Serine/arginine rich splicing factor 2 expression and clinic pathological features indicating a prognostic factor in human hepatocellular carcinoma patients

Abstract

BACKGROUND:

This research was aimed to study the expression of Serine/arginine rich splicing factor 2 (SRSF2) in tissues of hepatocellular carcinoma, and explore the relationship between the expression and the clinic pathological and prognosis of human hepatocellular carcinoma (HCC).

METHODS:

One hundred and fifty-three pairs HCC tissues and adjacent normal tissue were collected from January 2010 to March 2013. The expression of SRSF2 gene was detected by immunohistochemistry, western blotting and real-time quantitative polymerase chain reaction (PCR), and the relationship between the expression and the clinic pathological and prognosis of HCC being analyzed.

RESULTS:

In 153 cases of hepatocellular carcinoma, SRSF2 was highly expressed in 93 cases, low expression of 60 cases, immunohistochemistry score (6.50 $\pm$ 2.82), which was significantly higher than that in adjacent normal tissues (2.94 $\pm$ 1.23) ( $P<$ 0.05). The expression of SRSF2 in HCC was not associated with gender ( $\chi^{2}=$ 0.014, $P=$ 0.906), age ( $\chi^{2}=$ 0.007, $P=$ 0.931), tumor size ( $\chi^{2}=$ 3.566, $P=$ 0.059) and T stage ( $\chi^{2}=$ 2.708, $P=$ 0.100), and was significantly correlated with tumor differentiation ( $\chi^{2}=$ 9.687, $P=$ 0.007), lymph node metastasis ( $\chi^{2}=$ 4.827, $P=$ 0.028), distal metastasis ( $\chi^{2}=$ 9.235, $P=$ 0.002), tumor, node, metastasis (TNM) stage ( $\chi^{2}=$ 3.978, $P=$ 0.046), portal vein invasion and serum alpha-fetoprotein ( $\chi^{2}=$ 14.919, $P=$ 0.000). The expression of SRSF2 protein in hepatocellular carcinoma was positively correlated (r $=$ 0.704, $P<$ 0.05) with serum alpha-fetoprotein through Pearson analysis. The survival rates of SRSF2 overexpressing hepatocellular carcinoma were 74.19%, 44.09%, 26.88%, 24.73% and 21.51% at 1 year, 2 years, 3 years, 4 years and 5 years respectively, which were lower than those of SRSF2 low expression group (93.33%, 71.67%, 56.67%, 51.67% and 50.00%).

CONCLUSION:

SRSF2 is highly expressed in hepatocellular carcinoma and its expression increases with the degree of tumor differentiation and TNM staging. It is related to lymph node metastasis and metastasis of tumor cells, and is positively related to serum alpha fetoprotein content, and affects the postoperative survival time of HCC patients.

Keywords

SRSF2 hepatocellular carcinoma immunohistochemistry clinical pathology

Table 1
The base clinical data of the HCC patients

Indexes	Number/case	Proportion/%
Gender
Male	113	73.86
Female	40	26.14
Age (years)
$\geqslant$ 65	26	16.99
$<$ 65	127	83.01
Tumor size (cm)
$\leqslant$ 5	68	44.44
$>$ 5	85	55.56
Differentiation degree
Well differentiated	38	24.84
Moderately differentiated	69	45.10
Poorly differentiated	46	30.06
T stage
T1/T2	64	41.83
T3/T4	89	58.17
N poorly differentiated
N0	80	52.29
N1	73	47.71
M stage
M0	103	67.32
M1	50	32.68
TNM stage
I $+$ II	59	38.56
III $+$ IV	94	61.44
$+$	77	50.33
$-$	76	49.67
AFP (ng/ml)
$<$ 20	28	18.30
$\geqslant$ 20	125	81.70

