Ann Lab Med.  2020 Mar;40(2):155-163. 10.3343/alm.2020.40.2.155.

Upregulated Long Noncoding RNA LINC01234 Predicts Unfavorable Prognosis for Colorectal Cancer and Negatively Correlates With KLF6 Expression

• Jiang X
• Zhu Q
• Wu P
• Zhou F
• Chen J

• KMID: 2460913
• DOI: http://doi.org/10.3343/alm.2020.40.2.155

Abstract

BACKGROUND
LINC01234, a long noncoding RNA (lncRNA), is overexpressed in several cancers, including colorectal cancer (CRC). We investigated the role of LINC01234 in CRC development and confirmed its correlation with Krüppel-like factor 6 (KLF6), a tumor suppressor gene that is dysregulated in CRC.
METHODS
We tested mRNA levels using quantitative reverse transcription PCR (qRT-PCR). Tissue samples from patients with CRC, inflammatory bowel disease (IBD), hyperplastic polyp, and adenoma were included. Correlations between clinicopathological parameters, overall survival (OS) rate, and LINC01234 were analyzed using Kruskal-Wallis H test. Additionally, cell proliferation, apoptosis, and tumor formation in nude mice were tested to investigate the mechanism of LINC01234. Western blotting was used to determine protein levels.
RESULTS
LINC01234 expression was significantly upregulated in CRC tissues and CRC cell lines than in non-tumor tissues and normal epithelial cells, respectively. LINC01234 was associated with high tumor stage, larger tumor size, and metastasis. Patients with higher LINC01234 expression showed reduced OS. Cell proliferation was inhibited by LINC01234 knockdown, whereas apoptosis was enhanced. Mice injected with SW480 cells with LINC01234 knockdown displayed decreased tumor volume, weight, and Ki-67 levels compared with those injected with control cells. KLF6 was negatively regulated by LINC01234. Overexpression of KLF6 showed effects similar to those observed following LINC01234 knockdown on cell proliferation and apoptosis.
CONCLUSIONS
LINC01234 could be a prognostic biomarker in CRC patients. Upregulation of LINC01234 in CRC promotes tumor development through negative regulation of KLF6.

Keyword

Long noncoding RNA; LINC01234; Colorectal cancer; Krüppel-like factor 6; Prognosis

MeSH Terms

Adenoma
Animals
Apoptosis
Blotting, Western
Cell Line
Cell Proliferation
Colorectal Neoplasms*
Epithelial Cells
Genes, Tumor Suppressor
Humans
Inflammatory Bowel Diseases
Mice
Mice, Nude
Neoplasm Metastasis
Polymerase Chain Reaction
Polyps
Prognosis*
Reverse Transcription
RNA, Long Noncoding*
RNA, Messenger
Tumor Burden
Up-Regulation
RNA, Long Noncoding
RNA, Messenger

Figure

• 

  Fig. 1 LINC01234 is upregulated in CRC tissues and cells and associated with poor prognosis in CRC patients. (A) Quantitative analysis of lncRNA LINC01234. *P<0.001 vs CRC. (B) LINC01234 expression was analyzed in CRC tissues and the corresponding adjacent non-tumor tissues; data are presented as log2 fold change (CRC/NC; N=136). Median value of the log2 fold change is 1.736, and log2 fold changes were defined as follows: >1.736, high expression; <1.736, low expression. (C) Relative expression of LINC01234 in CRC cell lines and normal colon epithelial cell line NCM460 (N=10). †P<0.001 vs NCM460. (D) Patients with high LINC01234 expression (N=68) showed shorter OS (P<0.05) than those with low LINC01234 expression (N=68). (E) Analysis of the sensitivity and specificity of LINC01234 for distinguishing CRC tissues from normal and benign tissues by ROC curve analysis (cut-off=2.601). Data are presented as mean±SD.Abbreviations: CRC, colorectal cancer; NC, negative control (adjacent non-tumor tissues); IBD, inflammatory bowel disease; HP, hyperplastic polyp; AD, adenoma; OS, overall survival; ROC, receiver operating characteristic; AUC, area under the curve; CI, confidence interval.

