| Objective:Female physiological characteristics determine that fat is an indispensable part of female physiological function.Fat can secrete a variety of proteins,such as adiponectin,resistin,endolipin,chemerin,interleukin-6,etc.Ovary is an organ growing near adipose tissue,and ovarian cancer is mainly transplanted in abdominal cavity.The accumulation of lipids will put the human body in a state of continuous low-grade inflammation,under which the lipid proteins and adipokines released by inflammatory cells can activate the occurrence of female ovarian cancer,thus triggering the further development of female ovarian cancer.The defat of ovarian cancer cells can induce the secretion of adipokines.It plays an important role in the proliferation,migration and invasion of malignant tumor cells.At the same time,the defatalized fat cells provide energy to ovarian cancer cells and provide them with the fatty acids they need to grow,supporting the growth of cancer cells.The microenvironment mechanism of intraperitoneal implantation and metastasis of ovarian cancer remains to be further studied.Chemerin is an adipokine,which has significant effects on glucose and lipid metabolism and immune inflammatory response,and has been associated with many diseases,such as polycystic ovary syndrome,hypertension,obesity,malignant tumors,etc.Chemerin has received a lot of attention in recent years for its potential role in the development and progression of cancer and tumors.So far,most of the Chemerin receptors referred to have three types,and their multiple functions are mainly attributed to the fact that Chemerin-like receptor 1 CMKLR-1,widely distributed in many tissues,can significantly change the signal transduction mechanism.Studies have shown that high expression of CMKLR-1 may be related to survival of ovarian cancer patients.However,no studies have investigated whether Chemerin can affect the biological behavior of ovarian cancer through CMKLR-1.Ras homolog gene family member A RhoA,Rho-associated coiledcoil-forming protein kinase-1 ROCK1 is one of the downstream effectors of RhoA,It belongs to the serine/threonine kinase family and is involved in cell contraction,morphological change,proliferation,migration and other biological processes.The activity of ROCK1 can be activated by RhoA,and ROCK1 phosphorylates a variety of substrates,including actin and regulatory myosin light chain,and ultimately regulates cytoskeletal homeostasis,thereby affecting cell morphology and movement.RhoA/ROCK1 pathway has been reported to play an important role in the metastasis and invasion of tumor cells.By activating the RhoA/ROCK1 pathway,the transcriptional activity of a variety of genes can be regulated to promote the phosphorylation of actin(Myosin light chain MLC)and its assembly into a fibrous form,which may accelerate tumor development and metastasis.Some studies also found that RhoA/ROCK1 pathway could activate Serum response factor SRF to regulate the transcriptional activity of multiple genes,thus promoting the phosphorylation of actin MLC,and thus promoting the tumorigenesis and development.CMKLR-1 has been reported to activate RhoA/ROCK1 pathway and promote tumor progression.Whether Chemerin can promote RhoA/ROCK1 pathway,activate SRF transcription factor,promote MLC phosphorylation,promote free actin assembly into fibrosis,and promote the progression and metastasis of ovarian cancer through CMKLR-1 remains to be further studied.Epithelial Mesenchymal transition EMT(Epithelial Mesenchymal transition EMT)is a complex cellular procedure that is crucial for the early spread,invasion and metastasis of cancer cells.RhoA/ROCK1 activation can also induce EMT to play an important role in tumor cell metastasis,especially in ovarian cancer cells.This reveals the previously unproven role of RhoA/ROCK1 pathway-mediated EMTs in ovarian cancer metastasis.In this study,the expression of Chemerin and CMKLR-1 in high-grade serous ovarian cancer tissues and its correlation with clinical indicators of patients were analyzed,and the effects of Chemerin and CMKLR-1 on malignant behavior of SKOV-3 cells(proliferation,migration,invasion,EMT transformation)were explored.The regulation mechanism of CMKLR-1/RhoA/ROCK1 pathway on this process was analyzed.Methods: Part Ⅰ: 1.A total of 136 patients with high-grade serous ovarian cancer were selected.The expression of Chemerin and CMKLR-1 in ovarian cancer tissues was analyzed by immunohistochemical analysis and immunohistochemical pathological score,and statistical treatment was performed.Pearson multivariate analysis and correlation analysis with clinical indicators.2.Epithelial ovarian cancer cell lines SK-OV-3(experimental group)and OVCAR-3(control group)were selected as study objects.The effects of Chemerin at different concentrations on SK-OV-3 and OVCAR-3 cell morphology,cell survival,cell migration,cell proliferation and invasion were analyzed by CCK-8,Transwell and cell scratch tests,and Chemerin concentrations affecting SK-OV-3 and OVCAR-3 biological lines were determined.Part Ⅱ: Detection of Chemerin regulates actin assembly and EMT transformation through the CMKLR-1/RhoA/ROCK1 pathway.(1)mRNA of epithelial marker proteins Ecadherin and mesenchymal marker proteins N-cadherin and Vimentin of epithelial keratins EMT were detected by RT-PCR,so as to effectively detect the transformation of EMT.