1. Introduction

Alternative splicing (AS) refers to the process in which different mRNA splicing isoforms are produced through alternative splicing of the same mRNA precursor (different combinations of alternative splicing sites), and it plays a significance role in proteome diversity [1]. Most splicing factors are RNA binding proteins, which affect protein expressions by selection of different splicing sites, thus leading to alterations in protein expression levels and their functions [2]. In recent years, many studies [3, 4, 5] have pointed out that abnormal AS exists widely in many types of cancer cells, which significantly impacts cell cycle regulation, cell migration/invasion and other biological characteristics of cancer cells. More importantly, the specific splicing factors that are usually up-regulated in cancer cells not only promote cancer cell survival and disease progression, but also may be of predictive value on the prognosis of the patients [6, 7]. Lack of safe, efficient and easily controllable vehicles may be a key barrier to the development of gene therapy. Non-viral systems such as p53 gene delivery using non-viral methods attract more and more attention, especially chemical approaches [8]. Acid-labile amidized cationic polymers could make the polymers inert in the bloodstream but activate to recover such abilities as cellular ad- sorptive and endo/lysosome-lysing ability in the target sites by responding to the pH gradients in the tumoral interstitium or endo/lysosomes [9, 10]. Serine/arginine rich splicing factor 2 (SRSF2) protein is an important member of the Serine/arginine rich (SR) protein family. It plays an important role in transcriptional activation, RNA stability and mRNA translation through binding with enhancer sequences and acting as a splicing activator. Studies [11] have shown that the abnormal expression of SRSF2 is related to the genesis, development and adverse prognosis of myelodysplastic syndromes. In addition, animal experiments [12] have shown that SRSF2 plays a key role in liver development, and that the abnormal expression of SRSF2 leads to the death of the mice due to acute liver failure. However, there have been few reports on the expression of SRSF2 in human hepatocellular carcinoma (HCC) tissues and the correlation of its expression with clinical pathology or prognosis. In this study, we examined the expression level of SRSF2 protein in HCC tissues and the normal adjacent tissues through immunohistochemistry and Western blots, and analyzed the correlation between SRSF2 expression and hepatoma cell differentiation, histological grading as well as other clinic pathological features, thus providing certain theoretical basis for further studies of HCC pathogenesis and the development of new targeted drugs.

2. Materials and methods

2.1 Clinical specimens

Specimens of HCC tissues and the adjacent normal tissues were collected from 153 patients with hepatocellular carcinoma between January 2010 and May 2012 at Qilu Hospital Affiliated to Shandong University. None of the patients were treated with chemoradiotherapy or biological therapy before the resection, and all of them were diagnosed as HCC by pathological examination. The clinical data of the patients were shown in Table 1.

After the 153 specimens of HCC tissues and the adjacent normal tissues were removed by surgery, an appropriate proportion of each specimen was sent to the pathology department to be made into paraffin sections, while the remaining tissues with the size of a soybean were directly frozen in liquid nitrogen. This study was approved by the ethics Association of Qilu Hospital Affiliated to Shandong University. All the patients have signed informed consents and volunteered to join the study.

2.2 Reagents and instruments

Immunohistochemistry kit (BOSTER, Wuhan, China); Tissue total protein extraction kit (Sangon Biotech, Shanghai, China); Tissue total RNA extraction kit (QIGEN, Dusseldorf, Germany); Reverse-transcription kit (Takara, Tokyo, Japan); SRSF2 and GAPDH rabbit monoclonal antibodies, goat anti-rabbit secondary antibody (Abcam, Cambridge, USA).

2.3 Immunohistochemistry

The paraffin sections were subjected for immunohistochemical detection of SRSF2 expressions according to the instructions in the commercial kit. PBS was used as the negative control antibody. At least 5 visual fields were selected for assessment in each paraffin section, and the proportion of positive cells and the dyeing conditions were recorded (SRSF2 was expressed only in the nucleus). Immunohistochemical scoring criteria: the proportion of positive cells $\leqslant$ 10% $=$ 0 point, 10%–20% $=$ 1 point, 20%–50% $=$ 2 points, 50%–75% $=$ 3 points, $\geqslant$ 75% $=$ 4 points; Dyeing was colorless $=$ 0 point, canary yellow $=$ 1 point, pale brown $=$ 2 points, dark brown $=$ 3 points; Final score for each visual field $=$ the score of the proportion of positive cells X the score of dyeing conditions. The final score of each specimen (A) was determined as the average of the scores of all the visual fields. A $=$ 0–2 was assessed as negative expression, A $=$ 3–5 was assessed as weakly positive expression, A $=$ 6–8 was assessed as positive expression, and A $=$ 9–12 was assessed as strongly positive expression.

2.4 Western blot assay

The specimens frozen in liquid nitrogen were subjected for tissue total protein extraction by the commercial kit. Proteins were loaded for SDS-PAGE and transferred to a wet NC membrane (400 mA, 1.5 h). The membrane was blocked with 5% no-fat milk (1 h at room temperature), incubated with primary antibodies SRSF2 and GAPDH (ab204916 and ab9484, 4 ${{}^{\circ}}$ C overnight) and secondary antibodies (1 h at room temperature) before image development. The band density was analyzed by Image-J software.

