Silencing geranylgeranyltransferase I inhibits the migration and invasion of salivary adenoid cystic carcinoma through RhoA/ROCK1/MLC signaling and suppresses proliferation through cell cycle regulation

Geranylgeranyltransferase type I (GGTase‐I) significantly affects Rho proteins, such that the malignant progression of several cancers may be induced. Nevertheless, the effect and underlying mechanism of GGTase‐I in the malignant progression of salivary adenoid cystic carcinoma (SACC) remain unclear. This study primarily aimed to investigate the role and mechanism of GGTase‐I in mediating the malignant progression of SACC. The level of GGTase‐I gene in cells was stably knocked down by short hairpin RNA‐EGFP‐lentivirus. The effects of GGTase‐I silencing on the migration, invasion, and spread of cells were examined, the messenger RNA levels of GGTase‐I and RhoA genes of SACC cells after GGTase‐I knockdown were determined, and the protein levels of RhoA and RhoA membrane of SACC cells were analyzed. Moreover, the potential underlying mechanism of silencing GGTase‐I on the above‐mentioned aspects in SACC cells was assessed by examining the protein expression of ROCK1, MLC, p‐MLC, E‐cadherin, Vimentin, MMP2, and MMP9. Furthermore, the underlying mechanism of SACC cells proliferation was investigated through the analysis of the expression of cyclinD1, MYC, E2F1, and p21CIP1/WAF1. Besides, the change of RhoA level in SACC tissues compared with normal paracancer tissues was demonstrated through quantitative reverse‐transcription polymerase chain reaction and western blot experiments. Next, the effect after GGTase‐I silencing was assessed through the subcutaneous tumorigenicity assay. As indicated by the result of this study, the silencing of GGTase‐I significantly reduced the malignant progression of tumors in vivo while decreasing the migration, invasion, and proliferation of SACC cells and RhoA membrane, Vimentin, ROCK1, p‐MLC, MMP2, MMP9, MYC, E2F1, and CyclinD1 expression. However, the protein expression of E‐cadherin and p21CIP1/WAF1 was notably upregulated. Subsequently, no significant transform of RhoA and MLC proteins was identified. Furthermore, RhoA expression in SACC tissues was significantly higher than that in paracancerous tissues. As revealed by the results of this study, GGTase‐I shows a correlation with the proliferation of SACC through the regulation of cell cycle and may take on vital significance in the migration and invasion of SACC by regulating RhoA/ROCK1/MLC signaling pathway. GGTase‐I is expected to serve as a novel exploration site of SACC.

than that in paracancerous tissues.As revealed by the results of this study, GGTase-I shows a correlation with the proliferation of SACC through the regulation of cell cycle and may take on vital significance in the migration and invasion of SACC by regulating RhoA/ROCK1/MLC signaling pathway.GGTase-I is expected to serve as a novel exploration site of SACC.

| INTRODUCTION
Salivary adenoid cystic carcinoma (SACC) refers to a typically malignant cancer in the oral and maxillofacial regions (Ju et al., 2021;Wang, Fan, et al., 2021).Clinical research has suggested that SACC is characterized by slow growth, high recurrence rate, neurotropic damage, as well as the ease of distant metastasis (Li et al., 2022;Zhang, Wu, et al., 2021;Zhang, Zheng, et al., 2021).The mainstream clinical treatment of SACC has been the surgery in combination with radiotherapy and chemotherapy (Xie et al., 2022).However, due to the complex anatomical structure around the lesions, tumor recurrence, the damage to adjacent normal cells and serious adverse reactions are easy to occur, such that the treatment will have a poor postoperative effect (Lloyd et al., 2011;Zhang et al., 2020b).Thus, seeking novel treatment measures takes on critical significance in the treatment of SACC.
Rho protein family refers to a small molecular weight G protein in Ras protein superfamily, with the relative molecular weight ranging from 20 to 40 ku (Crosas-Molist et al., 2022;Zhang, Liu, et al., 2021).
In general, Rho protein family plays a certain role in cell maintenance polarity, cytoskeleton reorganization, cell adhesion, migration, proliferation, apoptosis, and many other biological activities (Tsubaki et al., 2021;Wang, Yuan, et al., 2021).However, Rho proteins, carboxyl terminal containing CaaX motif, lack the transmembrane and hydrophobic domain which traditional membrane-associated proteins cover, such that posttranslational modification is required to perform their biological functions (Glomset & Farnsworth, 1994).It is generally known that RhoA belongs to the Rho protein family, showing a close correlation with a wide variety of tumors (Zhang, Liu, et al., 2021).Existing research has suggested that RhoA is higher delivered in ovarian (Chen et al., 2021), breast (Liao et al., 2012), lung (Li et al., 2021), colorectal cancer (Wang et al., 2022), and the squamous cell carcinoma of oral cavity (Tian et al., 2021).Studies have shown that RhoA can not only indirectly affect the migration and invasion of tumor cells through a variety of molecular signaling pathways, but also affect the proliferation of tumor cells through the regulation of cell cycle (Jansen et al., 2018).As a transcription factor regulating downstream target genes, E2F1 plays an important role in the nucleus, and its abnormal expression can affect tumor progression (Wu et al., 2015).The expression of MYC oncogene is widespread, and its related mechanism is complex, which can regulate the occurrence and development of a variety of tumors.
Geranylgeranyltransferase type I (GGTase-I), first identified in 1991, refers to a heterodimer (Hinz et al., 2021).After the posttranslational modification by GGTase-I, the Rho proteins are localized at the plasma membrane and then achieve their normal functions (Shen et al., 2015).Accordingly, GGTase-I has a close correlation with the RhoA protein performing biological activities.Thus far, the roles of GGTase-I and RhoA and its downstream molecular targets in numerous cancers have been explored, whereas the role of GGTase-I and RhoA genes in oral cancers has been rarely investigated, especially SACC.
In this study, the effect and underlying molecular mechanism of specific inhibition of GGTase-I on cell migration, invasion, and proliferation were analyzed in SACC cells and tumors to identify the function of GGTase-I in SACC.

