The Regulatory Mechanismn Of RCS12 On Oral Inflammatory Osteolysis And Cancerous Invasion In Micevia The Phosphorylation And SUMOylation Of PTEN

ObjectiveInflammation plays a key component in the process of cancer progression,and there is an inseparable interaction between them.Inflammation and cancer are the two major research directions of oral diseases.Many scholars are getting more and more in-depth research on them.Recent studies have found that the phosphatase and tensin homolog deleted on chromosome 10(PTEN)is a critical regulator of tumorigenesis and bone remodeling.It plays an important regulatory role in various cell functions,including proliferation,apoptosis,autophagy,and migration,but the regulatory role of PTEN in oral inflammation is still unclear.Inflammatory osteolysis is a critical pathological feature of clinical inflammatory disease such as periodontitis which is one of the most common oral diseases and associated with alveolar bone resorption and tooth loosening in adults.The therapeutics for periodontitis are intended to block inflammation locally,but not enough for inflammatory bone destruction.However,many patients still suffer from bone loss under the anti-inflammation treatments.Therefore,more effective and economic treatments to reduce the inflammation and bone destruction are urgently needed.In the pathogenesis of periodontitis,osteolysis is predominantly caused by increased osteoclasts number and activity.The most determined cytokine involved in osteoclastogenesis is the receptor activator of nuclear factor ?B ligand(RANKL).RANKL can promote with proinflammatory cytokines.Thus,an ideal therapeutic strategy to prevent inflammatory bone destruction is to suppress pro inflammatory cytokines activation during osteoclastogenesis.Although it has been studied for many years,it is still inconclusive at the molecular mechanism level,and there is still a lack of direct and effective prevention and treatment methods.Recent studies have reported that PTEN can play a key role in rheumatoid arthritis osteolysis by effectively limiting the activity of phosphoinositide 3-kinase(PI3K),but its functional relevance in periodontitis remains unclear.Therefore,in-depth study of its pathogenesis,looking for direct and effective intervention targets,so as to formulate early and effective anti-inflammatory measures to prevent the loss of alveolar bone to the greatest extent,is of great and urgent significance for protecting adult periodontal health.Oral squamous cell carcinoma(OSCC)is the most common head and neck cancer(HNC)characterized by aggressive local invasion and metastasis.The poor prognosis of OSCC is considered due to the high occurrence of local invasion and metastasis as well as the lack of markers suitable for early detection.As an important inhibitor of human cancer,PTEN also plays a key role in the development of oral squamous cell carcinoma,and its downstream AKT/mTOR signaling pathway also plays an important regulatory role in tumorigenesis.But its potential molecular mechanism is still unclear.Recent studies have shown that different RGS proteins play specific roles in diverse cancer types.As the largest member of the RGS family,RGS12 has been found to be downregulated in prostate cancer in African Americans,but its role in other cancer types is still unknown.Based on the proteomics database of the Human Protein Atlas(https://www.proteinatlas.org)from HNC patients,the 5-year survival rate of patients with high RGS 12 expression is significantly better than that of patients with low expression.However,its potential molecular pathogenesis remains unclear.RGS 12 is a multi-domain multifunctional protein,including a PDZ domain,a phosphotyrosine binding(PTB)domain,an RGS domain,and a pair of Ras binding domains(RBD),and a Gai/o-Loco(GoLoco)motif.The PDZ domain is located at the N-terminal,which is a protein-protein interaction module.It plays an important role in most aspects of cell homeostasis and tumor growth,development,and metastasis,by specifically binding to the C-terminal PDZ binding motif(PBM)of the target protein.Coincidentally,PTEN possesses a a C-terminal PBM molecular structure,and loss of its activity leads to cancer susceptibility