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MicroRNA-638 inhibits cell proliferation by targeting phospholipase D1 in human gastric carcinoma.

Zhang J, Bian Z, Zhou J, Song M, Liu Z, Feng Y, Zhe L, Zhang B, Yin Y, Huang Z - Protein Cell (2015)

Bottom Line: From this research, we found that miR-638 was downregulated in GC tissues compared with corresponding noncancerous tissues (NCTs), and the DNA copy number of miR-638 was lower in GC than NCTs, which may induce the corresponding downregulation of miR-638 in GC.Subsequently, we identified that PLD1 is the target gene of miR-638 in GC, and silencing PLD1 expression phenocopied the inhibitory effect of miR-638 on GC cell proliferation.In summary, we revealed that miR-638 functions as a tumor suppressor in GC through inhibiting PLD1.

View Article: PubMed Central - PubMed

Affiliation: Wuxi Oncology Institute, The Affiliated Hospital of Jiangnan University, Wuxi, 214062, China.

ABSTRACT
MicroRNAs (miRNAs) are a type of small non-coding RNAs that are often play important roles in carcinogenesis, but the carcinogenic mechanism of miRNAs is still unclear. This study will investigate the function and the mechanism of miR-638 in carcinoma (GC). The expression of miR-638 in GC and the DNA copy number of miR-638 were detected by real-time PCR. The effect of miR-638 on cell proliferation was measured by counting kit-8 assay. Different assays, including bioinformatics algorithms (TargetScan and miRanda), luciferase report assay and Western blotting, were used to identify the target gene of miR-638 in GC. The expression of miR-638 target gene in clinical CRC tissues was also validated by immunohistochemical assay. From this research, we found that miR-638 was downregulated in GC tissues compared with corresponding noncancerous tissues (NCTs), and the DNA copy number of miR-638 was lower in GC than NCTs, which may induce the corresponding downregulation of miR-638 in GC. Ectopic expression of miR-638 inhibited GC cell growth in vitro. Subsequently, we identified that PLD1 is the target gene of miR-638 in GC, and silencing PLD1 expression phenocopied the inhibitory effect of miR-638 on GC cell proliferation. Furthermore, we observed that PLD1 was overexpressed in GC tissues, and high expression of PLD1 in GC predicted poor overall survival. In summary, we revealed that miR-638 functions as a tumor suppressor in GC through inhibiting PLD1.

No MeSH data available.


Related in: MedlinePlus

Identification of PLD1 as the target of miR-638. (A) Validation of the microarray results in both MKN-45 and SGC-7901 cells using qRT-PCR. A panel of 12 genes were indeed down-regulated by miR-638. (B) Proliferation assays performed on MKN-45 and SGC-7901 cells transfected with si-PLD1 or si-DEF6. Depleted PLD1 expression showed the most obvious growth repression effect. (C) Schematic of the wild-type (WT) or mutant-type (MT) 3′UTRs of the PLD1 plasmids. The complementary site of the seed region of miR-638 was selected for mutation. The free energy of hybrids between miR-638 and PLD1 was −21.1 kcal/mol. (D) Relative luciferase activity assays of luciferase reporter plasmids containing PLD1WT or MT 3′UTR were performed in cells (HEK-293T, MKN-45, and SGC-7901). Luciferase activity was determined 48 h after transfection and normalized to the Renilla luciferase activity. (E) The protein levels of PLD1 were determined by Western blotting in MKN-45 and SGC-7901 cells transfected with miR-638 mimic, miR-638 inhibitor or the corresponding NC. Beta-actin served as an internal control
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Fig3: Identification of PLD1 as the target of miR-638. (A) Validation of the microarray results in both MKN-45 and SGC-7901 cells using qRT-PCR. A panel of 12 genes were indeed down-regulated by miR-638. (B) Proliferation assays performed on MKN-45 and SGC-7901 cells transfected with si-PLD1 or si-DEF6. Depleted PLD1 expression showed the most obvious growth repression effect. (C) Schematic of the wild-type (WT) or mutant-type (MT) 3′UTRs of the PLD1 plasmids. The complementary site of the seed region of miR-638 was selected for mutation. The free energy of hybrids between miR-638 and PLD1 was −21.1 kcal/mol. (D) Relative luciferase activity assays of luciferase reporter plasmids containing PLD1WT or MT 3′UTR were performed in cells (HEK-293T, MKN-45, and SGC-7901). Luciferase activity was determined 48 h after transfection and normalized to the Renilla luciferase activity. (E) The protein levels of PLD1 were determined by Western blotting in MKN-45 and SGC-7901 cells transfected with miR-638 mimic, miR-638 inhibitor or the corresponding NC. Beta-actin served as an internal control

Mentions: To elucidate the mechanism underlying miR-638-mediated suppression of cell proliferation, we used two computer-aided algorithms, TargetScan and miRanda, to search for potential target genes of miR-638. Among the 12 potential tumor-related targets, 7 genes were significantly down-regulated in MKN-45 and SGC-7901 cells transfected with miR-638 and were considered to be the candidate targets of miR-638 (Fig. 3A). Based on these data and our previous results in CRC (Zhang et al., 2014), we focused on PLD1 and DEF6 for the subsequent analysis. The effects of the two candidate targets on cell proliferation were performed on MKN-45 and SGC-7901 cells using PLD1- or DEF6-specific siRNA. The results indicated that silencing PLD1 expression significantly decreased cell proliferation in both of MKN-45 and SGC-7901, which was not observed in the GC cells with silenced DEF6 expression (Fig. 3B). Taken together, these results suggest that PLD1 is a potential target gene of miR-638 in GC.Figure 3


MicroRNA-638 inhibits cell proliferation by targeting phospholipase D1 in human gastric carcinoma.

