Downregulation of RRS1 Inhibits Proliferation, Migration, And Epithelial-Mesenchymal Transition In Hepatocellular Carcinoma

Zhi Li Xingtai General Hospital of North China Healthcare Group Jitao Wang Xingtai People's Hospital Chengyu Liu Hebei Medical University Jinlong Li Xingtai People's Hospital Xiaochong Zhang Xingtai People's Hospital Xiaoling Zhao Xingtai People's Hospital Kunpeng Zhang Xingtai People's Hospital Zhongguang Zhen Xingtai People's Hospital Dengxiang Liu Xingtai People's Hospital Yong Liao (  dyliaoyong@163.com ) Xingtai People's Hospital


Introduction
Hepatocellular carcinoma (HCC) is one of the most common cancer and life-threatening malignancy in the world (1). Surgical resection is still the cornerstone in the treatment of HCC (2). However, the recurrence rate and remote metastasis after radical resection for HCC has been reported to reach 60%~70% in 5 years (1). Therefore, it is necessary to nding out the molecular mechanism of HCC invasion and migration, which is expected to develop more effective therapeutic strategies against this deadly cancer.
Epithelial-mesenchymal transition (EMT) is a biological process that epithelial cells lose their polarity and acquire the capacities of mesenchymal-like cells (3). It implies enhanced cellular invasion and epithelial plasticity. Previous studies have shown EMT is associated with HCC recurrence and metastasis (4)(5)(6). Therefore, it is important to clarify the molecular mechanism underlying EMT in HCC and to develop innovative strategies against HCC recurrence and metastasis.
Regulator of ribosome synthesis 1 (RRS1), rst identi ed in yeasts, is a eukaryotic conserved nuclear protein composed of 203 amino acids, which plays an important role in the biogenesis of ribosome (7)(8)(9)(10). It participates in the assembly of the 60s ribosomal subunit and escorts it out of the nucleus (11). In recent years, a growing number of reports have con rmed that RRS1 participates in the occurrence and development of many kinds of malignant tumors (12)(13)(14)(15)(16), but its mechanism in promoting tumors is not clari ed yet. A previous studies on 18 cases of hepatitis B virus (HBV)-related HCC shows that the mRNA expression abundance of RRS1 in HCC tissues is signi cantly higher than that in para-carcinoma tissues, and the expression level in HCC tissues is present at 3-to 4-fold increase than that in para-carcinoma tissues. The cellular functional experiment results were displayed in SMMC-7721 cells. The high expression of RRS1 might play a role in inhibiting apoptosis and promoting growth, and then maintain and support the malignant transformation and proliferation of cancer cells, but the function and carcinogenic mechanism of RRS1 in HCC are still uncertain.
In this study, we rst analyzed whether RRS1 affects cell proliferation, migration, and invasion in HCC.
Besides, we investigated the prognostic value of RRS1 in HCC patients. Finally, we revealed the molecular mechanism of RRS1 promoting HCC occurrence and development.

Materials And Methods
Patients and specimens All of the clinical specimens of HCC patients were retrieved from the para n specimens of the department of Pathology and Hepatobiliary surgery resection specimens of the Xingtai People's Hospital from 2015 to 2020, and we conducted retrospective analysis on its clinical pathological data and survival prognosis. This study was approved by ethics committee of Xingtai People's Hospital. Informed consent was obtained from every single patient in accordance with Declaration of Helsinki.
A pgCSiL-gFP lentivirus RNAi expression system (GeneChem Co., Ltd., Shanghai, China) were used for RRS1 knockdown. The effective targeting sequence for RRS1 (GCT GCC TTC ATT GAG TTT A) was selected by western blot analysis. A non-silencing shRNA sequence (TTC TCC GAA CGT GTT CAC GT) was used as a negative control, which targets no genes in humans, mice or rats as in NCBI RefSeq. The shRNA vectors and the lentiviral packaging plasmids pHelper1.0 and pHelper2.0 were cotransfected into the 293T cells using Lipofectamine 2000 (Invitrogen, Shanghai, China) to generate the respective lentiviruses. 3 days later, the viral stocks were collected from the 293T cells and were used to infect Huh7 cells. The Huh7 cells were infected with the RRS1-siRNA-lentivirus (Lv-shRRS1 group) or negative control lentivirus (Lv-shCon group) according to the recommended multiplicity of infection (MOI). The stem-loopstem oligos (shRNAs) were synthesized, annealed, and ligated into the pgCSiL-gFP lentivirus Rnai expression system. The lentiviral-based shRNA-expressing vectors were con rmed by DNA sequencing.

