miR-378d regulates polyploidization and malignant phenotype of tumor cells through AKT and RhoA

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 June 2021 doi:10.20944/preprints202106.0443.v1 © 2021 by the author(s). Distributed under a Creative Commons CC BY license. Studies have shown that stress such as hypoxia, chemotherapy, radiotherapy can lead to polyploidization of tumor cells, which play an important role in tumor heterogeneity and malignant phenotype. Paclitaxel (PTX) treatment promoted polyploid cancer cells (PCCs) formation, and miR-378d is sharply reduced in PCCs of esophageal squamous cell carcinomas (ESCC) cells, but miR-378d participation PCCs formation and the impact on the biological behavior of ESCC remains unclear. We analyzed the PCCs formation and biological behavior of ESCC cells in vivo and in vitro, and the related proteins regulated by miR-378d. Results showed that miR-378d expression was associated with good prognosis in ESCC patients. miR-378d inhibition promoted PCCs formation, heterogenicity, chemo-resistance, monoclonal formation, EMT, migration, invasion, stemness and metastasis of ESCC cells. miR-378d can target downregulated AKT1, and inactivating the AKT-β-catenin signaling pathway, miR378d and AKT can also regulated RhoA expression. AKT and RhoA regulated polyploidization and depolyploidization. Therefore, miR-378d expression is a good prognostic factor of ESCC patients and regulates polyploidization and malignant phenotype of tumor cells through AKT and RhoA.

differentiated, 50% moderately differentiated and 90.20% poorly differentiated colorectal cancers, furthermore, the accuracy of predicting lymph node metastasis was 85.71% (18/21) that PGCCs with budding emerged in the stroma of ovarian cancer [7] . Paclitaxel (PTX) is a first-line chemotherapy drug for ESCCs, and can stabilize microtubule polymers to block cell cycle during the G2-M phase, hindering the development of mitosis, thus forming PGCCs [6,8] . Studies have reported that AKT promote PTX resistance to multiple tumors. IL-22 enhanced the paclitaxel resistance of lung adenocarcinoma cells by promoting the expression of AKT and Bcl-2 [9] . Calpain-2 promotes NSCLC progression and contributes to the paclitaxel resistance by activating EGFR/pAKT pathway [10] . Inhibition of AKT by specific PI3K-AKT inhibitors (Wortmannin, and LY294002) synergistically increased the efficacy of the paclitaxel-induced apoptosis in ovarian cancer [11] . About 15.7% of AKT1 amplification is found in ESCC [12] , and a recent study has demonstrated that the AKT signaling pathway plays an important role in ESCC metastasis [13] . Xanthohumol significantly inhibits AKT kinase activity in an ATP-competitive manner and decreases tumor volume and weight in patient-derived xenografts (PDXs) that highly express AKT.
However, xanthohumol has no effect on PDXs that exhibit low expression of AKT in vivo [14] . AKT is also involved in cellular polyploidization, overexpression of AKT restored polyploidy in death effector domain-containing protein (DEDD) deficient mouse decidual cells [15] . Yap strongly induce acetyltransferase p300 mediated E3 ligase acetylation through AKT signaling, the Skp2 of acetylation is limited to the cytosol, leading to excessive accumulation of cyclin-dependent kinase inhibitor p27, resulting in the cessation of mitosis and subsequent cellular polyploidy [16] .
RhoA, a small GTPase, plays a variety of functions in regulating cell and developmental events, including cytokinesis, cell migration and phagocytosis [17] .
Cytokinesis is the last step of cell division, which divides heredity and cytoplasmic content into daughter cells. Cytokinesis failure can lead to polyploidy, genetic instability and cancer. Low level of local RhoA activation prevented the accumulation of actin in cleavage furrow and participated in the polyploidization of megakaryocytes (MK) [18] . microRNAs (miRNAs) play an important role in tumor chemotherapeutic resistance and tumor progression [19][20][21] , which also can be regulate PCCs formation by their target genes. In the present study, we induced PCCs by PTX and the miRNA expression profiles were analyzed compared with those of normal cultured cancer cells.
We found that miR-378d expression was significantly downregulated in PTX induced PCCs. However, miR-378d participation PCCs formation and the impact on the biological behavior of ESCC remains unclear.  A total of 318 patients had long-term follow-up results, and the mean survival time was 29 months (1-95.2 months).

