Evaluation Of Antisense Non-coding RNA In The INK4 Locus Potential In Hepatic Cancer Progression

[Objective]Hepatocellular carcinoma(HCC)is the sixth most frequently diagnosed cancer worldwide and is also responsible for the second most common cause of cancer-related mortality worldwide.HCC is a pathological type with the highest incidence of primary liver cancer.The incidence rate of HCC in China is the highest in the world,which is closely related to the high HBV infection rate in China.Despite the progress made in the tumorigenesis of HCC and therapeutic options,HCC is still poorly understood,and traditional therapies lack efficacy,often resulting in relapse shortly after clinical treatment.Therefore,it is important to identify novel approaches to diagnose and treat HCC.In the human genome,more than 90 percent of the sequences can be transcribed,but less than 2%of the sequences can be transcribed into the messenger RNA(mRNA)that encodes proteins.The other transcribable genomic sequences were transcribed into non coding RNA(ncRNA)without protein coding function.NcRNA is widely involved in all aspects of life phenomena,including growth,differentiation,development,immunity and apoptosis,and plays a significant regulatory role in tumor formation.LncRNAs are non-protein-coding transcripts with more than 200 nucleotides.LncRNAs have been reported to regulate gene expression during differentiation,proliferation,metastasis,and apoptosis.In addition,lncRNAs also have significant roles in the HCC tumorigenesis and it is expected to become a new target for early diagnosis and clinical efficacy judgment of HCC in the future.LncRNA AB209630 can inhibit HCC cell proliferation and metastasis.LncRNA TUG1 has been reported to regulate the Hedgehog pathway by sponging miR-132 in HCC.LINC00052 could also elevate EPB41L3 and inhibit HCC migration and invasion by binding miR-452-5p.Antisense noncoding RNA in the INK4 locus(CDKN2B antisense RNA1,ANRIL)has been identified from familial melanoma patients.Accumulating research has reported that ANRIL overexpression is a risk factor in several human malignancies including lung,gastric,breast,nasopharyngeal,and bladder cancers.ANRIL can directly combine with INK4b-ARF-INK4a gene cluster INK4B transcripts to form polycomb reactive complexes(PRC),and then inhibit INK4b-ARF-INK4a transcripts to p14ARF,p15 INK4b and p15 INK4a,and further affect cell cycle and promote tumor progression by activating Ras pathway.Although ANRIL has been recognized as a fatal oncogene in many cancers,the function of ANRIL in HCC development remains elusive.In recent years,microRNAs,which are small noncoding RNAs,have been reported to be silenced or elevated in many cancer types.MiRNAs could negatively regulate gene expression by directly targeting the 3’-untranslated region(3’UTR)of their target mRNAs.For example,miR-488 could suppress HCC cell proliferation and invasion by targeting ADAM9.Downregulation of miR-196a could inhibit HCC cell proliferation and invasion by targeting FOXO1.miR-708 could function as a tumor suppressor in HCC by targeting SMAD3.Extensive data have proven that miR-384 could play a tumor suppressive role in several cancer types such as colorectal cancer,lung cancer and prostate cancer.However,the exact molecular role of miR-384 in HCC remains to be investigated.One of the important functions of lncRNA is to "trick" RNA or protein away from the original target,so as to reduce its function.Highly up-regulated in liver cancer(HULC)can bind to and inhibit the activity of miR-372,showing an endogenous"sponge like" effect.The up regulation of HULC expression can significantly inhibit the expression of miR-372.In the present study,we focused on the biological roles of ANRIL in HCC progression.ANRIL was greatly elevated in HCC cells,whereas miR-384 was decreased significantly.In the process of HCC occurrence and development,whether the above "sponge like" effect also exist in ANRIL and miR-384,and further affects the process of hepatocyte carcinogenesisit is a problem which deserves thorough study.Signal transducer and activator of transcription(STAT)is a group of transcription factors that mediate cell signal transduction,and it is the key link of JAK-STAT signal transduction pathway.STAT3 signaling pathway plays a vital role in cell growth,differentiation,apoptosis,innate and acquired immune regulation,embryonic development and organ formation.STAT3 can be activated by a variety of carcinogenic proteins such as E1 protein,HCV core protein and non structural protein in pathological state.STAT3 activation can promote the deterioration of normal cells and block their apoptosis,and then promote the angiogenesis and proliferation of tumor cells.Blocking STAT3 activation and expression is of great value in the treatment of malignant tumors.A number of studies confirm that lncRNA can regulate the transcription and activity of miRNA through its "sponge like" effect on miRNA,and many miRNAs can play an anti-tumor role by inhibiting tumor related genes.In consideration of the difference of ANRIL,miR-384 and STAT3 expression in HCC cells,we speculated by checking the relevant website(http://www.noncode.org/index.php https://www.lncipedia.org/)and hypothesized that ANRIL could regulate HCC progression via targeting of miR-384 and STAT3.To explore the regulatory relationship among ANRIL,miR-384 and STAT3 provides a new way to further clarify the causes of HCC occurrence and development,and provides a new method for early diagnosis and treatment of HCC in clinical work.