| Patients and tissue samples
The tissue samples originated from 24 patients (i.e., 14 males and 10 females) who had not undergone any treatments before operation.
The above-described patients who were recruited from 2009 to 2019 were histopathologically and clinically diagnosed as primary SACC.After being removed from patients, SACC and normal paracancer tissues were fixed with 4% formaldehyde solution, frozen with liquid nitrogen, and finally ground into homogenate for subsequent tests.The experiment of this study gained approval from the Ethics and Humanities Committee of Qingdao Municipal Hospital.
All participants signed informed consent.

| Cell culture
The cells of SACC-LM and SACC-83 cells (provided by the Ninth 37°C.When SACC cells exhibited the confluence from 80% to 90%, all the cells were inoculated into a new cell nurturing flask at 1:4 for subculture and subsequent experiments.

| Cell transfection and stable cell lines construction
Genebank was logged in to obtain the genetic sequences of GGTase-I (serial number: NM_005023.3).Then, short hairpin RNA (shRNA) (target sequence: GGAACAGCTCATCCTTATG).In accordance with the sequences, one negative control shRNA was designed.
When exhibiting the confluence of 30%, all the cells were transfected with specific shRNA-GGTase-I-EGFP-Lentivirus targeting to knock down GGTase-I or scramble shRNA-EGFP-Lentivirus (synthesized from Guangzhou Yuanjing Biotechnology Co., Ltd.) at a multi-infection 50 times for 24 h in the culture medium supplemented with 5 mg/mL polybrene.The stable cells were selected through 2 mg/mL puromycin posttransfection.In addition, Lipofectamine 3000 was diluted and mixed with plasmid vectors pcDNA3.1-GGTase-Ior pcDNA3.1 (Miaolingbio lnc.) according to the instructions separately, and then added to the culture medium for transfection.In the experiment of this study, the experimental group (knockdown with shRNA-GGTase-I-EGFP-Lentivirus), negative control group (transfect with scrambled shRNA-EGFP-Lentivirus), blank control group (only transfect with EGFP-Lentivirus), pcDNA3.1-GGTase-I group and pcDNA3.1 group were covered.Lastly, the transfection efficiency of cells was estimated.

| Real-time polymerase chain reaction
The total RNA of tissue homogenate and cells of transfection groups was extracted by using TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa) and reversely transcripted into complementary DNA (cDNA) by applying the PrimeScript RT-PCR kit (TaKaRa) consistent with the manual.SYBR Green Polymerase Chain Reaction Master Mix (TaKaRa) was applied to determine the messenger RNA (mRNA) levels of GGTase-I, RhoA, and control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in a 20 μL reaction system.The primer sequences are listed in Supporting Information: Table S1.Lastly, the relative output of mRNA was calculated in accordance with the comparative threshold cycle (C t ) and ∆∆ 2 C t analysis method.
The group with the maximum silencing efficiency served as the experimental group to continue the subsequent experiments following the relative expression of GGTase-I mRNA.

| Cell invasion assay
The SACC cells were re-levitated in serum-free RPMI-1640.
Subsequently, the upper chamber covered with Matrigel was introduced into the SACC cells, each of which covered 2 × 10 5 cells.
The chemoattractant in the lower chamber was the medium supplemented with 20% FBS.After 48 h, the invaded cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet, and the images were captured under an optical microscope of ×200 times.Next, the number of cells penetrating the cell membrane was counted using the ImageJ software.

