX expression (Fig. 1E), suggesting that ZYX also affects the prognosis of HCC patients.

We obtained cancerous tissue and adjacent normal tissue from 17 patients with pathologically diagnosed HCC. Western blot results showed that the expression of ZYX was abnormally increased in HCC tissues (Fig. 1F). HE staining highlighted the pathological features of HCC tissues and adjacent normal tissues (Fig. 1G), and the IHC staining results showed that the expression of ZYX was significantly higher in HCC tissues than in normal tissues (Fig. 1H).

3.2 Construction of human hepatoma cell lines with stable overexpression or knockdown of ZYX

We selected six different human hepatoma cell lines and one normal human hepatocyte cell line for protein analysis. The results showed that, compared with the levels in normal human hepatocyte cell lines, the expression of ZYX mRNA was increased in BEL-7402, SK HEP-1, and MHCC97H human hepatoma cell lines, while it was lower in HCCLM3 and PLC/PRF/5 cells (Fig. 2A). Western blot experiments confirmed these results (Fig. 2B). We constructed stable PLC/PRF/5 and HCCLM3 lines overexpressing ZYX, and validated the stable transfection using qRT-PCR and western blot (Fig. 2C,D). At the same time, we used the hairpin interfering plasmid shZYX to transfect SK HEP-1 and Huh7 cell lines, and the detection of ZYX by qRT-PCR and western blot indicated that the expression of ZYX was reduced in these two cells (Figure. 2E, F).

3.3 ZYX affects HCC cell proliferation, cell cycle and apoptosis in vitro

To explore the effect of ZYX on the proliferation of HCC cells, we performed proliferation experiments using these four stably transfected cell lines. We found that when ZYX was highly expressed, cell growth was significantly accelerated; when ZYX expression was inhibited, the growth of hepatoma cells was significantly slowed down (Fig. 3A). EdU staining also showed the same results (Fig. 3B). We analyzed the cell cycle progression of HCC cells using flow cytometry. It was found that the proportion of HCC cells entering S phase increased when ZYX expression was increased, while inhibition of ZYX expression significantly decreased the proportion of cells entering S phase (Fig. 3C). This indicated that ZYX can promote the proliferation of HCC cells by regulating the cell cycle. We also explored the relationship between ZYX and apoptosis of HCC cells. When ZYX expression was knocked down, the proportion of apoptotic and necrotic cells increased significantly while overexpression of ZYX did not seem to have a significant effect on HCC cell apoptosis (Fig. S1A). At the same time, when ZYX expression increased, the expression of proteins related to cell proliferation and cell cycle such as Proliferating Cell Nuclear Antigen (PCNA), cyclin D1, cyclin-dependent kinase 2 (CDK2), and CDK4 were also increased (Fig. 4A), while the expression of apoptosis-related proteins did not change significantly (Fig. S1B). Knockdown of ZYX expression inhibited the expression of cyclin proteins (Fig. 4B), up-regulated the expression of apoptotic protein BAX, and decreased the expression of Bcl-2 (Fig. S1C). Subsequently, we performed cell colony formation experiments, which proved that the high expression of ZYX contributed to the colony formation of HCC cells, and when the expression of ZYX was inhibited, the colony formation rate of HCC cells also decreased significantly (Fig. 4C, D).

3.4 ZYX accelerates migration and invasion of HCC in vitro

We performed scratch time and invasion experiments to explore the effect of ZYX on the migratory and invasive abilities of HCC cells. In scratch experiments, we found that HCC cells overexpressing ZYX had faster scratch healing (Fig. 5A), indicating enhanced cell migration, whereas when ZYX expression was disturbed, the scratch repair was significantly slower (Fig. 5B). In transwell experiments, we found that HCC cells with high ZYX expression had stronger migratory and invasive abilities (Fig. 5C), and interfering with ZYX expression significantly attenuated these abilities, compared with control cells (Fig. 5D). Based on the above results, we concluded that ZYX could promote the migration and invasion of HCC cells. ZYX also significantly altered the expression of EMT-related proteins such as E-cadherin, Vimentin, MMP-9, MMP-2, and Snail. When ZYX expression was increased, an obvious increased of Vimentin, MMP-9, MMP-2, and Snail was observed in both cell lines compared with the control group, while on the contrary E-cadherin down-expressed. The expression of these proteins showed the opposite trend when ZYX expression was inhibited(Fig. 6).

