A Novel Molecule, Mir-216B, Is Involved In Pathogenesis And Progression Of Hepatocellular Carcinoma

Part I research in microRNA expression profiles in HCC plasma between HCC patients with family history and healthy volunteers without a family history.Purpose:To explore different microRNA expression profiles in plasma between HCC patients with family history and healthy volunteers without a family history.Methods:1, To select patients with hepatocellular carcinoma who had diognosed in liver surgerical department of Tongji Hospital, Huazhong University of Science and Technology and the direct family members (such as parents, siblings and children) are also suffering from HCC. During the research, everyone was informed the experimental purposes before the sign of an consent form, and then their peripheral blood were collected.2, The blood samples were collected into tubes containing anticoagulant component-EDTA. After the sample is collected, it is isolated with centrifugation at the air temperature and could be storaged at4℃within one hour, and then the supernatant plasma component extracted immediately stored at a low temperature of-80℃refrigerator.3, plasma samples were simultaneously extracted from plasma samples of healthy volunteers without family history of HCC, then stored in tanks containing liquid nitrogen, and transported by plane to Bo Hao Biological Co., Ltd. The latest database microRNA-based chip version16.0was used to screen between the two groups. The results obtained by the microRNA chip, were analysed through comparision of miRNAs expression profile between healthy volunteers and patients with a family history of liver cancer. in the plasma And several miRNAs which changes in expression were more than20-fold were selected. Access to a large literature, they has not yet been reported in HCC and deserved further study.Result:1, Three HCC patients with family history were collected and at least whose one direct family member are also suffered from hepatocellular carcinoma.2, After a high-speed centrifugation, the supernatant yellow transparent liquid extraction were extracted, and then this3ml samples extracted from plasma were stored at1.5mlEP tube which were treated by high temperature to free RNAse and enzyme, and stored at-80℃.3, After a rigorous comparison of these experiments, a total of46kinds of microRNA were detected of differential expression. the miRNAs which fold-change of expression value was over20fold were22kinds in all of three specimens. And the miRNAs that fold-change of expression value was more than20times were14kinds, which never reported in HCC research. In14miRNAs,12were overexpressed and2were down-regulated in blood plasma of patients with family history of HCC.Part Ⅱ Apparent differences in microRNA expression between the two groups in more HCC patients’ plasma samples verify the microarray result.Purpose:Verified chip results by RT-PCR method, and the sample scope was expanded, the introduction of non-family history HCC patients to verify the results obtained from the chips to support the practical and effective character of study.Methods:1, After selection of10patients, who will be conducted surgical treatment due to HCC, in November2012to April2013require, the patients were informed the experimental purposes and consents were acquired. Then peripheral blood samples10ml were collected. The same procedure was adopted to collect10cases of hemangioma patients without hepatitis B virus infection.2, The blood samples were collected into tubes containing EDTA anticoagulant component, then were isolated and stored at4℃within one hour after the sample is collected. After centrifugation, the supernatant component extracted from plasma wsa immediately stored at-80℃refrigerator.3, ABI Ambion’s mirVana TM PARIS Kit was used to get total RNA from plasma samples. Then the acquired RNA was dissolved in non-enzymatic RNA water and stored at-80℃condition.4, MiRNA samples were extracted using Toyobo Company ReverTra Ace qPCR RT Master Mix Kit. In this process, the stem-loop miRNA primers ordered from Invitrogen Ltd was joined during reverse transcription, and then the cDNA samples were obtained and stored at-20℃refrigerator.5, The cDNA samples acquired from previous step were used Toyobo’s SYBR(?)Green Realtime PCR Master Mix-Plus kit to make RT-PCR to detect miRNA expression in ABI Step-one RT-PCR machine.6,2-△△ct method was used for data standardization, then T test was used to compare differences in expression between the two groups, and the obtained results were compared to the result of chips to verify the reliability of the chip.Result:1, After a rigorous pathological examination screening after operation, blood samples were collected from HCC patients who were diagnosed with HCC. Then the patient was informed the study and consented for the experiment thus sign an agreement.10blood samples from patients with hepatocellular carcinoma were coded1to10. And10patients with hepatic hemangioma and without a family history of liver cancer and hepatitis B, whose blood samples were coded collected and according to HCC patients’ samples.2, After the multi-step centrifugation, following the use of free RNAse tubes and tips, each plasma samples were obtained about3ml as a yellow translucent liquid at a low temperature process.3, the appropriate plasma samples were taken, after a multi-step centrifugation, washing, purifying and dissolvment, each400μL plasma samples were obtained Total RNA samples30μl with concentration about1μg/μL. RNA was stored in free RNAse EP tube at-80℃.4, About1-2μL RNA samples obtained in the previous step, were added into Toyobo’s ReverTra Ace qPCR RT Master Mix reaction system, and the stem-loop miRNA primers used reverse transcription reaction was added.10μL cDNA sample was obtained from each RNA samples and stored at-20℃.5, cDNA samples obtained in the previous step were used Toyobo’s SYBROGreen Realtime PCR Master Mix-Plus kit and performed RT-PCR reaction in ABI Step-one RT-PCR machine.The result is recorded in the computer after standardization by the internal reference. RT-PCR experiments for each miRNA samples are repeated three times. 