| Objective:The Oral Squamous Cell Carcinoma(OSCC)is the most common malignant tumor in the head and neck.The Porphyromonas gingivalis(P.gingivalis)is a key oral pathogen,which can promote to form a Tumor micro-environment(TME)which is good for the growth of OSCC.Currently,lots of researches showed that in the TME of OSCC,P.gingivalis plays an important role in promoting the proliferation,differentiation,invasion and metastasis of tumor cells.This paper aims to study the relevant mechanism of P.gingivalis in the TME of OSCC to improve the development of OSCC through activated CXCL2 / CXCR2 axis.Methods:Part 1,1.A retrospective study was conducted on case and tissue specimens of 205 OSCC patients in the Department of Maxillofacial Oncology,Affiliated Stomatological Hospital of Xinjiang Medical University and the follow-up data were completed.Immunohistochemical staining was performed in accordance with the Helsinki Declaration on the use of human tissue specimens.Approved by the ethics committee of the First Affiliated Hospital of Xinjiang Medical University,the patient fully informed consent and signed the informed consent.Surgical tissue samples of 20 patients with OSCC were collected(all patients did not receive chemotherapy and radiotherapy before operation),and the corresponding non-tumor oral tissue samples(set as the control group)were taken.The expression level of P.gingivalis in the tissues of OSCC patients was detected by immunohistochemistry,and the difference between the expression levels of P.gingivalis and that of non-carcinomous oral tissues were compared.2.According to downloading GSE87539 and GSE138206 expression profile gene databases from GEO database,bioinformatics is adopted to analyze,and the probes were transformed into corresponding gene symbols based on the platform annotation information.The GSE87539 data set contained samples of normal oral epithelial cells infected with and without P.gingivalis.There were 3 groups samples in each group and 6 groups in total.The GSE138206 data set contained OSCC tissue samples and normal oral mucosa samples.There were 6 groups samples in each group and 12 groups in total.Using GEO2R(http: //www.ncbi.nlm.nih.gov/ geo/geo2r),Differential Expression Genes(DEGs)between GSE87539 and GSE138206 were screened.The difference was statistically significant when Log FC(fold change)>1 and adj.P-value<0.01.The expression levels of CXCL2 and TANs were detected by immunohistochemistry.All those methods were used to analyze the correlation between the immune expression levels of P.gingivalis,CXCL2 and TANs with clinical indicators and their impact on prognosis.3.The immunofluorescence double labeling technique was used to identify the location of P.gingivalis,CXCL2 and TANs in OSCC.Part 2,1.OSCC cell strain of TSCCA was cultured in vitro,and P.gingivalis strain(ATCC33277)was cultured under strict anaerobic conditions.TANs were isolated from peripheral blood of OSCC patients.The co-culture model was established with MOI=50(bacteria / cell).The TME cell model was constructed on the basis of co-culture model(TANs were added into the co-culture model,TANs and TSCCA were mixed in a ratio of 1: 1).2.The contents of CXCL2 in the supernatant of TSCCA,TSCCA + TANs,TSCCA + P.gingivalis and TSCCA + P.gingivalis + TANs were detected by Elisa testing.3.The different chemotactic ability of the supernatant to TANs was tested through the chemotactic experiment of TSCCA,P.gingivalis and TSCCA + P.gingivalis.4.Through the CCK8 method,cell scratch test and cell invasion test to detect the changes of biological behavior of TSCCA cells in four groups: TSCCA,TSCCA + P.gingivalis,TSCCA +TANs and TSCCA + P.gingivalis + TANs.5.After adding SCH527123 to test when blocking the CXCL2 / CXCR2 signal axis,the biological behaviors of TSCCA cells in TSCCA + P.gingivalis + TANs + SCH527123 group and TSCCA + P.gingivalis +TANs group were compared.6.SCC7 cell strain was cultured in vitro to establish the co-culture model,C57BL/6 mice was used to establish the tumor model,Different concentrations of SCH527123 were used for intervention,Control group(SCC7),low-dose intervention group(SCC7 + P.gingivalis + 0.05 n M SCH527123),high-dose intervention group(SCC7 + P.gingivalis + 0.20 n M SCH527123)and experimental group(SCC7+ P.gingivalis)were used to detect the changes of tumor models.Part 3,1.The expression of EMT related specific proteins and the activation of JAK1 / STAT3 signaling pathway in TME cell model were detected by q RT-PCR and Western Blot respectively.2.The lentiviral vectors of CXCL2 and CXCR2 were constructed.The two lentiviruses were co-infected into TSCCA cell strain.The flow cytometry was used to screen the positive cells expressing both lentivirus and lentivirus simultaneously.The positive cell clones were selected by limited dilution method,and then the cells were cultured in complete culture medium containing puromycin.3.The co-culture model and TME cell model were constructed by TSCCA cell strains infected with two kinds of lentivirus.The expression of E-cadherin and N-cadherin and the activation of JAK1 /STAT3 signaling pathway were detected at m RNA and protein levels by q RT-PCR and Western Blot.Result:Part 1,1.Among 205 OSCC patients,86 cases were weakly positive for P.gingivalis and 119 cases were strongly positive for P.gingivalis.The P.gingivalis was mainly expressed in the cytoplasm of tumor cells,but negative in non-carcinomous tissues.2.After the data set was standardized,26469 differential genes were identified in GSE87539 chip data set,and 9443 differential genes were identified in GSE138206.Among them,89 genes overlapped.CXCL2 was located in hub gene and its expression was significantly up-regulated.In 119 cases with high expression of