and causes continuous tumor progression.However,it remains unclear whether PTEN can be specifically recognized by the PDZ domain of RGS12,which aroused our interest in further exploration.In-depth research is needed to clarify the regulatory role and molecular mechanism of RGS12 and PTEN in oral cancer.Based on this,we proposed a new academic hypothesis:RGS12 associates with PTEN via PDZ domain to control the phosphorylation and SUMOylation of PTEN.On the one hand,it inhibits the downstream AKT/mTOR pathway,and on the other hand,it enhances the ability of DNA double-strand break repair in nucleus,which suppresses the progenesis and the aggressiveness of oral squamous cell carcinoma.This study aims to explore and reveal that the key nodes of the AKT/mTOR pathway mediated by RGS12 and PTEN can be used as potential therapeutic targets and prognostic biomarkers for oral squamous cell carcinoma.Methods1.Generation of RGS12 global knockout(CMVCre/+;RGS12fl/fl)mouse model1.1 To delete RGS12,we crossed RGS12fl/fl mice with homozygous CMV-Cre transgenic mice to generate CMVCre/+;RGS12fl/+ progeny,which were used for subsequent mating to produce heterozygous CMVCre/+;RGS12fl/fl mice.RGS12fl/fl mice were used as controls.1.2 Genomic DNA was extracted from tongue tissue to examine the deletion of RGS12.The level of RGS12 mRNA and the expression of RGS12 protein were measured by real-time quantitative PCR(qPCR)and Western blot(WB)to confirm that the RGS12 gene is efficiently deleted in this model.1.3 Statistical analysis was performed on the growth curves of RGS12 knockout mice and control mice.In addition,the histological staining of tongue tissue was compared and analyzed.2.PTEN Inhibits Inflammatory Osteolysis in Ligature-Induced Periodontitis via IL1 and TNF-?2.1 Generation of ligature-induced periodontitis mice model.2.2 Histological analysis was performed to assess the changes in inflammation and bone mass in vivo experiments.H?E staining was performed to assess the score of inflammation and bone destruction.In addition,samples were further analyzed by TRAP staining to assess osteoclast activity.2.3 The level of inflammatory cytokines(IL1,IL6,TNF-?)mRNA from mouse periodontal tissues,the bone formation gene markers,osteocalcin(OC)and alkaline phosphatase(ALP),and bone erosion markers,tartrate-resistant acid phosphatase(TRAP)and cathepsin K were measured by using qPCR.2.4 RAW264.7 macrophages were used to silence PTEN expression by siRNA and overexpression of PTEN respectively in vitro experiment,and then the level of inflammatory cytokines(IL1,IL6,TNF-?.)mRNA were detected by using qPCR.2.5 To further study the anti-inflammatory effects of PTEN,we made PTEN nanoparticles and injected the mixture into the gingiva.The level of inflammatory cytokines(IL1,IL6,TNF-?)and bone erosion markers(TRAP and cathepsin K)mRNA from mouse periodontal tissues were measured by using qPCR.3.RGS12 Represses Oral Cancer Invasion via the Phosphorylation and SUMOylation of PTEN3.1 To assess the expression of RGS12 in tongue mucosal epithelium and oral cancer tissues,human OSCC samples with different TNM stages and pathological grades and adjacent normal tongue tissue(NAT)samples were selected for IHC staining and scoring.3.2 To understand the role of RGS12 in tumorigenesis in vivo,we generated RGS12 global knockout mice and induce the mice to develop OSCC by oral administration of 4NQO in drinking water.H?E staining and Immunofluorescence(IF)staining were used to analyze the carcinogenesis process of OSCC.3.3 To get further insight into the role RGS12 in squamous cell carcinoma,we first confirmed the RGS12 expression in vitro.By comparing the control cells(HUVEC)and two squamous cell carcinoma cell lines(SCC4 and CAL27),the expression of RGS12 protein were determined by performing the WB.We transfected the SCC4 and CAL27 cells with shControl and shRGS12 plasmids and confirmed the protein levels by performing the WB.Matrigel invasion assays were performed in RGS12 silenced SCC4 and CAL27 cells in vitro.To examine the effect of RGS12 on cell proliferation,WST-1 assays were performed in SCC4 and CAL27 cells with and without RGS12 knockdown.3.4 To test the potential therapeutic effect of RGS12 on