MicroRNA-302a Inhibits Hepatoma Cells Proliferation And Promotes Apoptosis By Targeting MAP3K2 And PBX3

Hepatocellular carcinoma(HCC),as a common malignancy in China,has a high incidence and poor prognosis.Although great progress has been achieved in the treatment of HCC,including surgery,interventional therapy,radiotherapy,chemotherapy and molecular target treatment,the prognosis of HCC is still poor due to the metastasis and recurrence.Therefore,it is necessary to explore highly stable and specific molecular targets and provide the basis for early diagnosis of HCC and targeted therapy,in order to improve the prognosis of HCC.Meanwhile,elucidating the molecular mechanisms involved in HCC is essential for developing cancer prevention strategies and possible guiding disease management in the clinic.Accumulating evidence suggests that micro RNAs(mi RNAs)are involved in the initiation and progression of HCC.The 22 nt noncoding mi RNAs act as key regulators of various fundamental biological processes,such as development,differentiation,apoptosis,and cell proliferation,in which common pathways are shared with cancer.Bioinformation analyses estimate that mi RNAs may regulate as much as 30% of the human protein coding genes,including oncogenes and tumor suppressors,suggesting that these small RNAs may act to coordinate the interplay between complex signal transduction pathways.Several mi RNAs themselves have been demonstrated to serve as tumor suppressor genes or oncogenes in tumors.The mi R-302/367 cluster are located in chromosome 4q25 and contains several members such as mi R-302a/b/c/d and mi R-367.Studies have shown that mi R-302/367 plays a regulatory role in embryonic stem cell renewal and pluripotent maintenance through targeting cell cycle,transformation of the upper cortex and vesicle transportation.So far,more and more studies have confirmed that mi R-302/367 can exert different biological roles by targeting different target genes in tumor development.In prostate cancer and colon cancer tissues,the expression level of mi R-302 a is down-regulated,and the tumor cells are blocked in G1/S phase by targeting AKT1,resulting in the cell proliferation inhibition.Meanwhile,mi R-302/367 can down-regulate the expression of AKT1 and Cyclin D1 in cervical cancer cells to block cell proliferation.In ovarian cancer and breast cancer,mi R-302 a can respectively target SDC1 and CXCR4,thereby inhibiting tumor cellproliferation,invasion,migration and promoting tumor cell apoptosis.Further studies have shown that mi R-302 a can significantly increase the killing activity of cisplatin on testicular cancer cells,and reverse the malignant phenotype of melanoma tumor cells by inhibiting the activity of Notch4 signaling.However,the molecular mechanisms of mi R-302 a involves in the regulation of hepatocellular carcinoma activity remains unclear.In addition,molecular therapy can be used for the treatment of liver cancer,and molecular diagnosis can also be used in disease prevention.Therefore,it is particularly important to find effective genes diagnosis.In this study,the differential expression of mi R-302 a in different hepatoma cell lines,HCC and adjacent tissues were detected,meanwhile,the target relationship between mi R-302 a and MAP3K2 or PBX3 was verified.Furthermore,the role and regulation mechanism of mi R-302 a in hepatocarcinogenesis were studied,so as to provide effective means for liver cancer early diagnosis and clinical treatment.Part 1: mi R-302 a target genes prediction and target relationship verification Objective:(1)To detect mi R-302 a expression in normal hepatocytes and different hepatoma cell lines and the target genes of mi R-302 a were predicted according to the analysis of mi RNAs target genes bioinformatics database.(2)To construct mi R-302 a expression vector and dual luciferase reporter gene vector,and to verify the target relationship between mi R-302 a and MAP3K2,PBX3.(3)To study the differential expression of mi R-302 a,MAP3K2 and PBX3 in HCC and adjacent tissues,and to analyze the correlation between mi R-302 a and MAP3K2,PBX3 in HCC and adjacent tissues.Methods:(1)The expression level of mi R-302 a was detected in liver cancer cell lines L02,Bel-7402,SMMC-7721,PLC and Hep G2 using q PCR.