Sema4C Regulates Epithelial-Mesenchymal Transition (EMT) And Its Mechanism In Cervical Cancer

Background and objective:Despite improvments in diagnostic and screening techniques and the availability of vaccines,cervical cancer is still the third leading cause of tumor-related deaths in women worldwide,and pelvic lymph node metastasis is an important risk factor for cervical cancer recurrence or death.As FIGO 2018 Staging system,the results of pathology and imaging were used for staging,for the first time,and the clinical staging of cervical cancer was approached to the surgical pathological staging,and lymph node metastasis was included in the staging,which changed the diagnosis and treatment of cervical cancer.Early screening of cervical cancer makes it as accessible as possible to get early detection and early treatment,but due to the lack of effective biomarkers,lymphatic metastasis cannot be accurately evaluated before the first surgery,which has left some patients without standard treatment.Therefore,it is of great significance to find the mechanism pathways and candidate genes related to lymph node metastasis in cervical cancer.The pathological change of the tumor during the pathological EMT process leads to the transformation of cell morphology to mesenchymal cells,which is considered to play an important role in cancer progression and is associated with poor prognosis.EMT plays an important role in lymphatic metastasis of cervical cancer,and cervical cancer with EMT phenotype which shows tumor progression,improved invasion and metastasis ability,and is insensitive to chemotherapy drugs and radiotherapy.Therefore,some factors that can continuously activate cervical cancer cell EMT can be used as indicators to evaluate the prognosis of cervical cancer and as targets for the treatment of cervical cancer invasion and metastasis biomarker.Transcriptional growth factor-? family-induced EMT plays an important role in growth and development and tumor formation.TGF-?1 increases in extracellular levels in advanced cervical cancer,treating cervical cancer cell lines with TGF-?1not only led to morphological changes in EMT,but also downregulated the epithelial marker E-cadherin and up-regulated fibronectin and cytoskeletal fiber rearrangement.TGF-?1 in cervical cancer cells can activate multiple signaling pathways,such as MAPK,Smad,Wnt,TNF-? and NFKB signaling pathways,most of which are known to induce EMT in cervical cancer.The Semaphorins family is a secreted or transmembrane protein that was discovered many years ago as a factor that exerts axon-directing functions in the nervous system.Most proteins exert corresponding biological effects through their lingands Plexins and Neuropilins family,including:development,regulating cell movement,Angiogenesis,immune regulation,tumor formation and stem cell properties.Some members of the Semaphorin family are involved in the separation of the epithelial cell barrier and are related to tumor EMT.Sema3 E induces EMT accumulation in the nucleus by activating PI3 K / ERK / MAPK signal.Sema4 C plays an important role in the regulation of the directional growth of axons and the development of muscle ducts.it is expressed in human cervical cancer,endometrial cancer,breast cancer and prostate cancer and is associated with malignant behaviors such as lymphatic metastasis.Sema4 C overexpression in breast cancer cells up-regulates the transcription factor Snail Slug and sox-2,and Sema4 C is an important activator of the P38 MAPK signaling pathway during the differentiation of muscle cells.Previous studies have shown that the high expression of Sema4 C in cervical cancer tissues is related to malignant biological behavior,and EMT is closely related to the progression of cervical cancer.Therefore,this study investigated whether Sema4 C mediates the invasion and metastasis of cervical cancer cells by regulating epithelial-mesenchymal transition? TGF-?1 in cervical cancer cells can activate multiple pathways to promote EMT,including the P38 MAPK signaling pathway.Sema4 C is also an important activator of the P38 MAPK signaling pathway.However,there are no related reports in cervical cancer,so we further explore whether Sema4 C can regulate TGF-?1 induced cervical cancer epithelial mesenchymal transition by activating P38MAPK? Finally,the expressions of SEMA4 C,E-cadherin,and Vimentin in cervical cancer tissues are examined to explore the relationship between the three protein and cervical cancer prognosis.It provides theoretical basis and supplementary clinical data for Seam4 C to be aprognostic marker and therapeutic target for invasion and metastasis of cervical cancer.Materials and Methods:?.Effects of Sema4 C on the malignant biological behavior of cervical cancer cells:1.Collect cervical cancer cells Hela,C33 A,Siha,and detect the expression of mSema4 C and SEMA4 C protein by Realtime-PCR