Fig 1: TPX2 strengthened cell propagation, cell migration and invasion while weakened cell apoptosis in ovarian cancer cells which was regulated by miR-485-5p. (A) Western blot was employed to detect the protein level of TPX2 in SKOV3 and OVCAR3 cell lines after transfecting miR-485-5p inhibitor (inhibitor group), si-TPX2 (si-TPX2 group), negative control (NC group, si-TPX2 NC +inhibitor-NC) and co-transfecting miR-485-5p inhibitor and si-TPX2 (inhibitor+si-TPX2 group) and untreated cells (blank group). (B) CCK-8 assay was used to observe the cell proliferation in the si-TPX2, inhibitor, inhibitor+si-TPX2, NC and blank groups. (C) BrdU assay was used to observe the cell proliferation in the si-TPX2, inhibitor, inhibitor+si-TPX2, NC and blank groups. (D) Flow cytometry was employed to measure the cell apoptosis in the si-TPX2, inhibitor, inhibitor+si-TPX2, NC and blank groups. (E) Wound healing assay was employed to measure the cell migration in the si-TPX2, inhibitor, inhibitor+si-TPX2, NC and blank groups. Original magnification: ×100. (F) Cell adhesion assay was employed to measure the cell adhesion in the si-TPX2, inhibitor, inhibitor+si-TPX2, NC and blank groups. The cellular experiments were biologically repeated for three times, and the data were presented as mean ± standard deviation (SD). *P<0.05, **P<0.001 in contrast to blank group. ##P<0.001 in contrast to inhibitor group.
Fig 2: Identification of mRNA and miRNA of interest in this study. (A) The overlapping genes of the significantly upregulated genes from GSE119056 and GSE23392 data series downloaded from GEO database, and those from GEPIA database. (B) The STRING networking results of the 24 overlapping genes from (A). (C) The expression of TPX2 in ovarian cancer using GEPIA database. *P<0.01. (D) Expression of RHPN1-AS1 in ovarian cancer using GEPIA database. *P<0.01. (E) The common target miRNAs of TPX2 predicted by TargetScan Human 7.2 and of RHPN1-AS1 predicted by ENCORI Starbase database. FC, fold change; OV, ovarian cancer; T, tumor; N, normal.
Fig 3: The regulation of RHPN1-AS1 to OC progression was dependent on TPX2. (A) Western blot was employed to detect the protein level of TPX2 in SKOV3 and OVCAR3 cell lines with the transfection of si-RHPN1-AS1 (si-lnc group), miR-485-5p inhibitor (inhibitor group), co-transfecting si-RHPN1-AS1 and inhibitor (si-lnc+inhibitor group), negative control (NC group, siRNA NC+inhibitor-NC) and untreated cells (blank group). (B) Western blot was employed to detect the protein level of TPX2 in SKOV3 and OVCAR3 cell lines with the transfection of si-RHPN1-AS1 (si-lnc group), OE-TPX2 (OE-TPX2 group), co-transfecting si-RHPN1-AS1 and OE-TPX2 (si-lnc+OE-TPX2 group), negative control (NC group, siRNA NC+empty vector) and untreated cells (blank group). (C) CCK-8 assay was used to observe the cell proliferation in si-lnc group, OE-TPX2 group, si-lnc+OE-TPX2 group, NC group and blank group. (D) BrdU assay was used to observe the cell proliferation in si-lnc group, OE-TPX2 group, si-lnc+OE-TPX2 group, NC group and blank group. The cellular experiments were biologically repeated for three times, and the data were presented as mean ± standard deviation (SD). *P<0.05, **P<0.001 in contrast to blank group. ##P<0.001 in contrast to si-lnc group.
Fig 4: TPX2 was the downstream target gene of miR-485-5p. (A) The competitive region sequences between miR-485-5p and TPX2 was analyzed by TargetScan 7.2. (B) Luciferase reporter assay was used to measure the target relationship between miR-485-5p and TPX2. NC, miRNA mimic negative control. (C) RNA-pull down assay was used to measure the interacting relationship between miR-485-5p and TPX2. Bio-NC, bio-miRNA mimic negative control. (D) RT-qPCR analysis revealed that TPX2 mRNA expression was higher in OC tissues than that in adjacent normal tissues. (E) RT-qPCR analysis showed that TPX2 mRNA was higher in SKOV3 and OVCAR3 cell lines than that in normal ovarian epithelial cell by RT-qPCR. (F) Pearson's correlation analysis revealed that miR-485-5p had a negative relationship with TPX2. (G) RT-qPCR analysis revealed that TPX2 was increased in miR-485-5p inhibitor group compared with blank group. NC, miRNA inhibitor negative control. The cellular experiments were biologically repeated for three times, and the data were presented as mean ± standard deviation (SD). **P<0.001 in contrast to blank group.
Fig 5: HDAC1 knockdown represses the migratory, invasive and metastatic properties of CC cells via suppression of TPX2.A Cell migration and invasion upon HDAC1 silencing alone or with TPX2 overexpression in combination assessed by Transwell assay. B Program flowchart of tumor metastasis model developed in nude mice and HDAC1 silencing alone or with TPX2 overexpression in combination. C The number of nodules on the mouse liver surface. D The number of nodules inside the mouse liver. *p < 0.05. Data were compared by one-way ANOVA with Tukey’s post hoc test. The cell experiment was independently repeated three times. n = 6 for animal experiment.
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