ESCC organization source
All biopsies were immediately fixed in 4% buffered paraformaldehyde, routinely processed, and embedded with paraffin. Tumors were classified according to standard TNM staging guidelines of UICC (TNM Classification of Malignant Tumours, Eighth edition). The study protocol had been reviewed and approved by the local ethics committee. All patients gave written consent for their tissue samples. This research was approved by the ethics committee of Jining Medical University. Each patient signed an informed consent form.

Tissue microarray
Representative areas of the ESCC were marked on each hematoxylin-eosin (H&E) slide and tissue paraffin block, and the marked areas of tissue paraffin blocks were sampled for TMAs. TMAs were assembled with a tissue-arraying instrument (Beecher Instruments, Silver Springs, MD, USA) as described by Kallioniemi et al. [22] .

In Situ Hybridization
ESCC TMA was dewaxed in xylene, rehydrated in alcohol gradient, and washed two times with DEPC-PBS. The sections were treated with 2 μg/mL proteinase K (Roche) for 15 min at 37 °C and washed three times with DEPC-PBS. Then, the sections were acetylated 15 min at room temperature (acetic anhydride in DEPC-water, 6 N HCl, and triethanolamine) and subsequently washed three times with DEPC-PBS. Sections were prehybridized in hybridization buffer (50% formamide; 5× saline sodium citrate; pH 7.0; 100 μg/mL sheared salmon sperm DNA, 0.5 mg/mL yeast tRNA, and 1× Denhardt's solution) at 58 °C for 1 h before the buffer was replaced with hybridization solution containing miR probe. The miR-378d detection probes labeled with digoxin at 5′-end was from Boster (#MK10502). Probes were diluted in pre-hybridization buffer to a concentration of 5 nM and hybridized with the sections overnight at 58 °C according to the RNA melting temperature of probes. After hybridization, the sections were washed three times with 2 × SSC and 0.2 × SSC, permeabilized for immunostaining with 0.1% Triton X-100, and washed two times with PBS. Unspecific background was blocked with 5% swine serum diluted in PBS/BSA for 30 min.

PTX treatment
All cell lines were cultured in complete medium until the cells reached 90% confluence. Different concentrations of PTX were added to the different cells, which were then treated for 24 h. PTX was then withdrawn, the medium was replaced, and the cells were cultured until no significant cell death was observed.

Sequencing of miRNA and microarray analysis
The small RNA of TE-1 control and TE-1-PTX (9 days) treated with PTX was used for miRNA sequencing. The miRNA-sequencing libraries were constructed according to the protocol for the Illumina small RNA sample preparation kit.
Sequencing was performed on an Illumina HiSeq 2000 sequencer. Library construction and sequencing were performed at Genergy Biotech (Shanghai). miRNA expression was analyzed with miRdeep 2.0.0.7 [23] , and differentially expressed miRNAs were identified using an FDR cutoff value of 0.05. mRNA expression profiling was conducted with Roche NimbleGen Human 12 × 135 K Gene Expression Array by KangChen Bio-tech. Raw data were processed with RMA algorithm, and differential expression analysis was performed with R package limma37 (Version 3.22.7).

Cell transfection
Transfection of plasmids was performed using Lipofectamine TM 3000 reagent (Invitrogen, USA) according to the manufacturer's instructions. Transfection of miRNA mimics or inhibitors (Ribobio, China) was performed using Lipofectamine RNAiMAX (Invitrogen, USA) at a final concentration of 20 nM.