| Wound healing assay
All the cells were cultured on a six-well plate.Next, these cells were scratched with 200 μL pipette tip at the fusion degree ranging from 80% to 90%.After scratching, the nonadherent cells were translated by being rinsed with PBS for three times.The adherent cells were nourished in the culture medium containing 10% FBS.The healing of the scratches was identified under the microscope 24 h later.The wound-healing areas were assessed after 0 and 24 h with a microscope, and the results were analyzed using the Image J software.

| Cell viability assay
All experimental cells were seeded at a certain number of 2 × 10 3 cells per well, and the cells were cultured in the medium supplemented with 10% FBS for 0, 24, and 48 h.Subsequently, 10 µL/well of Cell Counting Kit-8 (CCK-8) was supplemented for 2 h.
Next, the 96-well plate was taken out from the light-proof box and placed into the microplate reader (Molecular Devices); the OD value of the respective well was read at a 450 nm wavelength.The differences between experimental groups were evaluated through numerical analysis.After the measurement, the 96-well plate was returned into the incubator for the next test point.The respective group had five holes at the respective test point.

| Cell cycle analysis and cell apoptosis assay
All of cells were inoculated in a six-well culture plate, the density of the respective well was 2 × 10 5 cells, and the total volume reached 2 mL/well.Subsequently, 1000g centrifugation was performed for 5 min, and the cells were fixed with precooled 70% ethanol at 4°C for 12 h.The fixed cells were exposed to propidium iodide (PI) without any light for 30 min.Afterward, the cell cycle was finally examined and investigated using the flow cytometry (Beckman Coulter) equipment, and the data results were calculated using the FlowJo V10 software.
Cell apoptosis was identified through Annexin V-FIPC/PI fluorescence double staining.The adherent cells were administrated with 0.25% trypsin (without EDTA) and centrifuged at 500g for 5 min.2 × 10 5 cells were taken from the respective group and then centrifuged at 500g for 5 min.After a pause in 500 μL 1× Annexin V Binding Buffer, they were incubated with 5 μL Annexin V-FIPC and 5 μL PI staining solution at ambient temperature in the dark for 15 min.Lastly, the ratio of apoptotic cells to total cell number was identified through flow cytometry (Beckman Coulter), and the result was determined using the FlowJo V10 software.

| Xenograft tumor model assay
A total of 24 specific pathogen-free BALB/C female nude mice with an average age of 4-6 weeks and a weight of 20-25 g were employed for the experiment.Both cell lines were injected, and the respective cell line comprised three groups (i.e., shRNA-GGTase-I, control shRNA, and untreated group) of mice (four mice per group).A total of 5 × 10 6 shRNA-GGTase-I and the control group cells were syringed on the left side of the mice, respectively.The mice body weight was recorded, and tumors size was examined after formation.
The size of the tumor was examined with calipers every 5 days, and the tumor volume was calculated according to the formula: , where A and B represent the large and small diameters of the tumor, respectively (Duxbury et al., 2004).After 26 days, the mice were anesthetized by inhalation and photographed by fluorescence imaging system with 484 nm blue light to analyze the growth of SACC in vivo.Then, the mice were dissected and the tumors were taken for WB to demonstrate the effect and underlying mechanism after GGTase-I knockdown.
Animal husbandry and experimental procedures had been approved by the Ethics and Humanities Committee of Qingdao University (Shandong, China) and experiments were performed in full compliance with the relevant ethical guidelines (Approval ID:2023108).

| STATISTICAL ANALYSIS
Data were analyzed with SPSS 16.0 (IBM SPSS Statistics; IBM-Corp.) and GraphPad Prism 7 (GraphPadSoftware Inc.).The results were indicated as mean ± SD, through either analysis of variance or t tests.
The p < .05were considered significant, and every experiment was performed three times.

| RhoA expression is elevated in SACC tissues
To analyze whether RhoA was overexpressed in SACC tissue specimens, we compared RhoA expression from SACC tissues with normal paracancer tissues by qRT-PCR and WB assays.RhoA expression was markedly enhanced in SACC samples (p < .01)(Figure 1).

| Test of transfection efficiency of cells
To investigate the effect of GGTase-I on the proliferation, migration, and invasion of SACC cells, the lentiviruses carrying GGTase-I silencing (sh-GGTase-I and sh-GFP) and plasmids overexpressing GGTase-I (pcDNA3.1-GGTase-I and pcDNA3.1)were transfected into SACC cells, separately.And the results of qRT-PCR and WB assays showed that the GGTase-I silencing and overexpression models were successfully constructed (Figure 2).