Cell proliferation
We used cell counting Kit-8 (CCK8) to detect cell viability. Untransfected group and cells transfected with shRRS1 were separately seeded into 96-well plates. The number of cells in each well is about 5×10 3 /100 μl. Three parallel holes were set for each group. Cell viability was tested at 24, 48, 72 and 96 h. Add 10μl CCK8 solution into each well and incubated at 37℃ 5% CO2 for 1 h, The optical density (OD) of each well was measured at 450 nm. Then calculate the average of every group and the proliferation curve was plotted based on the OD value.

Transwell analysis
Transwell chamber (Aperture: 8μm Costar Cambridge, Massachusetts, U.S.) was used to test cell migration. In the upper chamber coated with matrigel, and DMEM medium (500μl) containing 10% FBS was added to the lower chamber. The cells were suspended in DMEM medium and seeded into the upper chamber coated with matrigel. Then, DMEM medium(500μl) were added with 10% FBS into the lower chamber. After incubating under 37°C and 5% CO 2 for 24h, cells on the lower surface of the membrane were xed with methanol and stained with 0.1% crystal violet. The number of the migrated cells were counted under the microscope.

Immunohistochemistry
After being dewaxed with xylene, the para n sections were hydrated with gradient ethyl alcohol. Then, the sections were immersed in 3% H2O2 to inhibit endogenous peroxidase activity. The primary antibody was incubated for 1h at 37℃ and washed with PBS three times for 3 min each. The secondary antibody was incubated at room temperature for 30 min and washed with PBS three times for 3 min each. Then, stained by Diaminobenzidine (DAB) for 5 min, counterstained by hematoxylin and differentiate by 0.1% HCl alcohol for 1s. Finally, the section was dehydrated with gradient alcohol, transparent by xylene, and sealed with neutral gum. The staining intensity scoring standards are as follows: 0, no staining; 1+, light staining; 2+, moderate staining; 3+, heavy staining. The staining area scoring criteria are as follows: 0, no staining under the microscope; 1+, <30% tissue staining is positive; 2+, 30% ~ 60% tissue staining is positive; 3+, >60% tissue staining is positive. The expression of RRS1 was evaluated by the comprehensive score of staining intensity and area. The highest score is 6 points, and the lowest score is 0 points. The criterion for positive RRS1 staining is brown staining in the nucleus.
RNA extraction and real-time PCR.
Total RNA of HCC tissues, para-carcinoma tissues and cells were obtained by Trizol reagent. RNA (1μg) was reverse transcribed with PrimeScript™RT reagent Kit with gDNA Eraser (TaKaRa, Japan, RR047A).
Followed by real-time PCR detection using TaKaRa TB Green™ Premix Ex Taq™ (TaKaRa, Japan, RR820A). The primer sequences are as follows:

Western blot analysis
Protein of HCC tissues, para-carcinoma tissues and cells were extracted by RIPA lysis buffer with 1 mM PMSF. Then total protein content were quanti ed by using the BCA method. Equal amounts of proteins from tissues and cells were used for Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), and subsequently transferred onto the PVDF membrane. Block (proteins) at 37 ℃ by using 5% skim milk for 1h. Wash the membrane three times with TBST, and incubated with the primary antibody at 4 ℃ overnight. The next day, wash the membrane three times with TBST and incubate with the secondary antibody at 37 ℃ for 1 h. Blots were developed by using ECL plus. RRS1 (ab188161), E-Cadherin (ab231303), N-Cadherin (ab76011), Vimentin (ab20346), Snail (ab216347) primary antibodies were purchased from Abcam (Cambridge, MA, USA).

