Experimental Study Of Adipose-derived Stem Cells Targeting The Regulation Of HGF/c-Met Signaling Pathway To Promote The Proliferation And Invasion Of Cervical Cancer

Cervical cancer is one of the most common malignant tumors in women.This is a major health problem,especially in developing countries.The international agency for research on cancer(IARC)reports that more than 250,000 women die of cervical cancer each year,85%of which occur in low-and middle-income countries.Although great progress has been made in the treatment of cervical cancer,it is still necessary to find potential prognostic and diagnostic biomarkers to improve the treatment of patients with cervical cancer.Studying the molecular mechanisms of the occurrence and development of cervical cancer will help us to find new treatment options.Adipose derived stem cells(ADSCs)are derived from perivascular adipose tissue.ADSCs can secrete a variety of growth factors(GFS)including a variety of angiogenic factors and apoptotic factors,and which can promote the secretion of collagen I,III and fibronectin,promote the synthesis of collagen,and then play the role of antiinflammatory,anti-oxidation,anti-aging,damage repair and so on.ADSC can also differentiate into endothelial cells and participate in the formation of new vascular structures.In addition,the secretion of TGF-β1 promoted the immunoregulation,increased the deposition of extracellular matrix(ECM)and collagen tissues.ADSCs have been used in many clinical situations,especially in those tissues that are damaged and healed due to insufficient blood supply and chronic inflammatory state,such as those in radiotherapy.However,the safety of ADSC in the treatment process is more and more concerned.The main reasons for concern are their angiogenesis,antiapoptosis and immunomodulatory properties,which lead to the carcinogenicity of ADSCs.The coexistence of harmful and beneficial aspects has earned ADSCs the reputation of "double-edged sword".Previous studies have shown that ADSCs promote the occurrence and development of a variety of cancers,but the specific mechanism and molecular biological principle of ADSCs are not clear.There are many factors in tumor microenvironment that promote the occurrence and development of tumor,which are the result of multi-level interaction.Therefore,the next research on cervical cancer,mainly to explore the role of ADSCs in the tumor microenvironment on cervical cancer,further study the related signaling pathways involved,so as to find new targets of cervical cancer,and provide new markers for the treatment of cervical cancer.Met receptors belong to the family of growth factor receptors with tyrosine kinase activity,and together with the ligand hepatocyte growth factor(HGF),they participate in the proliferation,inhibition of apoptosis,movement and diffusion of cancer cells in the process of tumorigenesis and development.Met receptors are overexpressed in a variety of human cancers,including thyroid cancer,ovarian cancer,colorectal cancer,rhabdomyosarcoma,and renal cell carcinoma.Studies have also shown that hepatocyte growth factor(HGF)and its receptor met are significantly increased in ovarian cancer patients who relapse soon after chemotherapy.In cervical cancer,met receptor has been proved to promote the scattering and morphogenesis of tumor cells.Our previous studies have shown that HGF alone or in combination with matrix derived factor-1(SDF-1,the ligand of CXCR4 receptor)can induce the migration and cytoskeleton changes of cervical cancer cells.The data also showed that the activation of met and CXCR4 led to the translocation of β-Catenin to the nucleus,followed by the up regulation of CyclinDl expression.The purpose of this study is to investigate the effect of ADSCs on the growth of cervical cancer cells,explore its role in the occurrence and development of cervical cancer,and further explore whether HGF/c-Met is involved in the promotion of ADSCs on cervical cancer,and verify the relevant molecular mechanism through experiments.To provide new ideas for the diagnosis and treatment of cervical cancer.Part Ⅰ Effect of ADSCs on proliferation and invasion of cervical cancer cellsObjective:To identify ADSCs and investigate their effects on proliferation and invasion of cervical cancer cells.Methods:The surface antigens(CD29,CD105,CD90 and CD31)of ADSCs were detected by flow cytometry,the adipogenic capacity of ADSCs was detected by oil red O staining,and the fat related genes(leptin and PPAR-y)in ADSCs were detected by RT-PCR.CCK8 was used to detect the effect of ADSCs on the proliferation of cervical cancer cells,and transwell assay was used to evaluate the effect of ADSCs on the invasion of cervical cancer cells.Results:Flow cytometry showed that the markers CD29,CD105 and CD90 of