mRNA-lncRNA gene expression signature in HPV-associated neoplasia and cervical cancer

Cervical cancer is one of the most frequent cancers in women and is associated with human papillomavirus (HPV) in 70 % of cases. Cervical cancer occurs because of progression of low-differentiated cervical intraepithelial neoplasia through grade 2 and 3 lesions. Along with the protein-coding genes, long noncoding RNAs (lncRNAs) play an important role in the development of malignant cell transformation. Although human papillomavirus is widespread, there is currently no well-characterized transcriptomic signature to predict whether this tumor will develop in the presence of HPV-associated neoplastic changes in the cervical epithelium. Changes in gene activity in tumors reflect the biological diversity of cellular phenotype and physiological functions and can be an important diagnostic marker. We performed comparative transcriptome analysis using open RNA sequencing data to assess differentially expressed genes between normal tissue, neoplastic epithelium, and cervical cancer. Raw data were preprocessed using the Galaxy platform. Batch effect correction, identification of differentially expressed genes, and gene set enrichment analysis (GSEA) were performed using R programming language packages. Subcellular localization of lncRNA was analyzed  using Locate-R and iLoc-LncRNA 2.0 web services. 1,572 differentially expressed genes (DEGs) were recorded in the “cancer vs. control” comparison, and 1,260 DEGs were recorded in the “cancer vs. neoplasia” comparison. Only two genes were observed to be differentially expressed in the “neoplasia vs. control” comparison. The search for common genes among the most strongly differentially expressed genes among all comparison groups resulted in the identification of an expression signature consisting of the CCL20, CDKN2A, CTCFL, piR-55219, TRH, SLC27A6 and EPHA5 genes. The transcription level of the CCL20 and CDKN2A genes becomes increased at the stage of neoplastic epithelial changes and stays so in cervical cancer. Validation on an independent microarray dataset showed that the differential expression patterns of the CDKN2A and SLC27A6 genes were conserved in the respective gene expression comparisons between groups.

1. Ahmad A., Lin H., Shatabda S. Locate-R: subcellular localization of long non-coding RNAs using nucleotide compositions. Genomics. 2020;112(3):2583-2589. DOI 10.1016/j.ygeno.2020.02.011

2. Asano T., Hirohashi Y., Torigoe T., Mariya T., Horibe R., Kuroda T., Tabuchi Y., Saijo H., Yasuda K., Mizuuchi M., Takahashi A., Asanuma H., Hasegawa T., Saito T., Sato N. Brother of the regulator of the imprinted site (BORIS) variant subfamily 6 is involved in cervical cancer stemness and can be a target of immunotherapy. Oncotarget. 2016;7(10):11223-11237. DOI 10.18632/oncotarget.7165

3. Bao Y., Wang L., Shi L., Yun F., Liu X., Chen Y., Chen C., Ren Y., Jia Y. Transcriptome profiling revealed multiple genes and ECM-receptor interaction pathways that may be associated with breast cancer. Cell. Mol. Biol. Lett. 2019;24:38. DOI 10.1186/s11658-019-0162-0

4. Beatty G.L., Gladney W.L. Immune escape mechanisms as a guide for cancer immunotherapy. Clin. Cancer Res. 2015;21(4):687-692. DOI 10.1158/1078-0432.CCR-14-1860

5. Binnewies M., Roberts E.W., Kersten K., Chan V., Fearon D., Merad M., Coussens L., Gabrilovich D., Ostrand-Rosenberg S., Hed rick C., Vonderheide R., Pittet M., Jain R., Zou W., Howcroft T., Woodhouse E., Weinberg R., Krummel M. Understanding the tumor immune microenvironment (TIME) for effective therapy. Nat. Med. 2018;24(5):541-550. DOI 10.1038/s41591-018-0014-x

6. Chaiwongkot A., Buranapraditkun S., Oranratanaphan S., Chuen-Im T., Kitkumthorn N. Efficiency of CIN2+ detection by thyrotropin-releasing hormone (TRH) site-specific methylation. Viruse. 2023; 15(9):1802. DOI 10.3390/v15091802

7. Chen X., Wang X., Wei X., Wang J. EphA5 protein, a potential marker for distinguishing histological grade and prognosis in ovarian serous carcinoma. J. Ovarian Res. 2016;9(1):83. DOI 10.1186/s13048-0160292-1

