Preliminary Exploration Of The Mechanism Of CALD1 Influencing The Pathogenesis Of Ovarian Cancer

[Objective]In this study,we analyzed the difference in expression of CALD1（calmodulin binding protein）in ovarian cancer tissues and healthy tissues,constructed stable transfected cell lines of ovarian cancer with low expression of CALD1,analyzed the effects of CALD1 expres-sion on the morphology,structure and cell migration rate of ovarian cancer cells,and investi-gated whether CALD1 has potential clinical applications in the detection and treatment of ovar-ian cancer.[Method]A total of 9784 tumor tissue samples and 8320 healthy tissue samples were downloaded from TCGA（The Cancer Genome Atlas Project）and GTEx（Genotype-Tissue Ex-pression）database.（1）Using the R programming language,extract CALD1 expression infor-mation from the downloaded data.Then,perform an unpaired T-test analysis on tumor samples and corresponding healthy samples to detect CALD1 expression differences in tumor samples and normal tissues.（2）Using Gene MANIA and Metascape online analysis website,the protein network of CALD1 interactions was constructed,and KEGG（Kyoto Encyclopedia of Genes and Genomes）and GO（Gene Ontology）enrichment analysis of the protein network was per-formed to find the functional modules of CALD1 that may act on cells.（3）The interfering sh RNA1-3 of the CALD1 gene was designed,synthesized,and connected with the lentiviral shuttle vector p HBLV-U6-MCS-CMV-Zs Green-PGK-PURO construct p HBLV-CALD1-sh RNA1-Zs Green,p HBLV-CALD1-sh RNA2-Zs Green,and p HBLV-CALD1-sh RNA3-Zs Green three sets of recombinant plasmids.Each group of recombinant plasmids（Including negative control plasmid:p HBLV-NC-Zs Green）and lentiviral helper plasmids（p SPAX2,p MD2G）were co-transfected into 293T cells using Lipofiter^TM transfection reagent to obtain four groups of lentivirus HBLV-NC-Zs Green-PURO（Negative control）,HBLV-CALD1-sh RNA1-Zs Green-PURO,HBLV-CALD1-sh RNA2-Zs Green-PURO and HBLV-CALD1-sh RNA3-Zs Green-PURO,and virus titers were measured.（4）To select suitable hosts for lenti-virus infection,real-time fluorescence quantitative PCR was used to detect the expression of endogenous CALD1 m RNA in human ovarian cancer cells（SK-OV-3）and human ovarian ad-enocarcinoma cells（OVCAR-3）.The optimal MOI of the four groups of lentivirus-infected host cells was explored.The four groups of lentiviruses were used to infect the host cells ac-cording to the optimal MOI.（5）Real-time fluorescence quantitative PCR and Western blot were used to detect the difference in CALD1 expression in each group of cells,and the interference group that effectively reduced the expression of CALD1 in cells was screened out.（6）The group of cells that significantly interfered with CALD1 expression were continuously screened for positive cells using puromycin.Western blot was used to detect the stability of low CALD1protein expression in this group of cells.（7）Transwell invasion assay to analyze the changes in migration rate of CALD1 low expressing ovarian cancer cells.（8）Cytoskeleton was stained with rhodamine-labeled phalloidin to observe cytoskeleton changes in CALD1-low-expressing ovarian cancer cells.（9）Immunofluorescence was used to detect the focal adhesion protein Vinculin to explore the effect of low expression of CALD1 on the structure of focal adhesions in ovarian cancer cells.[Result]（1）CALD1 is abnormally expressed in various tumors,which may be related to the occurrence of tumors.Analysis of ovarian cancer data（427 ovarian cancer samples,88healthy samples）showed that the expression level of CALD1 was significantly lower than that of normal tissues potentially as a diagnostic marker for tumors.（2）The functions of CALD1and related genes were predominantly focused on regulating the cytoskeleton,creating focal adhesions,and other processes linked to cell movement and migration,according to GO and KEGG pathway enrichment analyses.It suggests that the level of CALD1 expression may influence tumor cell migration.（3）The sequencing results showed that the constructed p HBLV-CALD1-sh RNA1-Zs Green,p HBLV-CALD1-sh RNA2-Zs Green,p HBLV-CALD1-sh RNA3-Zs Green recombinant plasmids were successful.Each group of recombinant（Includes negative control plasmid:p HBLV-NC-Zs Green）and lentiviral helper plasmids（p SPAX2,p MD2G）was co-transfected into 293T cells,and apparent fluorescence was observed,indicating that the transfection was the transfection successful.After the virus was harvested,the lentivirus titers for each group were 2×10⁸ TU/m L,1.5×10⁸ TU/m L,2×10⁸ TU/m L,and 1.5×10⁸ TU/m L,re-spectively.（4）The expression of endogenous CALD1 m RNA in human ovarian cancer cells（SK-OV-3）and human ovarian adenocarcinoma cells（OVCAR-3）was detected by fluores-cence PCR.The results showed no significant difference in the expression of CALD1 m RNA between the two cells.Because SK-OV-3 cells grow faster and can effectively shorten the ex-perimental period,SK-OV-3 cells were selected as hosts for lentivirus infection.The optimal MOI of the four groups of lentivirus-infected SK-OV-3 cells showed that the optimal MOI of the four groups of lentivirus-infected SK-OV-3 cells was 5.（5）Four groups of lentivirus in-fected SK-OV-3 cells,and apparent fluorescence was observed at 72 h.The results of real-time fluorescent quantitative PCR and Western blot showed that HBLV-CALD1-sh RNA1-Zs Green-PURO-SK-OV-3 Compared with HBLV-NC-Zs Green-PURO-SK-OV-3 cells and SK-OV-3cells,the expression levels of m RNA and protein of CALD1 gene were significantly decreased.It was demonstrated that lentivirus-packed sh RNA1 was effective in reducing CALD1 expres-sion in SK-OV-3 cells.（6）The positive cells after puromycin selection were detected by West-ern blot,and the results showed that the expression of CALD1 protein was stably reduced.（7）Transwell results showed that the migration ability of HBLV-CALD1-sh RNA1-Zs Green-PURO-SK-OV-3 cells was significantly enhanced compared with HBLV-NC-Zs Green-PURO-SK-OV-3 cells and SK-OV-3 cell groups.（8）Cytoskeleton staining showed significantly fewer microfilaments on the cytoskeleton in the HBLV-CALD1-sh RNA1-Zs Green-PURO-SK-OV-3cell group compared to the HBLV-NC-Zs Green-PURO-SK-OV-3 cell and SK-OV-3 cell groups,and the cytoskeletal structure was loosely disordered.（9）The results of the cell adhesion spot protein Vinculin assay showed that HBLV-CALD1-sh RNA1-Zs Green-PURO-SK-OV-3 cells had significantly reduced adhesion spots compared to HBLV-NC-Zs Green-PURO-SK-OV-3cells and SK-OV-3 cell groups.[Conclusion]The expression of CALD1 was significantly reduced in ovarian cancer tis-sues compared to normal tissues.Reduced CALD1 expression in ovarian cancer cells resulted in fewer microfilaments on the cytoskeleton and a looser cytoskeleton;it was also found that the reduction of cell adhesion spots enhanced the migration rate of ovarian cancer cells and promoted the metastasis of ovarian cancer cells,indicating that CALD1 has potential clinical applications in the diagnosis and treatment of ovarian cancer.