KCTD12 may be one of the targeted regulatory genes of MSL1/MSL3.
MSL1 and MSL3 knockout (KO) 293T cell lines were established using CRISPR/Cas9 gene editing technology (27) (Fig. 1A). The effects of MSL1-KO (Fig. 1B) and MSL3-KO (Fig. 1C) on cell division were observed by cell immunofluorescence staining (Fig. 1D), and spindle multipolarity (green) was found in both MSL1-KO or MSL3-KO cells, suggesting the involvement of MSL1/MSL3 in cell division process. Based on the analysis of MSL1-KO and MSL3-KO gene expression profiles, a total of 2448 and 3431 differentially expressed genes (DEGs) were found in MSL1-KO and MSL3-KO cells respectively. Among them, 1817 DEGs were overlapped, and 34 genes in 1817 DEGs belong to the solute carrier (SLC) family genes, indicating the possibility of SLC family genes being regulated by MSL1/MSL3 (Fig. 1E, upper). Our results are consistent with the analysis conducted by Gao et al, who obtained 10 SLC family genes related to ferroptosis through intersecting SLC family genes and ferroptosis related genes (30). These 10 genes intersected with 34 SLC family genes coregulated by MSL1/MSL3, resulting in 4 overlapping genes (Fig. 1E, lower), demonstrating the involvement of MSL1/MSL3 in regulating the cell ferroptosis.
Venn diagram analysis was further performed using genes coregulated by MSL1/MSL3 (TCGA data) in different tumors and DEGs from RNA-Seq of MSL1-KO and MSL3-KO cells, and a total of 18 genes were overlapped (Fig. 1F). Among the 18 overlapping genes, KCTD12 aroused our curiosity because we have found in previous research that KCTD12 can inhibit the cell proliferation of OCM-1 (26). Based on TCGA database, lower expression of KCTD12 and higher expressed of MSL1/MSL3 were found in most tumor tissues, a negative correlation between MSL1/MSL3 and KCTD12 was speculated in COAD and KIRC tissues (Table 1 and Fig. 1G).
Table 1
Differentially expressed MSL1, MSL3, and KCTD12 genes in various tumor tissues
Tumors | KCTD12 (P-value) | Regulated type | MSL1 (P-value) | Regulated type | MSL3 (P-value) | Regulated type |
BRCA | 1.17E-51 | Down | 0.14378164 | ns | 0.000532 | Up |
LUAD | 2.14E-27 | Down | 0.00247984 | Up | 0.823263 | ns |
LUSC | 2.03E-29 | Down | 0.00394354 | Up | 0.317808 | ns |
UCEC. | 8.06E-21 | Down | 1.29E-07 | Up | 0.096974 | ns |
COAD | 1.45E-15 | Down | 7.30E-08 | Up | 3.88E-09 | Up |
KIRC | 3.43E-16 | Down | 1.15E-05 | Up | 5.33E-09 | Up |
READ | 8.76E-07 | Down | 0.15319792 | ns | 0.011912 | Up |
SKCM | 2.86E-07 | Down | 0.10306048 | ns | 0.000118 | Up |
BLCA | 1.81E-06 | Down | 0.04491664 | Up | 0.004616 | Up |
KIRP | 0.02023342 | Down | 0.00027205 | Up | 0.344453 | ns |
KICH | 0.05205218 | Down | 7.71E-09 | Up | 2.82E-08 | Up |
ESCA | 0.63818056 | ns | 4.03E-05 | Up | 0.000269 | Up |
HNSC | 0.66916146 | ns | 1.14E-12 | Up | 9.64E-07 | Up |
LIHC | 0.46169307 | ns | 2.60E-19 | Up | 2.01E-11 | Up |
STAD | 0.12251715 | ns | 5.89E-13 | Up | 2.12E-13 | Up |
PRAD | 0.43480534 | ns | 1.18E-07 | Up | 0.109063 | ns |
THCA | 0.70438965 | ns | 0.25788699 | ns | 0.14111 | ns |
CHOL | 0.00222305 | Down | 1.58E-08 | Up | 2.26E-09 | Up |
P-value, represents the difference in gene expression compared to normal tissues. BRCA, breast cancer; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; UCEC, endometrioid cancer; COAD, colon cancer; KIRC, kidney clear cell carcinoma; READ, rectal cancer; SKCM, skin melanoma; BLCA, bladder cancer; KIRP, kidney papillary cell carcinoma; KICH, kidney chromophobe; ESCA, esophageal cancer; HNSC, head and neck cancer; LIHC, liver cancer; STAD, stomach cancer; PRAD, prostate cancer; THCA, thyroid cancer; CHOL, cholangiocarcinoma. |
Downregulation of KCTD12 was verified in primary diagnosed colon cancer tissues
The data from TCGA database showed a significant downregulation of KCTD12 in both mRNA (Fig. 2A) and protein (Fig. 2B) level compared those to normal tissues. Moreover, low expression of KCTD12 was detrimental to the survival rate of colon cancer patients (Fig. 2C). Furthermore, paired tumor and adjacent tissues were collected from 40 patients (24 males and 16 females). H&E staining images of tumor and adjacent tissues are shown in Fig. 2D. In addition, the immunohistochemical staining of Ki67, p53, PMS2, MSH2, and MLH1 was shown in Fig. 2E, and those proteins are used as reference indicators for tumor diagnosis in routine clinical testing. As shown in Fig. 2F, KCTD12 mRNA levels revealed a significant difference between tumor and matched adjacent tissues. Also, a significant (> 2-fold decreased) downregulation of KCTD12 in 67.5% (27/40) of patients was detected (Fig. 2G). It is worth mentioning that the downregulation of KCTD12 expression was more pronounced in patients with distant metastasis (Fig. 2H).
