miRNA Roles: Sequence Analyses of Oncogenic and Tumor Suppressive miRNAs

Research Article

miRNA Roles: Sequence Analyses of Oncogenic and Tumor Suppressive miRNAs

Corresponding author:  Dr. Shigeru Takasaki, Toyo University, 1-1-1 Izumino Itakura-machi, Ora-gun Gunma, 374-0193, Japan, Tel: +81-276-82-9024; Email: s_takasaki@toyo.jp
Abstract
MicroRNAs (miRNAs) are small (∼25 nucleotides) noncoding RNA molecules thought to play an important role in regulating gene expression. Knowledge of the biological functions of most miRNAs is still limited, but these miRNAs are thought to regulate the gene expression in various diseases. In this paper the relations between the sequences of cancer-related miRNAs (both oncogenic and tumor suppressive) and those of control miRNAs in human beings are examined from the viewpoint of nucleotide frequencies at individual positions. Oncogenic and tumor suppressive miRNAs are involved in the overexpression/upregulation and underexpression/downregulation of cancers, whereas control miRNAs are not involved  in cancer development and progression. The 132 oncogenic, 111 tumor suppressive, and 1610 control miRNA sequences investigated in this work were collected from miRBase on the basis of the relations between miRNAs of Homo sapiens and various cancers in the literature. Statistical analyses of the positional nucleotide occurrence features revealed clear differences between the cancer-related and control miRNAs. This indicates that miRNAs can be used as biomarkers in human cancers.
Keywords: miRNA; Noncoding RNA; Gene Silencing; Cancer; Oncogenic; Tumor Suppressive; Significance Test; Biomarker
Introduction

MicroRNAs (miRNAs) are small (∼25 nucleotides) noncoding RNA molecules that regulate gene expression post-transcriptionally by base-pairing to mRNAs [1-5].Many miRNAs have recently been identified in various multicellular organisms and are evolutionally conserved. Although knowledge of the biological functions of most miRNAs is still limited, these molecules are thought to regulate the gene expression at various stages in diseases.

Animal microRNAs are typically transcribed as primary transcripts (pri-miRNAs) of varying length that in the  nucleus are processed by Drosha into stem-loop precursors consisting of ∼70 nucleotides. In the cytoplasm the precursor miRNA (pre-miRNA) is cleaved by the type Ⅲ RNase Dicer to a ∼25-nucleotide product. MicroRNAs regulate the expression levels of other genes by several mechanisms, generally reducing protein levels by cleaving mRNAs or repressing their translation [2].

Many miRNAs have been reported over the past several years to be related to the expression and progression of various cancers, and miRNA profiling has recently been considered a diagnostic and prognostic tool useful for indicating an oncogene or a tumor suppressor. That is, miRNAs may be biomarkers of various cancers [6-8].

In this paper, to investigate the relations between individual miRNAs and various cancers, oncogenic and tumor suppressive miRNA sequences (hereafter called cancerrelated miRNA sequences) and control miRNA sequences were collected from miRBase according to the relations between miRNAs and various cancers in the literature [9,10]. Examination of the individual nucleotide frequencies in these sequences at positions 1 to 22 revealed clear differences between the cancer-related and control miRNAs. Consideration of the positional nucleotide occurrences of cancer-related and control miRNAs from the viewpoint of statistical significance also indicates that there are clear differences between them and implies that miRNAs can be used as biomarkers in human cancers.
Table 1. Relations between human cancers and miRNAs.
GBM: glioblastoma multiforme, B-CLL: B cell chromic lymphocytic leukemia
AML: acute myeloid leukemia, ALL: acute lymphoblastic leukemia

Sequence Analyses of Oncogenic and Tumor Suppressive miRNAs in Cancers

Relations between Cancer-Related and Control miRNAs

It has been observed that miRNAs contribute to cancer development and progression. Croce and his colleagues analyzed 540 samples of lung, breast, stomach, prostate, colon, and pancreatic tumors and found that miRNAs are differentially expressed in normal tissues and cancers [11]. Other studies have also reported that miRNAs play roles in the expression of oncogenes and tumor suppressor genes. They can therefore be used as biomarkers in human cancers. In Table 1 miRNAs that are upregulated in various cancers are listed as oncogenic miRNAs and miRNAs that are downregulated in those cancers are listed as suppressive miRNAs [3,7,8,11-44]. It is clear from Table 1 that there are differences in occurrences of the oncogenic and suppressive miRNAs for the individual cancers.