Figure 1.

The expression of SRSF2 in HCC tissues and the adjacent normal tissues. A–E: immunohistochemistry detection of the strongly positive expression (A), positive expression (B) and weakly positive expression (C) of SRSF2 in HCC tissues, as well as the weakly positive expression (D) and negative expression (E) of SRSF2 in the adjacent normal tissues; (F) Van chart statistics of the immunohistochemical score of SRSF2 in different tissues; $P<$ 0.05 indicated significant differences between groups. (200 $\times$ ).

Figure 2.

The relationship between SRSF2 expression and clinicopathological features in HCC tissues. Legends: A: 4 cases of well-differentiated (WD) HCC tissues, moderately differentiated (MD) HCC tissues or poorly differentiated (PD) HCC tissues, respectively; B: 4 cases of HCC tissues with non-lymph node metastasis (NLNM) and lymph node metastasis (LNM), respectively; C: 4 cases of HCC tissues with no distant metastasis (NDM) or distant metastasis (DM), respectively; D: 4 cases of HCC tissues at TNM stage I, TNM stage II, TNM stage III or TNM stage IV, respectively. E: 4 cases of the negative expression of SRSF2 in paracancerous tissue (PT).

2.5 Statistical analysis

Statistical analysis was performed using the SPSS 20.0 statistical program. $t$ test was used to analyze the differences between the measurement data groups, while chi-square test was used to compare the differences between the enumeration data groups. Pearson analysis was used to examine the correlation between two indexes. $P<$ 0.05 was considered as statistically different.

3. Results

3.1 Clinical data of the 153 HCC patients

Among the 153 HCC patients, 113 were male and 40 were female; their age ranged from 34 to 75 years, with the mean age of (50.6 $+$ 10.2) years; 38 patients were with high differentiation, 69 patients were with middle differentiation and 46 patients were with moderate differentiation; tumor, node, metastasis (TNM) staging showed 26 cases at T1 phase, 38 cases at T2 phase, 33 cases at T3 phase and 56 cases at T4 phase; the serum alpha fetoprotein levels in 28 patients were lower than 20 $\mu$ g/L. Other general information was shown in Table 1.

3.2 Immunohistochemistry detection of SRSF2 protein expressions

The expressions of SRSF2 protein in the 153 cases of HCC tissues and the adjacent normal tissues were detected and scored by immunohistochemistry. The results showed that all the HCC tissues contained SRSF2 positive cells without any negative expression; the adjacent normal tissues showed no strongly positive or positive expressions of SRSF2, and all of them showed weakly positive or negative expressions of SRSF2; the immunohistochemical score of SRSF2 in HCC tissues was (6.50 $+$ 2.82), which was significantly higher than that in the adjacent normal tissues (2.94 $+$ 1.23) (Fig. 1, $P<$ 0.05).

3.3 The relationship between SRSF2 expression and the clinic pathological features of HCC tissues

The expressions of SRSF2 protein in the 153 cases of HCC tissues were detected and scored according to the procedures described in 1.3, and those with the immunohistochemical score $\geqslant$ 6 points were considered of high expression. The results showed that among the 153 HCC tissues, there were 93 cases with SRSF2 high expression and 60 cases with SRSF2 low expression; there was no significant difference between the HCC patients with SRSF2 high expression and those with SRSF2 low expression in gender ( $\chi^{2}$ $=$ 0.014, $P=$ 0.906), age ( $\chi^{2}=$ 0.007, $P=$ 0.931), tumor size ( $\chi^{2}=$ 3.566, $P=$ 0.059) or T staging ( $\chi^{2}=$ 2.708, $P=$ 0.100), while there were significant differences between the two groups in the degree of tumor differentiation ( $\chi^{2}=$ 9.687, $P=$ 0.007), lymph node metastasis ( $\chi^{2}=$ 4.827, $P=$ 0.028), distant metastasis ( $\chi^{2}=$ 9.235, $P=$ 0.002), TNM staging ( $\chi^{2}=$ 3.978, $P=$ 0.046), portal vein invasion ( $\chi^{2}=$ 5.931, $P=$ 0.015) and serum alpha-fetoprotein (sAFP) content ( $\chi^{2}=$ 14.919, $P=$ 0.000), as shown in Table 2; the expression level of SRSF2 protein in HCC tissues was positively correlated with the degree of tumor differentiation as well as TNM staging, and was higher in the HCC tissues with lymph node metastasis than those without metastasis, higher in the HCC tissues with distant metastasis than those without distant metastasis, as well as higher in the HCC tissues with portal vein invasion than those without invasion, as shown in Fig. 2.

