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Correlation of circular RNA abundance with proliferation--exemplified with colorectal and ovarian cancer, idiopathic lung fibrosis, and normal human tissues.

Bachmayr-Heyda A, Reiner AT, Auer K, Sukhbaatar N, Aust S, Bachleitner-Hofmann T, Mesteri I, Grunt TW, Zeillinger R, Pils D - Sci Rep (2015)

Bottom Line: Interestingly, the ratio of circular to linear RNA isoforms was always lower in tumour compared to normal colon samples and even lower in colorectal cancer cell lines.This negative correlation seems to be a general principle in human tissues as validated with three different settings.Finally, we present a simple model how circular RNAs could accumulate in non-proliferating cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynaecology, Molecular Oncology Group, Comprehensive Cancer Centre, Medical University of Vienna &Ludwig Boltzmann Cluster Translational Oncology, Vienna, Austria.

ABSTRACT
Circular RNAs are a recently (re-)discovered abundant RNA species with presumed function as miRNA sponges, thus part of the competing endogenous RNA network. We analysed the expression of circular and linear RNAs and proliferation in matched normal colon mucosa and tumour tissues. We predicted >1,800 circular RNAs and proved the existence of five randomly chosen examples using RT-qPCR. Interestingly, the ratio of circular to linear RNA isoforms was always lower in tumour compared to normal colon samples and even lower in colorectal cancer cell lines. Furthermore, this ratio correlated negatively with the proliferation index. The correlation of global circular RNA abundance (the circRNA index) and proliferation was validated in a non-cancerous proliferative disease, idiopathic pulmonary fibrosis, ovarian cancer cells compared to cultured normal ovarian epithelial cells, and 13 normal human tissues. We are the first to report a global reduction of circular RNA abundance in colorectal cancer cell lines and cancer compared to normal tissues and discovered a negative correlation of global circular RNA abundance and proliferation. This negative correlation seems to be a general principle in human tissues as validated with three different settings. Finally, we present a simple model how circular RNAs could accumulate in non-proliferating cells.

No MeSH data available.


Related in: MedlinePlus

Ratio of circular to corresponding linear RNAs in 31 matched normal mucosa (No) and colorectal tumour (Ca) tissues and in eleven colorectal cancer cell lines (CL).Expression of (i) five exemplarily chosen circular RNAs (circ0817, circ3204, circ6229, circ7374, and circ7780), (ii) four corresponding linear RNAs (CUL5, USP3, METTL3, and TNS4), and (iii) ratios of circular to linear RNAs (circRNA/linRNA); determined by RT-qPCR. Circ7780 was not detectable (n.d.) in cancer cell lines. Y-axes represent relative expression values normalised to housekeeping genes for the upper two panels and the ratios of circular RNAs to linear RNAs in the lower panel. Significance for multiple comparison of No versus Ca versus CL was assessed by Kruskal-Wallis rank sum tests (given p-values) followed by pairwise comparisons applying the post-hoc Nemenyi test with Chi-squared approximation (significant pairs indicated by blue brackets).
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f3: Ratio of circular to corresponding linear RNAs in 31 matched normal mucosa (No) and colorectal tumour (Ca) tissues and in eleven colorectal cancer cell lines (CL).Expression of (i) five exemplarily chosen circular RNAs (circ0817, circ3204, circ6229, circ7374, and circ7780), (ii) four corresponding linear RNAs (CUL5, USP3, METTL3, and TNS4), and (iii) ratios of circular to linear RNAs (circRNA/linRNA); determined by RT-qPCR. Circ7780 was not detectable (n.d.) in cancer cell lines. Y-axes represent relative expression values normalised to housekeeping genes for the upper two panels and the ratios of circular RNAs to linear RNAs in the lower panel. Significance for multiple comparison of No versus Ca versus CL was assessed by Kruskal-Wallis rank sum tests (given p-values) followed by pairwise comparisons applying the post-hoc Nemenyi test with Chi-squared approximation (significant pairs indicated by blue brackets).