OSCC,we stabley transfected the RGS12(RGS12 OE)in SCC4 cells and performed Matrigel invasion assays,scratch assays,and cell proliferation was assayed by WST-1.The overexpression of RGS12 was detected and confirmed by WB.3.5 We transfected the SCC4 cells with control pCMV,pCMV-RGS 12 and shRGS 12 plasmids and determined the expression of p-PTEN and total PTEN protein,the expression of p-AKT and total AKT protein,as well as the expression of p-mTOR and total mTOR protein by WB.To verify whether RGS12 affects SUMO1-PTEN associated DNA double-strand break repair,we detect y-H2AX level by performing IF staining.3.6 To verify whether PTEN is associated with RGS12,we performed IF staining to verify the expression of RGS12 and PTEN in both cytoplasm and nucleus.The strong affinity between RGS12 and PTEN further determined by Co-IP experiment.The protein interaction was also performed by Co-IP experiment after deletion of the PDZ domain of RGS12.3.7 To further determine the function of RGS12?PDZ and RGS12?RGS,we first transfected the control plasmid pCMV(Control),pCMV-RGS 12?PDZ,pCMV-RGS 12?RGS and pCMV-RGS12(RGS12 OE)in SCC4 cells.Then,we performed the cell Matrigel invasion assay and WST-1 proliferation assay.3.8 To further test the therapeutic effect of RGS12 in vivo,SCC4 cells with stable RGS 12 overexpression were subcutaneously injected into the hind leg of the immunodeficient NOD scid mice to detect tumor formation.We then harvested the tumors and confirmed the tumor volume and weight.Then the level of RGS12 mRNA were determined by using qPCR.Results1.Generation of RGS12 global knockout(CMVCre/+;RGS12fl/fl)mouse modelTo understand the in vivo role of RGS12 in tumorigenesis,we generated RGS12 global knockout(CMVCre/+;RGS12fl/fl)mice,which were born in expected Mendelian ratios.Genomic DNA was extracted from tongue tissue to examine the deletion of RGS12.The level of RGS12 mRNA was greatly reduced,and the expression of RGS12 protein was significantly decreased in CMVCre/+;RGS12fl/fl mice,which confirmed that the RGS12 gene was efficiently deleted in this model.2.1 Ligature-Induced Periodontitis and Inflammatory OsteolysisTo assess the changes in inflammation and bone mass,histological analysis was performed.Ligature-induced periodontitis mice showed a significant gingival swelling,a greater soft tissue thickness,and severe bone resorption compared to the unligated group.In addition,a wide range of inflammatory cells were shown around the root of the second molar and the inflammation score was significantly increased in ligature-induced periodontitis mice.Ten days after ligature,there was a significant difference in bone mass between the ligature-induced periodontitis group and the unligated group.The bone loss around the ligature were significantly increased in mice compared to unligated controls.To assess osteoclast activity,samples were further analyzed by TRAP staining.The result showed that TRAP-positive multinucleated cells increased significantly in the ligature-induced periodontitis group compared to the unligated group.2.2 Gingival mRNA Expressions of PTEN and Inflammatory CytokinesPTEN,as a regulator of the mitogen-activated protein kinase(MAPK)pathway and inflammatory cytokines,was found decreased in ligature-induced periodontitis.And the inflammatory cytokines such as IL1,IL6 and TNF-? mRNA expression levels were increased in the ligature-induced periodontitis group,especially the expression of IL1 and TNF-?.Inflammation was shown to cause excessive bone resorption as well as impaired bone formation.Thus,we determined the bone formation gene markers,osteocalcin(OC)and alkaline phosphatase(ALP),and bone erosion markers,tartrate-resistant acid phosphatase(TRAP)and cathepsin K.The results showed that the gene expressions of bone erosion makers TRAP and cathepsin K were significantly increased in the ligature-induced periodontitis,rather than bone formation markers OC and ALP.2.3 PTEN Regulated the Expression of Inflammatory CytokinesMacrophages play important roles in the pathogenesis of periodontitis by regulating the immune response and regulating tissue repair and bone loss.We first silence the PTEN expression in RAW264.7 cells through siRNA and found the same