and Western blot;2.By constructing LV-shSema4 C lentivirus,and selecting Hela cells as experimental cells,transfect the lentivirus to obtain stable Hela-shNC cells and Hela-shSema4 C cells;3.The effect of silencing Sema4 C on the proliferation of Hela cells was detected by CCK-8 method.The effect of silencing Sema4 C on the invasion ability of Hela cells was detected by invasion chamber experiment.The effect of silencing Sema4 C on Hela cell migration was detected by cell scratch assay.Cytoskeleton immunofluorescence detection of the effect of silencing Sema4 C on Hela cytoskeletal recombination.?.The mechanism of Sema4 C promoting the invasion and metastasis of cervical cancer by regulating epithelial-mesenchymal transition:1.Western blot was used to detect the expression of SEMA4 C,E-cadherin,Vimentin,P38 MAPK and p-P38 MAPK in Hela,Hela-shNC,Hela-shSema4 C cells and each group of cells after 72 h of incubation with 10 ng/mL TGF-?1;2.EISAL was used to detect the levels of FN secreted by Hela,Hela-shNC,Hela-shSema4 C cells and each group of cells after 72 h of incubation with 10 ng/mL TGF-?1;3.The fluorescence intensity of E-cadherin and Vimentin in each group of cells after Hela,Hela-shNC,Hela-shSema4 C and cells incubated with 10 ng/mL TGF-?1for 72 h were detected by immunofluorescence.?.The expression of Sema4 C,E-cadherin and Vimentin in cervical cancer and their relationship with prognosis:1.Collect clinical specimens of cervical cancer,and use immunohistochemical SP method to detect the expression of SEMA4 C,E-cadherin and Vimentin in 30 normal cervical tissues and 102 cervical cancer tissues;2.The expression differences of SEMA4 C,E-cadherin and Vimentin in normal cervical tissues and cervical cancer tissues were accurately tested by Fisher's exact test;3.The relationship between the expressions of SEMA4 C,E-cadherin and Vimentin and the clinical pathology of cervical cancer was measured by Chi-square test,Fisher's exact test and Kruskal-Wallis H test;4.Spearman correlation analysis among SEMA4 C,E-cadherin,and Vimentin expression level of clinicopathologic factors.5.The correlation between the expression of SEMA4 C,E-cadherin and Vimentin were analyzed by Spearman rank;6.Kaplan-meier method was used to construct the survival curve,and log-rank test was used for the difference comparison;Cox proportional hazard model was used for univariate analysis and multivariate analysis.Results:?.Effect of Sema4 C on the malignant biological behavior of cervical cancer cells:We selected the best RNA interference sequence of Sema4 C based on previous research.LV-shSema4 C lentivirus was constructed by Shanghai Genechem Co.,Ltd,and stable Hela-shNC and Hela-shSema4 C cells were obtained.In vitro proliferation of Hela cells was inhibited after silencing Sema4 C by CCK-8 method;Transwell experiments show that the invasion ability of Hela cells decreases after silencing Sema4 C.Cell scratch test showed that the migration ability was weakened after Sema4 C was silenced;Cytoskeleton experiment showed that cytoskeletal fibers and protrusions were significantly reduced after Sema4 C was silenced.Based on the above results,we speculated that the expression of Sema4 C in cervical cancer cells increased the proliferation ability of tumors and promoted invasion and migration.?.The mechanism of Sema4 C promoting the invasion and metastasis of cervical cancer by regulating epithelial-mesenchymal transition:We further explored the mechanism by which Sema4 C promotes invasion and metastasis of cervical cancer,and found that after silencing Sema4 C the expression of E-cadherin protein in Hela cells was up-regulated,the expression of Vimentin proteinwas down-regulated,and the cell secretion of FN was reduced.Immunofluorescence detection showed that after Sema4 C was silenced,the fluorescence intensity of E-cadherin in Hela cells increased,while that of Vimentin decreased.It can be seen that the expression of Seam4 C in cervical cancer cells has a regulatory effect on EMT,and it may promote the invasion and metastasis of tumors by regulating EMT.At the same time,we explored whether TGF-?1 has an effect on the expression of Sema4 C in cervical cancer cells.After incubating Hela cells with 10ng/mL TGF-?1for 72 h,the expression of SEMA4 C was up-regulated,the expression of E-cadherin was down-regulated,and there was no significant difference in Vimentin expression.After silencing Sema4 C,Hela cells began to show up-regulation of E-cadherin expression and down-regulation of Vimentin;but after 72 hours of TGF-?1 incubation,EMT also occurred in the cells,which was manifested by down-regulation of E-cadherin expression and up-regulation of Vimentin expression.Compared with Hela-shNC+TGF-?1 and Heal + TGF-?1 group cells,the expression of E-cadherin was up-regulated and the expression of Vimentin was not significantly in Hela-shSema4C+TGF-?1 