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MicroRNA-93 promotes the malignant phenotypes of human glioma cells and induces their chemoresistance to temozolomide.

Chen R, Liu H, Cheng Q, Jiang B, Peng R, Zou Q, Yang W, Yang X, Wu X, Chen Z - Biol Open (2016)

Bottom Line: Moreover, the upregulated miR-93 level was significantly associated with the advanced malignancy.We also found that upregulation of miR-93 promoted the proliferation, migration and invasion of glioma cells, and that miR-93 was involved in the regulation of cell cycle progression by mediating the protein levels of P21, P27, P53 and Cyclin D1.Knockdown of P21 attenuated the suppressive effects of miR-93 inhibition on cell cycle progression and colony formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Nanhua Hospital Affiliated to Nanhua University, Hengyang, Hunan 421001, China.

No MeSH data available.


Related in: MedlinePlus

MiR-93 directly targets p21. (A) Targetscan software data indicated that CDKN1A (P21) was a potential target of miR-93. (B) We constructed the wild-type (WT) P21 3′UTR containing the putative binding sequences (GCACUUU) of miR-93 and the mutant type (MUT) P21 3′UTR with the binding sequences were changed into ‘AAAAAAA’. We then inserted them into the pMIR- REPORT miRNA Expression Reporter vector, generating WT P21-3′UTR plasmid and MUT P21-3′UTR plasmid, respectively. HEK293 cells were co-transfected with WT P21-3′UTR or MUT P21-3′UTR plasmid, and miR-NC or miR-93 mimic, respectively. After co-transfection for 48 h, dual-luciferase reporter assay was performed to examine the luciferase activity in each group. *P<0.05. (C,D) U87 and SF126 cells were transfected with miR-93 inhibitor or mimic, respectively. Cells transfected with scramble miRNA (miR-NC) were used as control. Western blot was conducted to examine the protein expression of P21 (C), P27, P53 and Cyclin D1 (D) in each group. GAPDH was used as an internal reference. Data represented as mean±s.d; *P<0.05.
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BIO015552F4: MiR-93 directly targets p21. (A) Targetscan software data indicated that CDKN1A (P21) was a potential target of miR-93. (B) We constructed the wild-type (WT) P21 3′UTR containing the putative binding sequences (GCACUUU) of miR-93 and the mutant type (MUT) P21 3′UTR with the binding sequences were changed into ‘AAAAAAA’. We then inserted them into the pMIR- REPORT miRNA Expression Reporter vector, generating WT P21-3′UTR plasmid and MUT P21-3′UTR plasmid, respectively. HEK293 cells were co-transfected with WT P21-3′UTR or MUT P21-3′UTR plasmid, and miR-NC or miR-93 mimic, respectively. After co-transfection for 48 h, dual-luciferase reporter assay was performed to examine the luciferase activity in each group. *P<0.05. (C,D) U87 and SF126 cells were transfected with miR-93 inhibitor or mimic, respectively. Cells transfected with scramble miRNA (miR-NC) were used as control. Western blot was conducted to examine the protein expression of P21 (C), P27, P53 and Cyclin D1 (D) in each group. GAPDH was used as an internal reference. Data represented as mean±s.d; *P<0.05.