• 

  Fig. 2 Effects of LINC01234 knockdown on CRC cell viability and apoptosis in vitro. (A) and (B) LINC01234 expression level was determined by qRT-PCR following transfection of SW480 and HCT116 cells with si-LINC01234 (N=6) or si-NC. *P<0.001 vs Scrambled. (C) and (D) CCK-8 analyses were used to determine the viability of si-LINC01234-transfected SW480 and HCT116 cells. Values are from average of three independent experiments. †P<0.01 vs Scrambled. (E) Colony-forming analyses were conducted to determine the proliferation of si-LINC01234-transfected SW480 and HCT116 cells. Values are from three independent experiments. *P<0.001 vs Scrambled. (F) Flow cytometry was performed to analyze apoptosis rates in CRC cells transfected with si-LINC01234 after 48 hours. Values are from three independent experiments. *P<0.001, †P<0.01 vs Scrambled. (G) Western blot analysis of apoptosis-related proteins after transfection of SW480 and HCT116 cells with Scrambled siRNA, si-LINC01234 1#, or si-LINC01234 2#. GAPDH was used as an internal control. Data are presented as mean±SD.Abbreviations: CRC, colorectal cancer; qRT-PCR, quantitative reverse transcription PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CCK-8, Cell counting Kit 8.

• 

  Fig. 3 Down-regulation of LINC01234 suppresses tumor growth in vivo. (A) qRT-PCR analysis of LINC01234 expression in tumor tissues formed from SW480/sh-LINC01234, SW480/empty vector. *P<0.001, sh-LINC01234 vs Empty vector. (B) and (C) Tumor volume was calculated every four days after injection of SW480 cells stably transfected with sh-LINC01234 or empty vector (N=8). *P<0.001, †P<0.01, sh-LINC01234 vs Empty vector. (D) Tumors developed from sh-LINC01234-transfected SW480 cells showed lower Ki-67 protein levels than tumors developed from control cells. Scale bar, 100 µm. Data are presented as mean±SD.Abbreviation: qRT-PCR, quantitative reverse transcription PCR.

• 

  Fig. 4 LINC01234 represses KLF6 expression. (A) KLF6 expression in CRC tissues (N=76) compared with that in corresponding non-tumor tissues (N=76). KLF6 expression was examined by qRT-PCR and normalized to GAPDH expression. *P<0.01, NC vs CRC. (B) Co-expression analysis between LINC01234 and KLF6. (C) KLF6 mRNA levels were detected by qRT-PCR following the transfection of SW480 and HCT116 cells with si-LINC01234; the results were normalized to GAPDH expression. †P<0.001 vs Scrambled. (D) & (E) KLF6 protein levels were determined by western blotting following the transfection of SW480 and HCT116 cells with si-LINC01234. Values are from three independent experiments. †P<0.001 vs Scrambled. (F) KLF6 expression level in mouse tumors was determined by qRT-PCR formed from SW480/sh-LINC01234 or SW480/empty vector (N=8). ‡P<0.001, sh-LINC01234 vs Empty vector. Data are presented as mean±SD.Abbreviations: KLF6, Krüppel-like factor 6; qRT-PCR, quantitative reverse transcription PCR; CRC, colorectal cancer; NC, negative control (adjacent non-tumor tissues); GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

• 

  Fig. 5 Overexpression of KLF6 inhibits CRC cell proliferation and increases apoptosis. (A) mRNA level of KLF6 in SW480 and HCT116 cells transfected with pCMV-Tag2B-KLF6 was measured by qRT-PCR. (B)–(D) CCK-8 and colony-forming analyses were used to determine the viability of pCMV-Tag2B-KLF6-transfected SW480 and HCT116 cells. Values are from three independent experiments. (E) Apoptosis was determined by flow cytometry. Values are from three independent experiments. Data are presented as mean±SD. *P<0.001, †P<0.01.Abbreviations: KLF6, Krüppel-like factor 6; CRC, colorectal cancer; CCK-8, Cell Counting Kit 8; qRT-PCR, quantitative reverse transcription PCR.