Table 2
Immunohistochemistry detection of the relationship between SRSF2 protein expression and clinical pathology

Indexes	SRSF2		$\chi^{2}$	P
	High	Low
	expression	expression
	(case)	(case)
Gender
Male	69	44	0.014	0.906
Female	24	16
Age (year)
$\geqslant$ 65	16	10	0.007	0.931
$<$ 65	77	50
Tumor size (cm)
$\leqslant$ 5	47	21	3.566	0.059
$>$ 5	46	39
Differentiation degree
Well differentiated	32	6	9.868	0.007
Moderately differentiated	41	28
Poorly differentiated	20	26
T stage
T1/T2	34	30	2.708	0.100
T3/T4	59	30
Lymph node metastasis
Non-metastasis	42	38	4.827	0.028
Metastasis	51	22
Distant metastasis
Non-metastasis	54	49	9.235	0.002
Metastasis	39	11
TNM stage
I $+$ II stage	30	29	3.978	0.046
III $+$ IV stage	63	31
Portal vein invasion
$+$	57	20	5.931	0.015
$-$	36	30
AFP (ng/ml)
$<$ 20	8	20	14.919	0.000
$\geqslant$ 20	85	40

Figure 3.

Correlation between the expression of SRSF2 protein in HCC tissues and the serum alpha-fetoprotein (AFP) level.

Figure 4.

Relationship of SRSF2 expression and the postoperative survival rates of HCC patients.

3.4 Correlation between the expression of SRSF2 protein and the serum alpha-fetoprotein (AFP) level

Western blot was performed to determine the relative expressions of SRSF2 protein in the 153 cases of HCC tissues, and SPSS20.0 was used to analysis the correlation between SRSF2 expression in HCC tissues and the level of serum AFP in the patients by Pearson methods. The results showed that the relative expression of SRSF2 in HCC tissues was positively correlated with the level of serum AFP ( $r=$ 0.704, $P<$ 0.05). Please see Fig. 3.

3.5 Relationship between SRSF2 expression and prognosis in HCC patients

The 153 HCC patients were followed up for 5 years after operation, and their postoperative survival time was recorded. The results showed that the 1-year, 2-year, 3-year, 4-year and 5-year survival rates of the patients with SRSF2 overexpression were 74.19%, 44.09%, 26.88%, 24.73% and 21.51%, respectively, while the 1-year, 2-year, 3-year, 4-year and 5-year survival rates of the patients with SRSF2 low expression were 93.33%, 71.67%, 56.67%, 51.67% and 50.00%, respectively; suggesting that the HCC patients with SRSF2 low expression were correlated with better survival rates than those with SRSF2 overexpression, as shown in Fig. 4.

4. Discussions

Hepatocellular carcinoma is the fifth most common malignant cancer ranking after lung cancer, gastric cancer, esophageal cancer and breast cancer. There are a large number of HBV infected people in China, making it with high incidence of hepatocellular carcinoma. New HCC cases account for about 55% in the world every year [13]. According to the statistics, although the 1-year survival rate after HCC radical resection has increased from 39.3% to 87%, the 5-year postoperative survival rate is still only 15%–40%, and metastasis of cancer cells is one of the major causes of adverse prognosis. A number of studies on the molecular mechanism of cancer cell invasion and migration have indicated that the aberrant overexpression of splicing factors plays an important role in promoting the invasion/migration ability of cancer cells [3, 4, 5]. SR protein family is the most well-studied splicing factors: Li et al. [14] point out that SRSF10 regulates the production of lipin1 $\alpha$ , the most important regulator in adipogenesis, thus promoting adipocyte differentiation, and therefore is among the necessary proteins during adipocyte differentiation. SRSF2 is an important member of the SR protein family, which is localized on the human chromosome 17q25.1 and is encoded by 221 amino acids [11]. In this study, we found that the expression of SRSF2 in the 153 cases of HCC tissues was significantly higher than that in the adjacent normal tissues ( $P<$ 0.05), which was consistent with the results of Roessler et al. [15]. SRSF2 protein is widely expressed in many tissues and organs of the human body. In the liver, SRSF2 protein maintains the homeostasis of liver function by direct regulation of the transcription of multiple enzymes and metabolic transcription factors. Mice with liver-specific knockout of SRSF2 not only showed jaundice and morphological changes of hepatocytes, but also exhibited metabolism disorders of the liver, resulting in death of the mice in severe cases. These changes may be related to endoplasmic reticulum stress and oxidative stress in hepatocytes induced by SRSF2 deletion [12]. From these studies we can conclude that the abnormal expression of SRSF2 in the liver may be involved in the genesis and development of liver diseases through changes of the stress reactions in hepatocytes. Stress reactions are the major source of genomic instability, which is directly related to tumorigenesis [16]. This indicates that abnormal expression of SRSF2 may be one of the major molecular mechanisms underlying hepatocarcinogenesis.