results as those of in vivo experiment;the expressions of inflammatory cytokines IL1 and TNF-? were significantly increased.To further confirm this,we then forced overexpressed of PTEN in RAW264.7 cells.The results showed that overexpression of PTEN reduced the expression of IL1 and TNF-?.2.4 Overexpression of PTEN Inhibits the Inflammation In VivoTo further study the anti-inflammatory effects of PTEN,we made PTEN nanoparticles by mixing the 10?g PTEN plasmids with 20?l nanoparticles and injected the mixture into the gingiva three times/week for two weeks.After injection,we found that PTEN gene expression was increased while the inflammatory factors such as IL1,IL6 and TNF-? were decreased,especially the expression of IL1 and TNF-?.Moreover,osteoclast markers TRAP and cathepsin K were downregulated after exogenous PTEN injection.Conclusively,our results showed that nanoparticle packaged PTEN can alleviate inflammatory osteolysis.3.1 RGS12 expression level significantly decreases in human OSCC tissues in TNM stage and pathological grade dependent mannerTo assess the expression of RGS12 in tongue mucosal epithelium and oral cancer tissues,human OSCC samples with different TNM stages and pathological grades and adjacent normal tongue tissue(NAT)samples were selected for IHC staining and scoring.The results indicated that RGS12 protein was highly expressed in the NAT mucosal epithelium,but significantly decreased in OSCC samples.Moreover,as the TNM stage and pathological grade of the OSCC advanced,the expression level of the RGS12 protein decreased,indicating a negative correlation between RGS12 and OSCC tumorigenesis.3.2 Deletion of RGS12 significantly promotes the development of OSCC under 4NQO inductionTo understand the role of RGS12 in tumorigenesis in vivo,we generated RGS12 global knockout(CMVCre/+;RGS12fl/fl)mice and induce the mice to develop OSCC by oral administration of 4NQO in drinking water.Macroscopic images showed a smooth surface of the tongue mucosa in mice without 4NQO induction.However,after 4NQO induction,the CMVCre/+;RGS12fl/fl mice showed an increase in the range of tongue lesions compared with the control mice.H?E staining result showed the increased tongue lesion range,the unclear boundary,the obvious epithelial dysplasia,a large number of aggregated cancer nests,the severely destroyed basal and submucosal layers,and even the tumor invated into muscular layer in the CMVCre/+;RGS12fl/fl mice.However,in the control mice,the results showed the localized lesion range of the tongue and the relatively clear boundary without obvious epithelial dysplasia,aggregated cancer nest,and the invasion of basal,submucosal and muscular layers.The result of Immunofluorescence(IF)staining showed that the CK14+signal on the epithelial layer of the tongue mucosa was significantly enhanced and invaded into the muscular layer in 4NQO treated CMVCre/+;RGS12fl/fl mice,sugesting that deletion of RGS12 significantly promotes the development of OSCC.3.3 Knockdown of RGS12 promotes cancer cell migration and proliferationTo get further insight into the role RGS12 in squamous cell carcinoma,we first confirm the RGS12 expression in vitro.By comparing the control cells(HUVEC)and two squamous cell carcinoma cell lines(SCC4 and CAL27),we found that both the protein and mRNA expression of RGS12 were significantly lower in SCC4 and CAL27 cells.To further determine the decreased role of RGS12 in cancer cells,we transfected the SCC4 and CAL27 cells with shControl and shRGS12 plasmids and confirmed the protein levels by performing the western blot.Matrigel invasion assays were performed in RGS12 silenced SCC4 and CAL27 cells in vitro.The number of invading SCC4 and CAL27 cells was significantly increased in RGS12 silenced cells compared with that in the control shRNA transfected cells.To examine the effect of RGS12 on cell proliferation,WST-1 assays were performed in SCC4 and CAL27 cells with and without RGS12 knockdown.The results showed that knockdown of RGS12 significantly increased cell proliferation compared with the scrambled shRNA control.3.4 RGS12 associates with PTEN via PDZ domainTo determine the mechanism by which RGS12 regulates PTEN,we explored the molecular structure of