cells.Silencing Sema4 C can significantly inhibit the FN secretion induced by TGF-?1.Treatment with TGF-?1 will reduce the fluorescence intensity of E-cadherin,and has little effect on the fluorescence intensity of vimentin,but silencing Sema4 C will increase the fluorescence intensity of E-cadherin reduced by TGF-?1 treatment and reduce the fluorescence intensity of Vimentin.This is basically consistent with the results of Western blot.It can be known from this in the process of EMT induced by TGF-?1,E-cadherin is mainly down-regulated.Silencing Sema4 C can inhibit TGF-?1-induced EMT,and Seam4 C may play a certain adjuvant role in the process of TGF-?1-induced EMT.We examined the phosphorylation of P38 MAPK in Hela cells treated with TGF-?1.Western blot showed that Hela cells significantly increased the expression of p-P38 MAPK after 72 hours of treatment with TGF-?1,but the levels of p-P38 MAPK were significantly reduced after Sema4 C was silenced.This indicates that Sema4 C is involved in TGF-?1-induced EMT through the activation of P38 MAPK in cervical cancer cells.In summary,it is shown that down-regulation of Sema4 C can significantly reduce the P38 MAPK phosphorylation level of Hela cells induced by10 ng / mL TGF-?1,and then up-regulate the expression of E-cadherin and inhibit EMT of Hela cells.Thus,Sema4 C participates in TGF-induced EMT to promote invasion and metastasis of cervical cancer cells by phosphorylation of P38 MAPK.?.The expression of Sema4 C,E-cadherin and Vimentin in cervical cancer and their relationship with prognosisImmunohistochemical detection of SEMA4 C,E-cadherin and Vimentin expression in 102 cervical cancer tissues and 30 normal cervical tissues.It was found that the expressions of the three proteins in cervical cancer cells and normal cervical epithelial cells were statistically different.By analyzing the relationship between the three proteins and clinical parameters,it was found that the later the FIGO stage of cervical cancer,with deep stromal invasion,lymphovascular space invasion,positive surgical margin and lymph node metastasis,the higher the expression of Sema4 C.The later the FIGO stage of cervical cancer,the higher the differentiation grade,with deep stromal invasion,lymphovascular space invasion and pelvic lymph node metastasis,the lower the high expression of E-cadherin.The later of the FIGO stage of cervical cancer,the higher the differentiation grade,with deep stromal invasion,lymphovascular space invasion,lymph node metastasis,and the positive surgical margin,the higher the expression rate of Vimentin.SEMA4C and Vimentin were positively correlated with the FIGO stage,differentiation grade,deep stromal invasion,lymphovascular space invasion,and pelvic lymph node metastasis.E-cadherin is negatively associated with the FIGO stage,differentiation grade,deep stromal invasion,lymphovascular space invasion,and pelvic lymph node metastasis.Spearman correlation analysis found that E-cadherin was negatively correlated with the expression of Vimentin and SEMA4C;Vimentin was positively correlated with the expression of SEMA4 C,which was consistent with our previous detection results of cervical cancer cells.It was further confirmed that the high expression of SEMA4 C in cervical cancer affected the expression of E-cadherin and Vimentin,and was related to the EMT of cervical cancer.At the same time,the survival analysis of clinical data found that tumors with low expression or negative expression of SEMA4 C and Vimentin had betterprognosis than with high expression of SEMA4C;on the other hand,high expression of E-cadherin had better prognosis than patients with low expression and negative expression.Univariate and multivariate Cox regression analysis showed that lymphovascular space invasion,pelvic lymph node metastasis,positive margins and the SEMA4 C expression extent were independent prognostic factors affecting the overall survival of cervical cancer patients.Conclusions1.Seam4 C is commonly expressed in cervical cancer cell lines Hela,Siha,and C33 A,and its expression in cervical cancer cells promotes tumor proliferation,invasion and migration,and affects the reorganization of cytoskeletal proteins.2.Sema4 C is related to the EMT of cervical cancer cells,and may affect the invasion and metastasis of cervical cancer cells by regulating EMT;Sema4C promotes the invasion and metastasis of cervical cancer cells by acting on P38 MAPK phosphorylation and participating in EMT induced by TGF-?1.3.The overexpression of SEMA4 C and Vimentin in cervical cancer tissues and the low expression of E-cadherin are closely related to the decreased survival rate.Univariate and multivariate Cox regression analysis showed that lymphovascular space invasion,lymph node metastasis,positive surgical margin and SEMA4 C were independent prognostic factors for overall survival in patients with cervical cancer.