| Silencing GGTase-I inhibit growth while promoting death of SACC cells
To observe the effect of GGTase-I silencing on the proliferation of SACC cells, SCC-LM and SCC-83 cells were identified through a continuous 2-day CCK-8 assay and Flow cytometric analysis.As revealed by the result of CCK-8 assay, at different points in time, GGTase-I silencing restricted the proliferation of cells (P SACC- LM < 0.005, P SACC-83 < 0.005) (Figure 3a).Moreover, flow cytometry revealed that the transformation of cells in G1/S phase was inhibited, and the cells in S phase decreased after the silencing of GGTase-I (P SACC-LM < 0.01, P SACC-83 < 0.05) (Figure 3b).Furthermore,

| Silencing GGTase-I restrains activation of RhoA in SACC cells
To analyze the specific mechanism by which GGTase-I regulates RhoA, we examined the expression of RhoA protein and membrane RhoA protein by WB assay, respectively.WB results suggested that there was no significant change in RhoA protein levels in the respective group.(P SACC-LM > 0.05; P SACC-83 > 0.05).
However, the expression of RhoA membrane protein in the tested strains was downregulated significantly.In addition, the expression of RhoA membrane protein in pcDNA3.1-GGTase-I group was upregulated (P SACC-LM < 0.001, P SACC-83 < 0.001).As the results indicated that silencing GGTase-I has no effect on the genetic expression and the protein synthesis of RhoA during a certain period, whereas it effectively prevents its membrane association (Figure 5).were significantly increased after GGTase-I overexpression (P SACC- LM < 0.001, P SACC-83 < 0.001).While the expression of MLC was not changed significantly (P SACC-LM > 0.05, P SACC-83 > 0.05) (Figure 6).
Thus, as revealed by the above findings, the downregulation of

GGTase-I can inhibit the migration and invasion of SACC cells by
inhibiting RhoA/ROCK1/MLC signaling pathway.

| Silencing GGTase-I induces SACC tumors growth in the xenograft model
To explore the effect of GGTase-I on SACC growth, BALB/c nude mice were injected subcutaneously with GGTase-I-silenced cells or their control group.After 6 weeks, the mice were photographed by fluorescence imaging system with 484 nm blue light to analyze the growth of SACC in vivo (Figure 7a).Also, the average body weight of mice in control groups was not statistically different from that in GGTase-I-silenced groups (P SACC-LM > 0.05; P SACC-83 > 0.05) (Figure 7b).The volume of the tumors in GGTase-I-silenced groups was significantly smaller than that in control groups (P SACC-LM < 0.01, P SACC-83 < 0.01) (Figure 7c).Also, in the GGTase-I-silenced group, the expression of GGTase-I was reduced.The RhoA membrane protein was downregulated in the tumor tissue, ROCK1, p-MLC, MMP-2, MMP-9, CyclinD1, MYC, E2F1, and Vimentin proteins, while the expression of E-cadherin and p21 CIP1/WAF1 was increased (P SACC- LM < 0.01, P SACC-83 < 0.01) (Figure 7d).The above-described results further indicated that GGTase-I-silenced inhibited the growth of SACC, and the silencing of GGTase-I suppressed the RhoA/ROCK1/ MLC signaling pathway and cell cycle regulation in SACC cells.

