lncRNA H19 miR148a3p.docx

1、lncRNA H19 miR148a3pRegulation of laryngeal squamous cell cancer progression by the lncRNA H19/miR-148a-3p/DNMT1 axisABSTRACTLaryngeal squamous cell carcinoma (LSCC) is a highly aggressive malignant cancer. The regulation of LSCC progression by long non-coding RNA (lncRNA) was not well understood. I

2、n this study, we reported that the lncRNA H19 was upregulated in LSCC. The expression levels of H19 were inversely correlated with the survival rate of LSCC patients. Knockdown of H19 expression inhibited LSCC cell migration, invasion and proliferation. We identified microRNA miR-148a-3p as an inhib

3、itory target for H19. Overexpression of miR-148a-3p reduced LSCC migration, invasion and proliferation cell, while inhibition of miR-148a-3p did the opposite. The inhibition of LSCC progression induced by H19 knockdown required the activity of miR-148a-3p. We also identified DNA methyltransferase en

4、zyme DNMT1 as a target of miR-148a-3p. Cellular DNA methylation levels were inhibited by both miR-148a-3p overexpression and H19 knockdown. In summary, our study demonstrated that the lncRNA H19 promoted LSCC progression via miR-148a-3p and DNMT1.INTRODUCTIONHead and neck squamous cell carcinoma is

5、the sixth most common cancer in the world, among which the laryngeal squamous cell carcinoma (LSCC) is a highly aggressive malignancy 1. Although encouraging progress in the diagnosis and treatment for LSCC has been achieved in the past 20 years, the overall survival rate remains unfavorable. A rece

6、nt study has shown that the overall 1-and 2-year survival rates for LSCC patients without treatment are only 56.4% and 26.5%, respectively 2. Recurrence and metastasis are believed to be the major factors that to limit the successful treatment of LSCC 3. In order to develop effective therapy for LSC

7、C, the efforts towards understanding the underlying pathological mechanisms of LSCC have been intensified recently.Noncoding RNAs are subdivided into small ncRNAs ( 200 nt) based on their size. Small ncRNAs have been shown to act primarily as negative regulators of gene expression. A number of micro

8、RNAs, which belong to the small ncRNA family, have been demonstrated to function as oncogenes or tumor suppressor genes for LSCC in our previous studies 48. Long ncRNAs (lncRNAs) are poorly conserved among species 9,10, but accumulating evidences indicate that lncRNAs could play important roles in a

9、 variety of biological processes and may well be also involved in the development of cancer and other human diseases 11,12. Specifically, our previous studies have suggested the involvement of lncRNA HOTAIR in LSCC 13,14. The overall pathophysiological contribution of lncRNAs to LSCC, however, is la

10、rgely unknown.The lncRNA H19 is transcribed from a maternally expressed imprinted gene locus on human chromosome 11. The H19 gene encodes a 2, 600 nt capped, spliced, and polyadenylated noncoding RNA that is predominantly cytoplasmic 15,16. Although H19 has been intensively studied in the field of g

11、enomic imprinting, the biological function of H19 as a non-coding RNA has only recently begun to be elucidated. Accumulating evidence in recent studies has consistently demonstrated that the expression levels of H19 are upregulated in a variety of cancer types, including gastric cancer 17, esophagea

12、l cancer 18 colorectal cancer 1921, breast cancer 22,23, bladder cancer 24,25, and hepatocellular carcinoma 26,27. The upregulation of H19 in cancer tissues suggests its possible tumorigenic properties, although the detailed molecular mechanism remains to be investigated.In the present study, we fou

13、nd that the lncRNA H19 was upregulated in LSCC and that the expression levels of H19 were inversely correlated with the survival rate of LSCC patients. Consistently, we found that knockdown of H19 expression inhibited LSCC cell proliferation, migration and invasion. We identified microRNA miR-148a-3

14、p as a target for H19. The expression of miR-148a-3p was inhibited by H19, and the overexpression and the inhibition of miR-148a-3p were respectively associated with reduced and elevated LSCC proliferation, migration and invasion. Importantly, the inhibition of LSCC progression induced by H19 knockd

15、own required the activity of miR-148a-3p. We also determined that DNA methyltransferase enzyme DNMT1 as an inhibitory target of miR-148a-3p. Cellular DNA methylation was inhibited by both miR-148a-3p overexpression and H19 knockdown. Taken together, our study demonstrated that the lncRNA H19 could p

16、romote LSCC progression via miR-148a-3p and DNMT1, and that DNA methylation was involved in the regulatory mechanism.RESULTSH19 was upregulated in LSCC and was inversely correlated with patient survival rateSince lncRNAs have been implicated in the development of cancers, we first sought to examine

17、the landscape of lncRNA gene expression in LSCC with a hybridization microarray designed for lncRNAs. We found that the expression levels of H19 were significantly upregulated in LSCC tissue samples compared to those in normal tissues (p 0.001,Figure 1A). With primers specific to H19, we validated o

