The Role And Mechanism Of PLAC8 In The Occurrence And Development Of Hepatocellular Carcinoma

[Background and Objective]Hepatocellular carcinoma (HCC) continues to be a leading cause of cancer-related deaths worldwide and the third leading cause of cancer-related death. Nearly 1 million people die from HCC every year.Recently, although the clinical treatment of HCC has achieved a great development, the mechanism of tumor cell invasion and metastasis is still unclear. With the development of research on HCC signal pathway, people find that the activation or inhibition of signal pathway in HCC cell has a deep relationship with the occurrence and development of HCC.The suppression of molecular axis to cure cancer is the important breakthrough in recent years. The absence of ?-catenin by siRNA could block the cell cycle in G0/G1, thereby transfection inhibits HCC proliferation, which exert function in HCC treatment instead of complete cure. ?-catenin pathway in HCC could regulate cell growth, cycle, apoptosis, angiogenesis factor, telomerase and other pathways. Therefore, inhibition of ?-catenin pathway does play a role in HCC treatment.Pregnancy specific protein 8 (PLAC8) also called as onzin 8, rich in cysteine and originally found in human dendritic cell. PLAC8 gene is nearly 700 BP, its open reading frame length is about 348 BP, but some RNA blotting showed that in some organs its mRNA length is BP 4400-7000. PLAC8 molecular weight of 12.4 kDa, in some mammals restriction enzyme and its signal peptide binding sites are similar, and the amino acid sequence rich in cysteine occupy 10% of total length of, for example, in the full length human PLAC8, each of the 112 amino acids contains 15.5 cystine. In the full length of mouse PLAC8, each of the 115 amino acids contains 16 cysteine. Most of the cysteine forms a CXXC structure, forming the two sulfur bond inside the molecule, which is a special site that binds the substrate. Genomic analysis showed that the human PLAC8 gene is located in chromosome 4q13-4q21, which contains 5 exons, which are connected by GT-AG U2.Researchers found that PLAC8 is the biomarker in HCC progression, while the role of PLAC8 in HCC is definite and rarely studied. At the meantime, the reference factors to regulate expression of PLAC8 have no deep research.In the current study, the expression of PLAC8 was examined in HCC progression, in addition its cell function was investigated.[Methods]1. The expression of PLAC8 in HCC(1) The mRNA level of PLAC8 was detected in HCC patient liver compared with normal liver.RNA extraction from normal liver and HCC patient liver, and then cDNA synthesis by reverse transcription was performed. The expression level was examined by Real-time PCR.(2) The mRNA level of PLAC8 was detected in HCC tissues and adjacent tissueRNA extraction from HCC tissues and adjacent tissue, and then cDNA synthesis by reverse transcription was performed. The expression level was examined by Real-time PCR. (3) The protein level of PLAC8 was detected in HCC tissues and adjacent tissueHCC tissues and adjacent tissue in 136 patients was performed by IHC staining, and the protein level of PLAC8 was detected. (4) The influence of PLAC8 on survival of HCC patientSurvival time of 136 HCC patients was performed statistics, and creating graphs based on level of PLAC8 and survival time.2. The influence of PLAC8 on tumor growthOverexpression of PLAC8 in Huh 7 was orthotopicly implanted to established models.The Huh7 cell overexpressed PLAC8 or vector were injected in BALB/c (nu/nu) mice liver. The mice were euthanized after 4 weeks, and taken pictures for liver. We performed s statistics for tumor volume.3. The influence of PLAC8 on HCC cell biology characteristic(1) Influence of PLAC8 on HCC cell viabilityCell suspension was inoculated in 96-well plates, and cultured in incubator.10 uL CCK solution was added to each hole. The absorbance at 450 nm was examined by enzyme standard instrument.(2) Influence of PLAC8 on HCC cell clone formationCell in the logarithmic phase was digested and pipetted into individual cell by 0.25% trypsin, and then suspended in DMEM contained 10% FBS. The next step was that cell suspension was made gradient dilution. Cell in each group was incubated in 10 ml,37? dish contained pre-warmed medium at 50,100 and 200 cell/dish concentration. Dish was rocked to disperse cell, which cultured in incubator on 37?, 5% CO2 and saturated humidity about 2-3 weeks. Culture will be ended until clone formation was clearly observed. The supernatant was discarded, and then dish was washed by PBS twice. Cell was fixed in 4% paraformaldehyde for 15 mins, and fixative was throwed away. Then cell was stained by GIMSA for 10-30 mins followed by running water and drying air. The plates were inverted and superimposed a transparent grid film, visually direct counting cloning, or the microscope (low magnification) counts more than 10 cells clone multiples.