Mentions: As miRNAs play a role via regulating their targets expression (Areeb et al., 2015), we further investigated the putative targets of miR-93 by using several common miRNA analysis software, including Pictar, MicroRNA.org, and Targetscan, and P21 (encoded by CDKN1A) was predicated to be a target gene of miR-93 (Fig. 4A). To verify this prediction, we constructed the wild type (WT) P21 3′UTR containing the putative binding sequences (GCACUUU) of miR-93 and the mutant type (MUT) P21 3′UTR, within which the binding sequences were changed into ‘AAAAAAA’. We then inserted them into the pMIR- REPORT miRNA Expression Reporter vector, generating WT P21-3′UTR plasmid and MUT P21-3′UTR plasmid, respectively. HEK293 cells were co-transfected with WT P21-3′UTR or MUT P21-3′UTR plasmid, and miR-NC or miR-93 mimic, respectively. After co-transfection for 48 h, dual-luciferase reporter assay was performed, and our data showed that the luciferase activity was significantly decreased in HEK293 cells co-transfected with WT P21-3′UTR plasmid and miR-93 mimic compared to the control group (Fig. 4B). However, it was unchanged in the other groups, when compared to the control group (Fig. 4B). These data indicate that P21 is a direct target gene of miR-93. We further examined the effects of miR-93 on the expression of P21 in U87 cells. Our data showed that knockdown of miR-93 enhanced the protein expression of P21 in U87 cells, while overexpression of miR-93 led to a significant decrease in the protein levels of P21 in SF127 cells, when compared to the control groups, respectively (Fig. 4C). Accordingly, we suggest that miR-93 can negatively mediate the protein expression of P21 by directly binding to its 3′UTR in glioma cell lines. In addition, we also examined several other cell cycle-related proteins, and found that knockdown of miR-93 increased the protein levels of P27 and P53, but decreased the Cyclin D1 protein levels in U87 cells, while overexpression of miR-93 reduced the protein levels of P27 and P53, but upregulated the Cyclin D1 protein levels in SF126 cells, when compared to the control groups, respectively (Fig. 4D). These data are consistent with the result of cell cycle analysis in U87 and SF126 cells after knockdown or overexpression of miR-93. We further studied whether P21 acted as a downstream effecter of miR-93 in glioma cells. P21 siRNA was used to transfect U87 cells. After transfection, the protein levels of P21 were significantly decreased compared to the control group, indicating that the P21 siRNA is effective (Fig. 5A). We further set three groups using U87 cells: miR-93 inhibitor, miR-93 inhibitor+siRNA NC, and miR-93 inhibitor+P21 siRNA. After that, we examined the cell cycle distribution in each group. As indicated in Fig. 5B, cells in the G0/G1 stage were markedly decreased in the miR-93 inhibitor+P21 siRNA, while cells in the S stage were significantly increased in the miR-93 inhibitor+P21 siRNA, when compared to the other two groups, respectively. These data suggest that knockdown of P21 reversed the suppressive effects of miR-93 inhibition on the cell cycle progression in U87 cells. We then examined the colony formation capacities in each group. As indicated in Fig. 5C, the colony formation rate was significantly higher in the miR-93 inhibitor+P21 siRNA group, when compared with that in the other two groups, respectively. These results suggest that P21, as a downstream effecter of miR-93, had suppressive effects on the colony formation capacities of U87 cells.Fig. 4.


MicroRNA-93 promotes the malignant phenotypes of human glioma cells and induces their chemoresistance to temozolomide.

Chen R, Liu H, Cheng Q, Jiang B, Peng R, Zou Q, Yang W, Yang X, Wu X, Chen Z - Biol Open (2016)