| DISCUSSION
As the onset of SACC is not easily detected and often accompanied by distant metastasis, SACC patients are difficult to be cured (Du et al., 2022).Although improved imaging techniques such as CT and MRI have made the diagnosis of SACC more definite, the survival rate of SACC persons is not optimistic because of poor treatment (Hou et al., 2021;Yang et al., 2021).Accordingly, it is significant to reveal the mechanism that may influence SACC  Thus, the first step in posttranslational modification of Rho proteins and the precondition for activation of Rho proteins is GGTase-I.
Research shows that GGTase-I can regulate the migration, invasion, proliferation, and apoptosis of cancer cells, and that cancer development can be suppressed by inhibiting GGTase-I (Lau et al., 2011).Though it has been confirmed that GGTase-I is closely related to breast cancer, human brain glioma (Zhou et al., 2013), lung cancer (Mortazavi et al., 2015), and neurodegenerative diseases in recent years (Gao et al., 2016), there are still few studies on the role of GGTase-I in oral cancers, especially SACC.
In this study, the mRNA and protein communications of RhoA did not change significantly after GGTase-I knockdown in SACC cells, whereas the expression of RhoA membrane protein was downregulated prominently.As revealed by the above-mentioned results, the silencing of GGTase-I does not exert any effects on the genetic expression and the protein synthesis of RhoA during a certain period, whereas it effectively prevents its membrane association.As RhoA refers to an intermediate signal molecule existing in cells (Wang, Gao, et al., 2022).RhoA, an isomer of the Rho subfamily, has been extensively investigated as a cancer-related gene, and it can be considered a signaling switch (Kim et al., 2018;Svensmark & Brakebusch, 2019).When activated, it shows a significant correlation with metastasis, invasion, proliferation, and other cell behaviors (Mosaddeghzadeh & Ahmadian, 2021).Existing has confirmed that RhoA can be overexpressed in of tumor tissues, suggesting that RhoA is of great significance in the malignant progression of SACC (Smithers & Overduin, 2016).In this study, the level of RhoA in SACC tissues and normal paracancer tissues was analyzed, and the result suggested that RhoA can be overexpressed in SACC tissues.
This result confirmed that RhoA may be vital to the malignant progression of SACC.Thus, it is speculated that silencing GGTase-I inhibits the activity of RhoA protein, such that the malignant progression of SACC may be inhibited.
The data of this study revealed that the downregulation of GGTase-I inhabited the metastasis and invasion of cells compared with control cells.Also, the level of MMP-2 and MMP-9 was prominently reduced in vitro and in vivo after GGTase-I silencing, such that this inhibitory effect was further confirmed.MMP-2 and matrix and protein components and affecting the migration and invasion of cancers by regulating the proteolysis of vascular basement membrane and activating the metastasis pathway of endothelial cells (Yang, Wu, et al., 2021).Besides, recent research have demonstrated that they are overexpressed in SACC compared with the normal tissue, indicating that they are closely correlated with the aggressiveness of SACC (Zhao et al., 2016;Zhou et al., 2014).
However, it is necessary to illustrate how GGTase-I influences MMP-2 and MMP-9, and to conduct an in-depth exploration into the specific mechanism of GGTase-I regulating MMP-2 as well as the generation and activity of MMP-9.
Extensive research suggested that Rho/ROCK/MLC signaling pathway can mediate the level of them, thus enhancing cell migration (Jeong et al., 2019;Li et al., 2017;Schram et al., 2011;Tian et al., 2017).Rho-associated protein kinase (ROCK1 and ROCK2) refers to the optimal effector proteins for RhoA binding activity.
Previous research has revealed that activation of ROCK signaling pathway can affect the cell-cell adhesion as well as the migration ability of cells (Riento & Ridley, 2003).Existing research has reported that RhoA is localized on cell membranes where it can be activated, and then it facilitates invasion and metastasis by sending signals to ROCK and actomyosin (Hetmanski et al., 2019).RhoA also plays a certain role in vascular endothelial growth factor-induced tumor angiogenesis through ROCK signaling pathway, and it promotes distant metastasis of tumor cells (Bryan et al., 2010).It has been confirmed that when RhoA is in the GTP state, considerable substrates including MLC can be directly phosphorylated by ROCK1 (Hetmanski et al., 2019;Sawada & Liao, 2014).In addition, other studies have shown that p-MLC can mediate the activity of myosin to regulate the contractility of myosin, thus affecting the migration and invasion of tumor cells (Katoh et al., 2001).In this study, posttranslational modification of RhoA was inhibited by silencing GGTase-I, thus RhoA could not be correctly localized on the cell membrane and its activity was reduced.Ultimately, the expression of ROCK1 and p-MLC was reduced.It is indicated that GGTase-I could regulate the migration and invasion of SACC cells through RhoA/ ROCK1/MLC signaling pathway.
As is known to all, tumor migration and invasion involve a very complicated process, and there are many factors that affect the malignant procedure of neoplasm.In this study, we also focused on whether GGTase-I can mediate the migration and invasion of SACC through EMT.EMT represents the gradual degeneration of cells from epithelial phenotype to mesenchymal phenotype (Suarez-Carmona et al., 2017).During tumor genesis and development, EMT is considered as an early marker event of tumor migration and invasion, which significantly increases the degree of malignancy of tumor cells (Saitoh, 2018).When the EMT process of tumor cells is activated, the level of E-cadherin protein, the epithelial phenotypic marker, decreases, the level of Vimentin protein, the mesenchymal phenotypic marker, increases, the degree of tight connection between cells is reduced, and the capability of migration and invasion is strengthened (Hiramoto et al., 2017).In addition, EMT is a necessary factor for tumor metastasis in systemic tumors (Tam & Weinberg, 2013).The typical characteristics of EMT are epithelial adhesion, the loss of cytoskeleton markers, and the acquisition of migration stroma markers such as E-cadherin and vientin (Thiery, 2002;Yang & Weinberg, 2008).Jansen et al. showed that RhoA can affect EMT to regulate tumor invasion and metastasis (Jansen et al., 2018).At the same time, some studies suggest that EMT process exists when SACC is physically transferred (Dong et al., 2011;Gibbons & Creighton, 2018).In this study, our data suggest that the expression of E-cadherin protein is increased and that of Vimentin protein is decreased.Accordingly, the abovedescribed data demonstrated that GGTase-I silencing could also significantly restrain EMT in SACC.
The occurrence, invasion, and metastasis of tumors show a significant correlation with the cell proliferation which is primarily regulated by Cyclins and Cyclin-dependent kinases (CDKs) (Spring et al., 2017).A typical feature of malignant tumors is uncontrolled proliferation.The results of CCK-8 assay and the volume changes of xenograft tumors in mice as identified by fluorescence imaging system in the nontraumatic state suggested that the proliferation of SACC cells and tissues was inhibited by GGTase-I silencing.The expression of cyclinD1 and p21 CIP1/WAF1 was examined to further explore the mechanism of how cells and tissues proliferation can be regulated by GGTase-I.As the first proto-oncogene discovered (Bartkova et al., 1994), cyclinD1 is overexpressed in various tumor cells such as Esophageal squamous cell carcinoma (Zhang, Zhang, et al., 2020), colon cancer (Yang et al., 2020), and is a vital protein for the transition from G1 stage cells to S stage cells (Hulit et al., 2002).
The above results suggest that silencing GGTase-I is capable of inhibiting the modification of RhoA protein and thus affecting RhoA protein activity, rather than inhibiting RhoA protein expression.
Correspondingly, SACC proliferation is affected by inhibiting expression of CyclinD1, MYC, and E2F1 and promoting expression of p21 CIP1/WAF1 .
In brief, this study confirms that silencing GGTase-I is capable of suppressing the development of SACC cells, which may be partly attributed to the inhibitory effect of the RhoA/ROCK1/MLC signaling pathway.Furthermore, our subsequent research will investigate the specific molecular mechanism between GGTase-I and EMT in SACC cells, which can provide more insights into the effect and mechanism of GGTase-I in SACC, so as to propose novel therapeutic ideas and methods for the treatment of SACC.