8. Chen Z., Guo Y., Zhao D., Zou Q., Yu F., Zhang L., Xu L. Comprehensive analysis revealed that CDKN2A is a biomarker for immune infiltrates in multiple cancers. Front. Cell Dev. Biol. 2021;9:808208. DOI 10.3389/fcell.2021.808208

9. Dai J., Yu X., Han Y., Chai L., Liao Y., Zhong P., Xie R., Sun X., Huang Q., Wang J., Yin Z., Zhang Y., Lv Z., Jia C. TMT-labeling proteomics of papillary thyroid carcinoma reveal invasive biomarkers. J. Cancer. 2020;11(20):6122-6132. DOI 10.7150/jca.47290

10. Debaugny R., Skok J. CTCF and CTCFL in cancer. Curr. Opin. Genet. Dev. 2020;61:44-52. DOI 10.1016/j.gde.2020.02.021

11. Eklund C., Lagheden C., Robertsson K.D., Forslund O., Dillner J. Technical Report on the Global HPV LabNet DNA Genotyping Proficiency Panel 2019. International Human Papillomavirus (HPV) Reference Center, 2020

12. Fernandes A.T., Carvalho M., Avvad-Portari E., Rocha N., Russomano F., Roma E.H., Bonecini-Almeida M. A prognostic value of CD45RA+, CD45RO+, CCL20+ and CCR6+ expressing cells as ‘immunoscore’ to predict cervical cancer induced by HPV. Sci. Rep. 2021;11(1):8782. DOI 10.1038/s41598-021-88248-x

13. Fu D.Y., Wang Z.M., Wang B.L., Chen L., Yang W.T., Shen Z.Z., Huang W., Shao Z.M. Frequent epigenetic inactivation of the receptor tyrosine kinase EphA5 by promoter methylation in human breast cancer. Hum. Pathol. 2010;41(1):48-58. DOI 10.1016/j.humpath.2009.06.007

14. Gao Y., Zhou J., Mao J., Jiang L., Li X.-P. Identification of the Thyrotropin-Releasing Hormone (TRH) as a novel biomarker in the pro gnosis for acute myeloid leukemia. Biomolecules. 2022;12(10): 1359. DOI 10.3390/biom12101359

15. Gebrie A. Disease progression role as well as the diagnostic and prognostic value of microRNA-21 in patients with cervical cancer: a systematic review and meta-analysis. PLoS One. 2022;17(7):e0268480. DOI 10.1371/journal.pone.0268480

16. Guess J.C., McCance D.J. Decreased migration of Langerhans precursor-like cells in response to human keratinocytes expressing human Papillomavirus type 16 E6/E7 is related to reduced macrophage inflammatory protein-3α production. J. Virol. 2005;79(23):14852-14862. DOI 10.1128/JVI.79.23.14852-14862.2005

17. Hu Z., Zhu D., Wang W., Li W., Jia W., Zeng X., Ding W., Yu L., Wang X., Wang L., Shen H., Zhang C., Liu H., Liu X., Zhao Y., Fang X., Li S., Chen W., Tang T., Fu A., Wang Z., Chen G., Gao Q., Li S., Xi L., Wang C., Liao S., Ma X., Wu P., Li K., Wang S., Zhou J., Wang J., Xu X., Wang H., Ma D. Genome-wide profiling of HPV integration in cervical cancer identifies clustered genomic hot spots and a potential microhomology-mediated integration mechanism. Nat. Genet. 2015;47(2):158-163. DOI 10.1038/ng.3178

18. Jiang B., Xue M. Correlation of E6 and E7 levels in high-risk HPV16 type cervical lesions with CCL20 and Langerhans cells. Genet. Mol. Res. 2015;14(3):10473-10481. DOI 10.4238/2015.September.8.8

19. Karimzadeh M., Arlidge C., Rostami A., Lupien M., Bratman S., Hoffman M. Human papillomavirus integration transforms chromatin to drive oncogenesis. Genome Biol. 2023;24(1):142. DOI 10.1186/s13059-023-02926-9

20. Kober P., Bujko M., Olędzki J., Tysarowski A., Siedlecki J.A. MethylCpG binding column-based identification of nine genes hypermethylated in colorectal cancer. Mol. Carcinog. 2011;50(11):846-856. DOI 10.1002/mc.20763