Table 2. Control miRNAs.

On the other hand, the control miRNAs of Homo sapiens can be obtained from miRBase based on considering not related to various cancers. The samples of the obtained control miRNAs are listed in Table 2. The numbers of the oncogenic, tumor suppressive, and control miRNAs were respectively 132, 111, and 1610.

Differences between Cancer-Related and Control miRNAs

To analyze the differences between cancer-related and control miRNAs, the miRNA nucleotide sequences for the oncogenic/ tumor suppressive and control miRNAs listed in Tables 1 and 2 were collected from miRBase [9,10].From the obtained miRNA sequences, the frequencies of the four nucleotides (A, G, C, U) at positions from 1 to 22 were determined. These frequencies are listed in Tables 3(A), 3(B), and 3(C). Using Tables 3(A), 3(B), and 3(C), the total frequency ratios of individual nucleotides from positions 1 to 22 were determined for the oncogenic, tumor suppressive and control miRNA sequences. They are listed in Table 4.

As a whole, the ratios of the nucleotides A and U in the oncogenic and tumor suppressive miRNA sequences are higher than those in the control sequences, whereas the ratios of the nucleotides G and C in the cancer miRNA sequences are lower than those in the control sequences.

The ratios of individual nucleotide at positions from 1 to 22 for the oncogenic, tumor suppressive and control miRNA sequences are shown Figures 1(A), 1(B), and 1(C). There are differences in nucleotide distribution ratios at individual positions for oncogenic, tumor suppressive, and control miRNAs.

The relations between individual nucleotides at positions 1 to 22 of oncogenic, tumor suppressive, and control miRNAs were then analyzed as shown in Figures 2(A), 2(B), 2(C), and 2(D).

Although there are differences in individual nucleotides at positions 1 to 22, it is not clear what position and what nucleotide is clearly different from other ones. Therefore the distinct features of individual nucleotides among the oncogenic, tumor suppressive and control miRNAs were summarized as “high” and “low” (Table 5). It is clear that there are distinct different features of individual nucleotides among oncogenic, tumor suppressive, and control miRNAs.

In addition, the differences of individual nucleotides between oncogenic and control miRNAs, the differences of individual nucleotides between tumor suppressive and control miRNAs, and the differences of individual nucleotides between oncogenic and tumor suppressive miRNAs were examined based on Figures 2(A), 2(B), 2(C), and 2(D). They are summarized as follows:

Differences between Oncogenic and Control miRNAs

Nucleotide A: As a whole, the frequency of A in the oncogenic miRNAs is 4.6% higher than that in the control miRNAs. As shown in Figure 2(A), the especially higher positions are 2, 10, 13, 16, and 22.

Nucleotide G: As a whole, the frequency of G in the oncogenic miRNAs is 5.3% lower than that in the control miRNAs. As shown in Figure 2(B), the especially lower positions are 1, 4, 8, 9, 10, 12, and 13.

Nucleotide C: On the average, the frequency of C in the oncogenic miRNAs is 2% lower than that in the control miRNAs. As shown in Figure 2(C), however, there are clearly two kinds of positions with regard to the relative C frequency in the oncogenic miRNAs: the higher positions (i.e., positions  4 and 12) and the lower positions (i.e., positions 5, 9, 19, 20,and 22).

Nucleotide U: On the average, the frequency of U in the oncogenic miRNAs is 2.7% higher than that in the control miRNAs. As shown in Figure 2(D), there are two kinds of positions with regard to the relative U frequency in the oncogenic miRNAs: the higher positions (positions 9 and 21) and the lower positions (positions 2 and 18).

