Overexpression Of MiR-30a-5p Inhibits Cancer Progress By Targeting MTDH Protein

Hepatocellular Carcinoma(HCC) is the world’s fifth largest incidence of tumor with a situation still gradually increasing. When HCC diagnosis was made only 30% had surgery opportunity to remove the tumor, the postoperative five years survival rate was only about 30%~40%, seriously threatened patient’s life and health. Rapid growth of liver tumor, and easily spread to the lymph and blood vessels are important reasons of its poor prognosis. Gene therapy has been emerging as a promising intervention against HCC. However, the complexity of signaling pathways initiate and maintain the occurrence and progression of HCC, the poor understanding of underlying molecular mechanisms in HCC development impedes HCC therapy.MTDH is a single-pass transmembrane protein composed of 582 amino acid with a gene located at chromosome 8q22.5. Overexpression of MTDH could inhibit cancer cell apoptosis and increase invasiveness and metastasis. And many signaling pathways are regulated by MTDH such as nuclear factor-kappa B, Wnt/β-catenin, MAPK/ERK, and PI3K/AKT. MTDH expression in liver cancer, breast cancer patients has been reported. MTDH expression and clinical pathological characteristics and prognosis in thyroid carcinoma has not been reported. To investigate whether MTDH as the universality molecular for tumor targeted therapy, this part we test papillary thyroid carcinoma(PTC) MTDH expression and analyzed clinical pathological characteristics and prognosis, tried to explore MTDH as the universality of the malignant tumor molecular targeting therapy.It is well known that during cancer progression multiply oncoproteins upregulated, on the contrary, multiply tumor suppressing proteins downregulated. The mechanisms of special protein expression change are complicated and miRNAs are recognized to play important roles in epigenetic changes. By binding with target m RNA 3′-UTR, micro RNAs could effectively regulate protein expression. Recently, many reports have found miRNAs dysregulation in HCC and obviously miRNAs participated in malignant change of HCC. Some miRNAs functioned of promoting hepatic tumorigenesis were continued upregulated and silenced such miRNA could upregulate tumor suppressing gene expression. On the contrary, some miRNAs functioned of tumor suppressor were obviously downregulated.In the process of liver embryogenesis, miRNA-30 families expression gradually rise as the process of embryonic development. Bud reported that micro RNA-30 families frequently downregulated in HCC. These results prompt the miRNA-30 families and low differentiation and hint that miRNA-30 families might function of tumor suppressor in liver cancer. MiR-30a-5p overexpression in HCC cells could significantly downregulate MTDH protein expression, Luciferase activity assay showed that miR-30a-5p could directly bind the sequence of MTDH m RNA 3′-UTR in HepG2 cell. And relation analysis showed a reverse relation of MTDH level and miR-30a-5p in HCC tissues.PTEN is a well-known tumor suppressor gene, and loss of PTEN expression is associated with aggressive behavior and poor prognosis in cancer. Our result revealed that miR-30a-5p effectively increased PTEN protein expression which related to decreased MTDH protein expression. Western blot results showed that miR-30a-5p inhibited MTDH protein expression, thus promoted the PTEN protein expression, inhibited AKT phosphorylation activation. All these results indicated that miR-30a-5p might act as tumor suppressor by regulating MTDH/PTEN/AKT pathway in HCC. For the first time, our results indicated that miR-30a-5p functioned of tumor suppressor at least partly through inhibiting MTDH expression in HCC.PART ⅠObjective: The MTDH expression in liver cancer, breast cancer patients has been reported. MTDH expression and clinical pathological characteristics and prognosis in thyroid carcinoma have not been reported. To investigate whether MTDH as the universality molecular for tumor targeted therapy, this part we test papillary thyroid carcinoma(PTC) MTDH expression and analyzed clinical pathological characteristics and prognosis, tried to explore MTDH as the universality of the malignant tumor molecular targeted therapy.Methods1. Immunohistochemistry was adopted to detect MTDH expression in liver cancer, breast cancer and thyroid cancer tissues, and MTDH expression situation was compared with benign tissues.2. MTDH expression in the PTC and the relation with clinical pathological indicators were analyzed;3. High MTDH expression in PTC correlated with low worse DSS, Cox proportional hazards model analysis of the prognostic significance of MTDH expression in the PTC showed high MTDH expression was an independently worse prognosis factor.Results1. MTDH expression in liver cancer tissue was 66.67%(14/21), adjacent liver tissues positive MTDH positive expression rate was 20%(2/10);2. In mammary gland hyperplasia tissues, MTDH expression positive rate was 20%(2/10), the positive rate of MTDH expression in breast cancer was 72.7%(13/18);3. The positive expression rates of MTDH in PTC and ATC tissues