Lentivirus packaging and transduction
Vectors were packaged in 293FT cells using ViraPower Mix (Genepharma). After culturing for 48 h, lentiviral particles in the supernatant were harvested and filtered by centrifugation at 500 g for 10 min, and transfected into ESCC cells. The cells were then cultured under puromycin (10 μg/mL) selection for 2 weeks, after which real-time PCR was used to determine the level of miR-378d. Cell lines stably expressing miR-378dinhibitor or negative control (NC) vector were designated as Lv-miR-378d-inhibitor and Lv-miR-NC cells, respectively.

Western Blot
Cells were lysed in ice-cold RIPA buffer containing a protease-inhibitor cocktail (Roche). Protein content was quantified with a BCA protein assay kit (Thermo Fisher Scientific). About 30 μg of protein was subjected to electrophoresis, transferred onto PVDF membranes (Millipore), and blocked with 5% nonfat dry milk in Tris-buffered Proteintech) served as endogenous controls. The specific bands were visualized using secondary anti-rabbit or anti-mouse antibody (1:3000; Proteintech), enhanced chemiluminescence detection kit (Millipore), and FluorChem FC2 Multi-Imager II (Alpha Innotech).

Transwell migration and invasion assay
In vitro cell migration assay was performed using transwell chambers (8 mm pore size; Corning). Cells were plated in serum-free medium (2 × 10 4 cells per transwell).
Medium containing 15% FBS in the lower chamber served as a chemoattractant. After 48 h, the nonmigrating cells were removed from the top face of the filters by using cotton swabs, and the migratory cells located on the bottom side of the chamber were stained with crystal violet, air dried, photographed, and counted. Images of five random fields at 10× magnification were captured from each membrane, and the number of migratory cells was counted. Similar inserts coated with Matrigel were used to determine the cellular invasive potential in the invasion assay.

Matrigel 3D cell culture
Cells (5×10 3 /50 μL) were seeded onto 96-well plates with a round-bottom lid made of ultralow attachment polystyrene (#7007, Costar, USA). The cells were cultured overnight and found to form one sphere per well. After discarding the medium and adding 75 μL of melted Matrigel (BD, USA) to resuspend the cell sphere, the mixture was incubated for 30 min for settling. Finally, 200 μL full medium/well was added, and the medium was changed every other day.
To render the scaffold hydrophilic, inserts were first submerged in 70% ethanol for 1 min, then washed twice with sterile PBS and once with 10% FBS RPMI-1640 complete medium. In each experiment, 2 × 10 6 cells were seeded in either Alvetex® scaffold placed in a 12-well plate (covered by 3.5 ml of complete medium) . Cultured for 7 days.

Colony-formation assays
Cells were seeded onto six-well plates (5 × 10 2 cells per plate) and cultured for 10 days. The colonies were stained with 1% crystal violet for 30 s after fixation with 10% formaldehyde for 15 min and then imaged using the camera of an iPhone 5S (Apple, Inc., Cupertino, CA, USA).

Dual-luciferase reporter assay
In a typical procedure, 293T cells (3×10 4 cells per well) grown on a 24-well plate were co-transfected with luciferase reporter miRGLO-AKT1-3′UTR plasmid (WT or mutation type; Genepharma, Shanghai, China) (200 ng per well) and miR-378d (20 nM) using Lipofectamine™ 3000 (Invitrogen, USA). About 24 h later, a dualluciferase reporter assay kit (Promega, USA) was used to measure the luciferase and renilla activity of these samples according to the manufacturer's instructions.

F-actin cytoskeleton fluorescence staining
Cells were grown on laminin-coated glass cover slips, fixed in 4% paraformaldehyde, and stained with Phalloidin (Molecular Probes, USA). Cells were observed using a fluorescence microscope (Leica, Germany).

