Correction to “Exosomal miRNA-166-5p Derived From G-MDSCs Promotes Proliferation by Targeting ITM3E in Colorectal Cancer”

Abstract

Ma T, Jiang J, Shi M, Xu H. “Exosomal miRNA-166-5p Derived From G-MDSCs Promotes Proliferation by Targeting ITM3E in Colorectal Cancer,” Environmental Toxicology 39, no. 2 (2024):803–814, https://doi.org/10.1002/tox.23980.

All “ITM3E” need to be replaced with “ITM2B” in this article.

Specifically as follows:

The title “Exosomal miRNA-166-5p derived from G-MDSCs promotes proliferation by targeting ITM3E in colorectal cancer” was incorrect.

This should have read: “Exosomal miRNA-166-5p derived from G-MDSCs promotes proliferation by targeting ITM2B in colorectal cancer”. (page 803).

In the abstract, the text “This downregulation leads to the inhibition of integral membrane protein 2B (ITM3E) transcription, which in turn activates the PI3K/Akt signaling path way.” and “The primary results of this work show that exosomes produced from G-MDSCs and the miR-166-5p/ITM3E axis have therapeutic and diagnostic promise in colorectal cancer.” were incorrect.

This should have read: “This downregulation leads to the inhibition of integral membrane protein 2B (ITM2B) transcription, which in turn activates the PI3K/Akt signaling path way.” and “The primary results of this work show that exosomes produced from G-MDSCs and the miR-166-5p/ITM2B axis have therapeutic and diagnostic promise in colorectal cancer.”(page 803).

In the keywords, “ITM3E” was incorrect.

This should have read: “ITM2B”. (page 803).

In the background, “Integral membrane protein 2B (ITM3E) is an alternatively spliced protein that has been shown to enhance apoptosis and inhibit proliferation [20]. Several studies suggested that ITM3E could be useful as an anticancer agent [21]. Unfortunately, ITM3E has not been investigated in the context of colorectal cancer.” and “The primary results highlight the diagnostic and therapeutic promise of exosomes produced from G-MDSCs and the miR-166-5p/ITM3E axis in colorectal cancer. In colorectal cancer, exosomal miR-166-5p from G-MDSCs targets ITM3E to activate the PI3K/Akt pathway.” were incorrect.

This should have read: “Integral membrane protein 2B (ITM2B) is an alternatively spliced protein that has been shown to enhance apoptosis and inhibit proliferation [20]. Several studies suggested that ITM2B could be useful as an anticancer agent [21]. Unfortunately, ITM2B has not been investigated in the context of colorectal cancer.” and “The primary results highlight the diagnostic and therapeutic promise of exosomes produced from G-MDSCs and the miR-166-5p/ITM2B axis in colorectal cancer. In colorectal cancer, exosomal miR-166-5p from G-MDSCs targets ITM2B to activate the PI3K/Akt pathway.” (page 804).

In the 2.11 Luciferase reporter assay, the text “The pGL3-Basic luciferase reporter vector (Promega) was modified such that a DNA fragment encoding either the wild-type or mutant 3’-UTR of ITM3E could be subcloned downstream of the luciferase gene. 150 ng of empty vector or miRNA-166-5p, and 50 ng of firefly luciferase reporter expressing wild-type or mutant 3’-UTR of ITM3E fragment,” was incorrect.

This should have read: “The pGL3-Basic luciferase reporter vector (Promega) was modified such that a DNA fragment encoding either the wild-type or mutant 3’-UTR of ITM2B could be subcloned downstream of the luciferase gene. 150 ng of empty vector or miRNA-166-5p, and 50 ng of firefly luciferase reporter expressing wild-type or mutant 3’-UTR of ITM2B fragment,”. (page 805).

In the 2.12 Stable transfection with lentiviral vectors, the text “ITM3E overexpression plasmid, ITM3E shRNA, and negative control plasmid to infect 2 × 10⁵ cells in 6-well pans for 48 h.” was incorrect.

This should have read: “ITM2B overexpression plasmid, ITM2B shRNA, and negative control plasmid to infect 2 × 10⁵ cells in 6-well pans for 48 h.” (page 805).