Differences between Tumor Suppressive and Control miRNAs

Nucleotide A: On the average, the frequency of A in the tumor suppressive miRNAs is 2.3% higher than that in the control miRNAs. As shown in Figure 2(A), however, there are clearly two kinds of positions with regard to the relative A frequency in the tumor suppressive miRNAs: the higher positions (positions 3, 5, 7, 10, and 17) and the lower positions (positions 6, 11, 20, and 22).

Nucleotide G: On the average, the frequency of G in the tumor suppressive miRNAs is 1.5% lower than that in the control miRNAs. As shown in Figure 2(B), however, again there are two kinds of positions with regard to the relative G frequency: the higher positions (2, 8, 15, and 20) and the lower positions (3, 10, 13, 14, 16, and 21).

Nucleotide C: As a whole, the frequency of C in the tumor suppressive miRNAs is 5.7% lower than that in the control miRNAs. As shown in Figure 2(C), the lower positions are positions 1, 5, 6, 8, 9, 14, 15, 17, 18, and 19.

Nucleotide U: As a whole, the nucleotide frequency of the tumor suppressive miRNAs is 6% higher than that of the control miRNAs. As shown in Figure 2(D), the higher positions are 1, 6, 9, 13, 14, 18, 21, and 22.

Differences between Oncogenic and Tumor Suppressive miRNAs

Nucleotide A: On the average, the frequency of A in the oncogenic miRNAs is 2.3% higher than that in the tumor suppressive miRNAs. As shown in Figure 2(A), the higher positions are 1, 2, 6, 11, 13, 16, 18, and 22, whereas the lower positions are 3, 5, 10, 17, and 19.

Nucleotide G: As a whole, the frequency of G in the oncogenic miRNAs is 3.8% lower than that in the tumor suppressive miRNAs. As shown in Figure 2(B), the lower positions are 1, 2, 4, 8, 11, 12, 15, and 20, whereas the higher positions are 7, 14, 16, 17, 18, and 21.

Nucleotide C: As a whole, the frequency of C in the oncogenic miRNAs is 4.7% higher than that in the tumor suppressive miRNAs. As shown in Figure 2(C), the higher positions are 1, 4, 6, 8, 12, 14, 15, 17, and 18, whereas the lower positions are 21 and 22.

Nucleotide U: On the average, the frequency of U in the oncogenic miRNAs is 3.3% lower than that in the tumor suppressive miRNAs. As shown in Figure 2(D), the lower positions are 1, 6, 13, 14, 16, 18, and 22, whereas the higher positions are 4, 5, 8, 10, 12, 15, 19, and 20.

The above analyses revealed that there are differences in individual nucleotide occurrences at positions among oncogenic, tumor suppressive, and control miRNAs. Although there were differences in nucleotide distribution ratios, it is not clear that how nucleotide differences are significant statistically.

Positional Nucleotide Features of Cancer-Related and Control miRNAs

To analyze the significance of the nucleotide frequency differences statistically, two-sample population testing (not in pairs) was used for the number of nucleotides at individual positions and the total number of nucleotides in all positions. The following significance test was carried out.

where s is the site (position) 1 to 22, pas is the probability of each nucleotide a occurring at each site s (a = A, G, C, or U), pb is the occurrence probability of each nucleotide averaged over the entire target sequence population, P is the arithmetic mean of pas and pb, nas is the number of nucleotides at position s, and nb is the total number of nucleotides in all positions.

As the two-sided statistical test has two types of significance values, higher (upper) and lower levels of significance, they are expressed as follows:

Higher-significance nucleotide (v HN s ) and Lower-significance nucleotide ( v LN s ),

where H denotes higher, L denotes lower, and N is a nucleotide, v: 95-significance probability is 95% (level of significance = 0.05),

99-significance probability is 99% (level of significance = 0.01), and s: nucleotide position (site) ( i.e., 1–22).

Nucleotide frequencies at the individual positions listed in Tables 3(A), 3(B), and 3(C) were analyzed by using Eq. (1), and many higher-significance and lower-significance nucleotides were obtained. They are listed in Tables 6(A), 6(B), and 6(C).

