RGS19 activates the MYH9/β-catenin/c-Myc positive feedback loop in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common fatal cancers worldwide, and the identification of novel treatment targets and prognostic biomarkers is urgently needed because of its unsatisfactory prognosis. Regulator of G-protein signaling 19 (RGS19) is a multifunctional protein that regulates the progression of various cancers. However, the specific function of RGS19 in HCC remains unclear. The expression of RGS19 was determined in clinical HCC samples. Functional and molecular biology experiments involving RGS19 were performed to explore the potential mechanisms of RGS19 in HCC. The results showed that the expression of RGS19 is upregulated in HCC tissues and is significantly associated with poor prognosis in HCC patients. RGS19 promotes the proliferation and metastasis of HCC cells in vitro and in vivo. Mechanistically, RGS19, via its RGS domain, stabilizes the MYH9 protein by directly inhibiting the interaction of MYH9 with STUB1, which has been identified as an E3 ligase of MYH9. Moreover, RGS19 activates β-catenin/c-Myc signaling via MYH9, and RGS19 is also a transcriptional target gene of c-Myc. A positive feedback loop formed by RGS19, MYH9, and the β-catenin/c-Myc axis was found in HCC. In conclusion, our research revealed that competition between RGS19 and STUB1 is a critical mechanism of MYH9 regulation and that the RGS19/MYH9/β-catenin/c-Myc feedback loop may represent a promising strategy for HCC therapy.

h incubation time, the cells on the bottom surface of the filter membranes were fixed, stained and counted under a light microscope.

Cell proliferation and colony formation assay
For cell counting kit-8 (CCK-8) assays, transfected cell suspensions were seeded in 96well plates (1× 10 3 cells/well).CCK-8 solution (Dojindo, Japan) was added to each well at the indicated time points.After incubation for two hours at 37 °C, the absorbance at 450 nm was measured.For colony formation assays, 1 × 10 3 transfected cells were plated into 6-well plates.After incubating for 2 weeks, the culture medium was removed and the colonies were fixed with 4% paraformaldehyde (PFA) and stained with 0.5% crystal violet.

EdU assay
An EdU analysis kit (Beyotime, China) was used to perform EdU dye assays for cells according to the manufacturer's instructions.Then a fluorescence microscope (Nikon, Japan) was used to detect labelled cells, and the results were evaluated by Image-Pro Plus 6.0 software (Media Cybernetics, Inc., MD, USA).The results were calculated by the following process: percentage of proliferative cells (%) = the number of labelled cells/ the total cell number.

Terminal dUTP nick-end labeling (TUNEL) assay
We performed a TUNEL assay (Roche, Shanghai, China) according to the manufacturer's instructions to detect apoptosis levels in vivo and quantified TUNELpositive cells under microscopy.

Flow cytometric analysis
For apoptosis analysis, we used the Annexin V-FITC Apoptosis Detection Kit (Beyotime, China).Briefly, the indicated cells were suspended in 195μl of binding buffer, and 5μl Annexin Ⅴ-FITC and 10μl of PI were then added.The cells were incubated on the ice in the dark.The flow cytometric analysis was used to detect apoptotic cells.

Lentiviral construction and cell transfection
To generate stable RGS19-overexpressing and RGS19-knockdown cell lines, lentiviral vectors for human RGS19 regulation (Lv-RGS19, Lv-shRGS19) and the corresponding control vectors (Lv-NC) were obtained from HanBio (Shanghai, China).We also used Lipo2000 to transfect siRNA or plasmids targeting MYH9, β-catenin and c-Myc into HCC cell lines.

Immunohistochemical (IHC) staining
The IHC staining procedures were similar to what we previously reported 1 .The tissue sections were incubated with primary antibodies overnight.The next day, the sections were incubated with secondary antibodies (Vector Laboratories, Burlingame, CA, USA).Finally, tissue sections were stained with a diaminobenzidine (DAB) kit (Vector Laboratories) and counterstained with haematoxylin (Sigma-Aldrich, USA).The primary antibodies for IHC included RGS19, MYH9, β-catenin, Ki-67 and c-Myc.The results were based on the intensity and proportion of staining.The staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong).The positively stained cell proportion was defined as follows: 0 (negative), 1 (0.01-25%), 2 (25.01-50%), 3 (50.01-75%)and 4 (75.01-100%).The histologic score (H score) was calculated by multiplying the intensity score and proportion score.Tissue sections with an H score ≤4 were classified into the low expression group, and those with an H score >4 were classified into the high expression group.