AUTHOR CONTRIBUTIONS
Zengpeng Chi, Qimin Wang, Jiawei Zheng, and Zhenggang Chen were responsible for the conception and design of the study, data analyses, interpretation of the findings, and manuscript writing.Lei Tong, Jing Qiu, Fang Yang, and Qingyuan Guo contributed to the design of the study and assisted in the writing.Wenjian Li performed the data analysis and assisted with the writing.The author(s) read and approved the final manuscript.
People's Hospital Affiliated to Medical College of Shanghai Jiaotong University) were transplanted into RPMI-1640 (GIBCO).Subsequently, 10% fetal bovine serum (FBS) (Sciencell), 100 U/mL penicillin, and 100 U/mL streptomycin (GIBCO) were introduced into the medium.The cells were left in an environment of 5% CO 2 at CHI ET AL.
showed that silencing GGTase-I facilitated the apoptosis of SACC cells simultaneously (P SACC-LM < 0.01, P SACC-83 < 0.01) (Figure3c).Given the above-described phenotypic results, we explored the molecular mechanism of in vitro cell proliferation, and used WB to analyze the levels of cell cycle regulators in the G1/S transition, cally cyclinD1, MYC, E2F1 and p21 CIP1/WAF1 in SACC cells.Compared with other control groups, the level of cyclinD1, MYC, and E2F1 in SACC cells in the experimental group was lower, the protein level of p21 CIP1/WAF1 was notably increased.However, the opposite result was identified when GGTase-I was subjected to overexpression (P SACC-LM < 0.05, P SACC-83 < 0.01) (Figure3d,e).Thus, the above-mentioned data revealed that silencing GGTase-I in SACC cells inhibited cell proliferation by regulating cell cycle and facilitating apoptosis in vitro.F I G U R E 1 The expression of protein and mRNA of RhoA was elevated in SACC tissues.The data of qRT-PCR and WB assays showed that RhoA expression was markedly enhanced in SACC samples.*p < .05,**p < 0.01, ***p < .001,****p < .0001.mRNA, messenger RNA; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; SACC, salivary adenoid cystic carcinoma; WB, western blot.F I G U R E 2 Detection of transfection efficiency of cells.SACC cells were transfected with sh-GGTase-I, sh-GFP, pcDNA3.1-GGTase-I,and pcDNA3.1,separately.qRT-PCR and WB assays results showed that the GGTase-I silencing and overexpression models were successfully constructed.*p < .05,**p < .01,***p < .001,****p < .0001.qRT-PCR, reverse-transcription polymerase chain reaction; SACC, salivary adenoid cystic carcinoma; WB, western blot.C Cell ell B Biology iology I International nternational F I G U R E 3 Silencing GGTase-I inhibits proliferation and stimulates the apoptosis of SACC cells.(a) CCK-8 assay was used to examine the cell growth rates in BC, NC, and KD groups of SACC-LM and SACC-83.The data showed that the cell growth rate was significantly suppressed in shRNA-GGTase-I cells.(b) Cell cycle profile in BC, NC, and KD groups of SACC-LM and SACC-83 cells was analyzed by flow cytometry.The data showed that the transformation of cells in G1/S phase was inhibited, and the cells in S phase decreased after the silencing of GGTase-I.(C) The apoptosis in BC, NC, and KD groups of SACC-LM and SACC-83 cells was analyzed through flow cytometry.The data showed that silencing GGTase-I facilitated the apoptosis of SACC cells simultaneously.(d, e) WB assay was used to detect the protein expression of Cyclin D1, MYC, E2F1, and p21 in BC, NC, KD, p-NC, and p-G groups.The data showed that the protein expression of CyclinD1, MYC, and E2F1 in experimental cells was inhibited, the protein level of p21 CIP1/WAF1 was notably increased.However, the opposite result was identified when GGTase-I was subjected to overexpression.*p < .05,**p < .01,***p < .001,****p < .0001.CCK-8, cell counting kit-8; SACC, salivary adenoid cystic carcinoma; shRNA, short hairpin RNA; WB, western blot.4.4 | Silencing GGTase-I reduces the migration and invasion of SACC cellsTo further explore the mechanism how GGTase-I influences the biological behavior of SACC, we conducted related experiments in vitro.As revealed by the result of the scratch assay, the time taken for space closure of Design group was markedly longer than that of controls, and the migration capacity after GGTase-I knockdown was obviously reduced compared with the other two groups (P SACC-LM < 0.001, P SACC-83 < 0.001) (Figure4a).After GGTase-I silencing, the overall arrangement of migrating cells on the PET film was significantly reduced in the experimental group (P SACC-LM < 0.001, P SACC-83 < 0.001) (Figure4b).The above-described results indicate that GGTase-I modulates the migration and invasion of SACC cells in vitro.To analyze the molecular mechanism of invasion and migration of SACC cells, the expression of E-cadherin, Vimentin, MMP-2, and MMP-9 was examined through WB experiments.The protein levels of Vimentin, MMP-2, and MMP-9 in cells significantly declined in the GGTase-I-silenced groups compared with those in controls, the protein levels of E-cadherin were notably increased.However, the opposite result was identified when GGTase-I was subjected to overexpression (P SACC-LM < 0.01, P SACC-83 < 0.01) (Figure 4c,d).In brief, the above-mentioned data showed that GGTase-I might influence the migration and invasion of SACC cells by inhibiting epithelial-mesenchymal transition (EMT) and the level of MMP-2 and MMP-9.