Mentions: For differential gene expression of circRNAs between cancer and normal tissues, the individual 31 matched normal versus CRC samples were used for RT-qPCR analysis together with two housekeeping genes as normalisers. As inferred from RNA-seq data, the ratios of circRNAs to their corresponding linear transcripts (circRNA/linRNA) were reduced in CRC tumour tissues compared to normal colon mucosa tissues, i.e. mean log2 difference over all ratios circRNA/linRNA was -0.89 between normal and CRC tumour tissues, corresponding to a 46.0% reduction in the latter samples in linear space, irrespective of whether the linear transcripts were up- or down-regulated in cancer versus normal tissues (Fig. 3). Expression of the five circRNAs and the correlation to their linear counterparts (if present) was also assessed in eleven CRC cell lines with RT-qPCR. Cancer cell lines have some different characteristics compared to cancer tissues: usually they have a higher proliferation rate (if grown under optimal culture conditions) and are pure cancer cells, sometimes even monoclonal, without tumour stroma or microenvironmental cells. Interestingly, ratios of circRNA/linRNA were even smaller in CRC cell lines compared to colorectal cancer tissues, i.e. mean log2 difference over all ratios circRNA/linRNA was -1.97 between CRC tissues and CRC cell lines, corresponding to a 74.4% reduction in the latter samples in linear space (Fig. 3). The multiple comparison of normal mucosa versus CRC tumour tissue versus CRC cell lines resulted in highly significant p-values for all four ratios of circRNA/linRNA (circ0817/CUL5: p = 3.7e-08; circ3204/USP3: p = 6.4e-10; circ6229/METTL3: p = 2.2e-09; circ7374/TNS4: p = 1.7e-05). Also, all pairwise comparisons of the four ratios of circRNA/linRNA between normal mucosa, CRC tumour tissue, and CRC cell lines reached significance (post-hoc tests, Fig. 3).


Correlation of circular RNA abundance with proliferation--exemplified with colorectal and ovarian cancer, idiopathic lung fibrosis, and normal human tissues.

Bachmayr-Heyda A, Reiner AT, Auer K, Sukhbaatar N, Aust S, Bachleitner-Hofmann T, Mesteri I, Grunt TW, Zeillinger R, Pils D - Sci Rep (2015)

Ratio of circular to corresponding linear RNAs in 31 matched normal mucosa (No) and colorectal tumour (Ca) tissues and in eleven colorectal cancer cell lines (CL).Expression of (i) five exemplarily chosen circular RNAs (circ0817, circ3204, circ6229, circ7374, and circ7780), (ii) four corresponding linear RNAs (CUL5, USP3, METTL3, and TNS4), and (iii) ratios of circular to linear RNAs (circRNA/linRNA); determined by RT-qPCR. Circ7780 was not detectable (n.d.) in cancer cell lines. Y-axes represent relative expression values normalised to housekeeping genes for the upper two panels and the ratios of circular RNAs to linear RNAs in the lower panel. Significance for multiple comparison of No versus Ca versus CL was assessed by Kruskal-Wallis rank sum tests (given p-values) followed by pairwise comparisons applying the post-hoc Nemenyi test with Chi-squared approximation (significant pairs indicated by blue brackets).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4306919&req=5