In the 3.4 ITM3E is a miRNA-166-5p target gene in colorectal cancer cells, the title “ITM3E is a miRNA-166-5p target gene in colorectal cancer cells” was incorrect.

This should have read: “ITM2B is a miRNA-166-5p target gene in colorectal cancer cells” (page 807).

The text “The most consistent reduction in ITM3E expression was seen by RT-qPCR (Figure 5C). After 48 h of treatment with OE-miRNA-166-5p lentivirus, the protein level of ITM3E was shown to be reduced in both Caco-2 and SPC-A-1 cells using western blot tests (Figure 5D). We next used site-directed mutagenesis to alter the predicted miRNA-166-5p binding site in the ITM3E sequence, resulting in a reporter construct (Figure 5E). Mutant ITM3E, as predicted, reversed miRNA-166-5p's inhibitory effect on luciferase activity. Next, we looked at how much ITM3E was expressed when miRNA-166-5p was overexpressed in Caco-2 and SPC-A-1 cells. Overexpression of miRNA-166-5p substantially suppressed ITM3E expression (Figure 5F,G). Taken together, our results indicate that miRNA-166-5p targets the mRNA for ITM3E, reducing its expression levels.” was incorrect.

This should have read: “The most consistent reduction in ITM2B expression was seen by RT-qPCR (Figure 5C). After 48 h of treatment with OE-miRNA-166-5p lentivirus, the protein level of ITM2B was shown to be reduced in both Caco-2 and SPC-A-1 cells using western blot tests (Figure 5D). We next used site-directed mutagenesis to alter the predicted miRNA-166-5p binding site in the ITM2B sequence, resulting in a reporter construct (Figure 5E). Mutant ITM2B, as predicted, reversed miRNA-166-5p's inhibitory effect on luciferase activity. Next, we looked at how much ITM2B was expressed when miRNA-166-5p was overexpressed in Caco-2 and SPC-A-1 cells. Overexpression of miRNA-166-5p substantially suppressed ITM2B expression (Figure 5F,G). Taken together, our results indicate that miRNA-166-5p targets the mRNA for ITM2B, reducing its expression levels.” (page 809, 811).

In the FIGURE 5, the text “MiRNA-166-5p targets the protein ITM3E within colorectal carcinoma cells. Target genes of miRNA-166-5p predicted using different bioinformatics databases and RNA sequencing findings (A); (B) a Venn diagram of downregulated genes in Caco-2, SPC-A-1, and A549 cells. Western blot pictures of ITM3E proteins in colorectal cancer cells treated with OEmiRNA-166-5p lentivirus for 48 h (C) and the expression levels of four downregulated genes in 10 colorectal cancer cells (D). Diagram depicting the predicted miRNA-166-5p target location in ITM3E wild-type (WT) and mutant (Mut) 3′-untranslated regions (UTRs); Luciferase activity experiment in Caco-2 (F) and SPC-A-1 (G) cells transfected with ITM3E 3’-UTR (WT or Mut) carrying luciferase report plasmids and either control or miRNA-166-5p.” was incorrect.

This should have read: “MiRNA-166-5p targets the protein ITM2B within colorectal carcinoma cells. Target genes of miRNA-166-5p predicted using different bioinformatics databases and RNA sequencing findings (A); (B) a Venn diagram of downregulated genes in Caco-2, SPC-A-1, and A549 cells. Western blot pictures of ITM2B proteins in colorectal cancer cells treated with OEmiRNA-166-5p lentivirus for 48 h (C) and the expression levels of four downregulated genes in 10 colorectal cancer cells (D). Diagram depicting the predicted miRNA-166-5p target location in ITM2B wild-type (WT) and mutant (Mut) 3’-untranslated regions (UTRs); Luciferase activity experiment in Caco-2 (F) and SPC-A-1 (G) cells transfected with ITM2B 3’-UTR (WT or Mut) carrying luciferase report plasmids and either control or miRNA-166-5p.”(page 811).

In the 3.5 ITM3E inhibited colorectal cancer proliferation by repressing PI3K/AKT pathway activation, the title “ITM3E inhibited colorectal cancer proliferation by repressing PI3K/AKT pathway activation” was incorrect.

