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MiR-195 suppresses non-small cell lung cancer by targeting CHEK1.

Liu B, Qu J, Xu F, Guo Y, Wang Y, Yu H, Qian B - Oncotarget (2015)

Bottom Line: Our previous study showed high miR-195 plasma levels associated with favorable overall survival of non-smoking women with lung adenocarcinoma.We demonstrated that miR-195 expression was lower in tumor tissues and was associated with poor survival outcome.High expression of CHEK1 in lung tumors was associated with poor overall survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Epidemiology and Biostatistics, Key Laboratory of Breast Cancer Prevention and Therapy, Ministry of Education, Key Laboratory of Cancer Prevention and Therapy, Tianjin, National Clinical Research Center of Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China.

ABSTRACT
MiR-195 suppresses tumor growth and is associated with better survival outcomes in several malignancies including non-small cell lung cancer (NSCLC). Our previous study showed high miR-195 plasma levels associated with favorable overall survival of non-smoking women with lung adenocarcinoma. To further elucidate role of miR-195 in NSCLC, we conducted in vitro experiment as well as clinical studies in a cohort of 299 NSCLC samples. We demonstrated that miR-195 expression was lower in tumor tissues and was associated with poor survival outcome. Overexpression of miR-195 suppressed tumor cell growth, migration and invasion. We discovered that CHEK1 was a direct target of miR-195, which decreased CHEK1 expression in lung cancer cells. High expression of CHEK1 in lung tumors was associated with poor overall survival. Our results suggest that miR-195 suppresses NSCLC and predicts lung cancer prognosis.

No MeSH data available.


Related in: MedlinePlus

MiR-195 expression and effects on CHEK1 as a target of miR-195MiR-195 bound directly to the CHEK1 3′-UTRs and down-regulated its expression along with other proteins. (A) A Venn diagram shows 3 software which predict miRNA targets and identified 84 candidate genes which may interact with miR-195. (B) A significant inverse correlation was found in NSCLC between miR-195 and CHEK1 expression in TCGA. (C) A putative miR-195-binding site exists in the 3′-UTR of the CHEK1 mRNA, and 7-nucleotide deletion were generated in the binding site. (D) Transfection of miR-195 inhibited the firefly luciferase activity of the pMIR-REPORT-3′-UTR-CHEK1 (wt), but such inhibition was absent for the reporter which had deletion in the miR-195-binding site (del). MiR-NC was used as a negative control in all the experiments. The impact of miR-195 on CHEK1 expression was normalized and compared to those of negative miRNA (n = 3, p < 0.001). (E) The expression of miR-195 determined by RT-qPCR in three NSCLC cell lines was significantly increased following miR-195 transfection. (F) The protein level of CHEK1 was decreased in three NSCLC cell lines when transfected with miR-195 with beta-actin as a loading control. Two positive control Cyclin D1 and Cyclin E were detected as known targets of miR-195.
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Figure 4: MiR-195 expression and effects on CHEK1 as a target of miR-195MiR-195 bound directly to the CHEK1 3′-UTRs and down-regulated its expression along with other proteins. (A) A Venn diagram shows 3 software which predict miRNA targets and identified 84 candidate genes which may interact with miR-195. (B) A significant inverse correlation was found in NSCLC between miR-195 and CHEK1 expression in TCGA. (C) A putative miR-195-binding site exists in the 3′-UTR of the CHEK1 mRNA, and 7-nucleotide deletion were generated in the binding site. (D) Transfection of miR-195 inhibited the firefly luciferase activity of the pMIR-REPORT-3′-UTR-CHEK1 (wt), but such inhibition was absent for the reporter which had deletion in the miR-195-binding site (del). MiR-NC was used as a negative control in all the experiments. The impact of miR-195 on CHEK1 expression was normalized and compared to those of negative miRNA (n = 3, p < 0.001). (E) The expression of miR-195 determined by RT-qPCR in three NSCLC cell lines was significantly increased following miR-195 transfection. (F) The protein level of CHEK1 was decreased in three NSCLC cell lines when transfected with miR-195 with beta-actin as a loading control. Two positive control Cyclin D1 and Cyclin E were detected as known targets of miR-195.

Mentions: Using three miRNA databases, we identified a putative miR-195-binding site located in the 3′-UTR of CHEK1 mRNA (Figure 4A). To further validate the association between miR-195 and CHEK1, we analyzed the TCGA dataset and the result showed that the inverse correlation between miR-195 and CHEK1 was significant (r = –0.46, p < 0.0001) in NSCLC samples (Figure 4B). To confirm if miR-195 directly binds to this location in CHEK1, we cloned a full-length CHEK1 3′-UTR and inserted it into a luciferase reporter vector, downstream from the firefly luciferase gene. As for control, we made a mutant CHEK1 3′-UTR clone which had a 7-nucleotide deletion in the miR-195 binding site (Figure 4C), and the mutant clone was inserted into the same vector. Both vectors were transfected into lung cancer cells A549 and H1299 together with miR-195 or miR-NC. Our experiments showed that miR-195 significantly suppressed the luciferase activity in the CHEK1 wild type clone compared to miR-NC, but not in the mutant one (Figure 4D), suggesting that miR-195 directly binds to the 3′-UTR of CHEK1 mRNA. The transfection efficiency was examined with RT-qPCR, and our evaluation confirmed that miR-195 expression was increased substantially in the cell lines transfected with miR-195 mimic, but not in those with miR-NC (Figure 4E).