MiR-93 directly targets p21. (A) Targetscan software data indicated that CDKN1A (P21) was a potential target of miR-93. (B) We constructed the wild-type (WT) P21 3′UTR containing the putative binding sequences (GCACUUU) of miR-93 and the mutant type (MUT) P21 3′UTR with the binding sequences were changed into ‘AAAAAAA’. We then inserted them into the pMIR- REPORT miRNA Expression Reporter vector, generating WT P21-3′UTR plasmid and MUT P21-3′UTR plasmid, respectively. HEK293 cells were co-transfected with WT P21-3′UTR or MUT P21-3′UTR plasmid, and miR-NC or miR-93 mimic, respectively. After co-transfection for 48 h, dual-luciferase reporter assay was performed to examine the luciferase activity in each group. *P<0.05. (C,D) U87 and SF126 cells were transfected with miR-93 inhibitor or mimic, respectively. Cells transfected with scramble miRNA (miR-NC) were used as control. Western blot was conducted to examine the protein expression of P21 (C), P27, P53 and Cyclin D1 (D) in each group. GAPDH was used as an internal reference. Data represented as mean±s.d; *P<0.05.
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BIO015552F4: MiR-93 directly targets p21. (A) Targetscan software data indicated that CDKN1A (P21) was a potential target of miR-93. (B) We constructed the wild-type (WT) P21 3′UTR containing the putative binding sequences (GCACUUU) of miR-93 and the mutant type (MUT) P21 3′UTR with the binding sequences were changed into ‘AAAAAAA’. We then inserted them into the pMIR- REPORT miRNA Expression Reporter vector, generating WT P21-3′UTR plasmid and MUT P21-3′UTR plasmid, respectively. HEK293 cells were co-transfected with WT P21-3′UTR or MUT P21-3′UTR plasmid, and miR-NC or miR-93 mimic, respectively. After co-transfection for 48 h, dual-luciferase reporter assay was performed to examine the luciferase activity in each group. *P<0.05. (C,D) U87 and SF126 cells were transfected with miR-93 inhibitor or mimic, respectively. Cells transfected with scramble miRNA (miR-NC) were used as control. Western blot was conducted to examine the protein expression of P21 (C), P27, P53 and Cyclin D1 (D) in each group. GAPDH was used as an internal reference. Data represented as mean±s.d; *P<0.05.
Mentions: As miRNAs play a role via regulating their targets expression (Areeb et al., 2015), we further investigated the putative targets of miR-93 by using several common miRNA analysis software, including Pictar, MicroRNA.org, and Targetscan, and P21 (encoded by CDKN1A) was predicated to be a target gene of miR-93 (Fig. 4A). To verify this prediction, we constructed the wild type (WT) P21 3′UTR containing the putative binding sequences (GCACUUU) of miR-93 and the mutant type (MUT) P21 3′UTR, within which the binding sequences were changed into ‘AAAAAAA’. We then inserted them into the pMIR- REPORT miRNA Expression Reporter vector, generating WT P21-3′UTR plasmid and MUT P21-3′UTR plasmid, respectively. HEK293 cells were co-transfected with WT P21-3′UTR or MUT P21-3′UTR plasmid, and miR-NC or miR-93 mimic, respectively. After co-transfection for 48 h, dual-luciferase reporter assay was performed, and our data showed that the luciferase activity was significantly decreased in HEK293 cells co-transfected with WT P21-3′UTR plasmid and miR-93 mimic compared to the control group (Fig. 4B). However, it was unchanged in the other groups, when compared to the control group (Fig. 4B). These data indicate that P21 is a direct target gene of miR-93. We further examined the effects of miR-93 on the expression of P21 in U87 cells. Our data showed that knockdown of miR-93 enhanced the protein expression of P21 in U87 cells, while overexpression of miR-93 led to a significant decrease in the protein levels of P21 in SF127 cells, when compared to the control groups, respectively (Fig. 4C). Accordingly, we suggest that miR-93 can negatively mediate the protein expression of P21 by directly binding to its 3′UTR in glioma cell lines. In addition, we also examined several other cell cycle-related proteins, and found that knockdown of miR-93 increased the protein levels of P27 and P53, but decreased the Cyclin D1 protein levels in U87 cells, while overexpression of miR-93 reduced the protein levels of P27 and P53, but upregulated the Cyclin D1 protein levels in SF126 cells, when compared to the control groups, respectively (Fig. 4D). These data are consistent with the result of cell cycle analysis in U87 and SF126 cells after knockdown or overexpression of miR-93. We further studied whether P21 acted as a downstream effecter of miR-93 in glioma cells. P21 siRNA was used to transfect U87 cells. After transfection, the protein levels of P21 were significantly decreased compared to the control group, indicating that the P21 siRNA is effective (Fig. 5A). We further set three groups using U87 cells: miR-93 inhibitor, miR-93 inhibitor+siRNA NC, and miR-93 inhibitor+P21 siRNA. After that, we examined the cell cycle distribution in each group. As indicated in Fig. 5B, cells in the G0/G1 stage were markedly decreased in the miR-93 inhibitor+P21 siRNA, while cells in the S stage were significantly increased in the miR-93 inhibitor+P21 siRNA, when compared to the other two groups, respectively. These data suggest that knockdown of P21 reversed the suppressive effects of miR-93 inhibition on the cell cycle progression in U87 cells. We then examined the colony formation capacities in each group. As indicated in Fig. 5C, the colony formation rate was significantly higher in the miR-93 inhibitor+P21 siRNA group, when compared with that in the other two groups, respectively. These results suggest that P21, as a downstream effecter of miR-93, had suppressive effects on the colony formation capacities of U87 cells.Fig. 4.

Bottom Line: Moreover, the upregulated miR-93 level was significantly associated with the advanced malignancy.We also found that upregulation of miR-93 promoted the proliferation, migration and invasion of glioma cells, and that miR-93 was involved in the regulation of cell cycle progression by mediating the protein levels of P21, P27, P53 and Cyclin D1.Knockdown of P21 attenuated the suppressive effects of miR-93 inhibition on cell cycle progression and colony formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Nanhua Hospital Affiliated to Nanhua University, Hengyang, Hunan 421001, China.

No MeSH data available.


Related in: MedlinePlus