PTEN.We found that PTEN possesses a C-terminal four amino acid binding motifs(ITKV)which can be recognized by a specific set of PDZ domains from regulatory proteins.To verify whether PTEN is associated with RGS12,we first performed IF staining.The results showed that RGS12 and PTEN were expressed in both cytoplasm and nucleus,and overlapped in the expression location.Co-IP experiment further demonstrated a strong affinity between RGS12 and PTEN.The N-terminal PDZ domain of RGS12 is a protein-protein interaction module which can binds to the 3 or 4 amino acid binding motifs at the extreme carboxy terminus of the target protein.Notably,after deletion of the PDZ domain of RGS12,we found that RGS12 failed to bind PTEN,further confirming that PTEN can be specifically recognized and bound by the PDZ domain of RGS12.To further determine the function of RGS12?PDZ and RGS12?RGs,we first transfected the control plasmid pCMV,pCMV-RGS12?PDZ,pCMV-RGS12?RGS and pCMV-RGS12 in SCC4 cells.Then,we performed the cell invasion assay and proliferation assay.The results showed that RGS12?PDZ cannot inhibit the cancer cell migration and proliferation compared to RGS12 OE group.However,RGS12?PDZ can inhibit the the migration and proliferation of cancer cells compared with control.Thus,the PDZ domain of RGS12 may be a critical drug target in cancer studies.3.5 RGS12 regulates PTEN phosephorylation and SUMOylationPTEN is the target of multiple modes of post-translational modification,including phosphorylation,ubiquitination,sumoylation.The phosphorylation of PTEN(p-PTEN)negatively regulates its function as an antagonist of PI3K signaling.SUMO1(small ubiquitin-like modifier,SUMO)modification of PTEN(SUMO 1-PTEN)regulates tumorigenesis and nuclear SUMO-PTEN is required for the repair of DNA double-strand breaks.To further characterize the underlying mechanism that RGS12 negatively regulates OSCC tumorigenesis,we first test whether RGS12 affects post-translational modification of PTEN.We transfected the SCC4 cells with control pCMV,pCMV-RGS12 and shRGS12 plasmids and determined the p-PTEN and total PTEN protein by western blot.The results showed that p-PTEN was increased in RGS12 OE group while decreasd in RGS12 knockdown group,which suggest that RGS12 regulates p-PTEN in cancer cells.To further assess how RGS12 regulates the SUMOylation of PTEN,the IP and IB were performed.The results showed that the SUMOylation(SUMO1-PTEN)in SCC4 cells were increased after overexpression of RGS12.Furthermore,the phosphorylated RAC-alpha serine/threonine-protein kinase(AKT)/phosphorylated mechanistic target of rapamycin(mTOR)signaling pathway plays a key role in tumorigenesis which acts as a downstream of PTEN.Our results showed that the AKT/mTOR signaling were downregulated after overexpression of RGS12.Thus,our data demonstrated that RGS12 negatively regulates OSCC tumogenesis through controlling PTEN-related AKT/mTOR signaling pathway.To verify whether RGS12 affects SUMO1-PTEN associated DNA double-strand break repair,we detect?-H2AX level by performing IF staining.The results showed that bright area considered to be a densely packed heterochromatin reflecting significantly decreased after RGS12 overexpression compared to the control.3.6 Ectopic expression of RGS12 inhibits oral squamous cell carcinomaTo test the potential therapeutic effect of RGS12 on OSCC,we stabley transfected the RGS12(RGS12 OE)in SCC4 cells.Successful stable overexpression of RGS12 was confirmed by WB.Matrigel invasion assays results showed that stable overexpression of RGS12 reduced more than 50%in the number of invading cells,and significantly inhibited the recovery of wound area(45%recovery)following 48 hours post-scratch.Additionally,WST-1 assays revealed that stable overexpression of RGS12 significantly inhibited cell proliferation.To further test the therapeutic effect of RGS12 in vivo,SCC4 cells with stable RGS12 overexpression were subcutaneously injected into the hind leg of the immunodeficient NOD scid mice to detect tumor formation.The results showed a significant decrease in tumor volume and weight after overexpression of RGS12 compared to control groups.We then harvested the tumors and confirmed the RGS12 level by performing