MiR-195 suppresses non-small cell lung cancer by targeting CHEK1.

Liu B, Qu J, Xu F, Guo Y, Wang Y, Yu H, Qian B - Oncotarget (2015)

MiR-195 expression and effects on CHEK1 as a target of miR-195MiR-195 bound directly to the CHEK1 3′-UTRs and down-regulated its expression along with other proteins. (A) A Venn diagram shows 3 software which predict miRNA targets and identified 84 candidate genes which may interact with miR-195. (B) A significant inverse correlation was found in NSCLC between miR-195 and CHEK1 expression in TCGA. (C) A putative miR-195-binding site exists in the 3′-UTR of the CHEK1 mRNA, and 7-nucleotide deletion were generated in the binding site. (D) Transfection of miR-195 inhibited the firefly luciferase activity of the pMIR-REPORT-3′-UTR-CHEK1 (wt), but such inhibition was absent for the reporter which had deletion in the miR-195-binding site (del). MiR-NC was used as a negative control in all the experiments. The impact of miR-195 on CHEK1 expression was normalized and compared to those of negative miRNA (n = 3, p < 0.001). (E) The expression of miR-195 determined by RT-qPCR in three NSCLC cell lines was significantly increased following miR-195 transfection. (F) The protein level of CHEK1 was decreased in three NSCLC cell lines when transfected with miR-195 with beta-actin as a loading control. Two positive control Cyclin D1 and Cyclin E were detected as known targets of miR-195.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4496229&req=5

Figure 4: MiR-195 expression and effects on CHEK1 as a target of miR-195MiR-195 bound directly to the CHEK1 3′-UTRs and down-regulated its expression along with other proteins. (A) A Venn diagram shows 3 software which predict miRNA targets and identified 84 candidate genes which may interact with miR-195. (B) A significant inverse correlation was found in NSCLC between miR-195 and CHEK1 expression in TCGA. (C) A putative miR-195-binding site exists in the 3′-UTR of the CHEK1 mRNA, and 7-nucleotide deletion were generated in the binding site. (D) Transfection of miR-195 inhibited the firefly luciferase activity of the pMIR-REPORT-3′-UTR-CHEK1 (wt), but such inhibition was absent for the reporter which had deletion in the miR-195-binding site (del). MiR-NC was used as a negative control in all the experiments. The impact of miR-195 on CHEK1 expression was normalized and compared to those of negative miRNA (n = 3, p < 0.001). (E) The expression of miR-195 determined by RT-qPCR in three NSCLC cell lines was significantly increased following miR-195 transfection. (F) The protein level of CHEK1 was decreased in three NSCLC cell lines when transfected with miR-195 with beta-actin as a loading control. Two positive control Cyclin D1 and Cyclin E were detected as known targets of miR-195.
Mentions: Using three miRNA databases, we identified a putative miR-195-binding site located in the 3′-UTR of CHEK1 mRNA (Figure 4A). To further validate the association between miR-195 and CHEK1, we analyzed the TCGA dataset and the result showed that the inverse correlation between miR-195 and CHEK1 was significant (r = –0.46, p < 0.0001) in NSCLC samples (Figure 4B). To confirm if miR-195 directly binds to this location in CHEK1, we cloned a full-length CHEK1 3′-UTR and inserted it into a luciferase reporter vector, downstream from the firefly luciferase gene. As for control, we made a mutant CHEK1 3′-UTR clone which had a 7-nucleotide deletion in the miR-195 binding site (Figure 4C), and the mutant clone was inserted into the same vector. Both vectors were transfected into lung cancer cells A549 and H1299 together with miR-195 or miR-NC. Our experiments showed that miR-195 significantly suppressed the luciferase activity in the CHEK1 wild type clone compared to miR-NC, but not in the mutant one (Figure 4D), suggesting that miR-195 directly binds to the 3′-UTR of CHEK1 mRNA. The transfection efficiency was examined with RT-qPCR, and our evaluation confirmed that miR-195 expression was increased substantially in the cell lines transfected with miR-195 mimic, but not in those with miR-NC (Figure 4E).

Bottom Line: Our previous study showed high miR-195 plasma levels associated with favorable overall survival of non-smoking women with lung adenocarcinoma.We demonstrated that miR-195 expression was lower in tumor tissues and was associated with poor survival outcome.High expression of CHEK1 in lung tumors was associated with poor overall survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Epidemiology and Biostatistics, Key Laboratory of Breast Cancer Prevention and Therapy, Ministry of Education, Key Laboratory of Cancer Prevention and Therapy, Tianjin, National Clinical Research Center of Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China.

ABSTRACT
MiR-195 suppresses tumor growth and is associated with better survival outcomes in several malignancies including non-small cell lung cancer (NSCLC). Our previous study showed high miR-195 plasma levels associated with favorable overall survival of non-smoking women with lung adenocarcinoma. To further elucidate role of miR-195 in NSCLC, we conducted in vitro experiment as well as clinical studies in a cohort of 299 NSCLC samples. We demonstrated that miR-195 expression was lower in tumor tissues and was associated with poor survival outcome. Overexpression of miR-195 suppressed tumor cell growth, migration and invasion. We discovered that CHEK1 was a direct target of miR-195, which decreased CHEK1 expression in lung cancer cells. High expression of CHEK1 in lung tumors was associated with poor overall survival. Our results suggest that miR-195 suppresses NSCLC and predicts lung cancer prognosis.

No MeSH data available.


Related in: MedlinePlus