Cell-Viability Assay
CCK8 was used to assess cell viability. KYSE510 and TE-1 cells (1 × 10 4 ) were seeded onto a 96-well plate in quintuplicate per well. About 12 h later, the cells were incubated with a gradient concentration of therapeutic drugs for 48 h. The medium was removed, RPMI1640 (90 μL) and CCK8 (10 μL) were subsequently added to each well, and the mixture was incubated for 3 h at 37 °C. A microplate reader was used to measure the optical density (OD) at 450 nm. The degree of drug response for tumor cells was estimated by dividing the half-maximal inhibitory concentration (IC50).

Immunohistochemistry (IHC) and Immunofluorescence (IF)
IHC analysis was performed on the cell-block sections from the cultured cells by Immunofluorescence and confocal microscopy were performed as our previously reported [24,25] . Images were captured by using a Zeiss confocal microscope.

Sphere-formation assay
Sphere formation assay was performed as our previously reported [25,26] . The KYSE-150 and TE-1 cells were seeded in low-attachment six-well culture plates (Corning, NY, USA) at a density of 1 × 10 4 cells per well under serum-free conditions consisting of DMEM/F-12 (Life Technologies), 20 ng/mL epidermal growth factor (Invitrogen), 20 ng/mL basic fibroblast growth factor (Invitrogen), and 20 μL/mL B27 (Life Technologies). Images were captured under a microscope after 14 days, and the numbers of spheres (diameter ≥ 100 μm) in all wells were counted.

Liver transplantation
The animal protocol was approved by the ethical review committee of the Affiliated Hospital of Jining Medical University. Eight-week-old male BALB/c nude mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were anesthetized with 4% chloraldehyde hydrate (100 μL/10 g), and body temperature was maintained by heating blankets. The thoracoabdominal skin of nude mice was sterilized with 75% alcohol and iodophor, respectively. After cutting open the skin at the lower right of the cartilago to expose the lobe of the liver, 50 μL cell suspension (25 μL serumfree and 25 μL of Matrigel containing 5×10 5 cells) was injected into the liver capsule slowly. Then, the syringe was pulled out and the injection port was pressed for 2-5 min with an iodophor cotton ball. Finally, the incision was sutured layer by layer according to the anatomical structure. Mice were sacrificed 44 days after tumor-cell inoculation.
Afterwards, liver tissues, lung tissues, and abdominal-metastasis tumors were fixed in 4% saline-buffered formalin, embedded in paraffin, sectioned at 4 μm, and stained with H&E and IHC.

Statistical Analysis
Statistical analyses were performed using the SPSS 13.0 software package (SPSS, Chicago, IL, USA) and GraphPad Prism Software (version 6, La Jolla, CA, USA). For statistical comparison of two groups, two-sided Student's t test with the same variances was used. Differences between variables were analyzed by two-tailed or Fisher exact tests. Survival curves were plotted using the Kaplan-Meier method and compared with log-rank tests. Multivariate survival analysis was performed for all parameters found to be significant in univariate analysis using a Cox regression model. Comparisons between groups for statistical significance were performed with a two-tailed Student t test. Data are presented as the mean ± SD. P values < 0.05 were considered significant.