This should have read: “ITM2B inhibited colorectal cancer proliferation by repressing PI3K/AKT pathway activation” (page 812).

The text “The purpose of this research was to examine ITM3E expression and its role in colorectal cancer. At first, we analyzed the expression of the ITM3E transcript between colon tissues from healthy individuals (n = 34) and those from patients with colorectal cancer (n = 122). According to TCGA results, ITM3E levels were lower in colorectal cancer tissues than normal tissues (Figure 6A). Next, we performed RT-qPCR analysis on clinical samples and discovered that ITM3E expression was elevated and increased in more advanced pathological stages (Figure 6B). These findings point toward a potential role for ITM3E as a tumor suppressor gene in colorectal cancer. MiRNA-166-5p, which is highly concentrated in exosomes, has been demonstrated to control ITM3E. We used RT-qPCR to examine ITM3E levels in 10 different colorectal cancer cell lines in order to learn more about the potential function of extracellular factors in the downregulation of ITM3E in colorectal cancer tissues. The expression of ITM3E was found in NCM460 normal colon cells and colorectal cancer cell lines (Figure 6C). We treated Caco-2 cells for 48 h with mock, empty vector, and OE-ITM3E lentivirus and then performed RT-qPCR to learn more about the role of ITM3E in colorectal cancer cells. The treatment with OE-ITM3E lentiviral vector successfully increased ITM3E expression (Figure 6D). Proliferation of Caco-2 cells was slowed after treatment with OE-ITM3E lentivirus, as measured by the cell counting kit-8 (CCK-8; Figure 6E). Overexpression of ITM3E, as shown by RT-qPCR, resulted in lower Ki67 levels (Figure 6F). These findings demonstrate that overexpression of ITM3E slowed the spread of colorectal cancer by decreasing cell proliferation. We created a mouse model of colorectal cancer by injecting OE-ITM3E lentivirus-treated cells subcutaneously to corroborate our findings in vivo. Every week, tumor growth rates were recorded, and Kaplan– Meier estimates of overall survival were produced. ITM3E overexpression was associated with slower tumor development compared to the control group (Figure 6G). The longer-term survival rate (Figure 6H) favored the ITM3E overexpression group. Together, our data indicate that ITM3E is a tumor suppressor gene that is downregulated in colorectal cancer tissues. ITM3E overexpression slowed the growth of colorectal cancer cells.” were incorrect.

This should have read: “The purpose of this research was to examine ITM2B expression and its role in colorectal cancer. At first, we analyzed the expression of the ITM2B transcript between colon tissues from healthy individuals (n = 34) and those from patients with colorectal cancer (n = 122). According to TCGA results, ITM2B levels were lower in colorectal cancer tissues than normal tissues (Figure 6A). Next, we performed RT-qPCR analysis on clinical samples and discovered that ITM2B expression was elevated and increased in more advanced pathological stages (Figure 6B). These findings point toward a potential role for ITM2B as a tumor suppressor gene in colorectal cancer. MiRNA-166-5p, which is highly concentrated in exosomes, has been demonstrated to control ITM2B. We used RT-qPCR to examine ITM2B levels in 10 different colorectal cancer cell lines in order to learn more about the potential function of extracellular factors in the downregulation of ITM2B in colorectal cancer tissues. The expression of ITM2B was found in NCM460 normal colon cells and colorectal cancer cell lines (Figure 6C). We treated Caco-2 cells for 48 h with mock, empty vector, and OE-ITM2B lentivirus and then performed RT-qPCR to learn more about the role of ITM2B in colorectal cancer cells. The treatment with OE-ITM2B lentiviral vector successfully increased ITM2B expression (Figure 6D). Proliferation of Caco-2 cells was slowed after treatment with OE-ITM2B lentivirus, as measured by the cell counting kit-8 (CCK-8; Figure 6E). Overexpression of ITM2B, as shown by RT-qPCR, resulted in lower Ki67 levels (Figure 6F). These findings demonstrate that overexpression of ITM2B slowed the spread of colorectal cancer by decreasing cell proliferation. We created a mouse model of colorectal cancer by injecting OE-ITM2B lentivirus-treated cells subcutaneously to corroborate our findings in vivo. Every week, tumor growth rates were recorded, and Kaplan– Meier estimates of overall survival were produced. ITM2B overexpression was associated with slower tumor development compared to the control group (Figure 6G). The longer-term survival rate (Figure 6H) favored the ITM2B overexpression group. Together, our data indicate that ITM2B is a tumor suppressor gene that is downregulated in colorectal cancer tissues. ITM2B overexpression slowed the growth of colorectal cancer cells.” (page 812).