Zhang J, Bian Z, Zhou J, Song M, Liu Z, Feng Y, Zhe L, Zhang B, Yin Y, Huang Z - Protein Cell (2015)

Identification of PLD1 as the target of miR-638. (A) Validation of the microarray results in both MKN-45 and SGC-7901 cells using qRT-PCR. A panel of 12 genes were indeed down-regulated by miR-638. (B) Proliferation assays performed on MKN-45 and SGC-7901 cells transfected with si-PLD1 or si-DEF6. Depleted PLD1 expression showed the most obvious growth repression effect. (C) Schematic of the wild-type (WT) or mutant-type (MT) 3′UTRs of the PLD1 plasmids. The complementary site of the seed region of miR-638 was selected for mutation. The free energy of hybrids between miR-638 and PLD1 was −21.1 kcal/mol. (D) Relative luciferase activity assays of luciferase reporter plasmids containing PLD1WT or MT 3′UTR were performed in cells (HEK-293T, MKN-45, and SGC-7901). Luciferase activity was determined 48 h after transfection and normalized to the Renilla luciferase activity. (E) The protein levels of PLD1 were determined by Western blotting in MKN-45 and SGC-7901 cells transfected with miR-638 mimic, miR-638 inhibitor or the corresponding NC. Beta-actin served as an internal control
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4537476&req=5

Fig3: Identification of PLD1 as the target of miR-638. (A) Validation of the microarray results in both MKN-45 and SGC-7901 cells using qRT-PCR. A panel of 12 genes were indeed down-regulated by miR-638. (B) Proliferation assays performed on MKN-45 and SGC-7901 cells transfected with si-PLD1 or si-DEF6. Depleted PLD1 expression showed the most obvious growth repression effect. (C) Schematic of the wild-type (WT) or mutant-type (MT) 3′UTRs of the PLD1 plasmids. The complementary site of the seed region of miR-638 was selected for mutation. The free energy of hybrids between miR-638 and PLD1 was −21.1 kcal/mol. (D) Relative luciferase activity assays of luciferase reporter plasmids containing PLD1WT or MT 3′UTR were performed in cells (HEK-293T, MKN-45, and SGC-7901). Luciferase activity was determined 48 h after transfection and normalized to the Renilla luciferase activity. (E) The protein levels of PLD1 were determined by Western blotting in MKN-45 and SGC-7901 cells transfected with miR-638 mimic, miR-638 inhibitor or the corresponding NC. Beta-actin served as an internal control
Mentions: To elucidate the mechanism underlying miR-638-mediated suppression of cell proliferation, we used two computer-aided algorithms, TargetScan and miRanda, to search for potential target genes of miR-638. Among the 12 potential tumor-related targets, 7 genes were significantly down-regulated in MKN-45 and SGC-7901 cells transfected with miR-638 and were considered to be the candidate targets of miR-638 (Fig. 3A). Based on these data and our previous results in CRC (Zhang et al., 2014), we focused on PLD1 and DEF6 for the subsequent analysis. The effects of the two candidate targets on cell proliferation were performed on MKN-45 and SGC-7901 cells using PLD1- or DEF6-specific siRNA. The results indicated that silencing PLD1 expression significantly decreased cell proliferation in both of MKN-45 and SGC-7901, which was not observed in the GC cells with silenced DEF6 expression (Fig. 3B). Taken together, these results suggest that PLD1 is a potential target gene of miR-638 in GC.Figure 3

Bottom Line: From this research, we found that miR-638 was downregulated in GC tissues compared with corresponding noncancerous tissues (NCTs), and the DNA copy number of miR-638 was lower in GC than NCTs, which may induce the corresponding downregulation of miR-638 in GC.Subsequently, we identified that PLD1 is the target gene of miR-638 in GC, and silencing PLD1 expression phenocopied the inhibitory effect of miR-638 on GC cell proliferation.In summary, we revealed that miR-638 functions as a tumor suppressor in GC through inhibiting PLD1.

View Article: PubMed Central - PubMed

Affiliation: Wuxi Oncology Institute, The Affiliated Hospital of Jiangnan University, Wuxi, 214062, China.

ABSTRACT
MicroRNAs (miRNAs) are a type of small non-coding RNAs that are often play important roles in carcinogenesis, but the carcinogenic mechanism of miRNAs is still unclear. This study will investigate the function and the mechanism of miR-638 in carcinoma (GC). The expression of miR-638 in GC and the DNA copy number of miR-638 were detected by real-time PCR. The effect of miR-638 on cell proliferation was measured by counting kit-8 assay. Different assays, including bioinformatics algorithms (TargetScan and miRanda), luciferase report assay and Western blotting, were used to identify the target gene of miR-638 in GC. The expression of miR-638 target gene in clinical CRC tissues was also validated by immunohistochemical assay. From this research, we found that miR-638 was downregulated in GC tissues compared with corresponding noncancerous tissues (NCTs), and the DNA copy number of miR-638 was lower in GC than NCTs, which may induce the corresponding downregulation of miR-638 in GC. Ectopic expression of miR-638 inhibited GC cell growth in vitro. Subsequently, we identified that PLD1 is the target gene of miR-638 in GC, and silencing PLD1 expression phenocopied the inhibitory effect of miR-638 on GC cell proliferation. Furthermore, we observed that PLD1 was overexpressed in GC tissues, and high expression of PLD1 in GC predicted poor overall survival. In summary, we revealed that miR-638 functions as a tumor suppressor in GC through inhibiting PLD1.

No MeSH data available.


Related in: MedlinePlus