f3: Ratio of circular to corresponding linear RNAs in 31 matched normal mucosa (No) and colorectal tumour (Ca) tissues and in eleven colorectal cancer cell lines (CL).Expression of (i) five exemplarily chosen circular RNAs (circ0817, circ3204, circ6229, circ7374, and circ7780), (ii) four corresponding linear RNAs (CUL5, USP3, METTL3, and TNS4), and (iii) ratios of circular to linear RNAs (circRNA/linRNA); determined by RT-qPCR. Circ7780 was not detectable (n.d.) in cancer cell lines. Y-axes represent relative expression values normalised to housekeeping genes for the upper two panels and the ratios of circular RNAs to linear RNAs in the lower panel. Significance for multiple comparison of No versus Ca versus CL was assessed by Kruskal-Wallis rank sum tests (given p-values) followed by pairwise comparisons applying the post-hoc Nemenyi test with Chi-squared approximation (significant pairs indicated by blue brackets).
Mentions: For differential gene expression of circRNAs between cancer and normal tissues, the individual 31 matched normal versus CRC samples were used for RT-qPCR analysis together with two housekeeping genes as normalisers. As inferred from RNA-seq data, the ratios of circRNAs to their corresponding linear transcripts (circRNA/linRNA) were reduced in CRC tumour tissues compared to normal colon mucosa tissues, i.e. mean log2 difference over all ratios circRNA/linRNA was -0.89 between normal and CRC tumour tissues, corresponding to a 46.0% reduction in the latter samples in linear space, irrespective of whether the linear transcripts were up- or down-regulated in cancer versus normal tissues (Fig. 3). Expression of the five circRNAs and the correlation to their linear counterparts (if present) was also assessed in eleven CRC cell lines with RT-qPCR. Cancer cell lines have some different characteristics compared to cancer tissues: usually they have a higher proliferation rate (if grown under optimal culture conditions) and are pure cancer cells, sometimes even monoclonal, without tumour stroma or microenvironmental cells. Interestingly, ratios of circRNA/linRNA were even smaller in CRC cell lines compared to colorectal cancer tissues, i.e. mean log2 difference over all ratios circRNA/linRNA was -1.97 between CRC tissues and CRC cell lines, corresponding to a 74.4% reduction in the latter samples in linear space (Fig. 3). The multiple comparison of normal mucosa versus CRC tumour tissue versus CRC cell lines resulted in highly significant p-values for all four ratios of circRNA/linRNA (circ0817/CUL5: p = 3.7e-08; circ3204/USP3: p = 6.4e-10; circ6229/METTL3: p = 2.2e-09; circ7374/TNS4: p = 1.7e-05). Also, all pairwise comparisons of the four ratios of circRNA/linRNA between normal mucosa, CRC tumour tissue, and CRC cell lines reached significance (post-hoc tests, Fig. 3).

Bottom Line: Interestingly, the ratio of circular to linear RNA isoforms was always lower in tumour compared to normal colon samples and even lower in colorectal cancer cell lines.This negative correlation seems to be a general principle in human tissues as validated with three different settings.Finally, we present a simple model how circular RNAs could accumulate in non-proliferating cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynaecology, Molecular Oncology Group, Comprehensive Cancer Centre, Medical University of Vienna &Ludwig Boltzmann Cluster Translational Oncology, Vienna, Austria.

ABSTRACT
Circular RNAs are a recently (re-)discovered abundant RNA species with presumed function as miRNA sponges, thus part of the competing endogenous RNA network. We analysed the expression of circular and linear RNAs and proliferation in matched normal colon mucosa and tumour tissues. We predicted >1,800 circular RNAs and proved the existence of five randomly chosen examples using RT-qPCR. Interestingly, the ratio of circular to linear RNA isoforms was always lower in tumour compared to normal colon samples and even lower in colorectal cancer cell lines. Furthermore, this ratio correlated negatively with the proliferation index. The correlation of global circular RNA abundance (the circRNA index) and proliferation was validated in a non-cancerous proliferative disease, idiopathic pulmonary fibrosis, ovarian cancer cells compared to cultured normal ovarian epithelial cells, and 13 normal human tissues. We are the first to report a global reduction of circular RNA abundance in colorectal cancer cell lines and cancer compared to normal tissues and discovered a negative correlation of global circular RNA abundance and proliferation. This negative correlation seems to be a general principle in human tissues as validated with three different settings. Finally, we present a simple model how circular RNAs could accumulate in non-proliferating cells.

No MeSH data available.


Related in: MedlinePlus