SMP30 Regulates HCC Cell Migration via Affecting the Expression of MMPs and Binding to ROCK1


 Background: SMP30, as a calmodulin, is differentially expressed in hepatocellular carcinoma（HCC） tissues and adjacent tissues. Previous studies have indicated that the expression of SMP30 is closely related to the prognosis of patients with HCC. However, little is known about the detailed role of SMP30 on HCC. Methods: The SMP30 overexpression and silenced cell lines were constructed through lentivirus transfection，the effects of SMP30 on migration and invasion of HCC were observed by Transwell assay. Then subcloned cell line with different migration abilities were undergone four rounds of screening from SMP30 overexpressing cell lines, and the transcriptome and proteome were detected and analyzed. The interactions of proteins with SMP30 were validated by AP-MS and COIP-WB techniques. Results: High expression of SMP30 can effectively inhibit the migration and invasion of HCC, but has no significant effect on cell proliferation and cell cycle. The cell lines with higher metastatic abilities have significantly higher calcium ion concentrations than those with lower metastatic abilities and higher expression of MMP9, MMP14 and MMP15 proteins. There is an interaction between SMP30 and tumor metastasis-related protein ROCK1, which reduces the phosphorylation level of cytoskeleton-related MLC protein. The SMP30 regulatesthe downstream molecule MLC protein phosphorylation and affects the formation of cytoskeletal structure. Conclusion: On the one hand, SMP30 regulates the expression of MMPs protein by regulating the Ca2 + concentration; on the other hand, SMP30 affects the phosphorylation level of MLC protein by interacting with ROCK1 protein. The combined effect of the two aspects inhibits the metastatic ability of HCC.


Introduction
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, and the mortality rate is second only to gastric cancer and lung cancer [1] .The pathogenesis of HCC is closely related to metabolic diseases such as chronic viral hepatitis, alcoholic hepatitis and liver cirrhosis, but the mechanisms of its occurrence and metastasis of HCC is still unclear [2] .
Our research group has successfully screened out HCC related antigen-senescence marker protein 30 (SMP30) which was first identified from Guangxi human hepatocellular carcinoma cDNA expression library using a serological analysis of recombinant cDNA expression library (SEREX) approach [3] .The SMP30 protein is a novel calcium-binding protein that gradually decreases with age. The results of immunohistochemical analyses have demonstrated that SMP30 is generally showing low expression in tumor tissues and high expression in adjacent tissues. This is closely related to the shorter survival rate of patients. How this differential expression pattern affects the biological function of liver cancer cells is still not very clear [4] .The main objective of this study is to confirm the effect of SMP30 on HCC through cell behavior experiments and further investigating the mechanisms of SMP30 on HCC.

Cell proliferation assay
The 96-well cell culture plate was used to seed cells (1000cells/well) and incubated in different time (0,24,48,72,96 and 128hrs).The 1μl of CCK8 solution (Dojindo, Japan) was added to each well and incubated for 2hr in incubator. The absorbance values were measured daily by microplate reader at 450nm and each assay was repeated three times.

Cell migration and invasion assays
Cells were collected and the concentration was adjusted to 1×10 5 cells/ml with serum-free medium. Transwell plate was cultured at 37℃ in 5% CO 2 humidified atmosphere for 24hrs. The cells stayed in the upper and lower chambers were extendedly cultured separately. The two groups of cells were subjected to the above-mentioned repeated Transwell operations to finally select a subcloned cell line (SK-Down) with strong migration ability and a subcloned cell line with weak migration ability (SK-Up). 5 6. Homogeneous and heterogeneous adhesion tests of different migration ability of subcloned cell line.
For homogeneous adhesion ability test, the SK-Up and SK-Down cells were adjusted to 100/ml in DMEM medium, then added to a 96-well cell culture plate with 100μl per well. Cells were cultured at 37℃ until complete fusion. Culture medium was discarded and the cells were Total RNAs from SK-Up and SK-Down cells were extracted to convert cDNA by reverse transcription method and the cDNA library was constructed to prepare for the next sequencing analysis. The 10μl library 6 cDNA was mixed with 10μl NaOH for 5 minutes at room temperature and 980μl of pre-cooled hybridization buffer was added to the mixture.
The molecules in the library were combined with primers immobilized on the Flowcell for bridge PCR amplification to generate clusters. The generated Flowcell was transferred to the sequencing platform and the corresponding recipe was selected for sequencing.