6,2-△△ct method was used for data analysis, and then the data were compared with the microarray results and found that the data acquired in this part verify the chip result. The change in14kinds of miRNAs expression profile between10cases of hepatocellular carcinoma patients and10cases of hepatic hemangioma patients are associated with miRNA microarray results, but the degree of expression difference between these two groups was slightly less than the microarray results. After this verification, chip results were real and reliable this can be subjected to further study.Part III The expression level of miR-216b in tumor tissues and adjacent liver tissue of HCC patients and the correlation between miR-216b expression and clinicopathological parameters and prognosis. Purpose:Continue to use the RT-PCR method to study the correlation between miR-216b expression and clinicopathological parameters and prognosis were studied of in HCC patients.Methods:1, The miR-216b was chosen to apply next study.2,50patients with hepatocellular carcinoma were randomly selected from2008.4-2013.4who had suffered surgery in our hospital. Tumor tissue specimens and adjacent liver tissue was collected during operation, transported in liquid nitrogen cryogenic, packaging and stored at-80℃. The50specimens were homogenized, centrifuged and extracted the total RNA.3, Based on a large literature, miRNA-216b is selected for further study. RNA samples obtained in the previous step, were applied by Toyobo Company ReverTra Ace qPCR RT Master Mix Kit, added with stem-loop miRNA-216b primers to get the cDNA sample, and then stored at-20℃refrigerator. CDNA samples added with invitrogen’s miRNA-216b downstream and upstream primer, were applied with Toyobo’s SYBR(?)Green Realtime PCR Master Mix-Plus kit in ABI Step-one RT-PCR machine to conduct amplification, RT-PCR method was adopted to detect the expression of miRNA-216b.4, The above results,2-AAct method was used for data standardization, and comparison of expression of miR-216b between carcinoma and adjacent tissues was applied by student T-test.5, The correlation of expression of miRNA-216b and patients’ clinical and pathological data, such as pathological stage, tumor size, metastasis and other clinicopathological factors was researched by χ2test.6, For these patients, the investigation on patients survival and recurrence were followed, which studies the correlation of miRNA-216b expression and prognosis in HCC patients post-operation.Result:1, Through reading a large literature and it is found that miR-216b has never been reported in HCC. The results of chip also show that the miR-216b expression in blood plasma of HCC patients with a family history was most obviously different with that in plasma of healthy volunteers with no family history. It is reduced by about122fold. So miR-216b could be a tumor suppressor gene, so it was selected as target to continue study.2, Selected patients are aged between30-60, their fresh liver tumor tissue samples were collected during operation and stored at-80℃. Then total RNAs were extracted by trizol agent from tissue samples, each RNA sample volume was30μL and their concentration were about1μg/μL. The reverse transcription was next immediately or frozen at-80℃refrigerator.3, Using the ReverTra Ace qPCR RT Master Mix kit and miRNA-216b stem-loop primers in reverse transcription of each sample to achieve a particular cDNA10μL, and its concentration of about1μg/μL. After RT-PCR, the expression level of miR-216b in tumor tissues and the adjacent tissues were measured, and then were analysed through the T test. That showed that the adjacent tissues expression of miR-216b in75%of patients were significantly higher than tumor tissues in patients with primary hepatocellular carcinoma, while the average difference in expression level of miR-216b was approximately4.45times, and the difference was statistically significant (p<0.05).5, According to the expression of miR-216b in patients liver tissue with normal hepatic hemangioma, these specimens were set as a reference for comparison,50HCC patients were divided into high expression group and low expression group. Combined with clinical data, it is found that the tumor volume in high miR-216b expression group was significantly lower than the low expression group, and in this group, the portal thrombosis was also more found than the low expression group. Though chi-square test and it is statistically significant (p<0.05). The others remaining clinical data had no significant difference between the two groups.6, All of50patients were followed up and found that the postoperative patients with high expression of miR-216b have greater overall survival and five-year survival rates(65%and57%), and in patients with low expression of miR-216b the five-year overall survival and disease-free survival rate were37%and28%, seperately. And