Statistical analysis
Statistical analysis were performed by SPSS 22 software (Chicago, IL, USA). Chi square test were used to analyze the correlation between RRS1 expression and clinical-pathological parameters. Continuous variables were analyzed by t-test, and survival probability was detedted by Kaplan-Meier method. Differences between groups were statistically signi cant if P <0.05. Each group experiment was repeated three times.

RRS1 is highly expressed in HCC cancer tissues
We performed immunohistochemical staining on para n sections from 75 HCC tissues and paracarcinoma tissues to detect RRS1 expression. The results showed that the positive RRS1 protein staining was mainly located in the nucleus and was highly expressed in HCC tissues, while the paracancerous tissues were low (P <0.001) ( Figure 1A and 1B). Real-time PCR was performed to detect the RRS1 mRNA expression level of HCC and para-carcinoma tissues in 42 cases and it found that the expression level of RRS1 in HCC tissues was higher than that in para-cancerous tissues (P <0.05) ( Figure 1C and 1D).
Furthermore, western blot were used to detected protein expression of RRS1 in 16 cases. The results showed that the protein expression level of RRS1 in HCC tissues was signi cantly higher than that in para-cancerous tissues. (P <0.01) ( Figure 1E and 1F).

RRS1 is associated with a poor clinical prognosis in HCC patients
We divided the expression level of RRS1 in HCC tissues into a high expression group (n = 45) and a low expression group (n = 30) according to the "immunohistochemistry" method in Materials and Methods. The results showed that the expression of RRS1 was associated wirh the tumor size (P<0.01), clinical stage (P<0.01), and lung metastasis (P<0.05) of HCC (Table1).
We further analyzed the correlation between RRS1 expression and the clinical prognosis of HCC patients through the Kaplan-Meier survival curve. The results showed that the overall survival time of patients in the RRS1 high expression group was lower than that in the RRS1 low expression group (P <0.01) ( Figure  1G). Meanwhile, The recurrence-free survival time was also lower than that in the RRS1 low expression group (P <0.01) ( Figure 1H). Therefore, our results showed that the high expression of RRS1 in HCC is closely associated with the tumor malignancy and poor clinical prognosis.

RRS1 is highly expressed in HCC cells, and shRRS1 can effectively silence the expression of RRS1
We selected human normal liver cell line L02 and ve human hepatocellular carcinoma cell lines (SMMC-7721, HepG2, Hep3B, Bel-7402, Huh7) to detect the protein expression of RRS1 levels by western blot. The results showed that the expression of RRS1 in hepatocellular carcinoma cells (SMMC-7721, HepG2, Hep3B, Bel-7402, Huh7) is higher than that in normal liver cells (L02), and Huh7 cells showed the highest expression level of RRS1 in hepatocellular carcinoma cell lines (Figure 2A and 2B).
We further constructed shRRS1-RNA and transtected it into silenced the expression of RRS1 by transfect shRRS1-RNA into Huh7 cells to knockdown the expression of RRS1. To con rm transfection e ciency we observed the cells infected by shRRS1 and Lv-shCon vectors by uorescence microscope. We found that more than 80% of Huh7 cells express GFP, which indicates a high infection e ciency of shRRS1 ( Figure  2C). We veri ed the silencing effect of shRRS1 by real-time PCR and western blot. The results showed that shRRS1 can effectively knockdown the mRNA expression level of RRS1 (P <0.01) ( Figure 2D) and knockdown the protein expression level of RRS1 (P <0.01) ( Figure 2E and 2F).

Down-regulation of RRS1 can inhibit the growth and metastasis of Huh-7 cells
We silenced the expression level of RRS1 in Huh-7 cells and tested the proliferation ability of Huh-7 cells by CCK-8 assay. The results showed that compared with the negative control group, the proliferation ability of Huh-7 cells in the RRS1 knockdown group was suppressed (P <0.01) ( Figure 3A). Subsequently, we conducted cell migration experiments and the results showed that the migration of Huh-7 cells was inhibited after knockdown RRS1 expression ( Figure 3B). In addition, the migratory cells per eld and migration fold change of Huh-7 cells decreased after knockdown RRS1 expression ( Figure 3C and 3D). To identify whether EMT participate in the biological process in HCC migration and invasion, we further detected the expression of EMT-related proteins. The results showed that the expression of E-Cadherin in Huh-7 cells increased after knockdown the expression of RRS1. In contrast, the expression of N-Cadherin, Vimentin, and Snail decreased after knockdown the expression of RRS1 ( Figure 3E and 3F).

