Therapeutic Effect of Down-Regulation of MTH1 Gene Expression on HepG2 Hepatocellular Carcinoma in Nude Mice

The aim of this study was to establish an animal model of HepG2 liver cancer in nude mice, and to inhibit the expression of MTH1 gene in tumor cells by siRNA interference. To study the therapeutic effect of down-regulation of MTH1 expression on HepG2 tumor in nude mice, and to provide a new strategy for the treatment of HCC. Methods: Human HepG2 cells were cultured and transplanted tumor models were established under the armpit of right forelimb of nude mice. The successful nude mice were randomly divided into two groups: control group (CT) and siRNA transfection group (MTH1). The tumor volume of each group of nude mice was consistent. This experiment was mainly conducted by injecting siRNAs into tumor-bearing nude mice and transfection reagents in vivo. The survival time and living condition of nude mice were observed. The tumor inhibition was evaluated by measuring the volume and weight of solid tumor in nude mice. After the administration, The expression of mth1 and its related genes were detected by qPCR to determine the effect of QPCR on the mRNA expression of each gene. The relative expression of mth1 and related proteins in tumor tissues was detected by Elisa and Western blotting. The effect of siRNA on the protein level of each gene was evaluated by calculating the gray value and comparing the od value. Results: The animal model of HepG2 liver cancer in nude mice was successfully established, and the tumorigenesis rate was 100%. The siRNA transfection group had significant anti-tumor effect and significantly inhibited the growth of tumor cells. Survival rate of nude mice in each group was 100%. Compared with the control group, In the transfected group, the expression of mth1 mRNA and protein decreased significantly in the tumor tissues. The expression of caspase-3 protein was significantly increased in the transfected group. Conclusion: SiRNA-MTH1 can effectively silence the expression of MTH1 gene. Down-regulation of mth1 gene expression by siRNA can inhibit the growth, proliferation and apoptosis of HepG2 tumor cells.


Introduction
Primary liver cancer (HCC) is one of the most common malignant tumors in the world, its morbidity and mortality are among the highest in the world [1] . The liver cancer has the characteristics of concealment, long latency, rapid development, high malignancy and so on. Surgical treatment is the best method for the treatment of liver cancer [2] . However, because most patients were diagnosed only in the late stages of the disease, they missed the best time for surgical treatment. Therefore, the search for new therapeutic tools and drug targets is essential for the treatment of liver cancer [3] . MTH1 (mutT homolog1) is a homologous enzyme of MutT, which is widely distributed in the nucleus and mitochondria and participates in DNA damage and repair. especially in the repair of DNA damage of tumor cells. It is shown that MTH1 can scavenge the oxidized dNTPs, prevent them from participating in DNA replication and prevent DNA damage [4] . MTH1 is highly expressed in a variety of tumor cells, but not or rarely expressed in healthy tissue cells. MTH1 is considered to be an important repair gene in tumor cells [5,6] .
The occurrence and development of liver cancer is a complex multistage process. It is the long-term accumulation of multiple gene variants and is closely related to the increase of proto oncogene expression. Therefore, targeted therapy for proto-oncogene is more effective. The purpose of this study was to investigate the effects of simultaneous targeting proto-oncogene therapy on the biological behavior of HepG2 cells. We designed and synthesized siRNA sequences targeting MTH1 gene and transfected them into HepG2 cells in vitro. To investigate the effect of down-regulation of MTH1 gene expression on the proliferation, invasion and apoptosis of HepG2 cells. To provide a theoretical basis for gene targeting therapy of cancer.

MATERIALS AND METHODS Cell Culyures
HepG2 cells were grown in Roswell Park Memorial Institute medium (RPMI-1640) (Hyclone, GE Healthcare Life Sciences, Logan, UT, USA), containing 10% fetal bovine serum (Gibco TM ; Thermo Fisher Scientific, Inc, Carlsbad, CA, USA), 100U/ml penicillin and 100 µg/ml strepto-mycin, and incubated at 37˚C in a humidified atmosphere of 5% CO 2 . Cells in exponential growth phase were used throughout the experiments.

Establishment of Nude Mouse Model
After 6 days of inoculation, the tumor grows to a certain size. According to the size of tumor, they were divided into control group and siRNA-MTH1 transfection group. There were 6 mice in each group, and the tumor volume of each group was basically the same. The control group was given transfection reagent, SiRNA-MTH1 transfection group was given, each of them was 19.8 ug. Once every 3 days next to the tumor, lasting one month.

Observation on living condition of tumor-bearing nude mice
The activity, tumor growth, food intake and water intake were observed daily and the living conditions of the mice were recorded. The long and short diameters of the tumor were measured every 5 days, and the volume size of the tumor was calculated and recorded. V= 1 / 2 × ab 2 (a is long diameter and B is short diameter). Tumor growth inhibition rate (control group tumor weight-experimental group tumor weight / control group tumor weight × 100%).