the differences between the two groups were with statistical significance (p<0.01).Part IV The impact of miR-216b on cell proliferation and invasion in hepatoma cell line HepG2and SMMC7721.Purpose:Research the impact of miR-216b on cell proliferation and invasion in hepatoma cell line HepG2and SMMC7721.Methods:1, RT-PCR was used to detect the expression of miR-216b in hepatocellular carcinoma cell lines, and choose the cell lines with highest and lowest expression level to apply next experiment.2, Mediated by lipofectamine2000, the RNA interference was applied to transfected miR-216b mimics into HepG2cells and the expression of miR-216b was detected after transfection by using RT-PCR method. Similar step was applied to miR-216b inhibitor to transfect SMCC-7721cells, and the expression of miR-216b was detected after transfection by using RT-PCR method too.3, The CCK-8experiment was conducted to test the proliferation ability of HepG2cells and SMCC-7721cells after transfection.4, The soft agar colony formation assay was conducted to detect non-anchor proliferation ability of HepG2cells and SMCC-7721cells after transfection.5, The wound healing test was applied to detect the migration ability of transfected HepG2cells and SMCC-7721cells.6, Transwell assay was conducted to detect invasive ability of transfected HepG2a cells and SMCC-7721cells.7, HepG2cells and SMCC-7721cells were divided into four groups, grown in nude mice, then tumor formation assay was conducted in nude mice. After the formation of HepG2cells tumor, the miRNA mimics were given by intratumoral injection with concentration of100nM, and SMCC-7721cells tumor were given miRNA inhibitor by intratumoral injection with concentration of200nM. Tumor volume was measured during the injection. The tumor weight was measured after three weeks when the mice were killed, and then the expression of miR-216b was detected.Result:1, After the RT-PCR detecting, the miR-216b expression in HepG2cells was lowest and miR-216b expression was highest in SMCC-7721cells among several liver cancer cell lines, so these two cell lines were selected to conduct the next experiment.2, After the transfection of miR-216b mimics, compared with the control cells, the expression of miR-216b in HepG2cells was significantly increased; and after transfection of miR-216b inhibitor, compared with the control group, the expression of miR-216b in the SMCC-7721was significantly decreased. That demonstrated the effectiveness of these two RNA interfering agents.3, After mimics transfection, the proliferation of HepG2cells was observed on the third day. When transfected group compared with the control group, it has obviously been enhanced proliferation until the seventh day, the degree of enhancement was gradually increased. The difference between the two groups was statistically significant (p<0.05). After inhibitor transfection, the proliferation of SMCC-7721cells was observed on the third day. When transfected group compared with the control group, it was significantly inhibited proliferation until the seventh day, he degree of enhancement was gradually increased. The difference was statistically significant (p<0.05).4, After mimics transfection, non-anchored proliferation ability of HepG2cells was observed in two weeks. Two weeks after, compared between transfection group and the control group, the number and size of clones formed in control group are significantly higher than the trabsfected group. The difference was statistically significant (p<0.05). After inhibitor transfection, non-anchored proliferation ability of SMMC-7721cells was observed in two weeks. Two weeks after, compared between transfection group and the control group, the number and size of clones formed in transfected group are significantly higher than the control group. The difference was statistically significant (p<0.05).5, After mimics transfection, the ability of motility of HepG2cells was observed within48hours. Compared between the transfection group and the control group, scratches heal in the transfection group was significantly slower than the control group, and the difference between the two groups was statistically significant (p<0.05). After inhibitor transfection, the ability of motility of SMMC-7721cells was observed within48hours. Compared between the transfection group and the control group, scratches heal in the transfection group was significantly faster than the control group, and the difference between the two groups was statistically significant (p<0.05).6, After mimics transfection, the ability of invasion through matrigel of HepG2cells was observed within48hours. Compared between the transfection group and the control group, the number of cells that entered the lower chamber in the transfection group was significantly less than the control group, and the difference between the two groups was statistically significant (p<0.05). After inhibitor transfection, the ability of invasion through matrigel of SMMC-7721cells was observed within48hours. Compared between the transfection group and the control group, the number of cells that entered the lower chamber in the transfection group was significantly more than the control group, and the difference between the two groups was statistically significant (p<0.05).7, The nude mice were grown to21days, it is found through the growth curve that the growth rate of tumor in the subcutaneous of the inhibitor or the mimics group were significantly greater or smaller than the control group after21days (p<0.01); In21days, tumor diameter in mimics group (1.19±0.29cm3) and tumor diameter in inhibitor group (2.40±0.41cm3), to compared with the volume of the tumors in respective control group (1.60±0.21cm3) and (2.07±0.24cm3) were significantly decreased (p<0.01) and increased (p<0.01); the average tumor weight in mimics group(0.139±0.12) and the average tumor weight in inhibitor group (0.201±0.15), compared to the average weight of the tumors in the respective control groups (0.213±0.06) and (0.170±0.04), were significantly greater (p<0.01) or lower (p<0.01). Part V Mechanism of the impact of miR-216b on cell proliferation and invasion in HCC cell line HepG2cells and SMMC7721cells.Purpose:To explore Mechanism of the impact of miR-216b on cell proliferation and invasion in HCC cell line HepG2cells and SMMC7721cells.Methods:1, Because the important role of the3’UTR region in miRNA target genes, which could affect post-transcriptional expression of this gene. So the usage of miRbases and algorithms was adopted to predict the target genes of miR-216b.2, Kras was selected as the target genes of miR-216b and predicted the binding sites and nucleotide sequences to match miR-216b. Copy the the full length3’UTR region of the gene sequence and designed a variation Sequences as a control, luciferase assay was performed to determine whether miR-216b is the specific binding sequence. The usage of mimics or inhibitor was adopted to act on the sequence.3, RT-PCR and western blotting assay were performed to detect the mRNA expression levels and protein expression levels of KRAS after transfection of mimics or inhibitor.4, RNA interference technology was adopted to knockout Kras, thus to interfer the expression of KRAS, then adding the miR-216b mimics and inhibitor, after that the cell proliferation ability were observe in HepG2and SMMC-7721.5, To overexpress or knockout miR-216b, KRAS downstream genes and further studies of the mechanism of miR-216b were researched.Results:1, Based on a number of online databases and algorithms, it is predicted in five databases that miR-216b can act on the3’UTR of the KRAS gene, thus affects the KRAS protein, which in turn affect its downstream pathways. Then motility and proliferation of HCC were affected.2, By luciferase assay, we found that miR-216b mimics and inhibitor may be specific act on the sites in the3’UTR of the KRAS gene. And after base sequence point mutation in this binding site, the effect of mimics and inhibitor on the3’UTR of KRAS was suppressed.3, The usage of miR-216b mimics in transfection of HepG2cells can reduce the protein expression of the KRAS gene, and exibit a significant difference between the control cells and transfection cells. But the KRAS mRNA levels had no significant effect between two groups, indicating that miR-216b acts on the expression of KRAS after the transcription process. The usage of miR-216b mimics in transfection of SMMC-7721cells can increase the protein expression of the KRAS gene, and exibit a significant difference between the control cells and transfection cells. But the KRAS mRNA levels had no significant effect between two groups, indicating that miR-216b acts on the expression of KRAS after the transcription process too.4, After the KRAS gene was knockout, the inhibiting or promoting role on proliferation of miR-216b mimics or inhibitor on HepG2cells and SMMC-7721cells was disappeared, and there is no statistically significant difference between two groups. It indicates that miR-216b play a role in inhibiting the proliferation of HCC cells mainly by KRAS pathway.5, The usage of miR-216b mimics to transfect HepG2cells can reduce the expression of p-AKT and p-ERK which are the downstream gene of KRAS. The usage of miR-216b inhibitor to transfect SMMC-7721cells can increase the expression of p-AKT and p-ERK which are the downstream gene of KRAS. This indicates that miR-216b has an impact on the proliferation and metastasis of liver cancer by these signaling pathways.Statistical analysisThe results obtained in all experiments must be repeated three times, all the previously mentioned data were expressed as mean±tandard deviation represented in the form, and the application of SPSS16.0software for statistical analysis. The t-test or univariate analysis of variance (ANOVA) was applied to analyse the differences between the groups, and categorical variables be used chi-square test more generally, and the Kaplan Meier method were applied to analyze the survival rate, the p<0.05was considered a statistically significant differentiaton.Conclusion1, MiR-216b expression levels in the plasma of patients with a family history of hepatocellular carcinoma was significantly lower than in healthy volunteers. 2, The expression of miR-216b in patients tumor tissues with primary hepatocellular carcinoma was significantly lower than the adjacent liver tissue. And the expression of miR-216b in tumor tissues is closely related to the level of the patient’s tumor size, presence of portal vein tumor thrombus, and prognosis of postoperative.3, Inhibition or overexpression of miR-216b in vitro experiments and in nude mice in vivo experiments, the effects of the proliferation of HCC cell lines were enhanced or weakened.4, Inhibition or overexpression of miR-216b can affect the abilty of motility and invasion of HCC cell lines in vitro experiments.5, Mainly by direct regulating the KRAS gene, thus regulating the PI3K/ERK and AKT pathways, thereby miR-216b inhibits the ability of proliferation and invasion of HCC.