Effect And Mechanism Of LIMD2 On Proliferation And Metastasis Of Ovarian Cancer Cells

Ovarian cancer has the highest mortality rate among gynecological malignancies.The early clinical symptoms of ovarian cancer are not obvious,therefore,at the time of diagnosis most patients would have the advanced stage which has a low survival rate(five years).Due to unreliable biomarkers,the early diagnosis and prognosis of ovarian cancer are urgent clinical problems.In addition,despite the development of radiotherapy,chemotherapy,and targeted therapy,a considerable number of patients still find it difficult to benefit from this treatment due to the low response rate,side effects,and drug resistance.Therefore,there is an urgent need to identify reliable biomarkers for early diagnosis and prognosis,and potential protein therapeutic target molecules for ovarian cancer.Quantitative proteomics is used to study new biomarkers for cancer,cardiovascular and other diseases.By analyzing urine from ovarian cancer patients,scientists have found a protein molecular module that can distinguish between benign and malignant ovarian tumors WFDC2,PTMA,PVRL4,FIBA,and PVRL2).In this study,using Tandem Mass Tag(TMT)based quantitative proteomics,we compared the differences between early and advanced ovarian cancer patients.The results showed that there were 544 differentially expressed protein molecules,of which 455 had a higher expression and 89 had a lower protein expression in advanced ovarian cancer.The main processes induced by differential proteins include MAPK signaling pathway,innate immune response,type I interferon-signaling pathway,inflammatory response to antigen stimulation,and regulation of cell proliferation.The differential proteins from the KEGG pathway database are mainly concentrated in the NF-ΚB signaling pathway,the focal adhesion signaling pathway,cancer transcriptional regulation abnormality,and the metabolic pathway.We screened 40 protein molecules from the above data with the highest differential expression and found a potential target protein molecule LIMD2 using bioinformatics analysis.The GEPIA website showed that LIMD2 m RNA was highly expressed in ovarian cancer tissue compared to healthy ovarian tissues.We collected 60 paraffin sections of ovarian tissue(13healthy tissue,16 from stage I,15 from stage II,15 from stage III,and 1 from stage IV)and performed Immunohistochemistry detection of LIMD2 protein.The results indicated that LIMD2 was negative in healthy ovarian tissues and positive in ovarian cancer tissues.Through western blot and RT-PCR,we discovered that five human ovarian cancer cell lines(A2780,HO8910 PM,SKOV-3,HO8910,and OVCAR-3)expressed different levels of LIMD2 protein and LIMD2 mRNA.Immunofluorescence and cellular immunohistochemistry results showed that LIMD2 was mostly expressed in the cytoplasm and only a small amount was expressed in the cell membrane.To explore the functions and mechanisms of LIMD2,we performed small m RNA interference and overexpressed the LIMD2 gene in ovarian cancer cell lines,and studied its biological functions in vitro by CCK-8 assay,Transwell Migration Experiment(TME),and cell scratch experiment.The results showed that compared with control ovarian cancer cell lines,the proliferation and migration ability of LIMD2 silenced cell lines decreased while that of LIMD2 overexpressed cell lines was increased.In addition,the ovarian cancer nude mouse model showed that the LIMD2 silenced cells had a lower subcutaneous tumorigenicity and growth rate.The tumor metastasis ability in the abdominal cavity was reduced,and their numbers and sizes were smaller.Contrarily,ovarian cancer cell lines overexpressing LIMD2 enhanced subcutaneous tumorigenicity and proliferation;were easier to metastasize and grow in the abdominal cavity of nude mice.Furthermore,a large amount of ascites was often formed in the abdominal cavity of nude mice,and the number and size of tumors were larger.These studies show that the proliferation and metastasis of ovarian cancer cells can effectively be inhibited by silencing of LIMD2 gene and promoted by its overexpression in nude mice.To investigate how LIMD2 causes tumor malignancy,we used RNA-sequencing to sequence the LIMD2 silenced group and the control group.The results showed that the focal adhesion pathway,MAPK signaling pathway,TNF signaling pathway,extracellular matrix receptor interaction pathway,and apoptosis were all affected by LIMD2 silencing.This along with the previous proteomic results demonstrate that the focal adhesion pathway and MAPK signaling pathway plays a role in the progression of ovarian cancer malignancy.This suggested that the LIMD2 gene may be one of the causes of ovarian cancer deterioration.We then studied the relationship between the LIMD2 protein and