real-time PCR.The results showed RGS12 mRNA level in tumor from RGS12 OE group was still higher than control groups after subcutaneous tumor.DiscussionIn the ligation-induced periodontitis model,our in vitro and in vivo data highlight the anti-inflammatory role of PTEN presentation.The schematic structure of the predicted PTEN protein has been reported.The phosphoinositide 3-kinases can be inhibited by the protein and lipid phosphatase activity of PTEN.Here,we found the gene expression level of PTEN is responsible for inflammation regulation.However,we cannot neglect the effect of protein and its postmodification;thus,further study will focus on the protein and modification level.In our study,inhibition of PTEN expression with siRNA results in upregulation of IL1 and TNF-?,whereas the expression of IL6 was not significantly altered,suggesting that PTEN may exert anti-inflammatory effects in mouse periodontitis by modulating the expression of IL1 and TNF-?.NF-?B is generally considered to be the major regulatory pathway of genes encoding pro inflammatory proteins including IL1,IL6 and TNF-?.The expression of TNF-stimulated NF-?B-dependent genes can be blocked by PTEN,which supports our hypothesis that PTEN can play a role in regulating the expression of proinflammatory proteins IL1 and TNF-?.In addition,PTEN can regulate NF-?B via the PI3K/Akt pathway.Under physiological conditions,PI3K/Akt/PTEN signaling plays a key role in inflammation.As the common downstream molecules of NF-?B pathway,IL1,IL6 and TNF-? play a role in maintaining the cell homeostasis,further suggesting that PTEN may regulate the inflammatory response through the PI3K/Akt/NF-?B pathway.PTEN enforced overexpression in RAW 264.7 cells inhibited inflammation and osteoclast in our mouse model,indicating that PTEN may act as a critical regulator between inflammation and bone remodeling.Numerous studies have demonstrated that IL1 can stimulate osteoclast differentiation and activation.In addition,inhibiting IL1 significantly reduces bone erosions and cartilage degradation,whereas blocking TNF-? decreases inflammation.We therefore speculate that PTEN regulate the bone remodeling mainly through activation of IL1.However,no significant changes were found in osteoblast marker genes,suggesting that PTEN primarily regulates osteoclasts rather than osteoblasts during bone remodeling.All these data confirm that PTEN plays a controlling role in the process of oral alveolar bone remodeling,and it is determined that the new function of PTEN is a key factor in regulating periodontitis and bone remodeling.Recent studies have shown that different RGS proteins play specific roles in diverse cancer types.As the largest member of the RGS family,RGS12 has been reported to be lost selectively in African American prostate cancer(Wang et al.,2017).More importantly,based on the proteomics database of the Human Protein Atlas(https://www.proteinatlas.org)from HNC patients,the 5-year survival rate of patients with high RGS12 expression is significantly better than that of patients with low expression.In our study,we performed an IHC score analysis of RGS 12 expression in human OSCC tissues and found a significantly reduction in those OSCC tissues.Further analysis showed that the reduction in RGS 12 expression was in the TNM stage and pathological grade dependent manner,suggesting that RGS12 may serve as biomarker for OSCC diagonosis,treatment as well as prognostic analysis.The pathogenesis of OSCC is mainly due to the accumulation of multiple genetic mutations regulated by genetic predisposition.Our in vivo and in vitro data have demonstrated that RGS12 is oral cancer suppressor gene.To understand the role of RGS12 in tumorigenesis in vivo,we generated RGS12 global knockout(CMVCre/+;RGS12fl/fl)mice and induce the mice to develop OSCC by oral administration of 4NQO in drinking water.4NQO-induced OSCC model is a classical method for studying oral cancer.Vitale-Cross et al.compared different 4NQO-induced OSCC models,wherein Young et al.reported that some of the C57B1/6 mice failed to develop OSCC when a 50 ?g/mL dose of 4NQO was given in the drinking water and stimulated even up to 32 weeks.In contrast,Tang et al.reported a 