4. 6 |
Silencing GGTase-I inhibits RhoA/ROCK1/ MLC signaling pathway Existing research has confirmed that RhoA/ROCK1 pathway takes on critical significance in the development of SACC.In this study, it is supposed that GGTase-I downregulation suppresses the migration and invasion of SACC cells by inhibiting both RhoA/ROCK1 signaling pathway.Thus, we assessed the proteins expression of ROCK1, MLC, p-MLC in SACC cells.Also, the results showed that the expression levels of ROCK1 and p-MLC in SACC cells were significantly reduced after GGTase-I silencing (P SACC-LM < 0.01, P SACC-83 < 0.01), and both F I G U R E 4 Silencing GGTase-I reduces the migration and invasion of SACC cells.(a) Migration assay examined the cell migration ability in BC, NC, and KD groups of SACC-LM and SACC-83, respectively.The migration ability of GGTase-I silencing group was significantly inhibited.(b) Invasion assay examined the cell invasion ability in BC, NC, and KD groups of SACC-LM and SACC-83, respectively.The invasion ability of GGTase-I silencing group was significantly inhibited.(c, d) WB assay was used to detect the protein expression of MMP-2, MMP-9, E-cadherin, and Vimentin in BC, NC, KD, p-NC, and p-G groups.The data showed that the protein expression of MMP-2, MMP-9, and Vimentin in experimental cells was inhibited, the protein level of E-cadherin was notably increased.However, the opposite result was identified when GGTase-I was subjected to overexpression.*p < .05,**p < .01,***p < .001,****p < .0001.CCK-8, cell counting kit-8; SACC, salivary adenoid cystic carcinoma; RNA; WB, western blot.
germination and development and to explore a new approach to the treatment of SACC.GGTase-I is the enzyme formed by an α subunit and a β subunit(Reiss et al., 1990), participating in the activation of Rho proteins and guiding the correct positioning of Rho proteins on the cell membrane(Khan et al., 2013).The C-terminal CAAX motifs of Rho proteins are initial modified by farnesyltransferase (FTase) and GGTase-I.Next, they are all modified by Ras converting enzyme 1 (Rce1) to remove the three C-terminal amino acids (AAX).Lastly, the enzyme Icmt methylates the prenylated C-terminal (Winter-Vann & Casey, 2005).