3.5 ZYX affects the expression of AKT/mTOR signaling pathway proteins in hepatoma cells

The AKT/mTOR signaling axis is an important regulatory pathway in the initiation and development of HCC, and ZYX may play a role by regulating the expression of AKT/signaling pathway proteins. We examined changes in the expression of pathway proteins in stably transfected cells. As expected, the phosphorylation levels of AKT and mTOR in HCC cells were significantly upregulated when ZYX was overexpressed, while they were greatly reduced when ZYX expression was disturbed. However, the amounts of AKT and mTOR themselves did not change significantly (Fig. 7A). We used the AKT inhibitor MK2206(2.5ug/ml) and the AKT activator SC79(6ug/ml) to verify the relationship between ZYX and the AKT/mTOR pathway. Under the effect of MK2206, the proliferation rate of HCC cells overexpressing ZYX was significantly decreased, while SC79 enhanced the proliferation ability of HCC cells with low ZYX expression (Fig. 7B). The results of Transwell experiments showed that MK2206 reduced the migration and invasion rates of ZYX-overexpressing HCC cells. In HCC cells with knockdown of ZYX expression, their migratory and invasive abilities were restored under the action of SC79(Fig. 7C). MK2206 led to a decrease in AKT/mTOR phosphorylation levels, and SC79 up-regulated AKT/mTOR phosphorylation levels. What’s more, the changes in the expression of E-cadherin and CDK2 proteins showed the same trend as the exercise phenotype because of MK2206 and SC79(Fig. 7D).

3.6 ZYX promotes the proliferation of HCC cells in vivo

After interfering with ZYX expression, the tumorigenic rate of hepatoma cells reduced (Fig. 8A). The tumor growth rate in the ZYX interference group (shZYX) was lower than that in the control group; consequently, the tumors in this group were lower in volume (Fig. 8B). We performed IHC staining on the tumor model and found that the expression of Ki-67, and MMP-2 proteins in shZYX tumors was lower than that in control tumors (Fig. 8C). Western blot experiments showed that the expression of cyclin D1, CDK2, and MMP-2, as well as the phosphorylation of AKT and mTOR proteins in shZYX tumors was significantly decreased compared with the control group (Fig. 8D).

HCC is a malignant tumor characterized by high morbidity, high mortality, and poor prognosis. Its pathogenesis has always been a hot topic in medical research. The normal liver suffers from various liver injuries, undergoes multiple pathological changes such as hepatitis and liver cirrhosis, and eventually progresses to HCC, which is regulated by a huge and complex gene network[14, 15]. As we know, previous studies noted that a combination of early diagnosis and efficient therapy are key to successful clinical management of cancers[16], so finding the regulatory genes that play an important role in the development and progression of HCC is the most important topic at present. Here, our experimental evidence suggests that ZYX is a tumor-promoting protein in HCC. In our analysis, it was up-regulated in HCC tissues of most HCC patients, and high ZYX expression was associated with poor prognosis. We artificially induced the abnormal expression of ZYX in HCC cells with lentivirus, which increased its expression in PLC/PRF/5 and HCCLM3 cell lines and decreased its expression in SK HEP-1 and Huh7 cells, to explore the effect of ZYX on HCC more comprehensively.