Western blot
Tissues and cells were lysed with RIPA buffer containing protease and phosphatase inhibitors.The protein samples were separated on SDS-PAGE gels and transferred onto nitrocellulose membranes.Then, the membranes were incubated with primary antibodies overnight at 4 °C, probed with IRDye 800CW secondary antibodies (LI-COR, USA) and visualized by the Odyssey® Imaging System (LI-COR, USA).
Information of the primary antibodies is supplied in Supplementary Table 3.

Co-immunoprecipitation and Mass spectrum (MS)
Cells were lysed in IP lysis buffer (Beyotime, China), incubated for 30 minutes on ice and centrifuged at 12000×g for 15 minutes.Cell lysates were immunoprecipitated with antibodies against Flag and MYH9 or negative control IgG on a rotator at 4 °C overnight.The next day, the A/G plus agarose beads (Santa Cruze, USA) were added into precipitate the protein complex at 4 °C for 4h.Subsequently, samples were washed five times with IP buffer, and the beads were boiled in loading buffer.The protein was prepared for western blot or LC-MS/MS from Aksomics (Shanghai, China).The results of LC-MS/MS were shown in Supplementary Table 4.

CHX assay
The indicated cells were treated with CHX (20μM).These cells were collected at indicated time point and prepared for western blot analysis.Cells were transfected with scramble control, siRNA or plasmid, and cultured with MG132 (10μM) or CQ (10μM) for 6h.Then the cells were collected and prepared for western blot analysis.

In vitro ubiquitination assay
Cells were transfected with plasmids and siRNAs as indicated, with HA-Ub.After 48h of transfection, the cells were treated with MG132 (100nM) for 24h.The cell lysates prepared with IP lysis buffer (Beyotime, China) were used to immunoprecipitation for MYH9 proteins.The level of MYH9 ubiquitination were detected by anti-Ub or anti-HA antibody.

Immunofluorescent (IF) staining
The detailed procedures of IF have been previously described 2 .Briefly, cells were seeded in confocal dishes.After adherence to the dish, the cells were fixed with 4% paraformaldehyde and permeabilized with Triton X-100.Then, the cells were incubated with specific antibodies and counterstained with 4,6-diamidino-2-phenylindole (Vector, Laboratories).Confocal microscopy (Zeiss LSM 880) was used to take images.

Plasmid construction
RGS19 Flag , Ub HA and c-Myc Flag plasmids constructed with the pcDNA3.1(+)vector and MYH9 His plasmid constructed with the CMV-M14 vector were obtained from GeneChem (Shanghai, China).P1, P2, P3 truncations of RGS19, SH3-like, Motor, Tail truncations of MYH9 and the full length of STUB1 with its truncations were synthesized by Sangon (Shanghai, China).
His or His-tagged STUB1 and RGS19 were harvested via His-tag purification kit (Beyotime, China).The GST-RGS19 fusion protein was added to cell lysates of HEK-293T cells transfected with MYH9 His at 4 °C for 4h.Bound proteins were then analyzed via western blotting.

TOP/ FOP flash reporter assay
The TOP/ FOP-flash reporter and pTK-RL plasmids were co-transfected into cells.
After 48h transfection, the Dual-Luciferase Assay Kit (Promega) was utilized according to the instructions.We detected the activities of the both firefly and Renilla luciferase reporters.The TOP-Flash and FOP-Flash reporter activity levels were calculated as the relative ratio of firefly luciferase activity to Renilla luciferase activity.