(2)The target gene of mi RNAs can be predicted by bioinformatics software,and the results are analyzed by Target Scan,NCBI,mi Rbase,micrriorna.org and other bioinformatics softwares.(3)The over expression vector and interference vector of mi R-302a-3p were designed by mi RBase database.Using UCSC database and NCBI database to design dual luciferasereporter gene vectors.(4)The luciferase activity of mi R-302 a and target genes MAP3K2 and PBX3 were detected by dual luciferase reporter gene system.And the effect of mi R-302 a on the expression levels of MAP3K2 and PBX3 was detected at the cellular level.(5)Randomly selected 10 patients with liver cancer,collecting liver cancer and cancer adjacent tissue,observing tissue morphology with HE and real-time fluorescence quantitative PCR(q-PCR)methods was taken to detect the expression changes of mi R-302 a,MAP3K2 and PBX3 different tissues.Results:(1)QPCR detection results showed that the expression of mi R-302 a in hepatoma cell lines Hep G2 and SMMC-7721 was significantly lower than that in normal hepatocytes(P<0.01).(2)According to the analysis of mi RNAs target gene bioinformatics database,MAP3K2 and PBX3 were selected as mi R-302 a target genes.(3)The mi R-302 a mimics,inhibitor,mi R-sh NC expression vectors and dual luciferase reporter vectors were constructed successfully.MAP3K2-WT,PBX3-WT(wild type recognition sequences: AGCACTTT),MAP3K2-mut,PBX3-mut(mutant recognition sequences: ACACTCCA).(4)The mi R-302 a was co-transfected with MAP3K2-WT,PBX3-WT,mut,Si vectors,luciferase activity assay results showed that the luciferase activity of mi R-302a+MAP3K2-WT group was significantly lower than that of mi R-302a+MAP3K2-mut transfection group and mi R-302a+Si group(P<0.01).The luciferase activity of mi R-302a+PBX3-WT group was significantly lower than that of the other two groups(P<0.01),and there was no significant difference between other two groups(P> 0.05).(5)The results showed that the expression level of mi R-302 a in mi R-302 a mimics group was significantly higher than that in the control group and its level in mi R-302 a inhibitor group was significantly lower(P<0.01),indicating that mi R-302 a was overexpressed and interfered successfully in human Hep G2 and SMMC-7721 cells.Compared with the control group,the expression levels of MAP3K2 and PBX3 were significantly decreased,these indicators showed opposite trends in the mi R-302 a inhibitor group(P<0.01).(6)HE results showed that liver cancer tissues exhibited obvious cancer characteristics.The expression of mi R-302 a in HCC tissues was significantly lower than that of the adjacent tissues by q PCR detection(P<0.01);However,the MAP3K2 and PBX3 genes in HCC tissues were significantly higher than those in the adjacent group,and the protein levels kept the same trend(P<0.01).The expression level of mi R-302 a was negatively correlated with the expression of MAP3K2 and PBX3 in HCC and adjacent group.Conclusions:(1)The expression of mi R-302 a in hepatoma cell lines Hep G2 and SMMC-7721 was significantly lower than that in normal hepatocytes.MAP3K2 and PBX3 were predicted to be the target genes of mi R-302 a according to the bioinformatics database analysis.(2)Dual luciferase reporter gene system verification,q PCR and Western blot results showed that there was a target relationship between mi R-302 a and MAP3K2 or PBX3.(3)The expression levels of mi R-302 a,MAP3K2 and PBX3 were different in HCC and adjacent tissues,suggesting that mi R-302 a,MAP3K2 and PBX3 might be involved in the occurrence and development of HCC,which laid the foundation for further study of the regulation mechanism of mi R-302a/MAP3K2/PBX3 on HCC.Part 2: Effects of mi R-302 a on hepatoma cells proliferation and apoptosis by targeting MAP3K2 and PBX3Objective:To study the regulation mechanism of mi R-302 a on hepatoma cells proliferation and apoptosis.Methods:(1)MTT assay and flow cytometry were used to detect the effects of mi R-302 a on proliferation and apoptosis of hepatoma cells after transfection of Hep G2 and SMMC-7721 cells with mi R-302 a.(2)The Western blot methods were used to verify the effects of mi R-302 a on MAP3K2,PBX3,ALDH1,p-Erk,Erk,p-JNK,JNK,p-p38,p38,Cyclin A,Cyclin D,Caspase-3 and Bcl-2 protein levels.(3)The si MAP3K2 and si PBX3 were constructed and transfected into Hep G2 and SMMC-7721 cells.The MAP3K2 and PBX3 expression level was detected by q PCR andWestern blot and p-ERK,ERK,p-JNK,JNK,p-p38 n and p38 protein level was detected using Western blot method.