P.gingivalis,the expression of CXCL2 was weakly positive in 30 cases and strongly positive in 89 cases.The expression of TANs was weak positive in 38 cases and strong positive in 87 cases.Statistical analysis showed that the high expression of P.gingivalis,CXCL2 and TANs was significantly correlated with the poor prognosis of OSCC patients.3.The expression of P.gingivalis,CXCL2 and TANs in OSCC tissues were detected by immunofluorescence technique with single label and double label for verifying the location of its expression in OSCC.Part 2,1.TSCCA cell strain and P.gingivalis strain were cultured successfully and TANs were successfully extracted from peripheral blood of patients with OSCC.The 16 Sr RNA sequencing technology was used to sequence P.gingivalis.The sequencing results were compared with pubmed database,which showed that the coincidence rate was 99.3% in line with the standard strain ATCC33277.The laser confocal,q RT-PCR and Western Blot were used to verify the successful establishment of co-culture model.2.Tested by Elisa testing,the content of CXCL2 in the supernatant of TSCCA + P.gingivalis + TANs was significantly higher than that in the supernatant of TSCCA,TSCCA + P.gingivalis and TSCCA + TANs(P<0.001).3.The chemotactic ability of TSCCA + P.gingivalis supernatant group was significantly higher than that of TSCCA and P.gingivalis supernatant groups(P<0.001).4.The CCK8 method proliferation test,cell scratch test and cell invasion test were adopted to verify the proliferation,migration and invasion.TSCCA + P.gingivalis + TANs group was significantly higher than those of TSCCA,TSCCA + P.gingivalis and TSCCA +TANs groups(P<0.001).5.After the addition of SCH527123,CXCL2 / CXCR2 signal axis inhibitor,the proliferation,migration and invasion phenotypes of TSCCA + P.gingivalis + TANs + SCH527123 group were significantly lower than those of TSCCA +P.gingivalis + TANs group(P<0.001).6.The tumor animal model verified that P.gingivalis could promote the growth of tumor and SCH527123 intervention could significantly inhibit the growth of tumor.Part 3,1.The q RT-PCR was used to verify the m RNA expression levels of CXCL2(P<0.001),CXCR2(P<0.001),JAK1(P<0.05),STAT3(P<0.001)and N-cadherin(P<0.01)in TSCCA + P.gingivalis + TANs group and found that it was significantly higher than that in TSCCA group,while the m RNA expression level of E-cadherin(P<0.01)was significantly decreased.Western Blot was used to verify that the protein expression levels of CXCL2(P<0.001),CXCR2(P<0.001),JAK1(P<0.05),STAT3(P<0.001),p-JAK1(P<0.001),p-STAT3(P<0.001)and N-cadherin(P<0.001)in TSCCA + P.gingivalis + TANs group were significantly higher than those in TSCCA group,while the protein expression level of E-cadherin(P<0.001)was significantly decreased.2.The CXCL2-sh RNA and CXCR2-sh RNA lentiviral vectors were constructed,and the two lentiviruses were respectively infected into TSCCA + P.gingivalis co-culture cell model,which was set as the Knock down group.Meanwhile,the Blank group(co-cultured cell model without lentivirus infection)and NC group(infected with lentivirus only containing fluorescent protein)were established.The q RT-PCR was used to verify that the m RNA expression levels of CXCL2(P<0.001)and CXCR2(P<0.01)in Knock down group were significantly lower than those in Blank and NC groups.Western Blot analysis showed that the protein expression levels of CXCL2(P<0.001)and CXCR2(P<0.001)in Knock down group were significantly lower than those in Blank and NC groups.3.The CXCL2 /CXCR2-sh RNA co-infected TSCCA cell strain was successfully screened by flow cytometry,and the positive cell clones co-expressing CXCL2/CXCR2-sh RNA lentivirus were successfully selected by limited dilution method.4.The TSCCA cells co-infected with CXCL2 / CXCR2-sh RNA lentivirus were used to establish the cell models.The m RNA expression levels of JAK1(P<0.05),STAT3(P>0.05),N-cadherin(P>0.05)and E-cadherin(P>0.05)in TSCCA + P.gingivalis + TANs group were verified by q RT-PCR and the compared with TSCCA group that the difference was not significant.The protein expression levels of JAK1(P>0.05),STAT3(P>0.05),p-JAK1(P>0.05),p-STAT3(P>0.05)and E-cadherin(P>0.05)in TSCCA + P.gingivalis + TANs group were verified by Western Blot and the compared with TSCCA group that the difference was not significant except for the high expression of N-cadherin(P<0.05).At the same time,compared with the co-infected TSCCA + P.gingivalis + TANs group and the non-infected TSCCA + P.gingivalis + TANs group,the activation of JAK1 / STAT3 signaling pathway was significantly decreased and the phenotype of EMT was reversed significantly.Conclusion:1.The expression levels of P.gingivalis,CXCL2 and TANs in OSCC tissues were significantly higher than those in non-carcinomous tissues.The high expression of P.gingivalis,CXCL2 and TANs were associated with poor prognosis of OSCC patients.2.The content of CXCL2 in the supernatant of TSCCA+ P.gingivalis + TANs was significantly increased,and the chemotactic ability of TSCCA+ P.gingivalis supernatant was the strongest.3.The proliferation,migration and invasion of TSCCA+ P.gingivalis+ TANs group were the strongest.The CXCL2 / CXCR2 inhibitors could significantly inhibit the proliferation,migration and invasion of cells.4.The tumor animal model verified that P.gingivalis could promote the growth of tumor and SCH527123 intervention could significantly inhibit the growth of tumor.5.In TSCCA + P.gingivalis+ TANs group,JAK1 / STAT3 signaling pathway was activated and the phenotype of EMT was observed.The knockdown of CXCL2 / CXCR2 signal axis could significantly reduce the activation of JAK1 / STAT3 signaling pathway and the phenotype of EMT was reversed. |
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