MSCs were positive,while the markers CD31 of endothelial cells were negative.Moreover,ADSCs were observed as fibroblast like cells under light microscope.Oil red O staining showed the formation of lipid droplets in ADSCs,which confirmed the potential of ADSCs to differentiate into adipocytes.RT-PCR results showed that leptin and PPARγ were highly expressed in ADSCs.These results show that ADSCs have been successfully separated and can be used in subsequent experimental studies.CCK8 results showed that the supernatant of ADSCs and the co-culture of ADSCs with cervical cancer cells could significantly increase the proliferation of cervical cancer cells at 24 h,48 h and 72 h.Transwell assay showed that the supernatant of ADSCs and the co-culture of ADSCs with cervical cancer cells could significantly promote the invasive ability of cervical cancer cells.Conclusion:ADSCs can promote the proliferation and invasion of cervical cancer cells.Part Ⅱ Effect of ADSCs on cervical cancer cells through HGF/c-Met signaling pathwayObjective:To investigate the activation of HGF/c-Met signaling pathway in ADSCs,cervical cancer cells(cultured alone)and cervical cancer cells(co-cultured with ADSCs),and to detect whether the down-regulation of c-Met can inhibit the effect of ADSCs on cervical cancer cells.Methods:the concentration of HGF in the culture supernatant of ADSCs,cervical cancer cells(cultured alone)and cervical cancer cells(co-cultured with ADSCs)was detected by ELISA.The expression of c-Met and p-c-Met in cervical cancer cells was detected by western blot assay.The cervical cancer cells with stable low expression of c-Met were constructed and transfected by siRNA.After c-Met down-regulation,CCK8 was used to detect the effect of ADSCs on the proliferation of cervical cancer cells,and transwell assay was used to evaluate the effect of ADSCs on the invasion of cervical cancer cells.Results:ADSCs secreted more HGF than HeLa and CaSki cells,while ADSCs cocultured with HeLa or CaSki secreted more HGF than ADSCs cultured alone.HGF in the supernatant of HeLa or CaSki cells cocultured with ADSCs also increased significantly.Since HGF is an important cytokine derived from ADSCs,and HGF/cMet is involved in tumorigenesis and development,we studied the activation of c-Met.Western blotting showed that the phosphorylation ratio of c-Met was up-regulated in co cultured cervical cancer cells.Moreover,Western blotting confirmed that siRNA significantly reduced c-Met protein.CCK8 results showed that ADSCs supernatant and transfection control+ADSCs supernatant significantly increased the proliferation activity of cervical cancer cells,while siRNA-c-Met+ADSCs supernatant significantly inhibited the proliferation activity of cervical cancer cells.In the invasion experiment,ADSCs supernatant and transfection control+ADSCs supernatant groups showed an increase in invasion,while siRNA-c-Met+ADSCs supernatant group showed a decrease in invasion,indicating that the down-regulation of c-Met in siRNAc-Met+ADSCs supernatant group led to a decrease in invasion ability.Conclusion:ADSCs can promote the proliferation and invasion of cervical cancer cells by regulating HGF/c-Met signaling pathway.Part Ⅲ Effect of ADSCs on the growth of transplanted cervical cancer cellsObjective:To study the effect of ADSCs on the growth of cervical cancer xenograftsMethods:HeLa cells(1 × 107 cells suspended in 200 μL PBS)were subcutaneously injected into the anterior abdomen to establish cervical cancer xenotransplantation model.Then ADSCs supernatant and siRNA-c-Met+ADSCs supernatant were used to intervene in the tumor every week to detect the growth of the tumor,the expression of c-Met was detected by immunohistochemistry,and the expression of Bcl-2 and Bax was detected by Western blot.Results:HeLa cells were used to construct the transplanted tumor model,and then the effect of ADSCs culture supernatant on the transplanted tumor was observed.The tumor growth of the control group was rapid,but the tumor growth of the groups injected with ADSCs culture supernatant and lipo+ADSCs culture supernatant was faster.On the contrary,the tumor volume and weight of siRNA-c-Met+ADSCs group were significantly reduced compared with ADSCs culture supernatant and control group.Immunofluorescence results confirmed that the expression of c-met was inhibited by c-met siRNA treatment.Western blot analysis showed that compared with the control group,the expression of Bcl-2/Bax was significantly up-regulated in ADSCs culture supernatant and lipo+ADSCs culture supernatant,and c-Met siRNA treatment significantly alleviated this up-regulation.Conclusion:These results suggest that HGF secreted by ADSCs may play an important role in promoting the occurrence of cervical cancer through HGF/c-Met signaling pathway.