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I G U R E 5 The expressions of RhoA and membrane RhoA in experimental cells.WB assay was used to detect the protein expression of RhoA and membrane RhoA in BC, NC, KD, p-NC, and p-G groups of SACC-LM and SACC-83.The results showed that there was no change in RhoA protein expression after silencing and overexpressing GGTase-1.However, the expression of membrane RhoA was decreased and increased, respectively.*p < .05,**p < 0.01, ***p < .001,****p < .0001.WB, western blot.F I G U R E 6 Silencing GGTase-I inhibits RhoA/ROCK1/MLC signaling pathway in SACC-LM and SACC-83 cells.WB assay was used to detect the protein expression of main factors in RhoA/ROCK1/MLC signaling pathway in SACC-LM and SACC-83 cells.The data showed that the expression levels of ROCK1 and p-MLC in SACC cells were significantly reduced after GGTase-I silencing, and both were significantly increased after GGTase-I overexpression.While the expression of MLC was not changed significantly.*p < .05,**p < .01,***p < .001,****p < .0001.
indicated by the analysis of this study, after the synthesis of RhoA protein in the nucleus, it can be localized at the cell membrane to perform the corresponding biological functions through the posttranslational modification guided by the isoprene of GGTase-I; the expression inhibition of GGTase-I does not exert any effects on the gene expression and protein synthesis of RhoA in SACC cells, whereas it can affect the activity of RhoA in SACC cells, consistent with that in most tumor cells(Ullah et al., 2016).As discussed above, the posttranslational modification process guided by the isoprene of GGTase-I takes on critical significance in Rho membrane localization and activation.F I G U R E 7 Silencing the growth of GGTase-I induced SACC tumors in the xenograft model.(a) The growth of subcutaneous transplanted tumor was identified using the in vivo fluorescence imaging system.(b) The respective group material of body weight and tumor volume of mice.(c) Effects of lentivirus infection on tumorigenicity of cells in the respective group.(d) The tumor tissues were taken for WB assay to examine the effect and underlying mechanism after GGTase-I knockdown.The data showed that the expression of GGTase-I was reduced, and the RhoA membrane protein was downregulated in the tumor tissue, ROCK1, p-MLC, MMP-2, MMP-9, CyclinD1, MYC, E2F1, and Vimentin proteins, while the expression of E-cadherin and p21 CIP1/WAF1 was increased.*p < .05,**p < .01,***p < .001,****p < .0001.GAPDH, glyceraldehyde 3phosphate dehydrogenase.

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refer to the members of the Zn 2+ -dependent proteolytic enzyme family.As endopeptidase, they play a crucial part in a variety of tumors.They are capable of degrading a variety of extracellular has confirmed that the upregulation of p21 CIP1/WAF1 expression in cells is capable of inhibiting DNA replication, inhibiting the transformation of tumor cells in the G1/S stage, and restraining cell proliferation excessively, positively correlated with the treatment and prognosis of tumors.Simvastatin, an inhibitor of RhoA, can significantly up-regulate intracellular p21 CIP1/WAF1 and inhibit cell proliferation by suppressing RhoA expression(Yang, Wu, 2021).During our experiment, RhoA expression was not changed significantly, the level of CyclinD1 was notably decreased, and the expression of p21 CIP1/WAF1 significantly was upregulated after GGTase-I knockdown in vitro and vivo.Additionally, the level of MYC and E2F1 in SACC cells in the experimental group was lower.The results of GGTase-I overexpression were opposite in our study.MYC, widely recognized as a proto-oncogene, is involved in various growth-promoting signaling pathways and plays a pivotal role in the occurrence and development of tumors.MYC is highly amplified in numerous tumor types, and transient inhibition of MYC expression can reverse tumorigenesis in vivo(Dhanasekaran et al., 2022).E2F1 is a significant member of the E2F family, which consists of cell cycle-related transcription factors.It regulates the expression of numerous genes that are essential for cell progression into the S phase of the cell cycle, thereby participating in various cellular processes including cell cycle progression, differentiation, proliferation, apoptosis, and DNA repair and replication.Studies have demonstrated that E2F1 is greatly expressed in diverse tumors such