We further analyzed the relationship between SRSF2 expression and the clinicopathological features of HCC patients, and found that the expression of SRSF2 in HCC tissues was not correlated to sex, age, tumor size or T staging. However, the expression of SRSF2 in HCC tissues increased with the status of tumor differentiation as well as TNM staging, and was significantly increased in the HCC tissues with lymph node metastasis, distant metastasis or portal vein invasion, suggesting that overexpression of SRSF2 may be involved in the progression of HCC. In addition, Pearson correlation analysis showed that the relative expression of SRSF2 protein in HCC tissues was positively correlated with serum alpha fetoprotein ( $r=$ 0.704, $P<$ 0.05). AFPs are generated from embryonic liver cells, and the content of AFP in normal human serum is less than 20 ug/L. When hepatocytes become malignant, approximately 80% of HCC patients will restore the synthesis of alpha fetoproteins, which increase rapidly in the blood with the deterioration of HCC, and therefore can be used as one of the major specific indicators for the clinical diagnosis of HCC [17, 18]. High expression of AFP can promote the proliferation of HCC cells by activating the PI3K/Akt signaling pathway [19]. Chemotherapy drugs can reduce the content of AFP in the serum of HCC patients, thus improving the sensitivity of HCC cells to chemotherapeutic drugs as well as inhibiting the growth, invasion and migration of HCC cells [20]. The expression of SRSF2 was positively correlated to serum AFP content, suggesting that SRSF2 may participate in the progression of HCC by regulating the synthesis of AFP in hepatocytes.

Previous studies have pointed out that the abnormal expression of splicing factors is not only related to the genesis and development of multiple diseases, but also has certain predictive value for the prognosis of patients [6, 7]. In this study, we followed up the 153 HCC patients for 5 years, and found that the survival rates of the 93 HCC patients with high SRSF2 expressions were significantly lower than those of the 60 patients with low SRSF2 expressions 1–5 years post operation, suggesting that the expression of SRSF2 in HCC tissues could affect the postoperative survival of HCC patients.

To sum up, SRSF2 was overexpressed in HCC tissues. Its expression increased with tumor differentiation and TNM staging, and was related with lymph node metastasis as well as distant metastasis of tumor cells. Moreover, the expression of SRSF2 was positively correlated with serum content of alpha fetoprotein, and could affect the length of postoperative survival in HCC patients.

Footnotes

Acknowledgments

This research was supported by the Fund of the Key Research and Development Program of Shandong Province (2016GSF201180).

Conflict of interest

None declared.

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Serine/arginine rich splicing factor 2 expression and clinic pathological features indicating a prognostic factor in human hepatocellular carcinoma patients

Abstract

BACKGROUND:

METHODS:

RESULTS:

CONCLUSION:

Keywords

Table 1 The base clinical data of the HCC patients

2. Materials and methods

2.1 Clinical specimens

2.2 Reagents and instruments

2.3 Immunohistochemistry

2.4 Western blot assay

3. Results

3.1 Clinical data of the 153 HCC patients

3.2 Immunohistochemistry detection of SRSF2 protein expressions

3.3 The relationship between SRSF2 expression and the clinic pathological features of HCC tissues

Table 2 Immunohistochemistry detection of the relationship between SRSF2 protein expression and clinical pathology

3.5 Relationship between SRSF2 expression and prognosis in HCC patients

4. Discussions

Footnotes

Acknowledgments

Conflict of interest

References

Table 1
The base clinical data of the HCC patients

Table 2
Immunohistochemistry detection of the relationship between SRSF2 protein expression and clinical pathology