Luciferase assay
To investigate the regulatory effect of c-Myc on the transcriptional expression of RGS19, the full-length promoter region of RGS19 or mutated c-Myc binding sites of the RGS19 promoter were cloned into the luciferase reporter vector.Then, the WT-RGS19 promoter or MUT-RGS19 promoter reporter plasmids were co-transfected with con or c-Myc plasmids into HEK-293T cells.After transfection for 48h, the cells were harvested, and The Dual-Luciferase Reporter Assay system (Promega) was used to detect luciferase activity.

Chromatin immunoprecipitation assay (ChIP)
A ChIP assay kit was purchased from Millipore.Briefly, HCC cells were crosslinked with 1% formaldehyde, and the cell lysates were sonicated into 500bp fragments.The soluble material was then purified by centrifugation and mixed with a rabbit anti-c-Myc antibody or control rabbit IgG for immunoprecipitation.After that, the magnetic protein A/G beads were added, and the lysis were reverse rotation at 4°C for 24h.The recovered DNA fragments were used to detect the c-Myc binding sites by qPCR.The primers used for ChIP are listed in Supplementary Table 3.

Transcriptomics data analysis
Based on data from TCGA, mRNA expression profiles of HCC patients were applied in correlation analysis, differential expression analysis, survival analysis and Gene Set Enrichment Analysis (GSEA).

Hydrodynamic tail vein injection
Detailed methods have previously described 3,4 .Plasmid dissolved in 2 ml saline was injected into 6-week-old male C57BL/6 mice via the tail vein within 5-7 seconds.We prepared 13 μg of c-Myc, 10 μg of sgP53, 8.25 μg of Sleeping Beauty (SB) transposase and 10 μg of sgRGS19 or empty vector.Vectors for this experiment were prepared using the EndoFreeMaxi kit (Qiagen).The survival of mice and HCC incidence were analyzed.

In Vivo Efficacy Studies
Four-to six-week-old male BALB/C nude mice were perchased from Vital River Laboratory (Beijing, China) and were housed under specific pathogen-free conditions.
We performed animal experiments that were approved by the Animal Ethics Committee.

Supplementary Table 1.
Correlation between the clinicopathologic parameters and the expression of RGS19 in HCC.

For
the subcutaneous xenograft model, 3 × 106 transfected cells suspended in 150 μL PBS were injected into the flanks of the mice.Tumor size was measured weekly.After six weeks, the tumor was removed and its weight and volume were recorded.To establish the liver orthotopic xenograft implantation model, subcutaneous tumours were divided into 1 mm3 sections and were implanted into the liver.The growth of tumours was measured by bioluminescent signals once a week and six weeks later the mice were sacrificed.The pulmonary metastatic model was established by injecting 2 × 106 transfected cells into the tail vein of the mouse.The monitoring of metastatic nodules was achieved by bioluminescent signals.The mouse was sacrificed six weeks later.Representative images of Transwell migration and invasion assays with the indicated HCC cells.Scale bars: 100 μm.(g, h) Statistical analysis of the Transwell assays.(i) Representative images of bioluminescence of lung metastasis model from the indicated HCC cells.(j) Lung metastasis specimens and Statistical analysis of the number of metastatic nodules (n=7/group).All experiments were performed three times, and the data were presented as the means ±SD.*p<0.05;**p<0.01;***p<0.001.Full-length of STUB1 Myc and its truncated forms (P1, P2 and P3) with MYH9 His were co-transfected into HEK-293T cells.Cell lysates of 293T cells were immunoprecipitated with anti-Myc (against STUB1).Immunoblot analysis was performed with anti-His (against MYH9).
(a) Co-IP assays analysis of the interaction of MYH9 and RGS19 after STUB1 silencing in Huh7 cells.(b) Co-IP assays analysis of the interaction between MYH9 and RGS19 after STUB1 overexpression in HCCLM3 cells.(a) TOP/ FOP luciferase reporter assays were used to detect the transcriptional activity of Wnt signaling pathway in HCC cells with RGS19 overexpression.(b) Western blotting indicated the expression of β-catenin and c-Myc in HCC cells with RGS19 overexpression.

Table 2 .
The primers used in this study

Table 4 .
The results of LC-MS/MS