Higher- and Lower-Level Nucleotides of Oncogenic, Suppressive, and Control miRNAs

Higher- and lower-level nucleotides at individual positions in oncogenic and control miRNAs and in suppressive and control miRNAs are listed in Tables 7(A) and 7(B). Since the higher- and lower-level nucleotides at individual positions are those that have a larger influence on the upregulation or downregulation due to miRNAs, the coincidences between higher- and lower-level nucleotides of oncogenic and control miRNAs and the coincidences between higher- and lowerlevel nucleotides of suppressive and control miRNAs were examined. The coincidences between individual nucleotides of oncogenic and control miRNAs and the coincidences between individual nucleotides of suppressive and control miRNAs at individual positions are listed in Table 8. As the degrees of coincidences of each distinct nucleotide are not clear in the tables, they are shown in Figures 3(A) and 3(B).  Although one can infer from Tables 7 and 8 and Figures3(A) and 3(B) that there are differences in the coincidences between individual nucleotides of oncogenic/suppressive and control miRNAs, how many nucleotides are totally different in oncogenic/suppressive and control miRNAs is not clear. To clarify the differences quantitatively, the number of same and different nucleotides at individual positions were examined. The results are listed in Table 9. In the higher oncogenic miRNAs, the number of the same nucleotides is 7, whereas the number of the different nucleotides is 19. This means that the different nucleotides are 2.71 times more likely than the same nucleotides in the comparison with the oncogenic and control miRNAs. Similarly, in the lower oncogenic miRNAs, the different nucleotides are 3.14 times more likely than the same nucleotides. There are similar tendencies in the higher and lower suppressive miRNAs, i.e., the different nucleotides are respectively 2.11 times and 2.56 times more frequently than the same nucleotides. From the nucleotide position point of view, there is a tendency that there are many different nucleotides from positions 12 to 22 (Table 9).

 

Conclusions
In this paper the cancer-related (oncogenic and tumor suppressive) and control miRNAs of human being were analyzed and clear differences between the positional occurrences of cancer-related and control miRNAs were shown. This paper also considered the positional nucleotide occurrences of cancer-related and control miRNAs from the viewpoint of statistical significance. This result also indicates that there are clear differences between them and implies that miRNAs can be used as biomarkers in human cancers.
 References

1. Daniel WK Kao, Joseph P Fiorellini. An interarch alveolar ridge relationship classification. Int J Periodontics Restorative Dent. 2010, 30(5): 523-529.

2. Hom-Lay Wang, Khalaf Al-Shammari, HVC ridge deficiency classification: a therapeutically oriented classification. Int J Periodontics Restorative Dent. 2002, 22(4): 335-343.

3. Tinti C, Parma-Benfenati S. Clinical classification of bone defects concerning the placement of dental implants. Int J Periodontics Restorative Dent. 2003, 23(2): 147-155.

4. Studer S, Naef R, Scharer P. Adjustment of localized alveolar ridge defects by soft tissue transplantation to improve mucogingival esthetics: A proposal for clinical classification and evaluation of procedures. Quintessence Int. 1997, 28(12): 785- 805.

5. Khojasteh A, Morad G, Behnia H. Clinical importance of recipient site characteristics for vertical ridge augmentation: a systematcic review of literature and proposal of a classification. J Oral Implantol. 2013, 39(3): 386-398..

6. Kubiak EN, Beebe MJ, North K, Hitchcock R, Potter MQ. Early weight bearing after lower extremity fractures in adults. J AmAcad Orthop Surg. 2013, 21(12): 727-738.

7. Bel JC, Court C, Cogan A, Chantelot C, Piétu G et al. Unicondylar fractures of the distal femur. Orthop Traumatol Surg Res. 2014, 100(8): 873-877.

8.Bel JC, Court C, Cogan A, Chantelot C, Piétu G et al. Unicondylar fractures of the distal femur. Orthop Traumatol Surg Res. 2014, 100(8): 873-877.