Results:
3.1. miR-378d participates in the formation of polyploid cancer cells of ESCC in vitro and in vivo PTX is the first-line drug for ESCC, but drug resistance remains a problem. In this study, we used PTX to treat TE-1 cells. From days 2 to 9, the cells died continuously, and only a small number of large cells remained at day 9. These cells slowly proliferated until day 20. Studies have shown that paclitaxel treatment of tumor cells leads to PGCCs production, then the remaining PTX-treated cells at day 9 (TE-1-PTX) and DMSO-treated cells (TE-1-NC) ( Figure 1A) were used to perform DI analysis ( Figures   1B, 1C&1D) and detect miRNA differential expression by sequencing ( Figure 1E). The DI analysis results showed that large cells were significant enrichment in the TE-1-PTX, the volume of cells and nuclei increased significantly after PTX treatment ( Figure 1B), the large cells were significant enrichment even in the DI > 4 (8N) (Figure 1C), and analysis data showed that the ratio of cells DI > 2.5 (5N) were significant enrichment from 6% to 50% ( Figure 1D).
The remaining large cells and control cells were also used to detect miRNA differential expression by sequencing. miRNA gene-expression profile data showed that miR-378d was significantly downregulated in remaining large cells ( Figure 1E).
This result suggests that miR-378d play an important role in the formation of large cells. miR-378d has two different chromosomal in humans which named MIR378D1 and MIR378D2, the mature miRNA sequence is the same, and the CCLE data showed that    spheres in suspension state within 24h, but miR-37d inhibition promoted the spheres dissemination after 48h, while control group was still spheres ( Figure S2D). The spheres were transferred into the Matrigel and cultured for 8 days, miR-378d inhibition showed invasive growth ( Figure 2D). The invasive ability was further detected by 3D culture on 3D Alvetex®, a highly loose crosslinked polystyrene scaffold, and the data showed that miR-378d inhibition promoting cell invasion in 3D scaffold ( Figure 2E).

miR-378d loss expression promotes metastasis in vivo
Liver is a common metastasis site of ESCC, so liver-transplantation assay was performed to analyze the metastatic ability of tumor cells without miR-378d expression.
KYSE-150-miR-NC and KYSE-150-miR-378d-inhibitor cells were transplanted into subcapsular liver of BALB/c nude mice (hereafter denoted as NC-mice and In-mice, respectively). The experiment was terminated after 44 days. The body weight of Inmice was significantly lower than that of NC-mice at day 44 (P = 0.011, Figures 3A &   3B). Before the end of the experiment, NC-mice did not die (0/6), but two IN-mice (2/6) died at day 35 and 38, respectively ( Figure 3C). Although the number of liver nodules in In-mice was more than that in NC-mice, no statistical difference ( Figure 3D & 3E) was observed. Tumor cells basically metastasized to the abdominal cavity, and no metastasis was found in all lung tissues. Abdominal metastatic tumors appeared in 5/6 of the NC-mice and 6/6 of the In-mice ( Figure 3F). The number of abdominalmetastasis tumors in In-mice was more than that in NC-mice ( Figure 3G, P = 0.026), and the volume was also larger than that of the NC-mice. These data showed that loss of miR-378d expression promoted tumor metastasis.
CTNNB1 (β-catenin) oncogenic signature, which was the AKT regulated pathway. The protein expression levels of β-catenin and the downstream target genes vimentin and ALDH1A1 decreased in cells with miR-378d high expression ( Figure 4I), whereas miR-378d absent increased these protein levels ( Figure 4J). The EMT marker Ecadherin significantly upregulated in miR-378d high expression TE-1 cells, and downregulated in TE-1 cells with miR-378 inhibition ( Figure 4I&4J). The data showed that miR-378d absence activated the β-catenin pathway, and promoted the EMT of ESCC cells.
RhoA is a key regulatory protein for cytokinesis, we detected the RhoA expression    (Table 2). However, our data showed that miR-378d expression was significantly negatively associated with the overall survival rate ( Figure 6C; P =0.0009) of ESCC patients.