100%successful rate of OSCC model generation in C57BL/6 mice by giving a 100?g/mL dose of 4NQO in the drinking water and stimulated for 16 weeks,followed by 8-16 weeks regular drinking water,suggesting the importance of 4NQO dosage in generating the OSCC model.To ensure the success in creation of OSCC model with higher successful rate in C57BL/6 mouse strain,we followed Tang et al.method and obtained a satisfied outcome of the OSCC model generation in the control and RGS12 mutant mice,featured with multiple lesions,papillomas,and carcinomas over the surface of the tongue.Therefore,our data indicate that this is an optimal method to guarantee the successful rate of OSCC generation and an ideal model to disclose different progression of carcinoma in the original 4NQO-treated mice.RGS12 is a multidomain protein with the classical PDZ(PSD-95/Dlg/ZO-1)domain in the N-terminus.More than 250 proteins containing PDZ domain have been reported in the human proteome.As so many proteins possess multiple PDZ domains,their potential combinations with PBM containing proteins appear to be enormous.However,the recognition of PDZ domain is highly specific.Traditionally,PDZ domains have been classified as ?-? classes depending on the consensus sequences of PBMs.Studies have reported that the RGS12 N-terminal PDZ domain belongs to class I PDZ sequence(STTL)which is commonly thought to be specifically recognized and bound by C-terminal polypeptides class I PBM.Coincidentally,the last four amino acids at the C-terminus of the PTEN protein(residues Ile400-Thr401-Lys402-Val403-COOH;ITKV)constitute a PBM for class I PDZ domain.In our study,we identified the highly specific association between RGS12 and PTEN via PDZ domain,and that RGS12 activates PTEN through both phosphorylation and SUMOylation.Moreover,SUMO-PTEN is essential for tumorigenesis and is also important for DNA double-strand break repair based on homologous recombination.Consistently,we revealed a significant decrease in y-H2AX after RGS12 overexpression,indicating that RGS12 associates with PTEN to enhance the ability of DNA double-strand break repair in nucleus.Our results also showed that RGS12 can enhance the PTEN-related AKT/mTOR signaling to regulate the cell migration and proliferation.More importantly,our results showed continuous overexpression of RGS12 in tumor cells for four weeks can effectively inhibit tumor proliferation and invasion.It is suggested that exogenous administration of RGS12 on tumors may become a treatment strategy for oral cancer.Our results showed that the overexpression of RGS12 inhibited the cancer cell proliferation and invasion,which was impaired by PDZ domain mutation,but not by RGS domain mutation.These results suggest that PDZ domain plays important role in regulation of PTEN and downstream signaling,while RGS domain of RGS 12 may not be mainly involved in PTEN regulation or tumorigenesis.Given RGS proteins can inhibit G protein signaling through the RGS domain which interacts with the Gi and/or Gaq subunits and accelerates their GTP hydrolysis,we cannot exclude the regulation of G protein upstream of RGS 12 since the regulation of GPCRs on tumors is quite complicated.Thus,our further experiments will focus on the exploration of the upstream target proteins of RGS 12.ConclusionsIn summary,we report here that activation of PTEN is necessary for inhibition of inflammation and alveolar bone loss,which suppresses both processes via inhibiting the IL1 and TNF-? pathways in periodontitis.RGS12 is an important factor in the development of oral cancer,mainly regulating tumor proliferation,migration,and invasion;RGS12 associates and activates PTEN through its functional PDZ domain;RGS12 regulates the PTEN-related AKT/mTOR signaling and DNA repair via phosphorylation and SUMOylation of PTEN to inhibit OSCC tumorigenesis;the overexpression of RGS12 can inhibit the occurrence and development of OSCC.Thus,our studies indicated that RGS12 plays a key role in mouse oral inflammatory bone erosion and cancerous invasion via PTEN phosphorylation and SUMOylation.The key nodes of the AKT/mTOR pathway mediated by RGS12 and PTEN can be used as potential therapeutic targets and prognostic organism's markers for OSCC.