(4)The PBI-CMV3-MAP3K2 overexpression vector was constructed and expressed in mi R-302 a treated Hep G2 and SMMC-7721 cells.The expression level of MAP3K2 was detected by q PCR and Western blot.The proliferation and apoptosis of Hep G2 and SMMC-7721 cells were detected by MTT assay and flow cytometry.(5)The PBI-CMV3-PBX3 overexpression vector was constructed and expressed in mi R-302 a treated Hep G2 and SMMC-7721 cells.The expression level of PBX3 was detected by q PCR and Western blot.The proliferation and apoptosis of Hep G2 and SMMC-7721 cells were detected by MTT assay and flow cytometry.Results:(1)MTT and flow cytometry results showed that mi R-302 a could inhibit the proliferation of Hep G2 and SMMC-7721 cells and promote cell apoptosis(P<0.01).(2)Western blot detection results showed that,compared with the control group,the expression levels of ALDH1,p-Erk,p-JNK,p-p38,Cyclin A,Cyclin D and Bcl-2 were significantly decreased,the expression level of ERK,JNK and p38 remained unchanged,and Caspase-3 was significantly increased in mi R-302 a mimics group(P< 0.01).However,these indicators in the mi R-302 a inhibitor group showed an opposite trend(P<0.01).(3)The results of q PCR and Western blot showed that inhibition of MAP3K2 and PBX3 expression reduced the expression of p-ERK,p-JNK and p-p38(P<0.01).The expression levels of ERK,JNK,and p38 remained unchanged.(4)Overexpression of MAP3K2 or PBX3 in mi R-302 a mimics-treated cells restored Hep G2 and SMMC-7721 cell proliferation,while inhibiting mi R-302a-mediated apoptosis(P<0.01).Conclusions:miR-302 a could inhibit the proliferation of Hep G2 and SMMC-7721 cells and promote apoptosis partly through the MAP3K2/MAPK and PBX3/MAPK pathways.Objective:(1)To evaluate the effect of mi R-302 a on the expression of MAP3K2 and PBX3 in tumors.(2)To establish nude mice hepatoma model and to evaluate the effects of mi R-302 a on tumor formation and differentiation in nude mice.Methods:(1)The mi R-302 a mimics,inhibitor and mi R-sh NC were respectively transfected into human Hep G2 cells.The logarithmic growth phase cells were diluted by culture medium and inoculation in nude mice subcutaneous of the left upper limb.(2)The expression levels of MAP3K2 and PBX3 in the tumor were detected by q PCR and Western blot.(3)We weighed the weights of the tumors and the tumor volume was calculated.The effects of mi R-302 a on tumor formation was evaluated.Results:(1)Compared with the empty group,and the expression levels of MAP3K2 and PBX3 were decreased significantly(P<0.01)in mi R-302 a mimics group and significantly increased in the mi R-302 a inhibitor group(P<0.01).(2)The volume and weight of the tumor in mi R-302 a group were significantly smaller(P<0.05)and increased in the mi R-302 a inhibitor group(P<0.01).Conclusions:(1)Mi R-302 a could inhibit liver cancer formation obviously.(2)The mechanism of mi R-302 a effect on liver cancer was to regulate the MAP3K2 and PBX3 genes.Part 3: The mechanism study of mi R-302 a effects on hepatocarcinogenesis.Objective:(1)To evaluate the effect of mi R-302 a on the expression of MAP3K2 and PBX3 in tumors.(2)To establish nude mice hepatoma model and to evaluate the effects of mi R-302 a on tumor formation and differentiation in nude mice.Methods:(1)The mi R-302 a mimics,inhibitor and mi R-sh NC were respectively transfected into human Hep G2 cells.The logarithmic growth phase cells were diluted by culture medium and inoculation in nude mice subcutaneous of the left upper limb.(2)The expression levels of MAP3K2 and PBX3 in the tumor were detected by q PCR and Western blot.(3)We weighed the weights of the tumors and the tumor volume was calculated.The effects of mi R-302 a on tumor formation was evaluated.Results:(1)Compared with the empty group,and the expression levels of MAP3K2 and PBX3 were decreased significantly(P<0.01)in mi R-302 a mimics group and significantly increased in the mi R-302 a inhibitor group(P<0.01).(2)The volume and weight of the tumor in mi R-302 a group were significantly smaller(P<0.05)and increased in the mi R-302 a inhibitor group(P<0.01).Conclusions:(1)Mi R-302 a could inhibit liver cancer formation obviously.(2)The mechanism of mi R-302 a effect on liver cancer was to regulate the MAP3K2 and PBX3 genes.