Cyclin[17–19] and CDKs[20–22] drive the cell cycle, control Cells grow and proliferate, and signaling pathways targeting these proteins can improve a variety of cancers, including HCC. During G1 phase, in response to mitogenic stimuli, cells synthetize Cyclin D that promote the CDK4 activation. The CDK4-Cyclin D complexes catalyze the mono-phosphorylation of RB, a protein that in the hypo-phosphorylated state binds and inhibits E2F family transcription factors, drive cell cycle progression which occurs at G1-S transition and after that. This is also the first checkpoint of the cell cycle[23]. In late G1, CDK2 activates which further phosphorylates Rb, thereby promoting complete release of E2F factors, inducing their maximal action as transcriptional activators, and promoting cell cycle progression from G1 to S phase[24].PCNA lies at the center of the faithful duplication of eukaryotic genomes, loaded on chromatin to play a role, and is necessary for cellular DNA replication[25, 26]. Therefore, we determined the expression of proliferation-related proteins in human hepatoma cell lines stably overexpressing or knocking down ZYX and found that the expressions of PCNA, cyclin D1, CDK2 and CDK4 were upregulated with increasing ZYX expression and downregulated with decreasing ZYX expression. Combined with the results of flow cytometry, colony formation experiments and cell proliferation experiments, we can infer that ZYX affects cell cycle progression and can promote HCC cells from G1 phase to S phase, thereby increasing the efficiency of cell division and proliferation and promoting the progression of HCC. What’s more, when we knocked down the expression of ZYX, the apoptosis and necrosis of HCC cells increased. Over last decades, most antitumor therapeutic strategies have focused on apoptosis, triggering other programmed cell death such as necroptosis may achieve a better therapeutic efficacy[27]. In the absence of ZYX, HCC cells cannot be supported to complete normal growth and proliferation, which leading them to apoptosis and necrosis, which may indicate the ZYX plays a key role in cell growth and reproduction, and may be an effective target for HCC therapy. However, the mechanism of apoptosis and necrosis of HCC cells is very complex, and the same gene may not only promote apoptosis, but also inhibit apoptosis in some cases. Therefore, the effect of ZYX on HCC cell apoptosis may need more experiments to verify.

Results of the cell scratch and transwell assay showed that the high expression of ZYX could promote the migration and invasion of HCC cells. The epithelial-mesenchymal transition-related protein E-cadherin, Vimentin, and Snail are the most important mediators of cancer cell migration and invasion[28–30], and the importance of MMP family members in these processes is self-evident [31, 32]. MMP-2 and MMP-9 play a key role in the development of HCC[33–35]. Western blot results of the proteins E-cadherin, Vimentin, Snail, MMP-2, and MMP-9 demonstrated that ZYX could affect the expression of the proteins related to migration and invasion of HCC cells, and promote the progression of HCC.

We used stably transfected HCC cell lines with down-regulated ZYX expression to construct in vitro tumor models in nude mice. It was found that, compared with the control group, inhibition of ZYX expression reduced tumorigenicity and growth rate of hepatocellular carcinoma cells, and Western blot and immunohistochemical results showed the same trend as in vitro cell experiments. Therefore, we confirmed that ZYX, as an oncogene, mediates the proliferation of HCC cells in vitro. This also provided evidence for ZYX as a possible therapeutic target for HCC.

Similar to our results in HCC, several studies have found that ZYX is increased in diseases such as glioblastoma multiforme[8], colorectal cancer[9], oral squamous cell carcinoma[36], and melanoma[37]. ZYX is a component of focal adhesion FA, integrin and focal adhesion kinase (FAK) combine to form early focal complexes (FX), and then the involvement of ZYX promotes the development of FX into stable focal adhesions (FA)[38], mediates cell adhesion to the extracellular matrix, and modulates integrin protein-mediated extracellular matrix signaling[39] to affect downstream pathways related to cell motility, migration, invasion, survival, immunosuppression and apoptosis. There are studies have shown that ZYX may play a central role as an upstream regulator to regulate key cancer-related signaling pathways[40]. It has been reported that ZYX is closely related to the Hippo signaling pathway [41, 42], and may also activate the Jun signaling pathway [9], but its relationship with the AKT/mTOR pathway has never been reported. Cancer development involves multiple signaling pathways, among which the AKT/mTOR is a classic pathway mediating the progression of various cancers [43–45]. AKT/mTOR is an essential signaling pathway for regulating cellular glucose metabolism and maintaining cell growth and reproduction [46], and is a recognized oncogenic driver[47] which mediates metabolic imbalance and regulation of the cell cycle to benefit the survival and growth of cancer cells[48, 49]. The activation of AKT/mTOR signaling pathway plays a role in inhibiting apoptosis and protecting cardiomyocytes[50]. However, there is insufficient evidence to describe its relationship with ZYX and its role in HCC.