21. Li J., Zhou C., Zhou H., Bao T., Gao T., Jiang X., Ye M. The association between methylated CDKN2A and cervical carcinogenesis, and its diagnostic value in cervical cancer: a meta-analysis. Ther. Clin. Risk Manag. 2016;12:1249-1260. DOI 10.2147/TCRM.S108094

22. Li S., Zhu Y., Ma C., Qiu Z., Zhang X., Kang Z., Wu Z., Wang H., Xu X., Zhang H., Ren G., Tang J., Li X., Guan M. Downregulation of EphA5 by promoter methylation in human prostate cancer. BMC Cancer. 2015;15:18. DOI 10.1186/s12885-015-1025-3

23. Luan Y., Zhang W., Xie J., Mao J. CDKN2A inhibits cell proliferation and invasion in cervical cancer through LDHA-mediated AKT/ mTOR pathway. Clin. Transl. Oncol. 2021;23(2):222-228. DOI 10.1007/s12094-020-02409-4

24. Martin J.A., Wang Z. Next-generation transcriptome assembly. Nat. Rev. Genet. 2011;12(10):671-682. DOI 10.1038/nrg3068

25. Mazibrada J., Rittà M., Mondini M., De Andrea M., Azzimonti B., Borgogna C., Ciotti M., Orlando A., Surico N., Chiusa L., Landolfo S., Gariglio M. Interaction between inflammation and angiogenesis during different stages of cervical carcinogenesis. Gynecol. Oncol. 2008;108(1):112-120. DOI 10.1016/j.ygyno.2007.08.095

26. Nandi B., Pai C., Huang Q., Prabhala R., Munshi N., Gold J. CCR6, the sole receptor for the chemokine CCL20, promotes spontaneous intestinal tumorigenesis. PLoS One. 2014;9(5):e97566. DOI 10.1371/journal.pone.0097566

27. Okunade K.S. Human papillomavirus and cervical cancer. J. Obstet. Gynaecol. 2020;40(5):602-608. DOI 10.1080/01443615.2019.1634030

28. Puttipanyalears C., Arayataweegool A., Chalertpet K., Rattanachayoto P., Mahattanasakul P., Tangjaturonsasme N., Kerekhanjanarong V., Mutirangura A., Kitkumthorn N. TRH site-specific methylation in oral and oropharyngeal squamous cell carcinoma. BMC Cancer. 2018;18(1):786. DOI 10.1186/s12885-018-4706-x

29. Qi D., Li H., Wang S., Wang S., Zheng R., Liu N., Han B., Liu L. Construction of ceRNA network and key gene screening in cervical squamous intraepithelial lesions. Medicine (Baltimore). 2022; 101(48):e31928. DOI 10.1097/MD.0000000000031928

30. Robertson K.D., Jones P.A. Tissue-specific alternative splicing in the human INK4a/ARF cell cycle regulatory locus. Oncogene. 1999;

31. (26):3810-3820. DOI 10.1038/sj.onc.1202737

32. Royse K., Zhi D., Conner M., Clodfelder-Miller B., Srinivasasainagendra V., Vaughan L., Skibola C., Crossman D., Levy S., Shrestha S. Differential gene expression landscape of co-existing cervical pre-cancer lesions using RNA-seq. Front. Oncol. 2014;4:339. DOI 10.3389/fonc.2014.00339

33. Schubert M., Bauerschlag D., Muallem M., Maass N., Alkatout I. Challenges in the diagnosis and individualized treatment of cervical cancer. Medicina (Kaunas). 2023;59(5):925. DOI 10.3390/medicina59 050925

34. Siddiqi S., Matushansky I. Piwis and piwi-interacting RNAs in the epigenetics of cancer. J. Cell. Biochem. 2012;113(2):373-380. DOI 10.1002/jcb.23363

35. Soltanian S., Dehghani H. BORIS: a key regulator of cancer stemness. Cancer Cell Int. 2018;18:154. DOI 10.1186/s12935-018-0650-8