Reference

1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin. 2018; 68:7–30. PMID: 29313949.

2. Torre LA, Siegel RL, Ward EM, Jemal A. Global cancer incidence and mortality rates and trends—an update. Cancer Epidemiol Biomarkers Prev. 2016; 25:16–27. PMID: 26667886.

3. Davis CA, Hitz BC, Sloan CA, Chan ET, Davidson JM, Gabdank I, et al. The Encyclopedia of DNA elements (ENCODE): data portal update. Nucleic Acids Res. 2018; 46:D794–D801. PMID: 29126249.

4. Loewer S, Cabili MN, Guttman M, Loh YH, Thomas K, Park IH, et al. Large intergenic non-coding RNA-RoR modulates reprogramming of human induced pluripotent stem cells. Nat Genet. 2010; 42:1113–1117. PMID: 21057500.

5. Chisholm KM, Wan Y, Li R, Montgomery KD, Chang HY, West RB. Detection of long non-coding RNA in archival tissue: correlation with polycomb protein expression in primary and metastatic breast carcinoma. PLoS One. 2012; 7:e47998. PMID: 23133536.

6. Gupta RA, Shah N, Wang KC, Kim J, Horlings HM, Wong DJ, et al. Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature. 2010; 464:1071–1076. PMID: 20393566.

7. Meola N, Pizzo M, Alfano G, Surace EM, Banfi S. The long noncoding RNA Vax2os1 controls the cell cycle progression of photoreceptor progenitors in the mouse retina. RNA. 2012; 18:111–123. PMID: 22128341.

8. Sigova AA, Mullen AC, Molinie B, Gupta S, Orlando DA, Guenther MG, et al. Divergent transcription of long noncoding RNA/mRNA gene pairs in embryonic stem cells. Proc Natl Acad Sci U S A. 2013; 110:2876–2881. PMID: 23382218.

9. Kapusta A, Feschotte C. Volatile evolution of long noncoding RNA repertoires: mechanisms and biological implications. Trends Genet. 2014; 30:439–452. PMID: 25218058.

10. Cabanski CR, White NM, Dang HX, Silva-Fisher JM, Rauck CE, Cicka D, et al. Pan-cancer transcriptome analysis reveals long noncoding RNAs with conserved function. RNA Biol. 2015; 12:628–642. PMID: 25864709.

11. Ghaleb AM, Yang VW. The pathobiology of Krüppel-like factors in colorectal cancer. Curr Colorectal Cancer Rep. 2008; 4:59–64. PMID: 18504508.

12. He Z, Dang J, Song A, Cui X, Ma Z, Zhang Z. Identification of LINC01234 and MIR210HG as novel prognostic signature for colorectal adenocarcinoma. J Cell Physiol. 2019; 234:6769–6777. PMID: 30362555.

13. Yang F, Liu YH, Dong SY, Yao ZH, Lv L, Ma RM, et al. Co-expression networks revealed potential core lncRNAs in the triple-negative breast cancer. Gene. 2016; 591:471–477. PMID: 27380926.

14. Chen X, Chen Z, Yu S, Nie F, Yan S, Ma P, et al. Long noncoding RNA LINC01234 functions as a competing endogenous RNA to regulate CBFB expression by sponging miR-204-5p in gastric cancer. Clin Cancer Res. 2018; 24:2002–2014. PMID: 29386218.

15. Ghaffar M, Khodahemmati S, Li J, Shahzad M, Wang M, Wang Y, et al. Long non-coding RNA LINC01234 regulates proliferation, invasion and apoptosis in esophageal cancer cells. J Cancer. 2018; 9:4242–4249. PMID: 30519325.

16. Cho YG, Choi BJ, Song JW, Kim SY, Nam SW, Lee SH, et al. Aberrant expression of Krüppel-like factor 6 protein in colorectal cancers. World J Gastroenterol. 2006; 12:2250–2253. PMID: 16610031.