18、ur findings by qPCR analysis, and found that H19 levels were significantly higher (5.54-fold) in LSCC tumor tissues than those in adjacent non-neoplastic tissues (3.342 1.436 versus 0.596 0.259) (p 0.01,Figure 1B). Furthermore, we determined that the expression levels of H19 were significantly corre

19、lated with the progression of LSCC, including tumor grade, differentiation, neck nodal metastasis, and clinical stage (Table 1). Based on the levels of H19 expression, we categorized 82 LSCC patients into high (n= 41) and low (n= 41) H19 expression groups. With Kaplan-Meier analysis, we found that p

20、atients with high H19 expression had significantly poorer overall survival rate compared to those with low H19 expression (2= 8.704,p= 0.003) (Figure 1C). Taken together, these results indicated that H19 was upregulated in LSCC and was positively correlated with LSCC progression.Figure 1: H19 is upr

21、egulated in LSCC and is inversely correlated with patient survival rate. (A) Box plot of H19 expression levels in LSCC tissues and adjacent non-neoplastic normal tissues as determined by lncRNA-specific microarray analysis (p 0.001). (B) Real time PCR analysis of H19 expression levels in LSCC tissue

22、s and adjacent non-neoplastic normal tissues (*p 0.01). (C) The Kaplan-Meier overall survival rate curve for LSCC patients (n = 82) with high and low H19 expression levels (p = 0.003).Table 1: Relationship between H19 expression level and clinicopathologic parameters of LSCCCharacteristics (n)H19 le

23、velPSex0.638Male (55)3.295 1.580Female (27)3.440 1.142Age0.122 58 (41)3.590 1.426 58 (41)3.095 1.438T classification 0.01T12 (49)2.890 1.333T34 (33)4.014 1.357Differentiation0.017G1 (58)3.068 1.332G2 (24)3.993 1.553Lymph node metastasis 0.01Negative (52)2.825 1.281Positive (30)4.240 1.278Primary loc

24、ation0.103Supraglottic (35)3.639 1.357Glottic (47)3.121 1.482Clinical stage 0.01III (45)2.867 1.356IIIIV (37)3.92 1.351H19 knockdown inhibited LSCC cell migration, invasion and proliferationIn order to investigate the function of H19 in LSCC development, we knocked down the expression of H19 by tran

25、sfecting lentiviruses encoding control shRNA or H19 shRNA into Hep-2 cells, a well-established LSCC cell line. The H19 expression levels were significantly reduced by this treatment after 24 h (p 0.01,Figure 2A). We then performed wound healing cell migration assay on these cells. We found that the

26、migration of Hep-2 cells was significantly inhibited by H19 knockdown (p 0.05,Figure 2B). We also performed transwell assay to examine cell invasion ability, and found that compared to shRNA control, the shRNA targeting H19 led to significantly decreased number of transmembrane cells (p 0.01,Figure

27、2C). Moreover, by MTS assay, we discovered that H19 knockdown also significantly inhibited cell proliferation (p 0.05,Figure 2D). Furthermore, we generated LSCC stem cells (LSCC-SCs) from LSCC patient and knocked down H19 expression (Figure 2E). These LSCC-SCs were subjected to sphere formation and

28、MTS assays to examine whether H19 influences LSCC-SC proliferation. The results revealed that downregulated of H19 significantly suppressed LSCC-SC growth (Figure 2Fand2G). Taken together, decreased H19 expression led to impaired cell migration, invasion and proliferation in LSCC cells.Figure 2: H19

29、 knockdown inhibits LSCC cell migration, invasion and proliferation.(A) H19 expression levels in Hep-2 cells lentiviruses encoding control shRNA or H19 shRNA. (B) Wound healing cell migration assay, (C) Transwell cell invasion assay and (D) MTS cell proliferation assay in Hep-2 cells transfected wit

30、h lentiviruses encoding control shRNA or H19 shRNA. (E) H19 expression levels in LSCC-SCs transiently transfected with control siRNA or H19 siRNA. (F) Sphere formation in LSCC-SCs transfected with control siRNA or H19 siRNA. (G) MTS cell proliferation assay in LSCC-SCs transfected with control siRNA

31、 or H19 siRNA. (H) H19 expression levels in subcutaneous xenograft LSCC tumors transfected with lentiviruses encoding control shRNA or H19 shRNA. (I) Subcutaneous xenograft LSCC tumors developed in nude mice from Hep-2 cells transfected with lentiviruses encoding control shRNA or H19 shRNA. (J) Weig

32、ht quantification of tumor tissues depicted in (I). (K) Immunohistochemistry staining of BrdU in tumor tissues depicted in (I). Scale bar = 100 m. (L) Quantification of BrdU positive cells. *p 0.05 and *p 0.01 compared to the control group.In addition toin vitroexperiments, we used a mouse xenograft model to study the oncogenic role of H19 in LSCC developmentin vivo. All mice subcutane