A negative correlation between MSL1 and KCTD12 was confirmed in 293T and HCT116 cells
As an essential key subunit in the MSL complex, in subsequent studies, we take MSL1 as the main research object to explore its interaction with KCTD12. First, a negative correlation (cor = − 0.339) between KCTD12 and MSL1 in colon cancer cells (Fig. 3A) was clarified with the TIMER platform. Consistent with this, KCTD12 expression levels in both protein (Fig. 3B) and mRNA (Fig. 3C) were dramatically increased in MSL1-KO cells. Conversely, overexpressed MSL1 resulted in a dose-dependent decrease in KCTD12 protein and mRNA levels (Fig. 3D-3F), which supports the conclusion that MSL1 negatively regulates KCTD12. In addition, relative higher expression of KCTD12 in HCT116 colon cancer cells was detected, thus, it was selected for further mechanism studies (Fig. 3G and 3H).
SLC7A11 was identified as one of the potential targeted regulatory genes of MSL1 -KCTD12 axis.
The RNA-Seq was performed using lentiviral-mediated stably expressing KCTD12 HCT116 and OCM-1 cell lines (Fig. 4A). Obtained 284 and 107 DEGs (p < 0.001) from HCT116 and OCM-1 cells were conducted to Venn diagram analysis. Six overlapped genes including SLC7A11 were identified (Fig. 4B). DEGs from Flag-KCTD12 HCT116 cells (p < 0.01) were further visualized in volcano plot (Fig. 4C). SLC7A11 and SLC3A2 jointly constitute the important negative regulatory mechanism Xc-system of ferroptosis. To confirm the correlation between SLC7A11 and MSL1-KCTD12 axis, the protein and mRNA levels of SLC7A11 were first detected in MSL1-KO cells. As a result, higher expression of KCTD12 and decreased SLC7A11 in protein and mRNA levels were observed in MSL1-KO cells (Fig. 4D-4F). Conversely, dose-dependent declined KCTD12 and increased SLC7A11 in protein and mRNA levels were detected (Fig. 4H-4I). Moreover, compared to 293T cells, overexpressed KCTD12 in MSL1-KO cells resulted in decreased SLC7A11 and GPX4 (Fig. 4J, Lane 4), in contrast, shKCTD12 inhibited the upregulation of KCTD12 caused by MSL1-KO and the inhibitory effect of MSL1-KO on SLC7A11 expression (Lane 8, compared to Lane 7). Meanwhile, GPX4 proteins were modulated by MSL1-KCTD12 axis. Quantified protein levels for 4J were shown in Fig. 4K. The above results suggest the regulation of MSL1-KCTD12 axis on SLC7A11.
MSL1-KTD12 axis was involved in Erastin-induced ferroptosis by mediating SLC7A11 in HCT116 cells
Based on the facts that the MSL1-KTD12 axis regulates the expression of SLC7A11, the involvement of MSL-KCTD2 axis in ferroptosis was speculated. Therefore, the ferroptosis inducer Erastin was used in following experiments. Firstly, a median lethal dose of Erastin was determined through MTT assay (Fig. 5A), and 10 µM Erastin was used in subsequent experiments. And 10 µM Erastin induced ferroptosis can be effectively inhibited by the ferroptosis inhibitor Fer-1 (Fig. 5B). Furthermore, MSL1 protein and H4K16ac levels were dramatically decreased in Erastin exposed cells, meanwhile, high level of KCTD12, and decreased SLA7A11 and GPX4 were detected (Fig. 5C, Lane 2). As expected, these changes were reversed by the addition of Fer-1 (Lane 3). Quantified proteins were shown in Fig. 5D.
Next, the effect of KCTD12 on SLC7A11 protein level was tested in cells with or without Erastin treatment. Compared to without Erastin cells, overexpressed KCTD12 significantly reduced SLC7A11 and GPX4 protein level (Fig. 5E and 5F). Similar experiments were conducted in cells knocking down KCTD12, and a statistically significant increase of SLC7A11 and GPX4 proteins was found in Erastin induced ferroptosis cells (Fig. 5G, 5H). Taken together, KCTD12 negatively regulates the expression level of SLC7A11 and GPX4 in Erastin induced ferroptosis.