9.Lepley CR, Throckmorton GS, Ceen RF, Buschang PH. Relative contributions of occlusion, maximum bite force, and chewing cycle kinematics to masticatory performance. Am J Orthod Dentofacial Orthop. 2011, 139(5): 606-613.

10. Takaki P, Vieira M, Bommarito S. Maximum bite force analysis in different age groups. Int Arch Otorhinolaryngol. 2014, 18(3): 272-276.

11. Drăgulescu D, Rusu L, Dreucean M, Toth-Tascau M. Stress and deformation analysis induced by dental implants in mandible. Rev Med Chir Soc Med Nat Iasi. 2006, 110(1): 232-235.

12. Li Z, Kuhn G, von Salis-Soglio M, Cooke SJ, Schirmer M et al. In vivo monitoring of bone architecture and remodeling after implant insertion: The different responses ofcortical and trabecular bone. Bone. 2015, 81: 468-477.

13. Kopp S, Kuzelka J, Goldmann T, Himmlova L, Ihde S, Modeling of load transmission and distribution of deformation energy before and after healing of basal dental implants in the human mandible. Biomed Tech (Berl). 2011, 56(1): 53-58.

14. Stefan Ihde. Principles of BOI, Scientific and Practical Guidelines to 4-D Dental Implantology . Springer, Heidelberg, 2005.

15. Donsimoni JM, Dohan D. Les implants maxillo-faciaux à plateaux d’assise: Concepts et technologies orthopédiques, réhabilitations maxillo-mandibulaires, reconstructions maxillo- faciales, réhabilitations dentaires partielles, techniques de réintervention, méta-analyse. 1re partie : concepts et technologies orthopédiques. Implantodontie. 2004, 13(1): 13-30.

16. Ihde S, Kopp S, Gundlach K, Konstantinović VS. Effects of radiation therapy on craniofacial and dental implants: a review of the literature. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontics. 2009, 107(1): 56-65.

17. Konstantinović VS, Lazić V, Stefan I. Nasal epithesis retained by basal (disk) implants. J Craniofac Surg. 2010, 21(1): 33-36.

18.

19. Jemt T, Lekholm U, Adell R. Osseointegrated implants in the treatment of partially edentulous patients: a preliminary study on 876 consecutively placed fixtures. Int J Oral Maxillofac Implants, 1989, 4(3): 211-217.

20. Cawood JI, Howell RA. A classification of the edentulous jaws. Int J Oral Maxillofac Surg. 1988, 17(4): 232-236.

21. Atwood DA. The reduction of residual ridges: a major oral disease entity. J Prosthet Dent. 1971, 26(3): 266-279.

22. Atwood DA. Bone Loss of Edentulous Alveolar Ridges. Journal of Periodontology. 1979, 50(4): 11-21.

23. Seibert JS. Reconstruction of deformed, partially edentulous ridges, using full thickness onlay grafts. Part I. Technique and wound healing. Compend Contin Educ Dent. 1983, 4(5): 437-53.

24. Allen EP, Gainza CS, Farthing GG, Newbold DA. Improved technique for localised ridge augmentation. A report of 21 cases. J Periodontol. 1985, 56(4): 195-199.

25. Peñarrocha M, Carrillo C, Boronat A, Peñarrocha M. Retrospective study of 68 implants placed in the pterygomaxillary region using drills and osteotomes. Int J Oral Maxillofac Implants. 2009, 24(4): 720-726.

26. Konsensus für die dentale Implantologie: Beschreibung der Wege zur Erzielung der Osseointegration. The International Implant Foundation (IF), Munich.

27.

28.

29. Wang L, Ye T, Deng L, Shao J, Qi J et al. Repair of Microdamage in Osteonal Cortical Bone Adjacent to Bone Screw. PLoS one. 2014, 9(2): e89343.

Be the first to comment on " miRNA Roles: Sequence Analyses of Oncogenic and Tumor Suppressive miRNAs"

Leave a comment

Your email address will not be published.


*


Select Language