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Downregulation of six microRNAs is associated with advanced stage, lymph node metastasis and poor prognosis in small cell carcinoma of the cervix.

Huang L, Lin JX, Yu YH, Zhang MY, Wang HY, Zheng M - PLoS ONE (2012)

Bottom Line: Seven miRNAs, has-let-7c, has-miR-10b, has-miR-100, has-miR-125b, has-miR-143, has-miR-145 and has-miR-199a-5p were significantly down-regulated in advanced stage SCCC patients (FIGO IB2-IV) compared to early stage SCCC patients (FIGOIB1).Among, downregulation of six miRNAs, has-let-7c, has-miR-100, has-miR-125b, has-miR-143, has-miR-145 and has-miR-199a-5p were significantly associated with lymph node metastasis and reduced survival in SCCC.This study demonstrates that downregulation of 7 miRNA associated with advanced stage, 6 miRNAs with metastasis and 2 with poor prognosis in SCCC.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Oncology in Southern China, Cancer Center, Sun Yat-sen University, Guangzhou, People's Republic of China.

ABSTRACT

Background: Small cell carcinoma of the cervix (SCCC) is very rare, and due to the long time period required to recruit sufficient numbers of patients, there is a paucity of information regarding the prognostic factors associated with survival. MicroRNAs (miRNAs) have been used as cancer-related biomarkers in a variety of tumor types, and the objective of this study was to determine whether microRNA expression profiles can predict clinical outcome in SCCC.

Methodology/principal findings: Forty-four patients with SCCC who underwent radical hysterectomy between January 2000 and October 2009 were enrolled. Using the GeneCopoeia All-in-One™ Customized Human qPCR Primer Array, the expression profiles of 30 miRNAs associated with tumor metastasis was obtained from the formalin-fixed paraffin embedded samples of all 44 patients. Seven miRNAs, has-let-7c, has-miR-10b, has-miR-100, has-miR-125b, has-miR-143, has-miR-145 and has-miR-199a-5p were significantly down-regulated in advanced stage SCCC patients (FIGO IB2-IV) compared to early stage SCCC patients (FIGOIB1). Among, downregulation of six miRNAs, has-let-7c, has-miR-100, has-miR-125b, has-miR-143, has-miR-145 and has-miR-199a-5p were significantly associated with lymph node metastasis and reduced survival in SCCC. Kaplan-Meier survival analyses revealed that SCCC patients with low expression of has-miR-100 (P = 0.019) and has-miR-125b (P = 0.020) projected a significant tendency towards poorer prognosis.

Conclusions/significance: This study demonstrates that downregulation of 7 miRNA associated with advanced stage, 6 miRNAs with metastasis and 2 with poor prognosis in SCCC. Functional analysis of these miRNAs may enhance our understanding of SCCC, as altered expression of specific miRNAs may regulate the metastatic pathway and provide novel targets for therapy.

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Validation of the All-in-One™ Customized Human qPCR Primer Array detection system.(A) Amplification of 30 miRNAs (P1-P30, see Table S2; P24-P30 and SNORD44 are not show in this figure because of limit lane on a single agarose gel; lane1: FFPET human cervix, lane2: frozen human cervix, lane3: RT-minus control and lane4: negative control for each miRNA) and two reference genes (RNU6B, SNORD44) from cDNA synthesized from human cervix total RNA. All qPCR products were detected by electrophoresis on a 2.5% agarose gel, indicating a single product of the correct size for each target gene. (B) A significant correlation between the miRNA expression patterns in cDNA synthesized from formalin-fixed paraffin-embedded small cell carcinoma of the cervix samples (plat1: sample 1, sample 2, sample 3 and plat2: sample 1, sample 2, sample 3) was observed, R2 = 0.943, demonstrating a high reproducibility and reliable detection of miRNA expression levels.
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pone-0033762-g001: Validation of the All-in-One™ Customized Human qPCR Primer Array detection system.(A) Amplification of 30 miRNAs (P1-P30, see Table S2; P24-P30 and SNORD44 are not show in this figure because of limit lane on a single agarose gel; lane1: FFPET human cervix, lane2: frozen human cervix, lane3: RT-minus control and lane4: negative control for each miRNA) and two reference genes (RNU6B, SNORD44) from cDNA synthesized from human cervix total RNA. All qPCR products were detected by electrophoresis on a 2.5% agarose gel, indicating a single product of the correct size for each target gene. (B) A significant correlation between the miRNA expression patterns in cDNA synthesized from formalin-fixed paraffin-embedded small cell carcinoma of the cervix samples (plat1: sample 1, sample 2, sample 3 and plat2: sample 1, sample 2, sample 3) was observed, R2 = 0.943, demonstrating a high reproducibility and reliable detection of miRNA expression levels.

Mentions: Each primer set cross-linked to the All-in-One qPCR Primer Array was validated to amplify a single product of the correct size for each target gene from microRNAs isolated from human formalin-fixed paraffin-embedded and frozen cervix tissue (Figure 1A). The peak values of the amplification and melting curves indicated a single amplification product was obtained in each reaction (data not shown). A significant correlation was observed in the miRNA expression pattern of plat1 (SCCC FFPET sample 1, sample 2, sample 3) and plat2 (SCCC FFPET sample 1, sample 2, sample 3), with an R2 value of 0.943(Figure 1B). In addition, all PCR amplification products were confirmed by sequencing (Figure 2).Taken together, these results indicate that the All-in-One™ Customized Human qPCR Primer Array has a high specificity and sensitivity to detect miRNA expression levels in SCCC FFPET samples, with a high degree of reproducibility.