Detection of relative mRNA expression in tumor tissues
Real-time fluorescence quantitative PCR: To remove the total RNA by using Trizol reagent by conventional method. Total gold reverse transcription kit was used to reverse the RNA into cDNA. then the cDNA was amplified by QPCR. β-actin gene primers:

Determination of protein expression in tissues
The relative expression of MTH1 protein was detected by enzyme-linked immunosorbent assay (Elisa). The relative expression of apoptotic protein caspase-3 was detected by Western blot assay. The tumor tissue of 100mg was removed, and the blood stain was washed by PBS. The tissue was lapped in 1ml RIPA for the night and then centrifuged to extract the supernatant. The tissue protein solution. Then according to the instructions of Elisa kit and the method of Western imprinting.
Statistical treatment SPSS 17.0 statistical software was used to analyze the data. The experimental data are expressed as mean ±standard deviation, single factor analysis of variance was used for statistical analysis. P < 0.05 The difference is statistically significant.

Observation on general physiological conditions of tumor-bearing nude mice
HepG2 cells were all inoculated 7 days later, and the tumor formation rate was 100%. The survival condition of the mice was good, and there was no significant difference between the groups. With the development of the experiment, the tumor growth rate of siRNA transfected mice was slower than that of control group. In the later stage of experiment, the control group gradually suffered from loss of appetite and abnormal activity.
Effect of siRNA administration on the volume and quality of nude tumor 30 days after administration, the tumor size, volume, weight and growth rate of the two groups were compared. The tumor growth rate of nude mice in transfection group was significantly lower than that in control group (Fig.1A). The tumor size in the transfection group was significantly smaller than that in the control group (Fig.1B). The tumor weight in the transfection group was significantly lower than that in the control group ( fig. 1C). There were significant differences in volume and tumor weight between the transfection group and the transfection group. There was statistical significance (P < 0.05). The result is as follows.   showed that siRNA-MTH1 could inhibit the expression of proto-oncogene mth1. Fig. 2 The mRNA expression of MTH1 gene in tumor tissue in transfected group was significantly lower than that in control group, and the difference was statistically significant(*: P <0.05).

Detection of MTH1 and caspase-3 protein relative expression in tumor tissues
The expression of mth1 protein in the transfected group was significantly lower than that in the control group (Fig. 3A). The expression of caspase-3 protein in the transfected group was significantly higher than that in the control group (*: P <0.05). The relative expression level was detected by ELISA in tumor tissue. The expression of mth1 protein in the transfected group was significantly lower than that in the control group(**: P <0.01) (Fig. 3B). Fig. 3A Western blotting was used to detect the relative expression of MTH1 and caspase-3 protein in tumor tissues. The siRNA transfection group was compared with the control group, the expression of mth1 protein decreased significantly and the expression of caspase-3 protein increased significantly. Fig. 3B Elisa method was used to detect the relative expression of mth1 protein in tumor tissues. The expression of mth1 protein in the transfected group was significantly lower than that in the control group.

DISCUSSION
The best-known small, non-coding RNA that suppresses gene expression falls into three broad categories: Small RNAs miRNAs, small interference RNAs siRNAs, and interactions with PIWI protein RNAs piRNAs [7,8,9] . The mature forms of these RNAs are about 20-30 nucleotide nts, which are associated with many ago protein superfamily members and play a key role in RNA interference RNAi pathway [10][11][12] . It is generally believed that miRNA and siRNA mediate post-transcriptional gene silencing, guide the complement of ago and mRNA in cytoplasm to form silencing complex, induce transcriptional degradation and prevent post-transcriptional translation [8,11] . PIWI, a branch of the PiRNA and ago protein family, binds to the silent germ cell transposon and plays a role in many biological fertility [13,14] .
Although many preliminary studies have focused on the mechanism of gene silencing mediated by small and medium-sized RNAs in the cytoplasm, it has also been shown that many of these RNAs can also mediate epigenetic transcriptional silencing in the nucleus [15][16][17][18][19] . It is proved that ago protein is a key factor in the mechanism of nuclear RNAi, as well as its importance and versatility as a gene silencing complex. RNA-induced transcriptional silencing complex, mainly loaded with a small ribonucleic acid, can mediate cotranscriptional gene silencing [20,21] .
In this study, mth1 gene expression in tumor cells was silenced by RNAi technique, and the therapeutic effect of down-regulation of MTH1 expression on nude mice HepG2 hepatoma tumor was studied. The results showed that siRNA-MTH1 could silence the expression of MTH1 gene, and down-regulate the proliferation and invasion of HepG2 tumor after the expression of MTH1 gene was down-regulated. The ability of apoptosis was enhanced. In general, down-regulation of MTH 1 gene expression significantly inhibited the growth of tumor cells. At present, the majority of targeted immunotherapeutic drugs are antibody drugs and small molecular drugs. Small molecular drugs belong to chemical drugs, which have toxic side effects on the body, also cause damage to normal cells, and may also cause harm to people in the process of production. The antibody is a protein, high cost and long-term use of the body will cause immune response, but also produce side effects on the body. The purpose of this study is to find a substitute or method to reduce the side effects on the body and to effectively inhibit the proliferation of tumor cells at the same time. SiRNA is easy to produce and low in cost. It does not produce immune tolerance and is a biomolecules. It is metabolized quickly in vivo, so it has no side effects. This study is an attempt to search for siRNA anti-cancer drugs. The combination of siRNA and siRNA may enhance the anticancer effect, make it synergistic, and kill tumor cells to the maximum extent. Therefore, the use of RNAi targeted immunotherapy may become a hot topic in tumor therapy and an important part of accurate therapy.