Discussion
In this study, we found that RRS1 acted as a new tumor-promoting gene in HCC. It was associated with enhanced proliferation and invasion abilities. In addition, it could induce EMT in HCC cells.
RRS1 is a eukaryotic conservative nuclear protein made up of 203 amino acids (8). In secretory-de cient yeasts that nonsense mutations of an amino acid codon at the C-terminus of RRS1, the mutation of RRS1 signi cantly reduces the transcriptional inhibition of rRNA and ribosomal protein genes (17). It can be seen that RRS1 is an important protein in the signal transduction pathway of protein secretion and ribosome synthesis. In addition, RRS1 also acts as a messenger in the process of signal transmission and plays an important role in the occurrence of Huntington's disease (18,19). In normal human cells, RRS1 is located in the nucleus. In addition, it is found that RRS1 is located both in the nucleus and the periphery of the nucleus in the HeLa cell line (20). After further inhibiting the expression of RRS1 by RNAi technology, the cell cycle of HeLa cells was signi cantly delayed. Therefore, it is speculated that RRS1 may be related to the occurrence and development of cervical cancer and other tumors (20).
Song et al (15). con rmed that RRS1 is a new gene related to breast cancer and may play an important role in breast cancer proliferation and apoptosis. Mechanism studies have shown that RRS1 may activate the p53 pathway to promote breast cancer proliferation through RPL11/MDM2. Another study con rmed that RRS1 is involved in the progression of papillary thyroid carcinoma (14). Knockout of RRS1 can upregulate the expression of genes associated with cell apoptosis and metabolism, while down-regulating genes associated with cell proliferation and vascular development. This study con rmed the diagnostic value of RRS1 in thyroid cancer in children and adults, and the expression of RRS1 is positively correlated with age (14). However, the speci c mechanism is still unclear.
The effect of RRS1 on the clinical prognosis and invasion in HCC has not been reported yet. Our results showed that RRS1 is highly expressed in HCC and signi cantly negatively correlated with the poor prognosis. Moreover, RRS1 might participate in the EMT process of HCC cells. EMT is an important biological process involved in tumor invasion and metastasis, which induces the epithelial cells to lose their polarity and adhesive properties and enhances the ability of migration and invasion (3,4,21). EMT has been con rmed to be involved in the progression of HCC and is associated with a poor prognosis (4,16). To reveal the possible mechanism of RRS1 in the process of EMT in HCC, we transfected RRS1 shRNA to knockdown the RRS1 expression. We found that after knockdown RRS1, the protein levels of mesenchymal markers (N-cadherin, Vimentin, Snail) in HCC cell lines decreased signi cantly. In contrast, E-Cadherin increased signi cantly as an epithelial marker. Our data indicated that RRS1 induced the invasion of HCC cell lines, and further veri ed that RRS1 triggers EMT in cell lines, which plays a crucial role in HCC invasion and metastasis.
As far as we known, this is the rst study that clar ed the importance of RRS1 in HCC. However, this study still has some limitations. First, we need to further explore the regulation network of RRS1 and EMTrelated signals. Second, it is important to determine the upstream signal responsible for the overexpression of RRS1 in HCC. Finally, the clinical signi cance of RRS1 in HCC requires further veri cation in large cohorts.

Conclusions
All in all, this study revealed that RRS promotes HCC cell proliferation, migration and EMT. RRS1 was a promising prognostic indicator and therapeutic targets for HCC.

Declarations
Ethics approval and consent to participate Clinical data have been approved by the Ethics Committee of Xingtai People's Hospital and approved by the patients.

Consent for publication
All contributing authors agree to the publication of this article.

Availability of data and materials
All data are fully available without restriction.

Competing interests
The authors declare that they have no competing interests.