(3) The influence of PLAC8 on cell cycleThe influence of PLAC8 on Rb and CCND1 related cell cycle were examined by Western blot.4. The mechanism of PLAC8 inhibiting tumor progression(1) The influence of PLAC8 on Wnt/?-catenin axisThe relation between PLAC8 and c-Myc was detected by real-time PCR.. The relationship between PLAC8 and c-Myc in Hep3B detected by Western blot; Luciferase Reporter Assays detected the influce to ?-catenin by PLAC8 in Hep3B and HepG2.(2) The regulation of Akt/GSK3?/?-catenin signal pathway by PLAC8The influence test of PLAC8 to protein levels of p-Akt, Akt, p-GSK3?, GSK3p and ?-catenin by Western blot.5. The mechanism of PLAC8's down regulation in HCC(1) Real-time PCR detected mRNA levels of miR-185-5p in liver tissues and tumor tissues, respectively.(2) Correlation analysis between PLAC8 and miR-185-5p by Real-time PCR.(3) Luciferase Reporter Assays detected the modulatory effect of miR-185-5p to PLAC8 in Hep3B.(4) Western blot detected the modulatory effect of miR-185-5p to PLAC8 in Hep3B and HepG2.(5) The effect of miR-185-5p and PLAC8's relationship to cell viability in Huh7.6. Statistical analysisStatistical analyses were performed with SPSS (17.0 version), with a P value< 0.05 considered significant.[Results]1. The level of PLAC8 is down-regulated in HCC(1) mRNA level of PLAC8 in HCC is decreased.Real-time PCR showed that the mRNA level of PLAC8 in liver tumor tissues was significantly decreased compared to control group (p< 0.0001).(2) mRNA level of PLAC8 was dramatically decreased in liver tumor tissuesThe mRNA level of PLAC8 was detected in tumor tissues and adjacent tissues of HCC patients by Real-time PCR, we found that PLAC8 level in tumor tissues was significantly decreased.(3) Protein level of PLAC8 was down-regulated in tumor tissues of HCC patientsIHC presented that, PLAC8 level in 84 patients of total 136 was decreased compared with that in adjacent tissues,26 patients'PLAC8 level was unchanged and 26 patients'PLAC8 level was increased.(4) Low-level of PLAC8 in HCC patients along with high incidenceSurvival analysis indicated that patient with low level of PLAC8 suffered a high incidence. However, patient with high level of PLAC8 suffered a low incidence (p= 0.011).2. PLAC8 inhibits tumor growthThe xenograft HCC mouse model showed that mice with over-expressed PLAC8 suffered the decrease of tumor volume (p< 0.01).3. PLAC8 can inhibit the cell viability and proliferation of HCC(1) PLAC8 inhibited the cell viabilityLuciferase Reporter Assays showed that the increasingly of PLAC8 inhibited the cell viability in Huh7, SMMC-7721, HepG2 and Hep3B (p< 0.01); the inhibition of PLAC8 rescued the cell viability (p< 0.01).(2) PLAC8 inhibited cell proliferationClone formation indicated that overexpression of PLAC8 inhibited cell clone in Huh7 and SMMC-7721 (p< 0.01).(3) PLAC8 inhibited cell cycleWestern blot showed that overexpression of PLAC8 can inhibit protein level of Rb and CCND1.4. PLAC8 inhibits HCC by regulated Wnt/?-catenin and Akt/GSK3?/?-catenin(1) PLAC8 can inhibit Wnt/(3-catenin then inhibit HCCReal-time PCR and Western blot showed that PLAC8 was negatively correlated with c-Myc; Luciferase Reporter Assays showed that PLAC8 can inhibit ?-catenin in Hep3B and HepG2.(2) PLAC8 can inhibit Akt/GSK3?/?-catenin then inhibit HCCWestern blot showed that PLAC8 can inhibit the protein level of p-Akt and ?-catenin, increase the level of GSK3? in Hep3B and HepG2.5. miR-185-5p inhibits PLAC8 in HCC(1) miR-185-5p was significantly increased in HCCReal-time PCR indicated that miR-185-5p was significantly increased in liver tumor tissues.(2) miR-185-5p was negatively correlated with PLAC8Real-time PCR indicated that mRNA level of miR-185-5p was negatively correlated with PLAC8.(3) miR-185-5p inhibits PLAC8Luciferase Reporter Assays showed that miR-185-5p inhibits PLAC8; Western blot showed that protein level of PLAC8 was inhibited by miR-185-5p.(4) miR-185-5p inhibited PLAC8 and then promoted cell viability miR-185-5p and PLAC8 can interact with each other and then promoted cell viability.[Conclusion]1. PLAC8 is markedly decreased during the development of HCC.2. PLAC8 can inhibit tumor growth.3. PLAC8 inhibits cell viability, cell proliferation and cell cycle.4. PLAC8 inhibits HCC by regulate Wnt/p-catenin and Akt/GSK3?/?-catenin signal pathways.5. miR-185-5p can down-regulate the level of PLAC8.