36. Su Z.D., Huang Y., Zhang Z.Y., Zhao Y.W., Wang D., Chen W., Chou K.C., Lin H. iLoc-lncRNA: predict the subcellular location of lncRNAs by incorporating octamer composition into general Conflict of interest. Received July 21, 2023. Revised January 18, 2024. Accepted January 19, 2024. PseKNC. Bioinformatics. 2018;34(24):4196-4204. DOI 10.1093/bioinformatics/bty508

37. Sung H., Ferlay J., Siegel R.L., Laversanne M., Soerjomataram I., Jemal A., Bray F. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2021;71(3):209-249. DOI 10.3322/caac.21660

38. Suzuki R., Honda S., Kirino Y. PIWI expression and function in cancer. Front. Gene. 2012;3:204. DOI 10.3389/fgene.2012.00204

39. Walch-Ruckheim B., Mavrova R., Henning M., Vicinus B., Kim Y.J., Bohle R., Juhasz-Boss I., Solomayer E.F., Smola S. Stromal fibroblasts induce CCL20 through IL6/C/EBPβ to support the recruitment of Th17 cells during cervical cancer progression. Cancer Res. 2015;75(24):5248-5259. DOI 10.1158/0008-5472.CAN-15-0732

40. Wang X., Gao X.H., Hong Y., Li X., Chen H.D. Local hyperthermia decreases the expression of CCL-20 in condyloma acuminatum. Virol. J. 2010;7:301. DOI 10.1186/1743-422X-7-301

41. Wijetunga N.A., Belbin T., Burk R., Whitney K., Abadi M., Greally J., Einstein M., Schlecht N. Novel epigenetic changes in CDKN2A are associated with progression of cervical intraepithelial neoplasia. Gynecol. Oncol. 2016;142(3):566-573. DOI 10.1016/j.ygyno.2016.07.006

42. Xu C.Q., Zhu S.T., Wang M., Guo S.L., Sun X.J., Cheng R., Xing J., Wang W.H., Shao L.L., Zhang S.T. Pathway analysis of differentially expressed genes in human esophageal squamous cell carcinoma. Eur. Rev. Med. Pharmacol. Sci. 2015;19(9):1652-1661

43. Xu Y., Sun Y., Song X., Ren J. The mechanisms and diagnostic potential of lncRNAs, miRNAs, and their related signaling pathways in cervical cancer. Front. Cell Dev. Biol. 2023;11:1170059. DOI 10.3389/fcell.2023.1170059

44. Yamazaki T., Yang X., Chung Y., Fukunaga A., Nurieva R., Pappu B., Martin-Orozco N., Kang H.S., Ma L., Panopoulos A., Craig S., Watowich S., Jetten A., Tian Q., Dong C. CCR6 regulates the migration of inflammatory and regulatory T cells. J. Immunol. 2008;181(12): 8391-8401. DOI 10.4049/jimmunol.181.12.8391

45. Yan X., Hu Z., Feng Y., Hu X., Yuan J., Zhao S., Zhang Y., Yang L., Shan W., He Q., Fan L., Kandalaft L., Tanyi J., Li C., Yuan C.X., Zhang D., Yuan H., Hua K., Lu Y., Katsaros D., Huang O., Montone K., Fan Y., Coukos G., Boyd J., Sood A., Rebbeck T., Mills G., Dang C., Zhang L. Comprehensive genomic characterization of long non-coding RNAs across human cancers. Cancer Cell. 2015;28(4): 529-540. DOI 10.1016/j.ccell.2015.09.006

46. Yen M.C., Chou S.K., Kan J.Y., Kuo P.L., Hou M.F., Hsu Y.L. New insight on solute carrier family 27 member 6 (SLC27A6) in tumoral and non-tumoral breast cells. Int. J. Med. Sci. 2019;16(3):366-375. DOI 10.7150/ijms.29946

47. Yu Q., Lou X.M., He Y. Preferential recruitment of Th17 cells to cervical cancer via CCR6-CCL20 pathway. PLoS One. 2015;10(3): e0120855. DOI 10.1371/journal.pone.0120855

48. Zhao L., Zhang Z., Lou H., Liang J., Yan X., Li W., Xu Y., Ou R. Exploration of the molecular mechanisms of cervical cancer based on mRNA expression profiles and predicted microRNA interactions. Oncol. Lett. 2018;15(6):8965-8972. DOI 10.3892/ol.2018.8494