We found that when ZYX was highly expressed, the phosphorylation of AKT/mTOR was high, while when ZYX expression was inhibited, the phosphorylation levels of AKT/mTOR were significantly reduced. Validation of tumor proteins in vivo also yielded the same results. We then used MK2206 and SC79 for verification. MK2206 and SC79 counteracted the effects of ZYX overexpression and ZYX reduction on the proliferation, migration, and invasion abilities of HCC cells, respectively. These suggests that the abnormal expression of ZYX plays a role in HCC cells mainly by activating the AKT/mTOR signaling pathway. We used ZYX overexpression and interference plasmids to construct stably transfected hepatoma cell lines, confirmed the effect of ZYX expression on the motility of HCC cells, and the changes in AKT and mTOR phosphorylated proteins also revealed the relationship between ZYX and the AKT/mTOR signaling pathway for the first time. Based on the above results, we boldly speculate that ZYX may mediate the progression of HCC via the AKT/mTOR signaling pathway. But our study also has limitations. Although we found changes in the phosphorylation levels of AKT and mTOR, we did not analyze whether ZYX directly phosphorylates AKT/mTOR protein, or ZYX activates the AKT/mTOR signaling pathway by other means, which requires our more work to prove. In conclusion, our study has broadened the avenues for ZYX research in the field of cancer biology, and provided new targets and directions for the prevention and treatment of hepatocellular carcinoma as well.

Funding

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contribution

All authors contributed to the study conception and design. Conceptualization, methodology and funding acquisition were provided by Wenguang Fu and Yichao Du. Data curation, investigation and original draft was written by Tianying Cai. Investigation was performed by Junjie Bai. Visualization was performed by Peng Tan. Yonglang Cheng and Tongxi Li reviewed and edited the manuscript. Yifan Chen worked for validation. Jian Ruan and Lin Gao were responsible for supervision. All authors read and approved the final manuscript.

Data Availability

The datasets used and/or analyzed during this study are available from the corresponding author upon reasonable request.

Ethics approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Clinical Trial Ethics Committee of Affiliated Hospital of Southwest Medical University (approval number: KY2019053). Animal studies were reviewed and approved by the Southwest Medical University Laboratory Animal Ethics Committee (approval number: 20211119-062).

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Conflict of Interest

The authors declare no conflicting of interests.

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Zyxin promotes hepatocellular carcinoma progression via activation the AKT/mTOR signaling pathway

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Abstract

Figures

1 Introduction

2 Materials And Methods

2.1 Bioinformatic analysis

2.2 Patients and specimens

2.3 Cell culture

2.4 Preparation of lentivirus and screening of stable strains

2.5 Cell proliferation

2.6 EdU staining

2.7 Cell cycle and apoptosis

2.8 Colony formation

2.9 Scratch test

2.10 Transwell assay

2.11 Tumor xenografts

2.12 RNA extraction, reverse transcription and quantitative real-time PCR (qRT-PCR)

2.13 Western blot

2.14 Hematoxylin–eosin(HE) and immunohistochemistry(IHC) staining

2.15 Statistical analysis

3 Results

3.1 ZYX is overexpressed in HCC tissue

3.2 Construction of human hepatoma cell lines with stable overexpression or knockdown of ZYX

3.3 ZYX affects HCC cell proliferation, cell cycle and apoptosis in vitro

3.4 ZYX accelerates migration and invasion of HCC in vitro

3.5 ZYX affects the expression of AKT/mTOR signaling pathway proteins in hepatoma cells

3.6 ZYX promotes the proliferation of HCC cells in vivo

4 Discussion

Declarations

References

Supplementary Files

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