In the dissicussion, the text “ITM3E was shown to be a candidate. Based on our findings, miR-166-5p plays a key role in repressing ITM3E expression, and its knockdown resulted in ITM3E overexpression. The miR-166-5p immediately bound to the 3’-UTR of ITM3E, resulting in a decrease in ITM3E expression, as shown by luciferase reporter assays. Recent research has shown that ITM3E plays a crucial role in carcinogenesis. Our current findings additionally showed that ITM3E knockdown triggered the PI3K/Akt signaling pathway. Deguelin is a powerful inhibitor of PI3K and Akt that occurs naturally [34]. We showed that deguelin therapy partially reversed PI3K/Akt activation and boosted proliferation in colorectal cancer cells with downregulated expression of ITM3E. In addition, the proliferation-promoting effect of miR-166-5p was somewhat antag onized by overexpressing ITM3E. These results suggest that ITM3E,” and “We also uncovered the method by which miR-166-5p controls the PI3K/Akt pathway through interaction with ITM3E.” were incorrect.

This should have read: “ITM2B was shown to be a candidate. Based on our findings, miR-166-5p plays a key role in repressing ITM2B expression, and its knockdown resulted in ITM2B overexpression. The miR-166-5p immediately bound to the 3’-UTR of ITM2B, resulting in a decrease in ITM2B expression, as shown by luciferase reporter assays. Recent research has shown that ITM2B plays a crucial role in carcinogenesis. Our current findings additionally showed that ITM2B knockdown triggered the PI3K/Akt signaling pathway. Deguelin is a powerful inhibitor of PI3K and Akt that occurs naturally [34]. We showed that deguelin therapy partially reversed PI3K/Akt activation and boosted proliferation in colorectal cancer cells with downregulated expression of ITM2B. In addition, the proliferation-promoting effect of miR-166-5p was somewhat antag onized by overexpressing ITM2B. These results suggest that ITM2B,” and “We also uncovered the method by which miR-166-5p controls the PI3K/Akt pathway through interaction with ITM2B.”(page 812).

In the FIGURE 6, the text “Colorectal cancer tissues had lower levels of ITM3E, which slowed the growth of the tumor. Comparison of ITM3E expression in colorectal cancer and normal colon tissues (A); ITM3E expression in different stages of colorectal cancer and normal colon tissues (B); ITM3E transcription levels in 10 colorectal cancer cell lines (C); and ITM3E expression in Caco-2 cells (D) after infection with a mock, empty vector, or OE-ITM3E lentivirus. Mean ± SEM, *p < 0.05; ****p < 0.001.” was incorrect.

This should have read: “Colorectal cancer tissues had lower levels of ITM2B, which slowed the growth of the tumor. Comparison of ITM2B expression in colorectal cancer and normal colon tissues (A); ITM2B expression in different stages of colorectal cancer and normal colon tissues (B); ITM2B transcription levels in 10 colorectal cancer cell lines (C); and ITM2B expression in Caco-2 cells (D) after infection with a mock, empty vector, or OE-ITM2B lentivirus. Mean ± SEM, *p < 0.05; ****p < 0.001.”(page 813).

In the page 814, the text “How to cite this article: Ma T, Jiang J, Shi M, Xu H. Exosomal. miRNA-166-5p derived from G-MDSCs promotes proliferation by targeting ITM3E in colorectal cancer. Environmental Toxicology. 2024;39(2):803–814. doi:10.1002/ tox.23980” was incorrect.

This should have read: “How to cite this article: Ma T, Jiang J, Shi M, Xu H. Exosomal miRNA-166-5p derived from G-MDSCs promotes proliferation by targeting ITM2B in colorectal cancer. Environmental Toxicology. 2024;39(2):803-814. doi:10.1002/ tox.23980”. (The end of page 814).

We apologize for these errors.