were 37.2%(58/156) and 50%(3/6) respectively, and MTDH positive expression rates were both 10%(1/10) in MNG and thyroid adenoma tissues.4. High MTDH expression in PTC positive with larger tumor size(p = 0.030), high rates of lymph node(p = 0.041) and distant metastasis(p = 0.028), but no relation with the patient age, gender, tumor multicenter, extrathyroid invasion, and tumor grade. High MTDH expression was associated with desease specific survival rate(DSS)(p = 0.001). Cox regression analysis showed that high MTDH expression was an independent prognostic indicator for DSS in PTC patients( p = 0.035).ConclusionHigh MTDH expression was found in liver cancer, breast cancer and thyroid cancer tissues, high MTDH expression correlated with malignant pathological characteristics of patients and with poor prognosis, targeting MTDH therapy might provide important targets for tumor treatment.PART ⅡObjective: In this section, we selected miR-30a-5p, test its expression in liver cancer tissues and the adjacent carcinoma tissues, the biological information method was used to forecast miR-30a-5p targets, and the correlation of miR-30a-5p and MTDH expression in liver cancer tissues was analyzed.Methods1. The RT-q PCR method was used to detect miR-30a-5p expression in liver cancer cells, liver cancer tissues and the adjacent to carcinoma tissues;2. Bioinformatics technology showed MTDH might be miR-30a-5p targeting gene, Western blot confirmed that miR-30a-5p inhibited MTDH protein expression, Luciferase Assay validated MTDH was a miR-30a-5p targeting gene;3. Correlation analysis to analyze the relation of MTDH expression in liver cancer tissues and miR-30a-5p.Results1. MiR-30a-5p expression in liver cancer tissue was significantly reduced, the miR-30a-5p targeted MTDH protein expression in liver cancer tissues;2. MiR-30a-5p might target MTDH m RNA 3’-UTR;3. In liver cancer tissues, there is a negative correlation between miR-30a-5p and MTDH expression.ConclusionThese results indicated that miR-30a-5p might regulate MTDH expression by targeting MTDH m RNA 3’-UTR, and thus regulate the MTDH protein expression.PART ⅢObjective: MiR-30 family members mainly functioned as tumor inhibitor. Among them, one of the members of the family of miR-30a-5p commonly reported in other tumors, we selected miR-30a-5p as the research object, and detected its effect on liver cancer cell biology, and to explore the possible mechanism.Methods1. Lipofection2000 transfection technology to over-express miR-30a-5p in HepG2 and SMMC-7721 liver cancer cells, the CCK-8 and colony formation experiment to test proliferation, flow cytometry to detect cell apoptosis;2. Scratch experiment and Transwell invasion Assay were observed after miR-30a-5p express in hepatocellular carcinoma cell line HepG2 and SMMC-7721;3. With si RNA technology, silencing MTDH protein in liver cancer cell HepG2 and SMMC-7721, cell proliferation, colony formation, apoptosis, migration and invasion were observed;4. Western blot was used to detect PTEN protein, the explore miR-30a-5p regulation role of PTEN/AKT signal pathway.Results:1. MiR-30a-5p overexpression in HCC cells significantly inhibited cell proliferation, colony formation and induced apoptosis;2. MTDH was miR-30a-5p targeting gene. MiR-30a-5p upregulated PTEN protein expression and thus inhibited AKT activating by targeting MTDH;3. Silencing MTDH protein in liver cancer cell HepG2 and SMMC-7721 inhibited cell proliferation, colony formation, and induced apoptosis. MTDH si RNA also inhibited cell migration and invasion;4. Western blot results showed that miR-30a-5p inhibited PTEN/AKT pathway, overexpression of miR-30a-5p inhibited MTDH protein expression, thus promoted the PTEN protein expression, inhibited AKT phosphorylation activation.Conclusion: MiR-30a-5p might regulate through MTDH/PTEN/ AKT pathway, so as to inhibit HepG2 and SMMC-7721 hepatocellular carcinoma cells proliferation, colony formation, and promote tumor cell apoptosis. MiR-30a-5p also effectively inhibited liver cancer cell migration and invasion.PART Ⅳ Objective To investigate miR-30a-5p biologic effect on HepG2 cells inoculated subcutaneously into nude mice.Methods1. MiR-30a-5p was transferred into HepG2 cells and then inoculated subcutaneously into nude mice. And the control group transferred with miRNA NC. The tumor was observed.2. Immunohistochemistry was used to test MTDH, PCNA and TUNEL expression.3. Western blot was used to test MTDH, PCNA and p-AKT protein in tumor tissues from nude mice.Results1. Smaller tumor was observed in HepG2 cells transferred with miR-30a-5p mimic in nude mice. Compared with the control group, the tumor size and quantity of miR-30a-5p mimic group were reduced, the difference was statistically significant.(p <0.05).2. MiR-30a-5p significantly inhibited MTDH and PCNA expression in HepG2 tumor, however, higher TUNEL expression was found in miR-30a-5p mimic transferred group.3. And miR-30a-5p also inhibited AKT activation.ConclusionMiR-30a-5p could Inhibit the growth of HepG2 liver cancer tumor cells in nude mice. And this might be that miR-30a-5p could inhibit MTDH expression in tumor tissues.