Similarly, KCTD12, SLC7A11 and GPX4 proteins were assessed in cells treated with Erastin after transient transfection of Flag-MSL or pVLX-shMSL1. As shown in Fig. 5I, compared to DMSO group, Erastin resulted in a remarkable decrease of MSL1 and H4K16ac, and consistent with previous results, the decreased MSL1 upregulated KCTD12, which further led to a reduction in SLC7A11 and GPX4 protein levels (Lane 2 compared to Lane 1). However, once MSL1 was replenished in Erastin treated cells, the protein levels of KCTD12, SLC7A11 and GPX4 tended to the DMSO control group levels in a dose-dependent manner (Lane 3–4, compared to Lane 1). On the basis of Erastin induced reduction of MSL1, further knockdown of MSL1 promoted the upregulation of KCTD12 and downregulation of SLC7A11 and GPX4 by Erastin (Fig. 5J, Lane 3–4, compared to Lane 2). The numbers below the immunoblot image represented the quantified values compared to the DMSO control group. In summary, MSL1 regulated SLC7A11 and GPX4 by mediating KCTD12 in Erastin induced ferroptosis.
Both MSL1 and KTD12 regulated Erastin produced GSH, MDA and ROS levels in HCT116 cells.
Based on the potential role of the MSL1-KCTD12-SLC7A11 axis in Erastin induced ferroptosis, the levels of intermediate products ROS, GSH and MDA related to ferroptosis were detected in cells treated with Erastin. Compared to the control cells or transfection of pcDNA3.1 and shNT groups, there was no significant changes in GSH contents in KCTD12 overexpressed or knocked down cells (Fig. 6A,6B left panel), however, a significant decrease or increase was observed in overexpressing or knocking down KCTD12 cells (Fig. 6A,6B, right panel). In addition, the elevated MDA content induced by Erastin was suppressed by overexpressing Flag-MSL1 (Fig. 6C, left panel), while it was enhanced by knocking down MSL1 with pLVX-shMSL1 (Fig. 6C, right panel) in a dose-dependent manner.
The accumulation of lipid peroxides is an important cause of ferroptosis. Therefore, the reactive oxygen species (ROS) levels were assessed in Fig. 6D-6G experiments. Firstly, overexpression of KCTD12 dose dependently increased the ROS levels in cells treated with Erastin (Fig. 6D, upper), but the opposite effect was observed in cells knocking down KCTD12 (lower). The quantified fluorescence intensity was shown in Fig. 6E. In contrast, the ROS levels were negatively regulated by overexpressing or knocking down MSL1 in Erastin exposed cells in a dose dependent manner (Fig. 6F). The quantified fluorescence intensity was shown in Fig. 6G. The above results are sufficient to demonstrate the involvement of MSL1 and KCTD12 in regulating Erastin induced ferroptosis in HCT116 cells, and MSL1 and KCTD12 have opposite effects on ROS, GSH and MDA, which is consistent with the previous experimental results, once again indicating a negative regulatory relationship between MSL1 and KCTD12.
MSL1-KCTD12 axis synergistically regulated ROS, GSH and MDA levels produced by Erastin in HCT116 cells
Here, the effects of simultaneously changing the expression levels of MSL1 and KCTD12 on ROS, GSH, and MDA levels induced by Erastin were detected. Firstly, in cells exposed to Erastin, Flag-MSL1 and Flag-KCTD12 were overexpressed alone, or cotransfected, and 24 hours later, the ROS levels were detected. In line with the previous results, Erastin produced ROS can be visualized in cells transfected with pcDNA3.1 plasmid, and this ROS level was inhibited by transfection of MSL1 and enhanced by transfection with KCTD12. Conversely, the ROS levels promoted by KCTD12 can be reversed by adding MSL1 (Fig. 7A, left panel). Quantified integrated density of fluorescence was shown in Fig. 7B (left panel). However, opposite results were obtained when pLVX-shMSL1 and pLVX-shKCTD12 were transfected alone or cotransfected in Erastin exposed HCT116 cells (Fig. 7A, right panel). Quantified integrated density of fluorescence was shown in Fig. 7B (right panel). Similar experiments were also performed in detecting the GSH and MDA levels. As shown in Fig. 7C (upper panel), Erastin produced GSH level was promoted by overexpressing MSL1, while inhibited by knocking down MSL1. Contrary to the effect of MSL1, overexpression of KCTD12 reduced GSH produced by Erastin, while knockdown of KCTD12 increased GSH. If both were overexpressed or knocked down simultaneously, the effect on GSH was neutralized. The MDA content assessment was showed in Fig. 7C (lower panel). Higher MDA content was observed in Erastin treated cells (compared to the DMSO group), and this level was negatively regulated by MSL1 expression level, however, KCTD12 positively regulated the MDA content. When both MSL1 and KCTD12 were overexpressed or knocked down simultaneously, the effect on MDA was neutralized.