Discussion
Most research has primarily focused on miR-378a and miR-378b [27] , only a few studies have been performed on miR-378d and inflammation [28] or infection [29] . In the current work, miR-378d significantly decreased in PCCs, however, the role of miR-378d in ESCC remains unclear. In this study, we found that miR-378d inhibition promoted polyploid cancer cells formation, heterogenicity, chemo-resistance, monoclonal formation, EMT, migration, invasion, stemness and metastasis of ESCC cells. These results indicate that miR-378d is a tumor suppressor, and actually, miR-378d expression is a good prognostic factor for ESCC patients.
In this study, PTX was used to induce polyploid production, PTX is a mitotic inhibitor widely used in the treatment of cancer patients by inhibiting cell division and inducing apoptosis. A low dose (30 nM) of PTX treatment of esophageal cancer cells results in more than 90% of cells forming PCCs without apoptosis. While high doses of PTX treatment can lead to massive cell death, but a certain percentage of the surviving cells forming PGCCs, a more malignant cell type [6,8,30] .
PGCCs exhibit higher plasticity than traditional cancer stem cells and can differentiate into a variety of tissues, including adipose tissue, cartilage, bone and other stromal or fibroblast cells [30] . There are two asymmetric cell division patterns in the PGCCs: budding and rupture, which can occur alone or together. Budding usually occurs on PGCCs branches, while PGCCs cells containing multiple nuclei usually rupture, followed by the release of large numbers of small cells [31] . CD133 has been suggested as a broad-spectrum marker for cancer stem cells (CSCs). CD133 + cells are small, regular and round with small microvilli, in some fields, several giant cancer cells (GCCs) in the CD133 + cell group were identified under the light microscope, most of them were polynuclear cells in NPC cells [25]. In our study, CD133 + cells contained more large and small cells than CD133cells, the large cells are PGCCs and the small cells maybe the daughter cells of PGCCs, and medium size cells are non tumor-stem cells. Therefore, the clinical use of PTX should be re-evaluated.
PGCCs achieve rapid and malignant growth via efficient DNA replication and cell division [32] . PGCCs daughter cells promoted lymph node metastasis by expressing EMT related proteins [32] . Spectral karyotype (SKY) analysis found that PGCCs derived daughter cells obtained new cancer genome, and new chromosomal recombination, including deletion and translocation occurred in daughter cells [33] . These studies suggest that the PGCCs daughter cells have stronger migration and invasion ability than diploid tumor cells.
Our study showed that miR-378d inhibition promoted malignant ability of ESCC cells, but did not significantly increase the proportion of PCCs compared with paclitaxel treatment. Thus, like cancer stem cells, the proportion of PCCs in the cell population is not high, highly efficient generation of daughter cells may be an important reason for PCCs/PGCCs to promote malignant phenotype and tumor heterogeneity.
PI3K/AKT pathway are closely related with tumorigenesis, proliferation, growth, EMT, invasion, metastasis, stem-like phenotype and drug resistance of cancer cells, but PI3K or AKT inhibitors as monotherapy for different cancers have so far been limited [34] . In this study, miR-378d directly target regulate AKT1, and miR-378d inhibition activated the AKT-β-catenin signaling pathway, and promoted the EMT marker vimentin and CSCs marker ALDH1A1 expression, which may promote malignant phenotypes of ESCC cells.
Our data also showed that mi-378d inhibition decreased the RhoA expression.
RhoA is a key protein in regulating cytokinesis [35] , and cytokinesis is the last step of mitosis, cytokinesis failure causes polyploid formation. miR-378d inhibition may promote PCCs formation by reducing RhoA expression.
AKT or RhoA inhibition alone promoted PCCs formation, and the combination of AKT and RhoA inhibition significantly promoted PCCs production. Then we study the relationship between AKT and RhoA, AKT inhibition by MK2206 decreased the RhoA expression, we also found that β-catenin inhibition also reduced RhoA expression.
Therefore, our results suggest that miR-378d inhibition may promote polyploidization by inhibiting the expression of RhoA and promoting de polyploidization by promoting the activation of AKT and thus promoting the malignant phenotype of tumor.

Conclusions
miR-378d inhibition promoted PCCs formation, heterogenicity, chemo-resistance, monoclonal formation, EMT, migration, invasion, stemness and metastasis of ESCC cells. miR-378d can target downregulated AKT1, and inactivating the AKT-β-catenin signaling pathway, miR-378d and AKT can also regulated RhoA expression. AKT and RhoA regulated polyploidization and depolyploidization. Therefore, miR-378d expression is a good prognostic factor of ESCC patients and regulates polyploidization and malignant phenotype of tumor cells through AKT and RhoA.