the focal adhesion pathway.Western blot results showed that in LIMD2 silenced cell lines FAK,p-FAK,Rac1,and tensin2 were less expressed than in the control group.FAK inhibitor Y15 also inhibited the expression of FAK,p-FAK,and Rac1.In ovarian cancer cells overexpressing LIMD2,the expression of ɑ-Actinin,Vinculin,LIMD2,FAK,Rac1,and Tensin2 increased.In addition to focal adhesion-related proteins,expression of MMP9,MMP2,and other proteins related to tumor metastasis also increased.This suggests that LIMD2 may affect the progression of ovarian cancer cells through the Focal adhesion signaling pathway.Then we used immunoprecipitation to study the ovarian cancer cell lines with FLAG label and overexpression of LIMD2.The results showed that there is a direct interaction between LIMD2 and CISD2 and that they can bind together.Bioinformatics analysis exhibited a strong positive correlation between CISD2 and LIMD2 expression,and CISD2 overexpression in ovarian cancer was associated with poor prognosis.Western blot illustrated that the expression of CISD2 and FAK increased when LIMD2 was overexpressed.The expression of FAK was also inhibited when CISD2 was silenced by RNA interference in cell lines overexpressing LIMD2.The results showed that the overexpression of LIMD2 promoted the expression of CISD2,increasing the FAK protein expression.When CISD2 gene expression was inhibited,the FAK gene was also inhibited.This implied that LIMD2 could promote the expression of the FAK gene,but CISD2 silencing could inhibit this process.To study the application potential of LIMD2 in clinical diagnosis,we prepared LIMD2 monoclonal antibodies and polyclonal antibodies.Firstly,we cloned the optimized LIMD2 DNA sequence into PSMART-1 vector plasmid,then transformed the plasmid into E.coli BL21(DE3),and then induced LIMD2 protein expression with 0.1 m M IPTG.After two purifications by straw streptactin beads and nickel column,we obtained LIMD2 proteins with over 90% purity.Then,we immunized mice and rabbits with the purified LIMD2 protein to obtain five types of monoclonal antibodies(4F3B12,3E5C7,3F4B3,3F4C1,and 4F5F12)and a polyclonal antibody.The Western blot results illustrated that the five monoclonal antibodies and the polyclonal antibody could bind to the LIMD2 protein expressed in prokaryotes and ovarian cancer cell lines.Then we typed five monoclonal antibodies and found that 4F3B12 and 3E5C7 belonged to Ig G2 a,3F4B3 and 3F4C1 belonged to Ig G2 b,and 4F5F12 belonged to Ig G1 kappa subtypes.The ELISA results of LIMD2 antigens showed that the titer value of polyclonal antibody was higher than 128 K,the titer values for monoclonal antibodies 4F3B12,3E5C7,and 3F4B3 were more than 512 K,and for monoclonal antibodies,3F4C1 and 4F5F12 were more than 256 K.We tested the binding ability of the five monoclonal antibodies to proteins similar to LIMD2;LIMD1 and LIMS1 by cross-reaction.ELISA results indicated that the titer values of the 3F4B3 antibody against LIMD1-His were about 16 K and against LIMS1-GST was about 2K.The titer value of the 3F4C1 antibody against LIMD1-His was about 9K and that against LIMS1-GST was about 2K.The titer value of the 3E5C7 antibody against LIMD1-His was about 0.6k,and the titer against LIMS1-GST was invalid.The titer of the 4F3B12 antibody against LIMD1-His was about 0.8k,and against LIMS1-GST was about 0.4k.The titer values of the 4F5F12 antibody against LIMD1-His and LIMS1-GST were both invalid.Meanwhile,the results of Western blot showed that 4F3B12,3E5C7,3F4B3,3F4C1 and 4F5F12 could all bind to l IMD2 protein,and could not bind to LIMS1,an approximate protein of l IMD2.Among them,4F3B12,3E5C7,3F4B3 and 4F5F12 did not bind to LIMD1,while 3F4C1 could bind to LIMD1.Finally,we used five monoclonal antibodies and one polyclonal antibody to perform a western blot with LIMD2 protein expressed in E.coli and ovarian cancer cells.We found that all these five antibodies could not only bind to the recombinant LIMD2 protein expressed in prokaryotic cells,but also to the LIMD2 protein expressed in ovarian cancer cells.Subsequently,we used monoclonal antibody 4F5F12 and LIMD2 polyclonal antibody to perform immunohistochemistry on ovarian cancer surgical specimens.The results showed that LIMD2 was not expressed in normal tissues but was highly expressed in ovarian cancer tissues.In conclusion,LIMD2 protein has the potential to be a biomarker for ovarian cancer.Through focal adhesion signaling pathways,it enhances ovarian cancer proliferation and metastasis.Additionally,LIMD2 stimulates CISD2 expression,promoting the FAK pathway while CISD2 gene silencing inhibits the FAK pathway.The produced LIMD2 Mc Ab and Pc Ab can bind to both eukaryotic and prokaryotic expressed protein derivatives,providing a basis for its clinical detection and use.