Downregulation of six microRNAs is associated with advanced stage, lymph node metastasis and poor prognosis in small cell carcinoma of the cervix.

Huang L, Lin JX, Yu YH, Zhang MY, Wang HY, Zheng M - PLoS ONE (2012)

Validation of the All-in-One™ Customized Human qPCR Primer Array detection system.(A) Amplification of 30 miRNAs (P1-P30, see Table S2; P24-P30 and SNORD44 are not show in this figure because of limit lane on a single agarose gel; lane1: FFPET human cervix, lane2: frozen human cervix, lane3: RT-minus control and lane4: negative control for each miRNA) and two reference genes (RNU6B, SNORD44) from cDNA synthesized from human cervix total RNA. All qPCR products were detected by electrophoresis on a 2.5% agarose gel, indicating a single product of the correct size for each target gene. (B) A significant correlation between the miRNA expression patterns in cDNA synthesized from formalin-fixed paraffin-embedded small cell carcinoma of the cervix samples (plat1: sample 1, sample 2, sample 3 and plat2: sample 1, sample 2, sample 3) was observed, R2 = 0.943, demonstrating a high reproducibility and reliable detection of miRNA expression levels.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3306296&req=5

pone-0033762-g001: Validation of the All-in-One™ Customized Human qPCR Primer Array detection system.(A) Amplification of 30 miRNAs (P1-P30, see Table S2; P24-P30 and SNORD44 are not show in this figure because of limit lane on a single agarose gel; lane1: FFPET human cervix, lane2: frozen human cervix, lane3: RT-minus control and lane4: negative control for each miRNA) and two reference genes (RNU6B, SNORD44) from cDNA synthesized from human cervix total RNA. All qPCR products were detected by electrophoresis on a 2.5% agarose gel, indicating a single product of the correct size for each target gene. (B) A significant correlation between the miRNA expression patterns in cDNA synthesized from formalin-fixed paraffin-embedded small cell carcinoma of the cervix samples (plat1: sample 1, sample 2, sample 3 and plat2: sample 1, sample 2, sample 3) was observed, R2 = 0.943, demonstrating a high reproducibility and reliable detection of miRNA expression levels.
Mentions: Each primer set cross-linked to the All-in-One qPCR Primer Array was validated to amplify a single product of the correct size for each target gene from microRNAs isolated from human formalin-fixed paraffin-embedded and frozen cervix tissue (Figure 1A). The peak values of the amplification and melting curves indicated a single amplification product was obtained in each reaction (data not shown). A significant correlation was observed in the miRNA expression pattern of plat1 (SCCC FFPET sample 1, sample 2, sample 3) and plat2 (SCCC FFPET sample 1, sample 2, sample 3), with an R2 value of 0.943(Figure 1B). In addition, all PCR amplification products were confirmed by sequencing (Figure 2).Taken together, these results indicate that the All-in-One™ Customized Human qPCR Primer Array has a high specificity and sensitivity to detect miRNA expression levels in SCCC FFPET samples, with a high degree of reproducibility.

Bottom Line: Seven miRNAs, has-let-7c, has-miR-10b, has-miR-100, has-miR-125b, has-miR-143, has-miR-145 and has-miR-199a-5p were significantly down-regulated in advanced stage SCCC patients (FIGO IB2-IV) compared to early stage SCCC patients (FIGOIB1).Among, downregulation of six miRNAs, has-let-7c, has-miR-100, has-miR-125b, has-miR-143, has-miR-145 and has-miR-199a-5p were significantly associated with lymph node metastasis and reduced survival in SCCC.This study demonstrates that downregulation of 7 miRNA associated with advanced stage, 6 miRNAs with metastasis and 2 with poor prognosis in SCCC.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Oncology in Southern China, Cancer Center, Sun Yat-sen University, Guangzhou, People's Republic of China.

ABSTRACT

Background: Small cell carcinoma of the cervix (SCCC) is very rare, and due to the long time period required to recruit sufficient numbers of patients, there is a paucity of information regarding the prognostic factors associated with survival. MicroRNAs (miRNAs) have been used as cancer-related biomarkers in a variety of tumor types, and the objective of this study was to determine whether microRNA expression profiles can predict clinical outcome in SCCC.

Methodology/principal findings: Forty-four patients with SCCC who underwent radical hysterectomy between January 2000 and October 2009 were enrolled. Using the GeneCopoeia All-in-One™ Customized Human qPCR Primer Array, the expression profiles of 30 miRNAs associated with tumor metastasis was obtained from the formalin-fixed paraffin embedded samples of all 44 patients. Seven miRNAs, has-let-7c, has-miR-10b, has-miR-100, has-miR-125b, has-miR-143, has-miR-145 and has-miR-199a-5p were significantly down-regulated in advanced stage SCCC patients (FIGO IB2-IV) compared to early stage SCCC patients (FIGOIB1). Among, downregulation of six miRNAs, has-let-7c, has-miR-100, has-miR-125b, has-miR-143, has-miR-145 and has-miR-199a-5p were significantly associated with lymph node metastasis and reduced survival in SCCC. Kaplan-Meier survival analyses revealed that SCCC patients with low expression of has-miR-100 (P = 0.019) and has-miR-125b (P = 0.020) projected a significant tendency towards poorer prognosis.

Conclusions/significance: This study demonstrates that downregulation of 7 miRNA associated with advanced stage, 6 miRNAs with metastasis and 2 with poor prognosis in SCCC. Functional analysis of these miRNAs may enhance our understanding of SCCC, as altered expression of specific miRNAs may regulate the metastatic pathway and provide novel targets for therapy.

Show MeSH
Related in: MedlinePlus