17. Zhang Q, Tan XP, Yuan YS, Hu CM, He CH, Wang WZ, et al. Decreased expression of KLF6 and its significance in gastric carcinoma. Med Oncol. 2010; 27:1295–1302. PMID: 19967571.

18. DiFeo A, Feld L, Rodriguez E, Wang C, Beer DG, Martignetti JA, et al. A functional role for KLF6-SV1 in lung adenocarcinoma prognosis and chemotherapy response. Cancer Res. 2008; 68:965–970. PMID: 18250346.

19. Mukai S, Hiyama T, Tanaka S, Yoshihara M, Arihiro K, Chayama K. Involvement of Krüppel-like factor 6 (KLF6) mutation in the development of nonpolypoid colorectal carcinoma. World J Gastroenterol. 2007; 13:3932–3938. PMID: 17663506.

20. Liu JX, Li W, Li JT, Liu F, Zhou L. Screening key long non-coding RNAs in early-stage colon adenocarcinoma by RNA-sequencing. Epigenomics. 2018; 10:1215–1228. PMID: 30182733.

21. Xu Y, Jiang X, Cui Y. Upregulated long noncoding RNA PANDAR predicts an unfavorable prognosis and promotes tumorigenesis in cholangiocarcinoma. Onco Targets Ther. 2017; 10:2873–2883. PMID: 28652769.

22. Prigge ES, Toth C, Dyckhoff G, Wagner S, Müller F, Wittekindt C, et al. p16(INK4a) /Ki-67 co-expression specifically identifies transformed cells in the head and neck region. Int J Cancer. 2015; 136:1589–1599. PMID: 25104331.

23. Cho YG, Kim CJ, Park CH, Yang YM, Kim SY, Nam SW, et al. Genetic alterations of the KLF6 gene in gastric cancer. Oncogene. 2005; 24:4588–4590. PMID: 15824733.

24. Narla G, Heath KE, Reeves HL, Li D, Giono LE, Kimmelman AC, et al. KLF6, a candidate tumor suppressor gene mutated in prostate cancer. Science. 2001; 294:2563–2566. PMID: 11752579.

25. DiFeo A, Narla G, Hirshfeld J, Camacho-Vanegas O, Narla J, Rose SL, et al. Roles of KLF6 and KLF6-SV1 in ovarian cancer progression and intraperitoneal dissemination. Clin Cancer Res. 2006; 12:3730–3739. PMID: 16778100.

26. Hatami R, Sieuwerts AM, Izadmehr S, Yao Z, Qiao RF, Papa L, et al. KLF6-SV1 drives breast cancer metastasis and is associated with poor survival. Sci Transl Med. 2013; 5:169ra12.

27. Narla G, DiFeo A, Reeves HL, Schaid DJ, Hirshfeld J, Hod E, et al. A germline DNA polymorphism enhances alternative splicing of the KLF6 tumor suppressor gene and is associated with increased prostate cancer risk. Cancer Res. 2005; 65:1213–1222. PMID: 15735005.

28. Reeves HL, Narla G, Ogunbiyi O, Haq AI, Katz A, Benzeno S, et al. Kruppel-like factor 6 (KLF6) is a tumor-suppressor gene frequently inactivated in colorectal cancer. Gastroenterology. 2004; 126:1090–1103. PMID: 15057748.

29. Zhang B, Guo DD, Zheng JY, Wu YA. Expression of KLF6-SV2 in colorectal cancer and its impact on proliferation and apoptosis. Eur J Cancer Prev. 2018; 27:20–26. PMID: 29084019.

30. Cho YG, Choi BJ, Kim CJ, Song JW, Kim SY, Nam SW, et al. Genetic alterations of the KLF6 gene in colorectal cancers. APMIS. 2006; 114:458–464. PMID: 16856969.

31. Benzeno S, Narla G, Allina J, Cheng GZ, Reeves HL, Banck MS, et al. Cyclin-dependent kinase inhibition by the KLF6 tumor suppressor protein through interaction with cyclin D1. Cancer Res. 2004; 64:3885–3891. PMID: 15172998.