[Methods]1.Quantitative real-time polymerase chain reaction(qRT-PCR)assays were utilized to detect expressions of ANRIL,miR-384,and STAT3.2.CCK8 and EDU assays were employed to evaluate HCC cell proliferation.A flow cytometry assay was used to detect the HCC cell cycle and cell apoptosis.The scratch migration and Transwell invasion assays were performed to test cell migration and invasion,respectively.3.RIP and RNA pull-down assays were carried out to confirm the correlation between ANRIL and miR-384.The dual-luciferase reporter assay was used to prove the association between miR-384 and STAT3.Western blotting analysis was performed to examine protein levels of STAT3.4.IHC and HE staining were employed to detect Ki-67 and histopathology.[Results]1.LncRNA ANRIL increased while miR-384 decreased in HCC cellsFirst,ANRIL in HCC cells including SMCC7721,HepG2,MHCC-97H,SNU-449,HUH-7,and normal human liver cell line L02 was detected using a real-time PCR assay.ANRIL levels were significantly increased compared to L02 cells.In addition,miR-384 expression in HCC cells was remarkably decreased in comparison to normal human liver cells.These data demonstrate that ANRIL and miR-384 were negatively involved in HCC.2.Knockdown of ANRIL inhibited HCC cell proliferation in vitroNext,to investigate whether ANRIL can affect HCC cell proliferation,a CCK8 assay was employed in our study.SNU-449 and HUH-7 cells were infected with LV-NC or LV-shANRIL for 48 hours.ANRIL was greatly silenced by LV-shANRIL in HCC cells.We observed that HCC cell survival was strongly inhibited after ANRIL was decreased in HCC cells.In addition,the EDU assay indicates that HCC cell proliferation was repressed by LV-shANRIL.Also,knockdown of ANRIL greatly suppressed HCC cell-colony formation ability in SNU-449 and HUH-7 cells.These results demonstrate that ANRIL knockdown was able to inhibit HCC cell proliferation.3.ANRIL inhibition blocked HCC cell cycle in G1 phase and induced HCC cell apoptosisFlow cytometry was used to investigate the effect of LV-shANRIL on HCC cell cycle.ANRIL knockdown for 48 hours increased the percentage of cells in G1 phase and reduced those in S phase,which indicates that ANRIL inhibition blocked the cell cycle progression.Furthermore,apoptosis of HCC cells was significantly increased by LV-shANRIL.These data imply that ANRIL silence inhibited HCC cell cycle in the G1 phase and increased HCC cell apoptosis.4.Silence of ANRIL restrained HCC cell migration and invasionTo determine whether ANRIL was able to affect HCC cell migration and invasion capacity,a wound healing test and Transwell invasion assay were conducted.LV-shANRIL dramatically inhibited the wound closure in HCC cells.Additionally,invasion capacity of HCC cells was greatly repressed by LV-shANRIL.These results indicate that ANRIL silence can inhibit HCC cell migration and invasion in vitro.5.MiR-384 served as a direct target of ANRILTo evaluate the interaction between ANRIL and miR-384,HCC cells were infected with LV-shANRIL for 48 hours.MiR-384 was greatly increased by LV-shANRIL in HCC cells.Next,to test whether ANRIL can act as a sponge for miR-384,an RIP assay was performed,and ANRIL and miR-384 were more abundant in Ago2 pellets than in IgG pellets.In addition,an RNA pulldown assay using biotinylated miR-384(miR-384-bio)probe exhibited a higher level of ANRIL than control(NC-bio)or mutant miR-384(miR-384-bio-mut).Then,to observe whether miR-384 can modulate ANRIL levels,HCC cells were transfected with miR-384 mimics for 48 hours.Overexpression of miR-384 inhibited ANRIL expression.Moreover,we observed that miR-384 mimics repressed SNU-449 cell proliferation,inhibited cell cycle progression,and induced cell apoptosis significantly.In addition,miR-384 mimics can repress the migration and invasion of SNU-449 cells.These results indicate that ANRIL was able to regulate HCC development through targeting miR-384.6.STAT3 was a direct target of miR-384Furthermore,to explore the detailed mechanism of miR-384 in HCC progression,STAT3 was predicted as miR-384 target.The binding regions between miR-384 and STAT3 were indicated.Luciferase reporter plasmids of wild-type STAT3 and mutant STAT3 binding sites are shown in Fig.6B.Cotransfection of the luciferase reporter plasmid containing the wild type with miR-384 mimics decreased the luciferase reporter activity.In addition,STAT3 expression was inhibited by miR-384 mimics in HCC cells.Taken together,these results indicate that STAT3 can function as a direct target of miR-384.7.Downregulation of ANRIL inhibited HCC progression in vivoA HUH-7 cell nude murine xenograft model was established to determine whether ANRIL silence can inhibit HCC progression in vivo.Two groups were set up,and six mice were injected in the front dorsum with parental HUH-7 cells,while the other six mice were injected with HUH-7 infected with LV-shANRIL.Tumors were peeled from nude mice subcutis and demonstrated in Fig.7A.We observed that ANRIL inhibition greatly inhibited tumor growth in a time-dependent manner.Tumor weight was also reduced by LV-shANRIL compared with control.In addition,HE staining results were exhibited and the immunohistochemistry data revealed that Ki-67 was repressed by LV-shANRIL.Meanwhile,ANRIL knockdown inhibited STAT3 via sponging miR-384 in vivo.These data suggest that downregulation of ANRIL could inhibit HCC development via targeting miR-384 and STAT3 in vivo.[Conclusion]1.ANRIL expression was upregulated in HCC cells,including SMCC7721,HepG2,MHCC-97H,SNU-449 and HUH-7 cells,in comparison to the normal human liver cells L02.Knockdown of ANRIL suppressed HCC cell proliferation and induced cell cycle arrest and apoptosis.HCC cell migration and invasion capacity were inhibited by inhibition of ANRIL.2.Bioinformatics analyses revealed that ANRIL could interact with miR-384.MiR-384 was significantly decreased in HCC cells,and overexpression of miR-384 repressed HCC progression.STAT3 was predicted as a target of miR-384,and miR-384 can modulate STAT3 levels negatively in vitro.3.ANRIL is involved in HCC progression by direct targeting of miR-384 and STAT3.4.ANRIL could act as a potential candidate for HCC diagnosis,prognosis,and therapy.