(2)The protein levels of CMKLR-1/RhoA/ROCK1 and p-MLC in SK-OV-3 were detected by Western blot.The expression of EMT signature proteins E-cadherin,Vimentin and Ncadherin were detected.2.In order to clarify whether Chemerin enhances EMT conversion in SK-OV-3 cells in a RhoA/ ROCK1-dependent manner,SK-OV-3 cells were pretreated with RhoA specific inhibitor(C3T)and ROCK1 specific inhibitor(Y27632).The effective concentration of Chemerin(50ng/ml)was given for 24 h :(1)Experimental group: con,Chemerin,Chemerin+C3T,Chemerin+Y27632.(2)The expression levels of p-MLC,Ecadherin,N-cadherin and Vimentin in each group were detected by Western blot.Part III: To further verify whether CMKLR-1 is necessary for Chemerin to enhance the effects of RhoA/ ROCK1-mediated EMT and enhance SK-OV-3 cell migration/invasion,SK-OV-3 cells were transfected with CMKLR-1 si RNA(two interfering sequences)prior to Chemerin treatment.After 24 hours of Chemerin recombinant protein treatment: 1.The experiment was divided into 6 groups: NC,si-1,si-2,Chemerin+NC,Chemerin+si-1,and Chemerin+si-2.2.Cell migration ability was examined by cell scratch test.3.Transwell assay was used to investigate the invasion ability of cells.4.The protein expressions of CMKLR-1,RhoA,ROCK1,p-MLC,E-cadherin,N-cadherin and Vimentin in each group were detected by Western blot.Result: Part 1:1.Chemerin and CMKLR-1 are expressed in the tissue of high-grade serous ovarian cancer,and the expression sites are cancer plasma and cancer interstitium.Correlation analysis between pathological score and clinical indicators showed that there was a positive correlation between pathological score level and tumor stage,serum HE4 and CA125(P<0.05),but no significant correlation between pathological score level and age,BMI and serum CA199(P>0.05).2.CCK-8 experimental results showed that lowdose Chemerin(25ng/ml)did not affect the growth of SK-OV-3 and OVCAR-3 cells at any point in time,but high concentration of Chemerin significantly promoted the proliferation of SK-OV-3 cells at 48 h and 72 h.It was also suggested that the Chemerin concentration was 50ng/ml,the observed enhancement effect on SK-OV-3 proliferation was more significant,and high concentration(100ng/ml)of Chemerin inhibited the growth of SK-OV-3 cells after 72 hours of treatment.Chemerin did not respond to OVCAR-3 at any concentration 3.Different doses of Chemerin significantly accelerated the healing of SK-OV-3 cells after 24 h of cell scarring,indicating enhanced cell migration after Chemerin treatment(25,50 and 100ng/m L).Chemerin addition also had no significant effect on OVCAR-3 cell migration at any concentration.4.Transwell experiment showed that the invasion ability of SK-OV-3 cells was enhanced after treatment with Chemerin at different concentrations.Chemerin at 50ng/m L significantly enhanced the migration and invasion of SK-OV-3 cells,while different doses of Chemerin had no effect on OVCAR-3 cell invasion.Part Ⅱ: 1.RT-PCR analysis showed that mRNA levels of EMT epithelial marker proteins E-cadherin in SK-OV-3 cells decreased after Chemerin treatment,while mRNA levels of mesenchymal marker proteins N-cadherin and Vimentin increased.EMT activation.2.Western blot analysis showed that the addition of Chemerin increased the expression of CMKLR-1 in SK-OV-3 cells,promoted the expression of RhoA and ROCK1,and increased the phosphorylation level of MLC.3.Chemerin can activate EMTs by inhibiting E-cadherin expression while promoting Vimentin and N-cadherin expression.3.Phosphorylation level of RhoA/ROCK1 pathway MLC in inhibitor group: In SK-OV-3 cells pretreated with RhoA inhibitors C3T(C3 transferase)and ROCK1 inhibitor(Y27632),the expression of RhoA,ROCK1 and p-MLC was inhibited by C3 T and Y27632,while EMT was inhibited.After Chemerin treatment,RhoA,ROCK-1,p-MLC protein expression levels and EMT conversion in SK-OV-3 cells were inhibited.Part Ⅲ: 1.Western blot analysis showed that the expression of CMKLR-1 protein was significantly decreased in SK-OV-3 cell groups with CMKLR-1 gene silencing,and the expressions of RhoA,ROCK1 and p-MLC were also reversed by CMKLR-1 gene silencing.Similarly,the expression of CMKLR-1 was down-regulated and the EMT process was inhibited in SK-OV-3 cells treated with Chemerin compared with the control group.2.Transwell and cell scratch experiments also showed that,compared with the control group,CMKLR-1 silencing significantly reduced the invasion and migration ability of SK-OV-3 cells.Part Ⅳ: 1.mRNA levels of CMKLR-1,RhoA,ROCK1,N-cadherin and Vimentin in the Chemerin treatment group were significantly increased compared with the control group by RT-PCR.The mRNA level of E-cadherin protein,an epithelial marker,was significantly decreased.2.The protein expressions of CMKLR-1,RhoA,ROCK1,p-MLC,N-cadherin and Vimentin were increased,while the expression of E-cadherin was decreased in each group.Chemerin treatment was evaluated to induce the expression of CMKLR-1,enhance the phosphorylation level of MLC mediated by RhoA/ROCK1 pathway and initiate EMT conversion,promoting tumor progression.Conclusions:1.The high level of Chemerin and CMKLR-1 expression in human ovarian cancer suggests a high degree of malignancy.2.Chemerin can promote malignant behavior of SK-OV-3 cells.There was no obvious effect on OVCAR-3 migration,invasion and proliferation.3.In vivo and in vitro experiments,Chemerin was found to promote MLC phosphorylation and EMT activation through the CMKLR-1/RhoA/ROCK1 pathway,to promote the malignant behavior of ovarian cancer cells SK-OV-3.This pathway can be inhibited by CMKLR-1 gene silencing or by RhoA/ROCK1 inhibitors.Could be a new target for ovarian cancer treatment. |
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