Statistical Analysis
The SPSS 17.0 software was adopted for statistical analysis in this study.
The data obtained were expressed as mean±standard deviation (SD) and T test was used for comparison between groups. The P-value of less than 0.05 was considered having statistical difference.

The effects of SMP30
We have also successfully constructed SMP30 silenc (97L-shRNA) and negative controls ( Figure 2 investigate the effects of SMP30 on assay shows no significant abnormalities in cell proliferation after SMP30 is silenced ( Figure 2C).    Figure 8.Immunofluorescence was carried out to detect the stress fiber rearrangement. F-actin were stained by FITC-Phalloidine (red) and the nuclei were stained by Hoechst 33342 (blue).

Discussion
SMP30 is a new type of calcium-binding protein, which is involved in the regulation of various intracellular metabolic processes and is closely related to the occurrence and development of HCC [5] .In the early stages, our research group have confirmed through immunohistochemistry that the expression levels of SMP30 in HCC tissues are significantly lower than that in the corresponding paracancerous tissues. The expression level of SMP30 is closely related to tumor size, grade and prognosis of patients [6] .Previous studies have also confirmed that the proliferation and migration ability of HepG2 cells increased after SMP30 is silenced by shRNA [7] .
In HCC is a highly invasive tumor with intrahepatic proliferation and extrahepatic metastasis. Therefore, effective inhibition of tumor metastasis is very important for the prognosis of HCC [8][9] . SMP30 can effectively inhibit the migration and invasion of liver cancer cells, but its mechanism is not yet clear. Based on literature reports and our research, we speculate that SMP30 can play a role in inhibiting the migration and invasion of liver cancer cells through the following two aspects. On one hand, we have screened out subcloned cell lines with different migration abilities and performed transcriptome and proteome sequencing analysis.
Our studies have confirmed that the low level of SMP30 expression in SK-Down subcloned cell lines with strong metastasis ability led to the increase of intracellular Ca2+ concentration. Meanwhile, high concentration of Ca2+ induces cells to produce MMP9, MMP14 and MMP15, and participate in the regulation of extracellular matrix signaling pathway [10][11][12] . Therefore, SMP30, as a calmodulin, can activate the cellular calcium pump. When the expression level of SMP30 decreases, the activity of the cellular calcium pump decreases and the intracellular Ca 2+ concentration increases [13][14] . High concentrations of intracellular Ca 2+ promote the synthesis and release of MMPs family proteins and the MMPs protein family can degrade and damage collagen, fibrin, laminin and other extracellular matrix components, and play an important role in 18 cell invasion and metastasis [15][16][17] . On the other hand, we have screened ROCK1 protein as SMP30 interacting protein by affinity purification-mass spectrometry (AP-MS) and confirmed the existence of interaction between SMP30 and ROCK1 through Co-Immunoprecipitation-Western Blot (COIP-WB). The ROCK1, similar to Rho (Ras homolog), is a small molecule protein with GTPase activity and a helical structure，which can phosphorylate myosin light chain (MLC) to cause contraction of cell myofilament [18][19] .At the same time, ROCK1 can also phosphorylate MLCP. Phosphorylated MLCP cannot dephosphorylate MLC, and the contractile force of cell muscle filaments will be further increased [20][21] .Therefore, we speculate that ROCK1 binding to SMP30, resulting in weakened intracellular tension fiber synthesis, decreased MLC phosphorylation level and reduced cell migration ability.
We believe that SMP30 can effectively reduce the migration ability of