Fig 2: KO of FSTL1 promotes the proliferation, migration and invasion of cervical cancer cells and inhibits cell apoptosis. (A) MTT assay showed that the KO of FSTL1 began to significantly promote the proliferation of HeLa cells since the second day. However, the KO of FSTL1 started to significantly promote the proliferation of SiHa cells on the second day. (B) The colony formation assay indicated that the KO of FSTL1 promoted the single-cell proliferation of HeLa and SiHa cells. (C) The wound-healing assay indicated that FSTL1 accelerated the migration speed of HeLa and SiHa cells. (D and E) Invasion and migration of Transwell assay confirmed that the number of invasive and migratory cells in the FSTL1-KO group was significantly greater than that of the other two control groups. (F) Apoptosis assay revealed that the number of apoptotic cells in the FSTL1-KO group was significantly less than that in the other two control groups. Each experiment was repeated thrice. Data are expressed as the mean ± standard deviation and analyzed by one-way analysis of variance (ANOVA) from three independent experiments. KO, knockout; FSTL1, follistatin-like protein 1; NC, negative control.

Fig 3: Expression of FSTL1 is downregulated in cervical cancer tissues. (A) The expression of FSTL1 at the mRNA level was downregulated in cervical cancer tissues compared with that in the tumor-adjacent tissues or normal cervical tissues. The expression of FSTL1 at the mRNA level gradually increased in the cervical cancer tissues, tumor-adjacent tissues and normal cervical tissues. (B) The expression of FSTL1 at the mRNA level in the matched cancer tissues was downregulated compared with that in the tumor-adjacent tissues. (C) The expression of FSTL1 at the mRNA level in the non-matching cancer tissues was downregulated compared with that in the normal cervical tissues. (D) The expression of FSTL1 protein was downregulated in the cervical cancer tissues. Prostate cancer tissues served as the positive-control. PBS was replaced with the primary antibody in the negative-control group. A total of 84 cervical cancer tissues and 22 normal cervical tissues were detected by immunohistochemistry. The FSTL1 protein expression was found to be decreased in the cervical cancer tissues compared with that in the normal cervical tissues. FSTL1 protein was mainly localized in the nucleus. Data are expressed as the median (interquartile range) and analyzed by non-parametric test. FSTL1, follistatin-like protein 1.

Fig 4: Screening and verification of stable strains of cervical cancer cells overexpressing or knocking out FSTL1. (A and E) The OE or KO of FSTL1 lentiviral infection efficiency was >80% (b: green fluorescence g: bright). (B and F) Reverse transcription-quantitative PCR assay revealed that the expression of FSTL1mRNA was upregulated or downregulated in the FSTL1 OE group or the KO group compared with their corresponding NC group and the mock group (MOCK). (C) Western blotting revealed that the expression of FSTL1 protein was upregulated in the OE group compared with that in their corresponding NC and MOCK groups. (D) Quantified data of panel C. (G) The results of the KO and mutation detection assay revealed that the KO1 and KO3 targets were active, the KO2 target was inactive and the KO3 target activity was improved (M: ladder marker. CON: mock control corresponding to each experimental group. PC: positive-control group). (H) The sequencing of plasmids containing the three sgRNAs. (I) Western blotting revealed that the FSTL1 protein expression was downregulated in the KO groups compared with that in the corresponding NC and MOCK groups. (J) Quantified data of panel I. Data are expressed as the mean ± standard deviation and analyzed by one-way analysis of variance (ANOVA) from three independent experiments. FSTL1, follistatin-like protein 1; OE, overexpression; KO, knockout; NC, negative control; sgRNA, small-guide RNA.

Fig 5: FSTL1 may be involved in the regulation of the IGF-1R/PI3K/AKT/BCL-2 signaling pathway. (A) Disease and functional enrichment analyses revealed that differentially expressed genes of FSTL1 were mainly involved in the regulation of biological processes such as cancer development, damage and development of the body, gastrointestinal diseases and gene expression. (B) Heat map of cluster analysis of upregulated and downregulated differentially expressed genes in HeLa cells (it was based on all genes having been sorted using |Fold change |>2 and FDR <0.05). There were 881 differentially expressed genes in the OE group, of which 593 genes were upregulated and 288 genes were downregulated. (C) Kyoto Encyclopedia of Genes and Genomes and Ingenuity Pathway Analysis signaling pathway enrichment analyses revealed that the differentially expressed genes of FSTL1 were mainly enriched in the cancer transcriptional misregulation signaling, mitogen-activated protein kinase (MAPK) signaling, cancer signaling, P53 signaling and IGF-1 signaling pathways, among which the IGF-1 signaling pathway was significantly activated in this experiment. (D) The IGF-1R, PI3K, AKT, P-AKT, BCL-2 and BAX protein electropherogram. (E-H) The relative protein expression levels of IGF-1R, PI3K, AKT, P-AKT, BCL-2 and BAX in each group. Western blotting revealed that the OE of FSTL1 downregulated IGF-1R, PI3K, AKT, P-AKT and BCL-2, but upregulated BAX. The KO of FSTL1 restored these changes. Data are expressed as the mean ± standard deviation and analyzed by one-way analysis of variance (ANOVA) from three independent experiments. *P<0.05, compared with the control group. FSTL1, follistatin-like protein 1; OE, overexpression; KO, knockout; P-, phosphorylated.